Scientific Report 2003-2004 - Cleveland Clinic Lerner Research ...

From the Chairman, Board of GovernorsThe Cleveland Clinic Foundation’s Lerner Research Institute continues to exemplify the finest tradition of collaboration andcollegiality in the service of science and medicine. In 2002, the Lerner Research Institute was engaged in hundreds of investigativeprograms whose results will eventually impact medical knowledge and patient care. With this Scientific Report, we review and acknowledgethe importance of these endeavors and recognize the breadth and diversity of what is accomplished by our honored research group.The year 2002 saw a change of leadership in the Lerner Research Institute. Paul E. DiCorleto, Ph.D., chairman of the Departmentof Cell Biology, was appointed as the new chairman. George R. Stark, Ph.D., chairman for the past ten years, is now “DistinguishedScientist at The Cleveland Clinic,” concentrating his efforts on molecular biology research.Over the past decade, the Lerner Research Institute has become the fifth largest research institute in United States and a leadingrecipient of NIH and other prestigious grant awards. We look forward to an even higher national profile in coming years. To accomplishthis, we anticipate closer collaboration with outside partners who share our values and zeal for the translation of scientific knowledge tothe clinical realm.In 2002, The Cleveland Clinic was the grateful recipient of a $100 million gift for research and education from philanthropicpartners Alfred (1933-2002) and Norma Lerner and the Lerner family. Thanks to this outstanding generosity, we will be able to enhanceour research infrastructure and pursue a broader range of educational programs. Most notable among these will be the new ClevelandClinic Lerner College of Medicine of Case Western Reserve University, announced in 2002.The first new medical school to be founded in the United States in the past 25 years, the Lerner College of Medicine represents amajor collaboration between one of the nation’s top academic medical centers and a medical school renowned for its history of innovationin medical education and research. The College will address the critical shortage of qualified physician researchers through a curriculumthat integrates laboratory work with clinical experience to produce researchers who are also excellent patient care providers. The first classof 32 highly qualified candidates will be enrolled in 2004.The Lerner Research Institute and its staff will make an important contribution to the strength of the school’s academic program.Each area has a common commitment to results and the rapid movement of scientific discovery to clinical practice. Each, through its hardwork and passion for knowledge, contributes to the overall cause of health and medicine around the world.Sincerely,Floyd D. Loop, M.D.Chairman, Board of GovernorsThe Cleveland Clinic Foundation5

Paul E. DiCorleto, Ph.D.–The Lerner Research Institute Chairman’s OverviewPaul E. DiCorleto, Ph.D.Chairman,Lerner Research InstituteMy first year as Chairman of the Lerner Research Institute (LRI) has been both exciting andextremely positive. It has been a great privilege and honor to succeed George R. Stark, Ph.D., asChairman. Dr. Stark headed the LRI from 1992 until August 2002, a decade that will beviewed as a period of great growth of our institution, both in personnel and facilities. Dr. Starkadvanced the tradition and vision established and fostered by Irvine Page, Merlin Bumpus, and BernadineHealy, whose leadership shaped the Division of Research of the Cleveland Clinic Foundation (CCF) intothe modern, world-class institution that the LRI is today. Dr. Stark remains a very active member of ourfaculty as CCF’s first Distinguished Scientist.During the past year our research portfolio and external support has continued to climb dramaticallyand our number of publications in the highest impact, peer-reviewed journals has increased onceagain. With our growth during the past year, the LRI is now home to over 1,000 employees, including132 principal investigators, 166 junior faculty, 250 postdoctoral fellows, 140 predoctoral and undergraduatestudents, and 350 technologists and support personnel. All are contributing greatly to theresearch mission of CCF.A major event for the LRI and all of CCF in 2002 and 2003 has been the creation and developmentof the Cleveland Clinic Lerner College of Medicine (CCLCM). As discussed in greater detail below,this collaboration with Case Western Reserve University (CWRU) will create an unprecendented andextraordinary opportunity for advancement of both science and patient care and meshes beautifully withCCF’s mission to provide compassionate healthcare of the highest quality in a setting of education andresearch.Growth in our Research MissionThe opportunities to conduct world-class research at the LRI are built upon our success inexternal funding and the continuing internal support from CCF. External grant support continued to riseat double-digit rates. In 2002, NIH multi-year awards increased from 150 to 187--an 18.7% increase.The 2002 NIH award commitment increased by $7.6 million to a total of $55.1 million, 16% more thanthe previous year. Total external funding from all sources exceeded $100 million. CCF researchers wereawarded 47 new and renewed multi-year grants of more than $1 million each from external fundingContinued on Page 7NIH Grant Awards to CCF$60$50Dollars(millions)$40$30$20$10$019861987198819891990199119921993199419951996199719981999200020012002Year6

Continued from Page 6sources. The principal investigators who received the greatest amount ofnewly awarded NIH grant funding during 2002 were:• D. Geoffrey Vince, Ph.D., Biomedical Engineering, received a $4.3 millioncommittment over five years to pursue improvements in real-time ultrasounddetection and analysis of vulnerable atherosclerotic plaque.• George Muschler, M.D., Orthopaedic Surgery and Biomedical Engineering,received a five-year grant for $4.1 million to investigate the biologyof, and applications for, stem-cell therapy in bone reconstruction.• Stanley Hazen, M.D., Ph.D., Cell Biology, received two R01 grants totaling$3.0 million over five years to investigate the role of oxidation processesin the pathogenesis of two inflammatory diseases–asthma and atherosclerosis.Bruce R. Trapp, Ph.D., recipientof the 2003 CCF ScientificAchievement Award in BasicResearch• Bruce Trapp, Ph.D., Neurosciences, received $2.8 million toexpand upon his contributions in the area of neuron demyelinationand degeneration in multiple sclerosis. Dr. Trapp receivedthe Jacob Javits Award during 2002. This award willprovide an additional three years of funding, for a total of sevenyears of support. The Javits Award is the most prestigious honor bestowedby the National Institute of Neurological Disorders andStroke in support of biomedical research on the brain.The LRI added to its technological base during this past yearas well, using both CCF and NIH support. NIH Shared Equipmentgrants provided $2.8 million for major capital items. These includedthe purchase of a 600 MHz NMR spectrometer and a $1.5 millioncontribution toward a 900 MHz NMR spectrometer that will behoused in the Cleveland Center for Structural Biology. We alsopurchased a BIACORE 3000 instrument for the analysis of kineticand affinity parameters of biomolecule interactions, and a roboticssystem for use in proteomic approaches. In addition, we are in thefinal stages of construction of a new vivarium, started in 2002,which will expand our animal facilities by ~50,000 square feet and isscheduled to be opened in late Fall of 2003. This new space has beensorely needed to meet the growing demands of our investigators for additional mouse facilities.CCF scientists are an integralpart of the new ClevelandCenter for RegenerativeMedicine, a Northeast Ohioconsortium dedicated toadvancing stem-cell-basedtechnologies and therapies.The Center’s researchprogram has been funded by a$19.4 million grant from theState of Ohio to supportinvestigations such as thedevelopment of bone replacementcells from adult stemcells, the work of GeorgeMuschler, M.D., OrthopaedicSurgery and Department ofBiomedical Engineering.New Faculty in the LRIIn 2002, we successfully recruited Peter Cavanagh, Ph.D., from Penn State University to bethe new Chairman of the Department of Biomedical Engineering, our largest and most diverse department.Dr. Cavanagh was a Distinguished Professor of Kinesiology, Biobehavioral Health, Medicine, andOrthopaedics and Rehabilitation at Penn State. He was also the Director of the Penn State UniversityCenter for Locomotion Studies and Research Director of the Penn State Diabetes Foot Clinics. Dr.Cavanagh is actively involved in the American Diabetes Association and has served as national Chair ofthe ADA Council on Foot Care. He has been President of both the American and International Societiesof Biomechanics.Dr. Cavanagh’s research interests are presently focused on the lower extremity complications ofdiabetes and on bone mineral loss during long-term space flight. He has received numerous internationalawards for excellence and contributions to the science of biomechanics. In 1994, he received theBorelli Award, the highest honor awarded by the American Society of Biomechanics. He is a 1987recipient of the International Society of Biomechanics’ highest award, the Muybridge Medal, and haspresented the Wolffe and Dill Lectures to the American College of Sports Medicine. In 2002 he wasgiven the Pecoraro Award by the American Diabetes Association Foot Council.We are currently focused on recruiting individuals to chair the LRI Cell Biology Department,CCF’s Human Genetics/Genomics Institute, and a director for the Taussig Cancer Center. In addition,the CCF Board of Governors has approved the creation of a new Department of Stem Cell Biology andContinued on Page 87

Continued from Page 7Regenerative Medicine in the LRI, as discussed in greater detail below. A search for the inaugural chairof this department will begin shortly.Since our last report, six scientists have joined the Staff of the LRI. They are:• Peter Chumakov, Ph.D., Molecular Biology, from University of Illinois, Chicago• Cameron McIntyre, Ph.D., Biomedical Engineering, from Emory University, Atlanta, GA• Philip Pellet, Ph.D., Virology/ Molecular Biology, from the CDC in Atlanta, GA• Victor Perez, Ph.D., Ophthalmic Research, from the Massachusetts Eye and Ear Infirmary,Boston, MA• Jawhar Rawwas, M.D., Cancer Biology, from the Parker Hughes Cancer Center, St. Paul, MN• Jonathan Smith, Ph.D., Cell Biology, from the Rockefeller University, New York, NYScientific Progress and Programmatic Research InitiativesMembers of the ProstateCancer Group, the recipientsof the 2002 Cleveland ClinicResearch Program of the YearAward. See Pages 56-57.In 2002, LRI researchers published more than 500 peer-reviewed journal articles with about 10%appearing in journals designated among the top 15 “high-impact” science publications. These includedamong others Science, Cell, Nature, Nature Genetics and The New England Journal of Medicine.CCF’s thirty-three disease-focused research programs have been making great gains with increasedcommunication between basic scientists and clinical researchers, multiple new program grant applicationssubmitted, and many new CCF-sponsored national symposia. The first CCF “Research Program of theYear” Award was given for accomplishments in 2002. The award went to the Prostate Cancer Researchteam.Familial aggregation of prostate cancer has been appreciated for the pasthalf century, but it is only recently that progress was made in understanding thegenetic basis of familial forms of the disease. CCF scientists and cliniciansfeatured prominently in these recent advances, including Robert Silverman,Ph.D., Cancer Biology, who is the foremost expert on RNase L, a protein knownto cleave RNA and render it useless. In mammalian cells, RNase L suppressesviral replication as part of the interferon pathway. In a 2002 Nature Geneticsarticle by Carpten et al., germline mutations in the RNase L gene indicated linkagewith the hereditary prostate cancer 1 gene. Dr. Silverman’s research teamdemonstrated RNase L deficiencies in patients in this study, which was orchestratedat NIH by Dr. Jeffrey Trent. Later in 2002, a follow-up Nature Geneticsarticle by Graham Casey, Ph.D., et al., demonstrated that a particular RNase Lvariant is responsible for up to 13% of all prostate cancer cases. Dr. Casey is acolleague of Dr. Silverman’s in the Department of Cancer Biology. They workedclosely together in the study with Eric A. Klein, M.D., Glickman UrologicalInstitute.The Prostate Cancer team is pursuing 11 projects in a multi-departmental effort against thisdisease. The team is led by Skip Heston, Ph.D., Director, the George M. O’Brien Research Center forProstate Cancer, and Eric A. Klein, M.D., Head, Section of Urologic Oncology at the Clinic’s GlickmanUrological Institute. Contributing scientists included Andrei Gudkov, Ph.D., Chair, Molecular Biology,Edward Plow, Ph.D., Chair, Molecular Cardiology, Tatiana Byzova, Ph.D., Molecular Cardiology;Graham Casey, Ph.D., Robert Silverman, Ph.D., and Yan Xu, Ph.D., Cancer Biology; Robert Dreicer,M.D., Hematology and Medical Oncology; Jay Ciezki, M.D., and Arul Mahadevan, M.D., RadiationOncology; Jennifer Brainard, M.D., and Howard Levin, M.D., Anatomic Pathology; and RaymondTubbs, D.O., Chairman, Clinical Pathology.New Scientific PartnershipsWe have continued to build strong research collaborations with other academic institutions, aswell as corporations, within the region and around the world. CCF embraced a significant opportunityfor collaboration with CWRU, University Hospitals (UH), industrial partner Athersys, Inc., and otherorganizations to establish the Center for Stem Cell and Regenerative Medicine (CSCRM). This visionary8Continued on Page 9

Continued from Page 8initiative received $19.4 million in State of Ohio funding for the developmentof stem cell technology and its commercialization. The director of theCenter is Stanton Gerson, M.D., head of Hematology/Oncology at CWRUand UH and I am the current CCF co-director, until we recruit a chair forthe new Department of Stem Cell Biology and Regenerative Medicine in theLRI.CSCRM will initially focus on the use of adult stem cells to treatdiseases of the cardiovascular, hematopoietic, neurological, and musculoskeletalsystems. The State funds coming to CCF, which amount to ~ $9.3million, will be used in part to create laboratory space for the new stem cellbiology department. CCF scientists who are among the key co-investigatorsin establishing the center include Wendy Macklin, Ph.D., Department ofNeurosciences; Ronald Midura, Ph.D., Department of BiomedicalEngineering; George Muschler, M.D., Departments of OrthopaedicSurgery and Biomedical Engineering; Marc Penn, M.D., Ph.D., Departmentsof Cardiovascular Medicine and Cell Biology; Eric Topol, M.D.,Chief Academic Officer and Provost, Chair of the Department of CardiovascularMedicine, and Bruce Trapp, Ph.D., Chair, Department ofNeurosciences.I am also pleased to report that a new partnership has been formedbetween CCF and Technion University, Haifa, Israel. Five collaborativeresearch projects are being supported for two years by a philanthropist whois a great friend to both institutions, Mr. Stanley Zielony. These projects arefocused on translational research areas in the fields of cardiovascular andmusculoskeletal research.Partnering with CCF Innovations to CommercializeLRI DiscoveriesWith the help of CCF Innovations, directed by Christopher Coburnand Joseph Hahn, M.D., we have boosted levels of licensing and theestablishment of CCF spin-off companies to advance the discoveries madewithin our laboratories. In 2002, CCF staff disclosed 113 new inventions–an all time record, and CCF ranked first in Northern Ohio among majoracademic centers and in the top third nationally in commercializationrevenues (dollars generated per dollar of sponsored research). CCF secured$3.4 million in commercialization revenue and distributed $1.5 million to 32CCF inventors. CCF Innovations raised or facilitated $4.25 million in equityinvestments in CCF spin-offs and partner companies.Approximately $1.3 million in grants were secured from the State ofOhio, the Generation Foundation and the Codrington Foundation topromote the commercialization of CCF technologies. CCF Innovations hascontributed to the recruitment of several biotechnology companies thatrelocated to Northeast Ohio, including Quark Biotech and Simbionix, amedical technology company. In addition, three companies were launchedbased on intellectual property developed at CCF.LRI Educational MissionThe creation of the Cleveland Clinic Lerner College of Medicine(CCLCM) during this past year is truly an historic event for CCF. By creatingthis unique, specialized medical school program, CCF and CWRU are helpingto resolve a major national concern–the shortage of practicing physicianswho have a significant commitment to biomedical research. Staff membersfrom the LRI have been working with clinical colleagues to pioneer theContinued on Page 109

10Continued from Page 9development of a research-based curriculum that will be the hallmark of the CCLCM. Our goal is toeducate physician-scientists using a medical training format that more approximates graduate training inthe biomedical sciences than the classical medical school program. A new Department of MolecularMedicine was created within the CWRU School of Medicine to provide an administrative home for theLRI faculty contributing to the development and teaching of the innovative CCLCM curriculum.Though much effort has been extended toward the CCLCM, we have also moved forward withour graduate and postdoctoral training programs. In 2002, we recruited Marcia Jarrett, Ph.D. todirect the new Research Education Office in the LRI. This office facilitates recruitment of postdoctoraland predoctoral candidates to the LRI and enhances training, and ultimately placement, opportunities forpostdoctoral fellows. The office, including the website with a continuously updated job posting board, isa great resource for fellows and graduate students. Currently, we have ~120 graduate students with mostpursuing degrees at three area universities: Case Western Reserve University, Cleveland State University,and Kent State University.Our programs to generate excitement about biomedical science among younger students continueto draw from both private and public high schools in the area. The Clinic’s partnership with John HayHigh School and the Cleveland City School District creates opportunities for minority students toembrace science and research as a career opportunity. This is a nationally recognized program that hasflourished with funding from the NIH (1997-1999) and more recently the Howard Hughes MedicalInstitute (1999 through 2003).Challenges and Opportunities AheadThe future challenges that we face also represent opportunities to continue CCF’s climb into theupper echelon of academic biomedical research institutions. As individual scientists and as members ofteams, our future success is in our own hands. In the face of the reduced rate of NIH budget growth in2004 and beyond, we will need higher priority scores to obtain new grants and to be successful with ourcompetitive renewal applications. We willneed to pursue all relevant programsoffered by NIH, e.g., RFA’s, SPOR’s andprogram project grant applications. Wewill also need to look for alternativefunding sources and to partner with ourclinical colleagues in presenting comprehensiveand highly attractive programs forprivate companies and foundations tosupport. Senior members of the LRI mustbe committed to mentorship of juniorcolleagues to expedite the transition ofthese new faculty from seed funding toexternal support and independent status.While the LRI faces funding andother significant challenges in the nearfuture, I remain extremely optimistic. Wehave a great model for biomedicalresearch, just as our CCF clinical colleagueshave a great model for the practiceof medicine. When we come together, thesynergy benefits all. We also have theunwavering support of CCF leadershipand a stated commitment that the researchThe Lerner Research Institute Management Team: (first row from left) Bruce Trapp, Ph.D.,Chair, Neurosciences; Amiya Banerjee, Ph.D., Head, Section of Virology; Paul DiCorleto,Ph.D., Chair, LRI; Edward Plow, Ph.D., Chair, Molecular Cardiology; Thomas Hamilton,Ph.D., Chair, Immunology; Bryan Williams, Ph.D., Chair, Cancer Biology; Guy Chisolm,Ph.D., Interim Chair, Cell Biology; Andrei Gudkov, Ph.D., Chair, Molecular Biology; andremains a top priority of the institution.My enthusiasm and personal commitmentis further fueled by the outstandingperformance that I observe every day bythe hundreds of individuals that make upthe Lerner Research Institute.

BiomedicalEngineering

THE DEPARTMENT OFBIOMEDICALENGINEERINGCHAIRMANPeter R. Cavanagh, Ph.D.The Virginia Lois Kennedy ChairSTAFFLeonard A.R. Golding, M.D.Mark D. Grabiner, Ph.D.Linda M. Graham, M.D.Vincent C. Hascall, Ph.D.Raymond J. Kiraly, Ph,.D.*Cahir A. McDevitt, Ph.D.George F. Muschler, M.D.Ivan Vesely, B.E.Sc., Ph.D.ASSOCIATE STAFFSuneel S. Apte, M.B.B.S., D.Phil.Brian L. Davis, Ph.D.Ronald J. Midura, Ph.D.Guang H. Yue, Ph.D.Maciej Zborowski, Ph.D.ASSISTANT STAFFR. Tracy Ballock, M.D.Elizabeth Fisher, Ph.D.Aaron J. Fleischman, Ph.D.Melissa L. Knothe Tate, Ph.D.Véronique Lefebvre, Ph.D.Edward V. Maytin, M.D.Cameron C. McIntyre, Ph.D.Kimerly A. Powell, Ph.D.Shuvo Roy, Ph.D.William A. Smith, D.Eng., P.E.Antonie J. van den Bogert, Ph.D.D. Geoffrey Vince, Ph.D.PROJECT STAFFAnthony Calabro, Ph.D.Fernando Casas, Ph.D.Scott M. Colles, Ph.D.Susan E. D’Andrea, Ph.D.Kathleen A. Derwin, Ph.D.Kiyotaka Fukamachi, M.D., Ph.D.Csaba Fülöp, Ph.D.Mark S. Goodin, M.S.Helen E. Kambic, Ph.D.*Nikolay Kharin, Ph.D.Jingzhi Liu, Ph.D.Judith A. Mack, Ph.D.Scott McLean, Ph.D.Chizu Nakamoto, Ph.D.Yvonne Shao, Ph.D.Raj Shekhar, Ph.D.Wlodzimierz Siemionow, Ph.D.Aimin Wang, Ph.D.P. Stephen Williams, Ph.D.*Active Emeritus Staff12The Department of Biomedical EngineeringScientists, Engineers, and PhysiciansExploring Biology and MedicineAcross Ten Orders of MagnitudeThe discipline of biomedical engineeringseeks to apply engineering principles tosolve biomedical problems. At CCF, thisembodiment of biomedical engineering is evidentthrough active research programs in biologicalmicroelectromechanical systems (BioMEMS), thedesign and utilization of micro-computedtomography (micro-CT) and cardiac assist devices(including a total artificial heart and leftventricular assist systems), and the developmentof software to identify potentially lethalatherosclerotic plaque. Other studies, whichquantify images of the brain in multiple sclerosis,record and model the brain’s electrical activity,measure bone loss during space flight, studypeople with foot disease caused by diabetes, sortcells using quadrupole magnets, and buildmathematical models of athletes with cruciateligament damage, also represent traditional areasof the discipline. Tissue engineering of heartvalves and modeling of fluid flow in bone furtheremphasize the engineering base.However, the distinguishing characteristicsof CCF’s Department of Biomedical Engineering(BME) are its breadth and its links with bothfundamental biology and clinical medicine. BMEresearchers study the use of stem cells andelectrical stimulation for bone healing, identifymolecular mechanisms responsible for skeletaldevelopment and growth plate pathology, performfundamental studies of connective tissue biologyand matrix metalloproteases, study why vasculargrafts are rejected, and probe the molecularbiology of healing wounds in epidermis, kneemeniscus, and bone. Inventions by departmentmembers have been licensed to major medicalcompanies and are in clinical use or trialsworldwide. The focus of interest in the departmentextends over ten orders of magnitude—from structures of a few angstroms to more thana meter in length, from several DNA base pairs tothe entire human being. Most BME researchershave set their sights on at least one disease processthat will be better understood through theirscientific efforts and clinical activities orcollaborations. In addition to serving the researchcommunity, the department’ssuperb workshops, engineeringstaff, and facilities serve as amagnet for clinicians whowish to develop newengineering approaches totreatment. BME’s MedicalDevice Innovations groupcomprises engineers who workwith staff from manyacademic and clinicaldepartments to advancemedical devices from theconceptual stage to themedical marketplace. Morethan 18 patent disclosureswere filed by departmentmembers in 2002.The Department hasdistinguished roots at theCleveland Clinic in the formerDepartment of ArtificialOrgans, established within theDivision of Surgery in 1954and in which the first artificialkidney was developed. Theprior Department ofMusculoskeletal Research wasan outgrowth of a unit withinthe Department of OrthopaedicSurgery. In 1991,Artificial Organs was mergedwith MusculoskeletalResearch to form the newDepartment of BiomedicalPeter R. Cavanagh, Ph.D.The Virginia Lois Kennedy Chair Continued on Page 13

The Department of Biomedical EngineeringContinued from Page 12Engineering. BME is now the largest departmentof the Foundation’s Lerner Research Institute,with more than 250 personnel in 24 separateresearch laboratories and core facilities. In Fall2002, Peter R. Cavanagh, Ph.D., who studiesfoot disease in diabetes and bone loss duringspace flight, joined the Foundation as the newBME chairman. Dr. Cavanagh has active researchprograms funded by the National Institutes ofHealth and NASA, including a current flightexperiment on the International Space Station.Dr. Cavanagh is also Academic Director of theFoundation’s new Diabetic Foot Care Program,which is bringing amultidisciplinary approach to thecare of diabetic patients at risk forfoot disease and amputation.BME is organized into eightareas of intellectual focus:Biomechanics, Biomedical Devices,BioMEMS and Nanotechnology,Cardiovascular Bioengineering,Imaging, Neural Control, OrthopaedicBiology and Bioengineering,and Tissue Engineering and WoundHealing. Several staff memberscontribute to more than one area.The Orthopaedic Research Center, a jointenterprise between BME and the Department ofOrthopaedic Surgery, is also based in thedepartment. In fiscal year 2002, BME’s incomefrom research grants and contracts was $16.5million, with $12.5 million from federal sources;of that total, $8.8 million came from theNational Institutes of Health. Departmentmembers published more than 100 articles in2002 in journals such as Artificial Organs,Biomedical Microdevices, Clinical Orthopaedics,Computers in Medical Imaging, Gait & Posture,Journal of the American Medical Association, Journalof Biological Chemistry, Journal of Cell Science,Nature Cell Biology, New England Journal ofMedicine, and Science. A strong administrativeteam supports grant preparation and managementand editorial activities; BME’s educationalcoordinator works to integrate a range ofprograms that extend from high-school studentresearch projects, to summer Research Experiencefor Undergraduates programs, and conventionalgraduate education. BME graduatestudents come from the Biomedical Engineeringprogram at Case Western Reserve University(CWRU) or the Applied Biomedical Engineeringprogram at Cleveland State University. BMEstaff are actively engaged in educationalprograms at both institutions. Degree programs atthe M.S. and Ph.D. levels in Biomedical Engineeringare also offered to students enrolled inthe new Cleveland Clinic Lerner College ofMedicine of CWRU.The prime focus of several laboratories isin the area of Biomechanics. Brian Davis,Ph.D., studies lower-extremity biomechanics inrelation to forces applied to muscle, nerve, andbone. For amputee, arthritis and stroke patients,he has also developed an instrumented dual-tracktreadmill to permit real-time monitoring, analysisand rehabilitation of a patient’s gait. Work onstudies of 3-D pressure and shear loading on thediabetic foot vs. footwear and on countermeasuresto bone loss in astronauts are complementaryto those of the Cavanagh laboratory. SusanD’Andrea, Ph.D., is developing an instrumentedtreadmill to challenge thepostural control system ofastronauts as a countermeasureto neurovestibularadaptations ofmicrogravity whileproviding exercise tobenefit the musculoskeletalsystem. The work ofAntonie J. van den Bogert,Ph.D., correlates thedisciplines of mechanicalengineering, neuroscience,applied mathematics, andorthopaedics, using computational models of theneuromusculoskeletal system and novel experimentaltechniques in studying injury prevention,rehabilitation, and prosthetics. These methods areused to study prevention strategies againstanterior cruciate ligament (ACL) rupture and toquantify stability using feedback control ofposture and gait. Future applications involveprevention of falls in the elderly, treatment ofmovement disorders, intelligent prostheses,restoration of function after spinal cord injury,and development of biologically inspired robots.Mark D. Grabiner, Ph.D., (part-time BME Staffmember who is now at the University of Illinoisat Chicago) conducts studies related to falls andmobility in the elderly. Other BME staff whosework has a biomechanical emphasis are Drs.Kathleen Derwin, Guang Yue and Melissa KnotheTate (see below).Much of the activity in BiomedicalDevices is concerned with cardiac pumps. Froman NIH-funded Innovative Ventricular AssistSystem (IVAS) program, Leonard A.R. Golding,M.D., has patented CCF’s unique implantablecentrifugal (nonpulsatile) blood pump, the thirdgenerationCorAide TM Ventricular Assist System,which is nonthrombogenic without use ofanticoagulants; in April 2001, Arrow International,Inc., acquired the rights to the technologyto test the device as a bridge to transplant/recovery. Preclinical evaluations and validationsof this device continue at CCF. KiyotakaContinued on Page 14RESEARCH ASSOCIATESTodd C. Doehring, Ph.D.Ahmet Erdemir, Ph.D.Jon D. Klingensmith, Ph.D.Durba Mukhopadhyay, Ph.D.Anuja Nair, Ph.D.Yaling Shi, Ph.D.Patrick Smits, Ph.D.Robert P.T. Somerville, D.Phil.Xiaowei Su, Ph.D.Azita Tajaddini, Ph.D.JOINT STAFFWilliam J. Davros, Ph.D.Brian W. Duncan, M.D.Roy K. Greenberg, M.D.Urs O. Hafeli, Ph.D.Sandra S. Halliburton, Ph.D.David Huang, M.D., Ph.D.Joseph P. Iannotti, M.D., Ph.D.Ulf R. Knothe, M.D., Dr. Med.Patrick M. McCarthy, M.D.Robert F. McLain, Ph.D.Paul A. Murray, Ph.D.Emil P. Paganini, M.D.Stephen I. Reger, Ph.D.James D. Thomas, M.D.Jean A. Tkach, Ph.D.Martin S. Weinhous, M.D.ADJUNCT STAFFPeter M. Anderson, Ph.D.Ananth Annapragada, Ph.D.Ravi V. Bellamkonda, Ph.D.Joan M. Belovich, Ph.D.Jeffrey J. Chalmers, Ph.D.George P. Chatzimavroudis, Ph.D.Bradley D. Clymer, Ph.D.Patrick E. Crago, Ph.D.Alan Freed, Ph.D.Jaikrishnan R. Kadambi, Ph.D.Geoffrey Lockwood, Ph.D.Paul Malchesky, D. Eng.Gerald M. Saidel, Ph.D.Orhan Talu, Ph.D.Central image: Computersimulation of jump landings on anuneven surface.The computational model showshow an ankle sprain injury (front)can be prevented through subtlechanges in landing technique(middle and back). From thelaboratory of Ton van den Bogert,Ph.D., Section of Biomechanics.13

The Department of Biomedical EngineeringModel of the total artificial heartfrom the laboratory of WilliamSmith, D. Eng., P.E., Section ofBiomedical Devices14Continued from Page 13Fukamachi, M.D., Ph.D., supports the researchefforts of CCF’s Kaufman Center for HeartFailure, co-directed by Patrick M. McCarthy,M.D. (Department of Thoracic and CardiovascularSurgery) and James B. Young, M.D. (Departmentof Cardiovascular Medicine). The majorfocus is on cardiovascular dynamics of cardiacdevices and animal and bench testing of surgicalinterventions to treat heart failure. This groupfocuses on testing various devices, including oneto treat dilated cardiomyopathy by changing theshape of the left ventricle (Myosplint tm ) andcatheter-type ventricular assist devices (enabler tmand Impella tm ). Based on years of research intononpulsatile blood flow, the Cardiac Assist andReplacement Laboratory, headed by William A.Smith, D.Eng., P.E., is (a) developing a family ofblood pump devices (total artificial heart withinternal battery, ventricular assist devices rangingfrom long-term adult to miniature pediatricapplications—all based on MagScrew TM technology),(b) defining test methods to accurately/repeatably characterize them, (c) developing arational design philosophy for rotary bloodpumps’ performance, efficiency, size, lowhemolysis level and minimal deposition, (d) usingacoustic methods for diagnostic monitoring ofblood pumps, and (e) refining external supportpump systems, including an emergency cardiopulmonarybypass/extracorporeal membraneoxygenation system, funded by the Departmentof Defense, a catheter pump for minimallyinvasive surgery, and an external-use version ofone internal system. These efforts also involvework funded through the NIH’s Small BusinessInnovation Research mechanism.The rapidly evolving field of BioMEMSand Nanotechnology provides several diverselines of investigation. Aaron J. Fleischman, Ph.D.,and Shuvo Roy, Ph.D., use microelectronics,microfabrication and micromachining technologiesas enabling technology to improve medicaldiagnostics and therapies by reducing device sizeand cost. Their collaborative studies involveengineering micro-/nanometer-sized features fortissue engineering, protein analyses, assays, andcell interrogation; among the applications beingdeveloped are miniaturized versions of drugdelivery systems, transducers for ultrasoundimages, and in situ telemetrically monitoredpressure/temperature sensors for minimallyinvasive surgery/follow-up. Maciej Zborowski,Ph.D., investigates magnetic flow cell sorting forvarious diagnostic and therapeutic applications,such as rapid screening for cancer cells in blood orblood-forming stem-cell transplantation (withCCF’s Taussig Cancer Center) and in model cellsystems of human peripheral lymphocytes,cultured cell lines, and samples donated bypatients, such as bone marrow. Continuousmagnetic flow sorting is a high-speed, gentleprocess, with high specificity and high recoveryof sorted fractions via cell tagging (e.g., via aniron-doped polymeric nanoparticle developedwith Bar-Ilan University in Israel). Cell TrackingVelocimetry, developed with the Ohio StateUniversity, can analyze individual cell velocitiesof hundreds of cells at a time, yielding data aboutthe population average and dispersion, based onquadrupole and dipole magnetic fields, which cansort some 10 million cells/second with 70%recovery of target cells and be optimized forincreased fractionation resolution and speed. P.Stephen Williams, Ph.D., builds mathematicalmodels of field-flow fractionation usingquadrupole magnets. His work informs the designof devices for cell separation.Several BME investigators work in the areaof Cardiovascular Bioengineering in studiesof blood vessels and heart valves, especially ininteraction with implanted prostheses. Linda M.Graham, M.D., seeks to design longer-lived tissueengineeredvascular grafts. Her group investigates,at the molecular level, how smooth-muscle cells(SMCs) and collagen affect cell proliferation andingrowth into prosthetic grafts, including: 1) themolecular mechanisms involved in the posttranscriptionalregulation of collagen secretion bygraft SMCs, 2) the mechanism by which oxidizedLDL inhibits endothelial cell migration, and 3)the effect of hypercholesterolemia on endothelialcell ingrowth onto prosthetic grafts in vivo. ScottColles, Ph.D., focuses on the role of glutathioneperoxidase and lipid oxidation products in thedevelopment of vascular disease. Lipid oxidationproducts are thought to be major factors in theContinued on Page 15

The Department of Biomedical EngineeringContinued on Page 14development of various vascular diseases includingatherosclerosis. Roy Greenberg, M.D., joint staffwith the Department of Vascular Surgery, focuseson the development of novel techniques andendovascular devices (e.g., stents and stent grafts)to treat aortic aneurysms and dissections, asignificant threat to the aging population. His aimis to ward off the main complication ofendovascular repair, the development of early orlate endoleak, which remains undetected byconventional clinical methods. The Heart ValveLaboratory team led by Ivan Vesely, Ph.D., studiesthe structure/function relationship of heart valvetissues to determine failure mechanisms ofmanufactured replacement heart valves, with theaim of developing a bioprosthetic valve thatcompletely mimics the natural valve’s function. Thegroup uses materials testing, mathematicalmodeling, microscopy, biochemical analysis, andcell culture, along with micromechanical testing,video image processing, and extensions to Fung’soriginal Quasi-Linear Viscoelastic theory. Incollaboration with NASA researchers, they aredeveloping advanced soft-tissue models forsimulating robotic surgery in a virtual-realitytraining system. The group also addresses geneticand biomechanical characteristics of aorticvalves affected by myxomatous mitral valvedisease, a condition characterized by thickeningof valve tissues and stretching of leaflets andchordae, causing the valve to leak. The group isalso creating tissue-engineering implants ofelastin, collagen and glycosaminoglycanssynthesized by cells in culture or purified fromtissues, then manipulated to mimic the aorticvalve’s normal structural framework. Dr.Geoffrey Vince’s work also contributes to thisarea of emphasis.Researchers in the Whitaker ImagingLaboratory specialize in micro-CT, magneticresonance imaging (MRI), and ultrasound.Kimerly Powell, Ph.D., uses high-resolutionmicro-CT, a 3D x-ray imaging technology, toevaluate bone microarchitecture in early bone lossand bone formation in small-animal models ofosteoporosis, as well as to monitor the effects ofvarious treatments longitudinally. with a goal ofhelping the aging population at risk for bone loss.Future goals include modes that can be usedinteractively in reviewing, localizing, andquantifying information obtained from variousimaging modalities at different spatial resolutions.Elizabeth Fisher, Ph.D., uses MRI to quantifybrain atrophy in patients with multiple sclerosisand predict the course of disease. The Fishergroup is (a) participating in a 5-year longitudinalstudy of clinical, MRI, immunologic, andpathologic correlates of brain atrophy in MSpatients and a 10-year follow-up image-analysisstudy to relate images of pathology to specificdomains of cognitive impairment, (b) exploringthe relationships between clinical and MRIvariables and time course of changes in the MRImeasurements via a novel postmortemimaging protocol indonated brains, and (c)developing and refiningsoftware (now in clinicaltrials) to incorporate brainatrophy measures into clinicalpractice. Raj Shekhar, Ph.D.,works on real-time 3D(RT3D) image acquisition, anew trend in ultrasoundimaging that eliminates motion artifacts and enablesrapid, more accurate imaging of dynamic anatomy(e.g., heart) in the operating room or at remotelocations. Improved visualization and imageprocessing software will allow tracking of an organ’sshape over time and alignment of 3D images withimproved speed and accuracy for serial comparisonof various image modalities. The goal of D.Geoffrey Vince, Ph.D., is to improve intravascularultrasound (IVUS) imaging to achieve a precisetomographic assessment of the coronary arteryanatomy in vivo in real time. His group seeks toeliminate three major limitations (time consumption,reduced resolution, and variability in the data setcaused by the operator) by taking into accountultrasound radiofrequency signals (backscatter). Thegroup has custom-developed software that usesspectral analysis methods to determine plaquecomposition from IVUS images and to display a“Virtual Histology” map, which has been licensedto Volcano Therapeutics (Laguna Hills, CA) and isundergoing trials in Europe. In collaboration withthe BioMEMS technology of Drs. Shuvo Roy andAaron Fleischman, the group will design and buildhigh-frequency IVUS transducers comprisingtraditional ceramic and novel polymeric materialsand assess how well these transducers perform forhigh-frequency harmonic imaging.The topic of Neural Control is addressed inContinued on Page 16Image at left: Human femurdepicted through the use of highresolutionmicro-CT, a 3D x-rayimaging technology, to evaluatebone microarchitecture in earlybone loss in osteoporosis. ByKimerly Powell, Ph.D., theWhitaker Imaging Laboratory.Below: 3-Dimensional tomographicassessment of the humancoronary artery anatomy in vivo inreal time. By Geoffrey Vince,Ph.D., the Whitaker ImagingLaboratory.15

The Department of Biomedical Engineering16Continued from Page 15both human studies and mathematical models.Guang H. Yue, Ph.D., is pursuing a variety ofexperimental studies (cortical control of fingermovements vis-a-vis brain electrical activity,training the nervous system to improve motorfunction in disabled patients without actualmuscle training, fatigue in patients with neurologicaldisorders, and functional magneticresonance imaging (fMRI) and movement-relatedcortical potentials in stroke patients). Among theresearch questions under study are how the braincontrols voluntary motor action and how thecentral nervous system, including the brain,adapts to various acute and chronic perturbations,such as fatigue, immobilization, training, aging,microgravity, injury or disease, with a viewtoward initiating more effective treatment ofmovement disorders, designing better rehabilitativetreatments, and reducing health care costs.This work is highly collaborative with the Clinic’sPhysical Medicine and Rehabilitation group.Cameron C. McIntyre, Ph.D., leads a newprogram involving model-based analysis of highfrequencydeep brain stimulation (DBS), inconjunction with results from PET/fMRIexperiments, to better treat parkinsonian andother movement disorders. Using the techniquesof computational neuroscience and electromagneticfield modeling, his group’s goal is toaugment experimental investigation in DBS ofthe parkinsonian nonhuman primate as well asimprove the electrode targeting and postoperativeparameter selection processes in humans. Bycoupling results from a number of sources, thegroup is creating a theoretical framework thatenhances understanding of the effects of DBSand provides a virtual testing ground for newstimulation paradigms that will yield maximumtherapeutic benefit and minimal side effects.A strong research program in OrthopaedicBiology and Bioengineering ishighlighted by integration with CCF’s OrthopaedicResearch Center. Suneel Apte, M.B.B.S.,D.Phil., investigates extracellular matrix as well asthe metalloproteases that remodel it and modifyAt left: Six images of progressive normal skeletaldevelopment in the mouse. From the laboratory of SuneelApte, M.B.B.S., D. Phil., the Section of OrthopaedicBiology and Bioengineering.Above at right: Island in the storm; trabecular bone fromthe femoral neck of an osteoporotic patient. Islands ofbone tissue with viable osteocytes (grey areas with stellateshaped cells) juxtaposed to empty spaces (black)corresponding to areas of bone removed by osteoclastsduring progression of osteoporosis. From the laboratoryof Melissa Knote Tate, Ph.D., the Section ofOrthopaedic Biology and Bioengineering.cellular behavior through extracellular proteolysis.These include enzymes of the MMP andADAMTS family. He uses transgenic mice tostudy how the body utilizes these molecules fordevelopmental processes such as skeletogenesisand lung development. This fundamental work iscomplemented by studies of the roles of thesemolecules in arthritis, inflammation and cancer.R. Tracy Ballock, M.D., focuses on translationalstudies of the growth plate in relation tochildhood obesity (especially in slipped capitalfemoral epiphysis, an obesity-related hip disease inchildren) by studying molecules called peroxisomeproliferator-activated receptors (PPARs), whichare also expressed in bone and cartilage andinterfere with thyroid hormone receptor (TR)-mediated gene transcription. This work employshuman tissue and a rat model of physeal cartilageformation and uses an instrumented surgicalstaple to measure compressive forces generated bythe physis growing against the staple. KathleenDerwin, Ph.D., probes the interface betweentendon and ligament biology and biomechanics asapplied to the design of tissue-engineeredmaterials. A key aspect of this research involvestissue engineering of tendon and ligamentsubstitutes using fibroblasts seeded onto naturalextracellular matrices.Vincent C. Hascall,Ph.D., studies thestructure, functionand metabolism ofproteoglycans,especially aggrecan,which helps tissuesresist compressiveloading in cartilage. Amajor focus is onhyaluronan, whichforms scaffolds for molecules involved in cartilageformation, oocyte fertilization, skin keratinization,colon and lung smooth muscle cells’ response toviral stimuli, and abnormal matrices synthesized inresponse to elevated glucose in vascular anddiabetic pathologies. Melissa L. Knothe Tate, Ph.D.,explores the signaling/timing of interactionsbetween bone-cell types, in regard to growth,adaptation, and repair of musculoskeletal tissuesand bone. Her group has developed innovativemethods to study mechanical load-induced fluidflow and mass transport through tissue, as well astheoretical computer models to predict flowpatterns under simulated conditions and explicatethe relationship between mechanical loadingparameters and fluid dynamics in bone. She also hasan interest in the spaces through which extravascularfluid flows to develop drug-delivery systems forskeletal tissues and for new bioactiveendoprostheses designed to optimizeosseointegration. Véronique Lefebvre, Ph.D., usesContinued on Page 17

The Department of Biomedical EngineeringContinued from Page 16molecular biology, cell biology, and mouse geneticengineering approaches to study the roles of Soxtranscription factors during development and inpathologies of the skeleton and hematopoieticsystem. Her group is determining how these Soxfactors control cell fate and differentiation inspecific cell lineages and how they act on targetgenes and interact with other factors to enhance orrepress transcription. Cahir A. McDevitt, Ph.D.,studies tissue and animal models to explore woundhealing in the knee joint meniscus, especially therelationships of networks of varying collagen typesand cell forms that are quiescent until wounding,whereupon mRNA levels for type I and type VIcollagen and other matrix proteins dramaticallyincrease, with the wound crevice becomingpopulated by cells that appear to come from thesuperficial zone and that can migrate into acellularareas created by apoptosis of resident cells. RonaldJ. Midura, Ph.D., pursues studies of bone remodeling,involving bone-matrix production and/ormineralization as regulated by cytokines/hormonesin normal/pathologic states. He concentrates onthe role of parathyroidhormone (PTH) in (a)maintaining Ca 2+ levels inblood by reabsorbing it fromkidney and releasing it frombone and (b) exertingsomatotrophic effects onbone formation when usedtherapeutically. His group hasfound that PTH dramaticallyaffects an osteoblast’s abilityto produce select matrixmacromolecules, alters theirassembly into an extracellular matrix, and regulatesmatrix mineralization. George F. Muschler, M.D.,is developing more effective, less invasive methodsto treat fractures/deformities, using techniques ofcell and molecular biology, growth factor expressionand action, cell matrix interaction, imageprocessing, and biomechanics. His group has (a)devised minimally invasive methods to harvest andrapidly collect bone stem cells, (b) achieved boneregeneration with fully implantable devices that usethe process of distraction osteogenesis toregenerate bone segments and lengthen limbswithout cumbersome and painful frames, (c) avoidharvesting bone from one site and transplanting itto another by exploring synthetic materials (calciumphosphate ceramics, purified collagen preparations,and some polymers) in combination with growthfactors; and (d) developed a segmental caninemodel for efficient, sensitive evaluation ofcomposite graft materials for spinal fusion.Investigators from Biomechanics (Drs. Cavanagh,Davis, van den Bogert), Neural Control (Dr. Yue)and Imaging (Dr. Powell) are also active participantsin this group.Tissue Engineering and Wound Healingis an area of research in which BME departmentmembers from Orthopaedic Biology (Drs. Derwin,McDevitt, Midura, and Muschler) and CardiovascularBiomechanics (Dr. Vesely) are also active.Edward V. Maytin, M.D., Ph.D., pursues studies inhealing of skin wounds. He studies CCAAT/Enhancer Binding Proteins via an artificial skinmodel in which keratinocytes grow on a collagenraft floating at the air-liquid interface to simulate invivo conditions that promote epidermal stratificationand differentiation-related gene expression. Hisgoals are to study epidermal homeostasis, mechanismsof ultraviolet light damage to skin, extracellularhyaluronan’s role in regulating epidermal cells,and photodynamic therapy for skin cancer and otherhyperproliferative diseases.BME’s depth and breadth of expertise aregreatly enhanced by contributions from staffmembers from other departments who have jointappointments. Among the departments (andindividuals) represented are Cardiovascular Medicine(James Thomas, M.D.), the Center for AnesthesiologyResearch (Paul Murray, Ph.D.),the Cole Eye Institute (DavidHuang, M.D., Ph.D.), Nephrologyand Hypertension/Dialysis (Emil P.Paganini, M.D.), OrthopaedicSurgery (Joseph Iannotti, M.D.,Ph.D., Ulf Knothe, M.D., Dr. Med.,Robert F. McLain, Ph.D), Pediatric/Congenital Heart Surgery (Brian W.Duncan, M.D.), Radiation Oncology(Urs Hafeli, Ph.D., MartinWeinhous, M.D.), Radiology(William Davros, Ph.D., Sandra S.Halliburton, Ph.D., Jean A. Tkach, Ph.D.), Thoracicand Cardiovascular Surgery (Patrick McCarthy,M.D.), Vascular Surgery (Roy Greenberg, M.D.), andPhysical Medicine and Rehabilitation (Stephen I.Reger, Ph.D.). In addition to these individuals, 14scientists from other institutions have adjunctappointments in BME.The Department of Biomedical Engineeringis committed to investigation, innovation, andthe translation of scientific discoveries intopractical applications that enhance patient care.By providing a forum in which engineers, basicscientists and physicians can interact, thedepartment plays a key role in the LernerResearch Institute and in the Foundation as awhole, advancing the mission to promoteexcellence in research, education, and patientcare.Centeral image: Proliferation ofHuman CTPs and Expressionof Alkaline Phosphatase onLoaded Coralline HA disks, day9 culture. From the laboratory ofGeorge F. Muschler, M.D., theSection of Orthopaedic Biologyand Bioengineering.17

BIOMECHANICSTHE CAVANAGHLABORATORYRESEARCH ASSOCIATEAhmet Erdemir, Ph.D.SENIOR ENGINEERSJohn Bednarek, M.S.E.E.Tammy M. Owings, D.Eng.ENGINEERAndrea Rice, M.S.TECHNOLOGISTSKerim Genc, M.S.Kiran Gumma, B.S.RESEARCH NURSEPaul Tokar, R.N.GRADUATE STUDENTSSachin Budabhatti, M.S.Marc T. Petre, B.S.COLLABORATORSJan Apelqvist, M.D. 1Georgeanne Botek, D.P.M. 2Andrew J. Boulton, M.D. 3Brian L. Davis, Ph.D. 4Brian G. Donley, M.D. 2Byron J. Hoogwerf, M.D. 5Peter K. Kaiser, M.D. 6Michael P. Recht, M.D. 2, 7Susan J. Rehm, M.D. 8James J. Sferra, M.D. 2Anders Stenstrom, M.D., Ph.D. 1Jan S. Ulbrecht, M.B.B.S. 9Loretta Vileikyte, M.D. 31Dept. of Orthopaedics,University Hosp., Lund,Sweden2Dept. of Orthop. Surgery, CCF3Dept. of Medicine, ManchesterRoyal Infirmary, Manchester,U.K.4Dept. of Biomed. Eng., CCF5Dept. of Endocrinol., Diabetes& Metabolism, CCF6Cole Eye Inst., CCF7Depts. of E-Radiol. andRegional Radiol. Practice,CCF8Dept. of Infectious Dis., CCF9Penn. State Univ., UniversityPark, PAThe Department of Biomedical EngineeringFoot Complications of Diabetes and MusculoskeletalChanges During Space FlightEstablished in September 2002, thelaboratory has as its primary foci thestudy of lower-extremity disease indiabetes mellitus and the exploration ofmusculoskeletal changes during space flight.At the time of writing, Dr. Cavanagh is in theprocess of recruiting his research team andestablishing new collaborative relationshipswithin the Cleveland Clinic Foundation.Foot Complications of DiabetesLower-extremity amputation in peoplewith diabetes continues to be a major publichealth problem. More than 65,000 amputations areperformed annually ondiabetic patients in theUnited States, and, despiterecent efforts, this number isincreasing. Ulceration in theneuropathic foot is a majorprecursor of amputation, andthus identification of riskfactors, together with primaryand secondary prevention offoot ulceration, are key goalsof our research program. Tounderstand the mechanicalstresses that occur duringfoot-shoe interaction and thatcause many plantar ulcers, weare developing three-dimensional finite-elementmodels of the interface between foot and shoe.Our group also has an interest in magneticresonance imaging of the foot, and we haverecently found evidence of remarkable atrophyin all the intrinsic muscles of the neuropathicsubjects when compared to nondiabetic controls.Although sensory neuropathy is often emphasizedin considerations of diabetic foot pathology,our results show that the consequences ofmotor neuropathy in the feet are profound inpeople with diabetes.Neuropathic patients experience problemswith gait and posture. They also suffer more fallsand fractures. We are using gait analysis techniquesto explore both the role of the foot as asensory organ and the contributions of proprioceptionto the control of movement. As Dr.Cavanagh also serves as Academic Director of theDiabetic Foot Care program for the Cleveland ClinicFoundation, his research program is closely integratedwith the activities of CCF ‘s newly establishedDiabetic Foot Clinic.Musculoskeletal Changes during Space FlightThe loss of bone mineral in the lowerextremities is widely viewed as one of the criticalfactors that may limit long-term human habitation ofspace. Decrements in muscle function as a result ofprolonged exposure to microgravity also haveimportant implications for performance and safetyduring space missions. A major aim of ongoingprojects is to investigate the role thatload reduction and reduced muscleactivity may play in the loss of bonemineral and muscle strength. Twoexperiments are currently under way,one ground based and the other on theInternational Space Station (ISS).Our ground-based study isdesigned to examine the efficacy ofexercise in microgravity. Tests areconducted on a simulator in whichhuman subjects walk and run whilesuspended in a harness apparatus toPeter R. Cavanagh, Ph.D. simulate microgravity conditions. ThisThe Virginia Lois Kennedy Chairarrangement is being used to examinethe biomechanics and perceived comfortof exercise in microgravity; ultimately, the data will becoupled with a robotic simulation of the exerciseusing human cadaver limbs to measure bone strainduring simulated microgravity exercise.As part of the in-flight experiment, we arecharacterizing the comparative load on the lowerextremities during entire days of working on Earthversus on the ISS. We are using instrumentation fromthe Human Research Facility (http://hrf.jsc.nasa.gov).Pre- and post-flight estimates of bone mineral density,muscle cross-sectional area, and joint torques providea perspective against which the consequences ofchanges in activity profiles can be judged. The resultsof this research will provide an understanding of therole of mechanical stress in in-flight osteopenia andimportant information to assist in the design ofexercise countermeasures to bone and muscle loss.Cavanagh, P.R., Boulton, A.J., Sheehan, P., Ulbrecht, J.S., Caputo, G.M., and D.G. Armstrong (2002)Therapeutic footwear in patients with diabetes [letter]. JAMA 288:1231; discussion 1232-1233.Lloyd, T., Beck, T.J., Lin, H.M., Tulchinsky, M., Eggli, D.F., Oreskovic, T.L., Cavanagh, P.R., and E.Seeman (2002) Modifiable determinants of bone status in young women. Bone 30:416-421.McCrory, J.L., Baron, H.A., Balkin, S., and P.R. Cavanagh (2002) Locomotion in simulated microgravity:gravity replacement loads. Aviat. Space Environ. Med. 73:625-631.Bus, S.A., Yang, Q.X., Wang, J.H., Smith, M.B., Wunderlich, R., and P.R. Cavanagh (2002) Intrinsicmuscle atrophy and toe deformity in the diabetic foot: a magnetic resonance imaging study. DiabetesCare 24:1444-1450.Piazza, S.J., Erdemir, A., Okita, N., and P.R. Cavanagh (2003) Assessment of the functional method ofhip joint center location subject to reduced range of hip motion. J. Biomech. (in press).18http://www.lerner.ccf.org/bme/cavanagh/

Examples of devices that are being designedare (i) a device to simultaneously measurepressure and shear forces at specific sitesunder the feet of patients with distal peripheryneuropathy; (ii) rehabilitation devices to enhancethe ambulatory capabilities of patients withmusculoskeletal problems, and (iii) novel exercisedevices for astronauts to use on the InternationalSpace Station.Foot injuriesCalcaneal fractures, Achilles tendonruptures, posterior tibialis insufficiency and manyother musculoskeletal injuries of the foot greatlyaffect patients’ mobilityand, consequently, theirquality of life. Throughfunding from theDepartment of Defense,the focus of our studies ison understanding themechanisms of variousfoot injuries, includingmetatarsal stress fracturesand hindfoot impactinjuries. Throughcollaborations withcolleagues in the OrthopaedicResearch Center,new models are beingdeveloped to optimizesurgical approaches usedto correct foot pathologies.Shear and pressure measurement deviceFoot ulceration, a diabetic complicationthat is difficult to treat, results in significantmorbidity and, in many cases, precedes limbamputation. It has been reported that 20% of alldiabetic patients in hospitals have been admittedfor foot problems. Previous research hasestablished the significance of nerve damage anda compromised vascular system in the etiology ofdiabetic foot ulcers.In recent years, the importance ofmechanical factors such as pressure and frictionalforces has also been established, but to date, thesetwo loading conditions have never been measuredsimultaneously. As a consequence, the true threedimensionalloads that are applied to the sole ofthe foot have never been quantified. Therationale behind the current research is that by (i)quantifying localized skin loads and (ii) obtainingnoninvasive measurements of tissue properties,the factors leading to diabetic skin ulceration willThe Department of Biomedical EngineeringLower Extremity Biomechanical AnalysisAims to Stem Bone Loss in Space,Aging and Diseasebe more fully understood.RehabilitationAn NIH-funded research and developmenteffort focuses on the design of an instrumenteddual-track treadmill. The device will permit realtimemonitoring, analysis and rehabilitation of apatient’s gait. The walking surface will beconnected to force sensors that permit bothvertical and longitudinal shear loads to bemeasured. These data will be combined in asoftware system to produce detailed informationon the kinetics of the lower limb joints ofamputee, arthritis and stroke patients.Countering bone loss inastronautsBone demineralizationis a well-documentedphysiologic effect of spaceflight. Animal experimentsdone in 1G have indicatedthat (i) certain bone strainand strain rates stimulatebone deposition, and (ii)repetitive loading of thelower extremity canincrease osteonal boneformation even as proximallyas the vertebralcolumn. In a previous CCFstudy, “Exercise Countermeasuresfor Astronauts,”Brian L. Davis, Ph.D.the merits of performingjumping exercises in microgravity were investigated.We are now designing a countermeasuredevice to optimize bone deposition while keepingvibration effects on the International SpaceStation below thresholds set by NASA.BIOMECHANICSTHE B. DAVISLABORATORYINVESTIGATORSSusan E. D’Andrea, Ph.D.Azita Tajaddini, D.Eng.Julie Perry, M.S.Gail Perusek, M.S.Jennifer Kuznicki, B.S.GRADUATE STUDENTSYan Chen, Ph.D.Nelson Morales, M.S.Solomon Praveen, Ph.D.UNDERGRADUATE STUDENTSRandy BlyLuke JanikLindsay KellerAri LevineMary OrtolanoAbby WaltersCOLLABORATORSPeter R. Cavanagh, Ph.D. 1Brian Donley, MD. 2Helen E. Kambic, Ph.D. 1Mark Luciano, M.D. 3Cahir A. McDevitt, Ph.D. 1James Redhed, C.P.O. 4James Sferra, M.D. 2Ton van den Bogert, Ph.D. 11Dept. of BiomedicalEngineering, CCF2Dept. of Orthopaedic Surgery,CCF3Dept. of NeurologicalSurgery, CCF4Dept. of Orthotics andProsthetics, CCFKao, P., Davis, B.L., and P.A. Hardy (1999) Characterization of the calcaneal fatpad in diabetic and non-diabetic patients using magnetic resonance imaging. Magn.Reson. Imaging 17:851-857.Perusek, G.P., Davis, B.L., Sferra, J.J., Courtney, A.C., and S.E. D’Andrea (2001)An extensometer for global measurement of bone strain suitable for use in vivo inhumans. J. Biomech. 34:385-391.Perry, J.E., Davis, B.L., and M.G. Luciano (2001) Quantifying muscle activity innon-ambulatory children with spastic cerebral palsy before and after selective dorsalrhizotomy. J. Electromyogr. Kinesiol. 11:31-37.Perry, J.D., Hall, J.O., and B.L. Davis (2002) Simultaneous measurement of plantarpressure and shear forces in diabetic individuals. Gait Posture 15:101-107.Praveen, S.S., Hanumantha, R., Belovich, J.M., and B.L. Davis (2003) Novel hyaluronicacid coating for potential use in glucose sensor design. Diabetes Technolo.Ther. 5:393-399.19

BIOMECHANICSThe Department of Biomedical EngineeringAnalysis of Musculoskeletal Function forInjury Prevention and RehabilitationTHE VAN DEN BOGERTLABORATORYPROJECT STAFFScott G. McLean, Ph.D.RESEARCH FELLOWSElizabeth C. Hardin, Ph.D.Kiyonori Mizuno, M.D.RESEARCH ENGINEERAnne Su, M.Sc.SENIOR RESEARCH TECHNICIANXuemei Hiuang, M.S.GRADUATE STUDENTSRudolph Pienaar, M.Sc. 1Jerôme Hausselle, B.S. 21Applied Biomed. Eng.program, Cleveland StateUniv., Cleveland, OH2Mechanical Engineering,Cleveland State Univ.,Cleveland, OHUNDERGRADUATE STUDENTGurpreet DhillonBiomedical Engineering, CaseWestern Reserve University,Cleveland, OHCOLLABORATORSJack T. Andrish, M.D. 1Brian L. Davis, Ph.D. 2Mark D. Grabiner, Ph.D. 2,3Richard R. Neptune, Ph.D. 41Dept. of Orthopaedic Surgery,CCF2Dept. of Biomedical Engineering,CCF3Dept. of Kinesiology, Univ. ofIllinois, Chicago4Dept. of Mech. Engineering,Univ. of Texas, AustinHuman movement is generated by muscleforces, which are in turn controlled bythe central nervous system. At the sametime, joints and ligaments are subject tomechanical stresses that can lead to injury. Ourlaboratory aims to understand these processesthrough computational models of theneuromusculoskeletal system as well as throughnovel experimental techniques. We apply thisknowledge to injury prevention, rehabilitation,and prosthetics. This work spans the disciplinesof mechanical engineering, neuroscience, appliedmathematics, and orthopedics.Sport injuriesRupture of the anteriorcruciate ligament (ACL) in theknee is a common and serioussport injury. Apart from acuteeffects, this injury often causesosteoarthritis within 10-20years. Our hypothesis is thatACL injury can be preventedthrough a combination ofimproved neuromuscularcontrol and altered frictionalproperties of footwear. Wehave developed a threedimensionalmodel that canperform dynamic landing tasksthat put the knee joint at riskfor injury. After “training” themodel to reproduce a fast sidestepping movement,several ACL injuries can be generated byperforming thousands of simulations accordingto the subject’s variability in neuromuscularcontrol. We then quantify the effect of variousprevention strategies on the incidence of injury.Antonie J. van den Bogert, Ph.D.A similar approach is used for the study of footand ankle injuries during landings on unevensurfaces.In conjunction with this computationalwork, we are performing experiments on cadaverspecimens to determine how the strain in the ACLdepends on the interactions between large threedimensionalforces and torques applied to theknee joint.Control of posture and gaitNeuromuscular control is not onlyimportant for sport injuries, but also is a necessarycomponent of seemingly simple tasks such asstanding and walking. Using computer simulations,we can quantify the effectof neuromuscular control on gaitstability and have shown thatfeedback control is needed togenerate an efficient, stablewalking gait. In postural control,we have applied the theory ofreinforcement learning (RL) tothe control of a multi-linkinverted pendulum. Our goal is todesign an adaptive neuralcontroller for human posture thatcan learn to perform a step torecover balance after a largeperturbation. Walking may thenoccur naturally as a sequence ofbalance recoveries. This work hasimplications for prevention of falls in older adultsand for treatment of movement disorders. Otherpotential applications of this work are inintelligent prostheses, function restoration afterspinal cord injury, and the development ofbiologically inspired robots.Wright, I.C., Neptune, R.R., van den Bogert, A.J., and B.M. Nigg (2000) The influence of foot positioningon ankle sprains. J. Biomech. 33:513-519.Neptune, R.R., Wright, I.C., and A.J. van den Bogert (2000) The influence of orthotic devices andvastus medialis strength on patella-femoral loads during running. Clin. Biomech. 15:611-618.Bellchamber, T.L., and A.J. van den Bogert (2000) Contributions of proximal and distal moments to tibialrotation during walking and running. J. Biomech. 33:1397-1403.Wilson, A.M., McGuigan, M.P., Su, A., and A.J. van den Bogert (2001) Horses damp the spring in theirstep. Nature 414:895-899.van den Bogert, A.J., Pavol, M.J., and M.D. Grabiner (2002) Response time is more important thanwalking speed for the ability of older adults to avoid a fall after a trip. J. Biomech. 35:199-205.20

The Department of Biomedical EngineeringMicro-Mechanism Designs Aimed forDevelopment of Biomedical ApplicationsBIOMEMSANDNANOTECHNOLOGYRecent progress in microelectromechanicalsystems – the microelectronics,microfabrication and micromachiningtechnologies known collectively as MEMS – isbeing applied to biomedical research areas and hasbecome a new field of research unto itself,known as BioMEMS. The technology is originallybased upon the same technology that has beenused to makecomputer chips evermore powerful andless expensive. MEMStechnology hasenabled low-cost,high-functionalitydevices in somecommonly used areas,such as inexpensiveprinter cartridges forAaron Fleischman, Ph.D.ink jet printing andchip-based accelerometersresponsible fordeployment of automotive airbags.BioMEMS applies these technologies andconcepts to diverse areas in biomedical researchand clinical medicine. BioMEMS is an enablingtechnology for ever-greater functionality and costreduction in smaller devices for improved medicaldiagnostics and therapies. BioMEMS technologywill enhance catheter-based procedures byproviding pressure sensing, imaging, drug deliveryand tissue sampling, all via tiny biochips occupying

THE ZBOROWSKILABORATORYPROJECT SCIENTISTP. Stephen Williams, Ph.D.SENIOR RESEARCH ENGINEERLee R. MooreLEAD TECHNOLOGISTDiane R. LeighRESEARCH TECHNICIANBoris KligmanSTUDENTSLeonora Felon 1Francesca Carpino 21Co-Op Program, Univ. ofToledo, OH2Visiting graduate student,Univ. of Bologna, ItalyCOLLABORATORSBIOMEMSANDNANOTECHNOLOGYBrian J. Bolwell, M.D. 1Ernest C. Borden, M.D. 2Jeffrey J. Chalmers, Ph.D. 3Aaron Fleischman, Ph.D. 4Mauricio Hoyos, Ph.D. 5Shlomo Margel, Ph.D. 6George F. Muschler, M.D. 4Shuvo Roy, Ph.D. 4Alan N. Schechter, M.D. 71Bone Marrow Transplant.Progr., Taussig Cancer Ctr.,CCF2Dept. of Cancer Biology andTaussig Cancer Center, CCF3Dept. of Chemical Engineering,Ohio State Univ.,Columbus, OH4Dept. of Biomed. Eng., CCF5Ecole Supérieure dePhysique et ChimieIndustrielles, Paris, France6Dept. of Chemistry, Bar-IlanUniversity, Israel7Laboratory of ChemicalBiology, NIH/NIDDK,Bethesda, MD=======================Maciej Zborowski, Ph.D.=======================Our laboratory investigates novel methodsof cell separation for medical applications.The current effort focuses onmagnetic flow cell sorting, for diagnostic andtherapeutic applications. The potential diagnosticapplications include rapid screening for rare,unusual cells such as cancer cells in blood or fetalcells in maternal blood. The potential therapeuticapplications include cell therapies, such ashematopoietic (blood-forming) stem celltransplantation. Theseapplications are pursuedin collaboration with theCCF’s Taussig CancerCenter.Magnetic flowcell sorting is studied inmodel cell systems ofhuman peripherallymphocytes, culturedcell lines, and samplesdonated by patients:bone marrow, peripheralblood primed forapheresis, and umbilicalcord blood. Theadvantage of continuousmagnetic flow sorting isthat it is a high-speed,gentle process, with thepotential for highspecificity and highrecovery of sortedfractions. The specificity of sorting depends onthe specificity of the monoclonal antibodies andmagnetic agents used for cell tagging. One suchagent, an iron-doped polymeric nanoparticle, isbeing developed for magnetic cell sorting incollaboration with Bar-Ilan University in Israel.The mechanics of cell sorting in a flow, inThe Department of Biomedical EngineeringMagnetic Flow Cell Sorting Screens forCancer Cells, Offers Optionsfor Stem Cell TherapiesMaciej Zborowski, Ph.D.the presence of a magnetic field, is poorlyunderstood. We study cell motion using a uniquesystem, Cell Tracking Velocimetry (CTV),developed in collaboration with the Ohio StateUniversity. The system allows us to analyzeindividual cell velocities of hundred of cells at atime, leading to important information about thepopulation average and dispersion. The characteristiccell velocities are correlated with the physicaland biological properties of the cell, such as cellsize and cell surface markerexpression. The cell velocitiesmeasured by CTV are thebasis for the magnetic flowsorter design.The current flow sorterdesigns are based on quadrupoleand dipole magneticfields. The laboratory-scaleprototype of the quadrupolesorter produces sorting speedsin excess of a ten million cellsper second, enriches rare cellsa hundred-fold, and achievesa 70% recovery of the targetcells. The dipole cellfractionator separates cellsamples into eight fractions,characterized by different cellmobilities and different cellsurface marker expression.These capabilities areimportant in cell biologyresearch and in clinical applications. Thequadrupole sorter design is being scaled up for celltherapy applications, requiring sorting speeds onthe order of ten million cells per second, atenrichment and recovery rates comparable tothose of the laboratory prototype; the dipolefractionator design is being optimized forincreased fractionation resolution and speed.Moore, L.R., Rodriguez, A.R., Williams, P.S., McCloskey, K.E., Bolwell, B.J., Nakamura, M., Chalmers,J.J., and M. Zborowski (2001). Progenitor cell isolation with a high-capacity quadrupole magnetic flowsorter. J. Magnetism Magnetic Materials 225:277-284.Comella, K., Nakamura, M,, Melnik, K., Chosy, J., Zborowski, M., Cooper, M.A., Fehniger, T.A.,Caligiuri, M.A., and J.J. Chalmers (2001) Effects of antibody concentration on the separation of humannatural killer cells in a commercial immunomagnetic separation system. Cytometry 45:285-293.Hoyos, M., McCloskey, K.E., Moore, L.R., Nakamura, M., Bolwell, B.J., Chalmers, J.J., and M.Zborowski (2002) Pulse-injection studies of blood progenitor cells in a quadrupole magnet flow sorter.Separation Sci. Technol. 37:745-767.Zborowski, M., Moore, L.R., Williams, P.S., and J.J. Chalmers (2002) Separations based on magnetophoreticmobility. Separation Sci. Technol. 37:3611-3633.Chosy, E.J., Nakamura, M., Melnik, K., Comella, K., Lasky, L.C., Zborowski, M., and J.J. Chalmers(2003) Characterization of antibody binding to three cancer-related antigens using flow cytometry andcell tracking velocimetry. Biotechnol. Bioeng. 82:340-351.22

The Department of Biomedical EngineeringBIOMEDICALDEVICESLeonard A.R. Golding, M.D.Nonpulsatile Blood Pump at Heart of CCF’sInnovative Ventricular Assist ProgramFor nearly a quarter century, the ClevelandClinic Foundation (CCF) has been a strongcontributor in the development ofventricular assist technology. A major milestonearising from these efforts in late 1995 wasfunding from the National Heart, Lung, andBlood Institute, which awarded our collaborativeteam a contract for $4.3 million over 5years, under the Innovative Ventricular AssistSystem (IVAS) Program. The IVAS program wasa cooperative effort involving experts inindustry, academia, and clinical practice. Centralto this project is the CCF’s unique implantablecentrifugal (nonpulsatile) blood pump developedand patented in the Department of BiomedicalEngineering.The NIH program resulted in the ThirdGeneration CorAide TM Ventricular Assist System,which proved to be nonthrombogenic withoutthe use of anticoagulants. In April 2001, ArrowInternational, Inc., acquired the rights to thetechnology and is funding a 3-year program totake the system into clinical trials. The laboratoryis active in characterization system tests and finalchronic implant evaluations prior to human use.In December 2002, Arrow received regulatoryapproval in Europe for the first clinical trial ofthe device as a bridge-to-transplant/recovery.Golding, L.A., Medvedev, A., Massiello, A., Smith, W.A., Horvath, D., and R. Kasper (1998) ClevelandClinic continuous flow blood pump: progress in development. Artif. Organs 22:447-450.THE GOLDINGLABORATORYSURGICAL TEAMKazuyoshi Doi, M.D.Kiyotaka Fukamachi, M.D., Ph.D.Yoshio Ootaki, M.D., Ph.D.BIOMEDICAL ENGINEERSAlexander Massielllo, M.S.MECHANICAL ENGINEERDavid Horvath, M.S.RESEARCH TECHNICIAN/Q.A.Stephen Benefit, A.A.S.COLLABORATIONArrow International, Inc.,Reading, PA=====================LeonardA.R.Golding,M.B.,B.S.,F.R.A.C.S.,F.R.C.S.(C)=====================Golding, L., Smith, W. Horvath, D., and A. Medvedev (2000) Rotodynamic pump development. In: H.Matsuda, ed. Rotary Blood Pumps: New Developments and Current Applications. Tokyo, Japan: Springer-Verlag, pp. 47-56.Ochiai, Y., Golding, L.A., Massiello, A.L., Medvedev, A.L., Gerhart, R.L., Chen, J.F., Takagaki, M., andK. Fukamachi (2001) In vivo hemodynamic performance of the Cleveland Clinic CorAide blood pump incalves. Ann. Thorac. Surg. 72:747-752.Golding, L.A. (2002) Cleveland Clinic centrifugal blood pump. ASAIO J. 48:578; discussion 578-579.Gerhart, R.L., Horvath, D.J., Ochiai, Y., Krogulecki, A.Y., and L.A. Golding (2002) The effects of impacton the CorAide ventricular assist device. ASAIO J. 48:449-452.Fukamachi, K., Ochiai, Y., Doi, K., Massiello, A.L., Medvedev, A.L., Horvath, D.J., Gerhart, R.L., Chen,J.F., Krogulecki, A.Y., Takagaki, M., Howard, M.W., Kopcak, M.W. Jr., and L.A. Golding (2002) Chronicevaluation of the Cleveland Clinic CorAide left ventricular assist system in calves. Artif. Organs26:529-533.Ochiai, Y., Golding, L.A., Massiello, A.L., Medvedev, A.L., Horvath, D.J., Gerhart, R.L., Chen, J.F.,Krogulecki, A.Y., Takagaki, M., Doi, K., Howard, M.W., and K. Fukamachi (2002) Cleveland ClinicCorAide blood pump circulatory support without anticoagulation. ASAIO J. 48:249-252.23

THE W. SMITHLABORATORYNanette Businger, B.S.Fernando Casas, Ph.D.Dave Dudzinski, B.S.Lei Gu, D. Eng.Christine Flick, B.S.Ryan Klatte, B.S.Lei Gu, D. Eng.Markus Lorenz, M.S.Wenfeng Lu, M.S.Viviane Luangphakdy, M.S.Andrew Reeves, M.S.John Sankovic, M.S.Stephan Weber, M.S.COLLABORATORSH. Ming Chen, Ph.D. 1Ji-Feng Chen, B.S.Brian Duncan, M.D. 2Brian Farrell, M.S. 3Kiyotaka Fukamachi, M.D., Ph.D. 3Jai Kadambi, Ph.D. 4Raymond J. Kiraly, M.S, .M.E.Patrick McCarthy, M.D. 5John Player, M.S. 3Nicholas Vitale, M.S.M.E. 1Qun Zhou, B.S.1Foster-Miller Technologies,Inc., Albany, NY2Dept. of Pediatric andCongenital Heart Surgery3Foster-Miller, Inc., Boston, MA4Case Western ReserveUniversity5Dept. of Thoracic andCardiovascular Surgery,CCFCase Western ReserveUniversity24BIOMEDICALDEVICESBlood Pump Technology Leads toDevelopment of a Familyof Blood Pumping DevicesPrior research at The Cleveland ClinicFoundation has shown that chronic survival withnonpulsatile blood flow is possible. Thepurpose of this research is to explore engineeringtechnologies that will advance the design, developmentand application of this new form of cardiac assistdevice.One aspect of the program is development oftest methods to accurately and repeatably characterizenonpulsatile blood pumps.Determination of the “index ofhydrolysis” is one important test.Unfortunately, comparing resultsfrom laboratory to laboratory (oryear to year within one laboratory)is difficult because the criticalparameters and tolerances havenever been quantified. To create atest standard with a scientific basis,we are working to determine howvariations in test characteristics mayaffect pump results. A morefundamental understanding offactors relating to blood fragilitymay also be obtained.Another research area ispump design technology. Industrial rotary pumptechnology was developed based on water as theworking medium. Blood is a significantly differentfluid. Blood pumps are also in a very different range ofpressure and flow, as compared to commercial pumpapplications. The goal of this research is to develop arational design philosophy for rotary blood pumps.The ultimate goal is to achieve a balanced optimum ofperformance, efficiency, size, low hemolysis andminimal deposition.A third research area is diagnostic monitoringof blood pumps. Permanent blood pump implantswill require means to detect failures well before theyoccur, so that replacement can be arranged in goodLorenz, M., and W.A. Smith (2002) Rotodynamic pump scaling. ASAIO J. 48:419-430.Doi, K., Smith, W.A., Harasaki, H., Takagaki, M., Ochiai, Y., Howard, M.W., Weber,S., Byerman, B.P., Massiello, A.L., Vitale, N., Donahue, A., Hirschman, G., and K.Fukamachi (2002) In vivo studies of the MagScrew total artificial heart in calves.ASAIO J. 48:222-225.McCarthy, P.M., and W.A. Smith (2002) Mechanical circulatory support—a long andwinding road. Science 295:998-999.Smith, W.A., Fukamachi, K., Weber, S., Harasaki, H., Doi, K., Schenk, S., Vitale,N., Hirschman, G., and A. Donahue (2002) The Cleveland Clinic/Foster Miller Mag-Screw pulsatile blood pump program. Annu. Int. Conf. IEEE Eng. Med. Biol. Proc.2002, vol. 2.Weber, S., Doi, K., Massiello, A.L., Byerman, B.P., Takagaki, M., Fukamachi, K.,Donahue, A., Chapman, P., Hirschman, G., Vitale, N., and W.A. Smith (2002) Invitro controllability of the MagScrew total artificial heart system. ASAIO J. 48:606-611.The Department of Biomedical Engineeringtime. The laboratory is part of an effort to developacoustic methods to detect the early stages of pumpmalfunction.Device Design and DevelopmentTotal Artificial Heart. The total artificial heart(TAH) program at CCF is the continuation of a longtermeffort. The CCF’s TAH design uses pusher-platepumps with biolized surfaces. A continuouslyreciprocating actuator is packagedbetween the two ventricles. The rate isvaried to maintain the left ventricle at anominal 90% of full stroke. An internalbattery provides short-term power, anda transcutaneous energy transmissionsystem (TETS) provides primarypower from a wearable external batterypack.The CCF’s TAH uses theMagScrew system of Foster-MillerTechnologies, Inc., the latest generationof blood pump driver. In operation,the MagScrew system is similar inconcept to an ordinary nut-and-screwsystem, except force is transmittedWilliam A. Smith, D.Eng., P.E.magnetically rather than by threadcontact, thus eliminating friction and wear. A thinsealing wall can also be interposed between “nut” and“screw,” permitting the motor and bearings to behermetically sealed in their own compartment.Combined with the CCF pumps, the system has provencapable of 9 L/min with 15 mm Hg atrial pressure.Ventricular Assist Devices. Our program ispursuing a number of approaches to a ventricular assistdevice (VAD). The MagVAD (based on the MagScrewactuator described above for the TAH) uses the rightpump changer of the TAH. However, the MagVAD is asingle-chamber assist system that will unload a failingventricle. Like the TAH, the MagVAD shows very highsensitivity to inlet pressure and can, if necessary, entirelyreplace the function of one ventricle.The inherently controlled bearing (ICB) pump isa VAD that uses a magnetic-bearing-supported rotaryblood pump. It promises to provide a simple, reliableapproach to an LVAD for those hearts that need asignificant increment of flow to support a satisfyinglifestyle.The “MiniMixedFlow” pump technology is ahighly miniaturized magnetic bearing rotary bloodpump technology, which is being pursued to developpartial assist adult LVAD’s, RVAD’s, and implantablepediatric pumps.Support Pumps. A number of external supportpump systems are being developed. These includean emergency cardiopulmonary bypass/extracorporealmembrane oxygenation system, funded by theDepartment of Defense; a catheter pump forminimally invasive surgery; and an external-useversion of the ICB system. Each of these pumpsfills a particular and crucial niche in the medical/surgical armamentarium.

The Department of Biomedical EngineeringHealing of Prosthetic Vascular GraftsCARDIOVASCULARBIOENGINEERINGSynthetic grafts are used widely in vascularreconstructive surgery, but their long-termpatency, especially in small-vessel or lowflowapplications, is limited. Smooth-muscle-cellaccumulation and matrix deposition adjacent tothe anastomoses mayprogress to intimalhyperplasia and graftfailure. We haveshown that smoothmuscle cells onprosthetic grafts arecharacterized by asynthetic, proliferativephenotype that isdistinctly differentfrom the contractilephenotype of arterialsmooth muscle cells.The graft smoothmuscle cells producehigh levels of growthfactors, and this maystimulate the ongoingmigration of arterialsmooth muscle cellsonto grafts as well assmooth-muscle-cellproliferation in thearea of the anastomosis.In addition, thegraft smooth musclecells secrete higher levels of collagen than aorticsmooth muscle cells. We are currently investigatingthe regulation of collagen synthesis. Prostheticgrafts in humans, unlike in experimentalanimals, never develop a complete endotheliallining, and therefore, they remain relativelythrombogenic compared with normal bloodvessels. Low-density lipoprotein (LDL) oxidizedLinda M. Graham, M.D.by Dacron graft-activated monocytic cellsinhibits endothelial cell migration in vitro, and theinhibition can be prevented by certain antioxidants.We are currently investigating themechanism by which oxidized LDL inhibitsendothelial cell migration,focusing on the effect ofoxidized LDL on intracellularcalcium concentration,cell membranefluidity, and cytoskeleton.In addition, the effect ofhypercholesterolemia, andthe effect of antioxidants,on endothelial cellingrowth onto prostheticgrafts in vivo is beingstudied. Identification ofthe mechanisms by whichhypercholesterolemiaimpairs prosthetic grafthealing will allow developmentof effective therapiesto improve graft patency.Active areas ofinvestigation include: 1)the molecular mechanismsinvolved in the posttranscriptionalregulationof collagen secretion bygraft SMC, 2) the mechanismby which oxidizedLDL inhibits endothelial cell migration, and 3)the effect of hypercholesterolemia on endothelialcell ingrowth onto prosthetic grafts in vivo. Abetter understanding of the changes in cellfunction on prosthetic grafts will be used todesign a better vascular graft using tissueengineering principles.THE GRAHAMLABORATORYPROJECT SCIENTISTScott M. Colles, Ph.D.POSTDOCTORAL FELLOWSAmitabha Chakrabarti, Ph.D.Pinaki Chaudhuri, Ph.D.Dongmei Zhang, Ph.D.TECHNICIANSAmanda FinanXuemei GaoJunqing Shen, B.S.STUDENTSNamisha JainCOLLABORATORSDerek S. Damron, Ph.D. 1Paul L. Fox, Ph.D. 21Center for AnesthesiologyResearch, CCF2Department of Cell Biology,CCFvan Aalst, J.A., Pitsch, R.J., Absood, A., Fox, P.L., and L.M. Graham (2000) Mechanism of Dacron-activatedmonocytic cell oxidation of low density lipoprotein. J. Vasc. Surg. 31:171-180.Absood, A., Furutani, A., Kawamura, T., and L.M. Graham (2002) Differential PDGF secretion by graftand aortic SMC in response to oxidized LDL. Am. J. Physiol. Heart Circ. Physiol. 283:H725-H732.Ghosh, P.K., Vasanji, A., Murugesan, G., Eppell, S.J., Graham, L.M., and P.L. Fox (2003) Membrane microviscosityregulates endothelial cell motility. Nat. Cell Biol. 4:894-900.Chaudhuri, P., Colles, S.M., Damron, D., and L.M. Graham (2003) Lysophosphatidylcholine inhibits endothelialscell migration by increasing intracellular calcium and activating calpain. Arterioscler. Thromb.Vasc. Biol. 23:218-233.25

CARDIOVASCULARBIOENGINEERINGTHE GREENBERGLABORATORYINVESTIGATORKenneth Ouriel, M.D.Dept. of Vascular Surgery, CCFRESEARCH FELLOWSStephan Haulon, M.D.Jamal Khwaja, M.D.Ellis Sampram, M.D.Gene Tanquilut, MDTECHNICAL SUPPORTTamara BurtonDaniel YoungENGINEERSJames Foster, B.S.M.E.Davorin Skender, B.S.Karl WestRoy K. Greenberg, M.D.Vascular Surgery Focuses on theEndovascular Treatment ofAortic Aneurysms and Aortic DissectionsThe Vascular Surgery Laboratory, which hasrecently expanded, focuses on twoapproaches to endovascular treatment:mechanical devices and drug therapy fordissolving thrombus.Endovascular grafting of the aorta hasemerged as the treatment of choice for certainpatients with an abdominal aortic aneurysm. Therisks associated with open surgery are exponentiallyincreased with the number and severity ofmedical comorbidities, to the point where therisk of open operation is prohibitive. The use ofendovascular technology to site an aortic grafteliminates many of the risks ofopen abdominal surgery, inparticular, the physiologicalstresses associated with aorticcross-clamping.Currently being developedare endovascular branch vesseldevices for the repair of aorticaneurysms involving thehypogastric, renal, SMA andceliac arteries. Universalapplication of endografttechnology is limited by theanatomy of aorta, especially theextent and distribution of theaneurysmal disease itself.Aneurysm repair requires anappropriately designed deviceand technical maneuvers tailoredto the individual patient’s need.Considering the wide variety ofaneurysm anatomy, branched stent grafting forthese complex aneurysms is now a valuableoption for patients who are deemed to be at highrisk for conventional aneurysm repair. Thesedevices are being developed and prototyped withthe aid of solid and surface modeling using Pro/E, along with analytical calculations andGreenberg, R.K., Srivastava, S.D., Ouriel, K., Waldman, D., Ivancev, K., Illig, K.A.,Shortell, C., and R.M. Green (2000) An endoluminal method of hemorrhage control andrepair of ruptured abdominal aortic aneurysms. J. Endovasc. Ther. 7:1-7.Greenberg, R.K., Lawrence-Brown, M., Bhandari, G., Hartley, D., Stelter, W., Umscheid,T., Chuter, T., Ivancev, K., Green, R., Hopkinson, B., Semmens, J., and K. Ouriel(2001) An update of the Zenith endovascular graft for abdominal aorticaneurysms: initial implantation and mid-term follow-up data. J. Vasc. Surg. 33(2Suppl):S157-S164.Greenberg, R.K. (2002) Abdominal aortic endografting: fixation and sealing. J. Am.Coll. Surg. 194(1 Suppl):S79-S87.Ouriel, K., Greenberg, R.K., and D.G. Clair (2002) Endovascular treatment of aorticaneurysms. Curr. Probl. Surg. 39:242-345.Kwan, D., Dries, A., Burton, T., Bhandari, G., Young, D., Green, R., Ouriel, K., andR.K. Greenberg (2003) Thrombus characterization with intravascular ultrasound: potentialto predict successful thrombolysis. J. Endovasc. Ther. 10:90-98.The Department of Biomedical Engineeringexperimental data collection and interpretation usingMatlab. Specific interest in device development willbe in the effects of pulsatile flow on the device’sgraft material/alloy construction.The laboratory has also worked with theBiological Resources Unit (BRU) to institute acompliant setting in which Good LaboratoryPractice (GLP) studies can be conducted in a fullyequipped animal angiography suite with state-ofthe-artfluoroscopic imaging and intravascularultrasound. A Quality Assurance (QA) office wasestablished to independently monitor each GLPstudy to assure the quality and integrity of thedata in conformance with 21CFR Part 58. StandardOperating Procedures wereestablished, implemented and areregulated by the QA office. Wehave completed three GLPstudies working with companieson developing innovative stentsand stent grafts and are planningon performing more GLP studiesthis year.The other focus of thelaboratory is the study of drugtreatment for thrombusdissolution. Administration ofthrombolytic agents intooccluded arteries and veins hasbecome one of the mostfrequent methods to dissolvethrombus. Along with lyticregimes used in the clinicalmanagement of peripheral arterial ischemia, newdrugs and catheter devices are being explored andcompared within in vitro and in vivo models.We have developed an in vitro perfusionsystem, simulating the human arterial system in aclinical setting. Thus we are able to control suchvariables as the size and consistency of thethrombus and the hemodynamic conditionspresent while maintaining flow rates, meanpressures and temperature. Our goals are todetermine the speed of flow restoration, quantifyembolic debris and evaluate the chemicalcompleteness of thrombolysis. Some of ouranalysis is performed with the use of radioactivematerials, 125 Iodine-labeled fibrinogen and111Indium oxyquinoline, to label platelets andanalyze the effects of lytic agents on the thrombus.The laboratory has also created a reproduciblechronic thrombus in vivo model in the caninesarteria system. Both in vitro and in vivo models willallow for future research of innovative thrombolyticdrugs in conjunction with device cathetersfor studying thrombolysis.Roy K. Greenberg, M.D.26

The Department of Biomedical EngineeringFeatures of Natural Heart Valves FormBasis for Bioprosthetic ReplacementsCARDIOVASCULARBIOENGINEERINGThe Heart Valve Laboratory’s mainapproach is to study the structure/function relationship of heart valve tissuesto determine failure mechanisms of manufacturedreplacement heart valves. Our goal is todevelop a bioprosthetic valve that completelymimics the function of the natural valve. Otherapproaches include materials testing, mathematicalmodeling, microscopy, biochemical analysis,and cell culture.Micromechanical testing has been used todetermine the functional relationships betweenthe fibrosa and the ventricularis and the rolesplayed by collagen and elastin. Selectiveenzymatic degradationhas been used to removecertain valve tissues’structural proteins(collagen, elastin, andglycosaminoglycans) andmeasure the mechanics ofthe resulting material todetermine how eachconstituent contributes tothe mechanics of thewhole.We use videoimage processing tomeasure biaxial strains ofintact valve materials.Such alternative materialstesting techniques areneeded to describecompletely the materialproperties of such highlydeformable and anisotropicfibrous materials.Mathematicalmodeling allows us to analyze the viscoelasticnature of heart valve tissue and to establish acloser link between testing and analysis.Difficult-to-measure material parameters canthereby be estimated, constitutive modelsverified, and difficult-to-perform tests simulated.We have already developed extensions to Fung’soriginal Quasi-Linear Viscoelastic theory thatenable us to extract QLV parameters fromconventional, medium-speed materials tests thatare remarkably predictive of the long-term cyclicloading behavior of heart valve tissue. Incollaboration with NASA researchers, we aredeveloping advanced soft-tissue models forsimulating robotic surgery in a virtual realitytraining system.The mechanical, microscopic andbiochemical techniques developed on aorticvalves have recently been applied in studyingmyxomatous mitral valve disease, a conditioncharacterized by thickening of valve tissues andIvan Vesely, B.E.Sc., Ph.D.stretching of leaflets and chordae, causing thevalve to leak. Although the condition can becorrected surgically, the outcomes are not alwayssatisfactory, and the disease itself is notunderstood. By understanding the causes of thedisease, we can better treat the patient. Fromstudies of specific biochemical and mechanicalchanges that occur in these tissues, our resultssuggest it is a disease more of the chordae thanthe leaflets, as was previously thought. We arealso exploring the genetic determinants of thedisease.A long-term project is ongoing to engineera viable tissue valve implant consisting ofelastin, collagen andglycosaminoglycans likethose found in naturaltissues. These moleculescan be synthesized by cellsin culture or purified fromtissues, then manipulatedto mimic the aortic valve’snormal structural framework.This process is calledtissue engineering, and ourapproach is specificallyfocused on replacingconnective tissues notcapable of repair on theirown. We have fabricatedtissue-engineered mitralvalve chordae and are nowapplying this technology tothe development of themore complex aortic valve.This research isfunded by grants from theNIH, the U.S. Army, andthe Mareb Foundation. We are actively seekingways of augmenting our research funds byparticipating in technology transfer and inresearch contracts for the biomedical industry.THE VESELYLABORATORYSENIOR ENGINEERSJ. Edward Barber, B.Sc.Evelyn O. Carew, Ph.D.ENGINEERMargaret Adamczyk, Ph.D.POSTDOCTORAL FELLOWTodd Doehring, Ph.D.GRADUATE STUDENTSRahila Ansari , M.S.Jun Lo, M.S.Kalpesh Patel, B.S.Lawrence Rittman, B.S.Yaling Shi, M.S.Metin Yavuz, M.S.COLLABORATORSDelos M. Cosgrove, M.D. 1Brian P. Griffin, M.D. 2Vincent C. Hascall, Ph.D. 3Ronald J. Midura, Ph.D. 3Norman B. Ratliff, M.D. 41Dept. of Thoracic andCardiovascular Surgery, CCF2Dept. of CardiovascularMedicine, CCF3Dept. of Biomedical Engineering,CCF4Dept. of Anatomic Pathology,CCFCarew, E.O., Barber, J.E., and I. Vesely (2001) Role of preconditioning and recoverytime in repeated testing of aortic valve tissues: validation through quasilinearviscoelastic theory. Ann. Biomed. Eng. 28:1093-1100.Lee, T.C., Midura, R.J., Hascall, V.C., and I. Vesely (2001) The effect of elastindamage on the mechanics of the aortic valve. J. Biomech. 34:203-210.Ramamurthi, A.. and I. Vesely (2002) Smooth muscle cell adhesion on crosslinkedhyaluronan gels. J. Biomed. Mater. Res. 60:195-205.Mills, W.R., Barber, J.E., Skiles, J.A., Ratliff, N.B., Cosgrove, D.M., Vesely, I.,and B.P. Griffin (2002) Clinical, echocardiographic, and biomechanical differences inmitral valve prolapse affecting one or both leaflets. Am. J. Cardiol. 89:1394-1399.Shi, Y., Ramamurthi, A., and I. Vesely (2002) Towards tissue engineering of a compositeaortic valve. Biomed. Sci. Instrument. 38:35-40.27

THE WHITAKERBIOMEDICAL IMAGINGLABORATORYTHE FISHERLABORATORYINVESTIGATORSAndrew Cwik, B.S.Bernhard Sturm, Ph.D.Robert Holtman, M.S.GRADUATE STUDENTKunio Nakamura, B.S.COLLABORATORSJeff Cohen, M.D. 1Robert Fox, M.D. 1Mike Phillips, M.D. 2Richard Rudick, M.D. 1Lael Stone, M.D. 1Jean Tkach, Ph.D. 2Bruce Trapp, Ph.D. 31Mellen Ctr. for MS Treatmentand Research, Dept. ofNeurology, CCF2Div. of Radiology, CCF3Dept. of Neurosciences, CCFThe objectives of the neuroimaging researchprogram are to develop new imagingtechniques that provide relevant markersand predictors of disease progression in multiplesclerosis (MS) and other neurological diseases.Our research is focused on quantitative methodsfor measurement of tissue injury andneurodegeneration from magnetic resonanceimages (MRIs) of the brain. We also seek thebest ways to apply these methods to improveroutine monitoring and therapy evaluation, aswell as to gain new insights into pathologicprocesses.To quantify brainMRIs, we are workingon the development andvalidation of imageanalysis software forautomated imagesegmentation andregistration, multiprotocollesionquantification, andanatomic labeling. Amajor area of interest inour laboratory isaccurate and reliablemeasurement of brainatrophy, a marker ofirreversible tissuedamage. Application ofa new brain-atrophymeasurement methodhas demonstrated thatatrophy occurs in theearly stages of MS, iscorrelated to concurrentand future disability,and can be slowed bytreatment. Ongoing projects include a longitudinalstudy of brain atrophy, which aims todetermine the clinical, MRI, immunologic, andpathologic correlates of brain atrophy in MSpatients. This 5-year study involves the evalua-The Department of Biomedical EngineeringImaging Provides New Views on MultipleSclerosis and NeurodegenerationElizabeth Fisher, Ph.D.tion of MS patients and normal healthy volunteerswith MRI and clinical examinations. We areworking on improved methods for combinationof information from different types of imagesand more sensitive methods for detection oflesion changes over time. The relationshipsbetween clinical and MRI variables and timecourse of changes in the MRI measurements willbe investigated. A post-mortem imaging protocolhas been incorporated into an MS brain donationprogram to relate the different types of MRImeasurements to the underlying pathology. Othercollaborative research projects include a followupstudy to investigatethe predictive value ofMRI measurements overthe course of 10 years inMS patients and theanalysis of images todetermine associationsbetween specific domainsof cognitive impairmentand MRI measures ofpathology in patients withMS. Software developedin our laboratory is beingused in several clinicaltrials of new treatmentsfor MS, and efforts areunder way to incorporatebrain atrophy measurementsinto clinicalpractice.The newneuroimaging centerlocated at CCF’s MellenCenter will enable us toextend our efforts fromimage analysis into imageacquisition. Future plans include the investigationof newer imaging techniques that will providemore specific information about underlyingdisease processes in MS and other neurologicaldisorders.Fisher, E., Cothren, R.M., Tkach, J.A., Masaryk, T.J., and J.F. Cornhill (1997) Knowledge-based 3D segmentationof MR images for quantitative MS lesion tracking. SPIE Medical Imaging 3034:599-610.Rudick, R.A., Fisher, E., Lee, J.-C. and J. Simon (1999) Use of the brain parenchymal fraction to measurewhole brain atrophy in relapsing-remitting MS. Mutiple Sclerosis Collaborative Research Group. Neurology53:1698-1704.Fisher, E., Rudick, R.A., Cutter, G., Baier, M., Miller, D., Weinstock-Guttman, B., Mass, M.K., Dougherty,D.S., and N.A. Simonian (2000) Relationship between brain atrophy and disability: an 8-year follow-upstudy of multiple sclerosis patients. Multiple Sclerosis 6:373-377.Meier, D., and E. Fisher (2002) Parameter space warping: shape-based correspondence between morphologicallydifferent objects. IEEE Transactions on Medical Imaging 21:31-47.Fisher, E.. Rudick, R.A., Simon, J.H., Cutter, G., Baier, M., Lee, J.C., Miller, D., Weinstock-Guttman, B.,Mass, M.K., Dougherty, D.S., and N.A. Simonian (2002) Eight-year follow-up study of brain atrophy in patientswith MS. Neurology 59:1412-1420.28

The Department of Biomedical EngineeringQuantitative Microscopy andHigh-Resolution Imaging of BoneTHE WHITAKERBIOMEDICAL IMAGINGLABORATORYOsteoporosis, a disease characterized bylow bone mass and loss of structuralintegrity of bone tissue, can ultimatelylead to bone fracture. Osteoporosis is responsiblefor 1.5 million fractures annually inAmerica. The annual cost for treatingosteoporotic fractures worldwide is estimatedto be 10-15 billion U.S. dollars. Significantmorbidity and mortality are associated withosteoporotic fractures, and 80% of thoseaffected by osteoporosis are women.Micro-computed tomography (micro-CT)is a three-dimensional (3D) x-ray imagingtechnology used to evaluatethe trabecular structure ofcancellous bone both in vivoand ex vivo specimens fromhumans and animals. As anoninvasive imagingmodality, it is suitable formonitoring varioustreatment effects in smallanimals over time. It hasbeen used to study in vivochanges in trabeculararchitecture of the tibia inOVX rats and the effects ofsubsequent hormonereplacementtherapy inestrogen-depletedosteopenic rats. Our grouphas recently designed anddeveloped a high-resolutionmicro-CT imaging system toevaluate the microarchitectureof bone ex vivoin specimens and in vivo insmall animal models.Working closely with basic and clinicalscientists, we are developing novel highresolutionimaging and post-processingtechniques that will aid in evaluating early bone lossand bone formation in small-animal models ofosteoporosis and in monitoring the effects ofvarious treatments longitudinally. We propose todevelop: 1) high-resolution 3D fluorescencemicroscopy and micro-CT imaging techniques foranalyzing in vivo and excised bone over largeregions of interest; 2) intra- and multimodalityregistration technique for spatially aligninglongitudinal micro-CT images and 3D colorhistology images of trabecular bone; 3) real-timevisualization and graphic manipulation tools forsimultaneously displaying 3D color-histology and4D micro-CT images;and 4) novel measuresto identify early boneloss/formation fromdirect measurement oflongitudinal micro-CTimages of bonetrabecula. Theseimaging tools willallow bone researchersto visualize two ormore spatially aligned3D micro-CT imagesets as well as the 3Dcolor histology withthe micro-CT imagessimultaneously. Mostimportantly, it willprovide a mechanismfor interactivelyreviewing, localizing,and quantifyinginformation obtainedfrom different imagingmodalities at differentspatial resolutions.Kimerly A. Powell, Ph.D.THE POWELLLABORATORYRESEARCH ENGINEERLarry Latson, M.S.COLLABORATORSSuneel S. Apte, Ph.D. 1Bradley Clymer, Ph.D .2Ronald J. Midura, Ph.D. 1George F. Muschler, M.D. 1Don Stredney 31Dept. of BiomedicalEngineering, CCF2Dept. of Electrical Engineering,Ohio State University,Columbus, OH3The Ohio SupercomputerCenter, Columbus, OHLatson, L., Powell, K.A., Sturm, B., Schvartzman, P.R., and R. D. White (2001) Clinical validation of anautomated boundary tracking algorithm on cardiac MR images. Int. J. Cardiac Imaging 17:279-286.Obuchowski, N.A., Graham, R.J., Baker, M.E, and K.A. Powell (2001) Ten criteria for effective screening:their application to multislice CT screening for pulmonary and colorectal cancers. AJR Am. J. Roentgenol.176:1357-1362.Sivaramakrishna, R., Obuchowski, N.A., Chilcote, W.A., and K.A. Powell (2001) Automatic segmentationof mammographic density. Acad. Radiol. 8:250-256.Sturm, B., Powell, K., Stillman, A.E., and R.D. White (2002) Registration of 3D CT angiography MRimages in coronary artery disease patients, Int. J. Cardiovasc. Imaging, accepted.Muschler, G.F., Nitto, H., Matsukura, Y., Boehm, C., Valdevit, A., Kambic, H., Davros, W., Powell, K., andK. Easley (2003) Spine fusion using cell matrix composites enriched in bone marrow-derived cells. Clin.Orthop. (407):102-118.29

THE WHITAKERBIOMEDICAL IMAGINGLABORATORYTHE VINCELABORATORYINVESTIGATORSDevyani Bedekar, B.S.Jon D. Klingensmith, Ph.D.Barry D. Kuban, B.S.Anuja Nair, B.Eng., Ph.D.COLLABORATORSAaron J. Fleischman, Ph.D. 1Steven E. Nissen, M.D. 2Shuvo Roy, Ph.D. 1E. Murat Tuzcu, M.D. 21Dept. of BiomedicalEngineering, CCF2Dept. of CardiovascularMedicine, CCFIntravascular ultrasound (IVUS) is becomingaccepted as an imaging technique that allowsprecise tomographic assessment of thecoronary artery anatomy in vivo. Clinical studieshave documented the sensitivity of IVUS indetecting atherosclerosis and in quantifying themorphology of coronary arterial lesions. Moreimportantly, IVUS can potentially quantify thestructure and composition of normal andatherosclerotic coronary arteries in the clinicalsetting rather than relying on histological data,which can only be obtained at autopsy.Many studies evaluating the efficacy ofIVUS in determining plaque composition havebeen limited by theirreliance on digitizingvideotape, which hasthree major limitations:(1) it is verytime consuming andtherefore not feasiblefor near real-timeanalysis; (2) it reducesthe resolution of theimage to that ofvideotape (approximately330 µm); and(3) parameters such asgain and intensity canbe adjusted by the operator, thereby addingvariability to the data set. More recent studieshave realized the importance of gaining access tothe ultrasound backscattered signal, often referredto as the radiofrequency (RF) signal or “backscatter”.Spectral analysis of the unprocessedultrasound signal allows a more detailed interro-The Department of Biomedical EngineeringIntravascular Ultrasound Offers InnovativeView on Atherosclerotic Plaque Buildupand Treatment Strategy OptionsTHETHEVINCELABO-RAgation of various vessel components thandigitization of videotape.We have developed software that usesspectral analysis methods to determine plaquecomposition from IVUS images and display a“Virtual Histology” map. This analysis tool hasbeen licensed to Volcano Therapeutics (LagunaHills, CA) and is currently undergoing trials inEurope.Harmonic ImagingOver the past several years, the technologyfor IVUS, in response to clinical pressure, hasmoved towards lower-profile probes withimproved handling. Not only does a lower-profileprobe allow one to exploremore of the coronary tree,but it is also less likely todisturb potentially unstableplaque at a stenosis site.Intravascumovtowardslower-profiprobes withImage quality has beenmuch less an issue thancatheter profile, and in fact,newer catheters usingsmaller, unfocused elementsactually offer poorerimaging performance thantheir predecessors.In collaboration withDrs. Shuvo Roy and Aaron Fleischman, wepropose to design and build high-frequencyultrasound transducers comprising traditionalceramic and novel polymeric materials fabricatedusing MEMS technology. The ability of thesetransducers to be used for high-frequencyharmonic imaging will be assessed.D. Geoffrey Vince, Ph.D.Nair, A., Kuban, B.D., Tuzcu, E.M., Schoenhagen, P., Nissen, S.E., and D.G. Vince (2002) Coronaryplaque classification using intravascular ultrasound radiofrequency data analysis. Circulation 106:2200-2206.Klingensmith, J.D., and D.G. Vince (2002) B-spline Methods for interactive segmentation and modeling oflumen and vessel surfaces in three-dimensional intravascular ultrasound. Comput. Med. Imaging Graph. 26429-438.Tajaddini, A., Kilpatrick, D., and D.G. Vince (2002) A Novel Experimental Method to Estimate Stress-StrainBehavior of Intact Coronary Arteries Using Intravascular Ultrasound (IVUS). Journal of Biomechanical Engineering125(1):120-123, 2002Klingensmith, J.D., Tuzcu, E.M., Nissen, S.E., and D.G. Vince (2003) Validation of an automated systemfor luminal and medial adventitial border detection in three-dimensional Intravascular ultrasound. Int. J.Cardiovasc. Imaging 19:93-104.Vince, D.G., Nair, A., Klingensmith, J.D., Moore, M.P., and V. Burgess (2003) Radiofrequency tissue characterization.In: Waksman R, Serruys P, eds. Handbook of the Vulnerable Plaque. London: Martin DunitzLtd., 2003.30

Deep brain stimulation (DBS) of the thalamus orbasal ganglia represents an effective clinicaltreatment of several medically refractorymovement disorders, including Parkinson'sdisease and essential tremor. However, understandingof the mechanisms of action of DBSremains elusive. It is presently unclear whatelectrode designs and stimulation parameters areoptimal for maximum therapeutic benefit andminimal side effects. The goal of this laboratoryis to couple results from functional imaging,neurophysiology, and neuroanatomy to create atheoretical framework that enhances ourunderstanding of the effects of DBS andprovides a virtual testing ground for newstimulation paradigms.We are working to develop a quantitativeunderstanding of the effects of DBS using thetechniques of computational neuroscience andelectromagnetic field modeling. Our goal is toaugment experimental investigation in DBS ofthe parkinsonian non-human primate as well asimprove the electrode targeting and postoperativeparameter selection processes in thehuman. Our modeling process consists of threebasic steps. First we develop models of theelectric field generated by DBS electrodes. Weestimate the tissue electrical properties of thebrain region surrounding the electrode usingdiffusion tensor MRI. We then create a 3Drendering of the DBS electrode and surroundingtissue medium and solve for the electric fieldusing the finite element method. Our resultsshow that minor alterations in either theelectrode position in the brain or geometry of thestimulating contact can strongly affect the shapeof the field and subsequent neural response tostimulation. The second step consists ofcoupling the electric field to models of individualneurons. The neuron models consist of geometriesbased on 3D reconstructions, and ionchannel biophysics derived from experimentalrecordings. The neuron models are positioned inthe field and their response is measured as aThe Department of Biomedical EngineeringNew Program Investigates Deep BrainStimulation for Movement DisordersUsing Computational Modelingfunction of the stimulation parameters. Usingthese techniques, we have developed stimuluswaveforms that enable selective activation oftargeted neuronal populations surrounding theelectrode. The final step in our modeling processconsists of applying the stimulation effectspredicted at the single cell level to large scaleneuronal network models. The therapeutic effectsof DBS probably lie in its ability to disruptpathological network oscillations within differentsections of the brain. We are working tounderstand the origin of these oscillatory patternsin network models that consist of hundreds ofinteracting neurons. We apply the effects of DBSto our network models and address how thestimulation changes interactions between nuclei.Our results show that DBS can dramaticallyenhance the firing of nuclei upstream anddownstream from the site of stimulation and weare working to couple our results to PET/fMRIexperiments during DBS in the human.DBS technology is in its infancy. We areusing computer modelingcoupled to experimental andclinical investigation to buildthe foundation for the developmentof the next generation ofDBS devices. Our goal is todevelop a computationalframework that will generateexperimentally testablehypotheses on the mechanismsof DBS and provide a testingground for new electrodedesigns and stimulationparameters. In turn, we hope toimprove DBS for the treatmentof movement disorders andprovide fundamental technologynecessary for the application ofDBS to new clinical arenas suchas epilepsy and obsessivecompulsive disorder.NEURAL CONTROLTHE MCINTYRELABORATORYCOLLABORATORSNitish V. Thakor, Ph.D. 1Jerrold L. Vitek, M.D., Ph.D. 2Warren M. Grill, Ph.D. 3André Parent, Ph.D. 4Ali Rezai M.D. 5Mike Phillips, M.D. 61Dept. of Biomed. Engineering,Johns Hopkins Univ.Sch. of Med., Baltimore, MD2Dept. of Neurology, EmoryUniv. Sch. Of Med., Atlanta,GA3Dept. of Biomed. Eng., CaseWestern Reserve Univ.,Cleveland, OH4Dept. of Anatomy, Univ. ofLaval, Québec, Canada5Div. of Neurosurgery, CCF6Div. of Radiology, CCFCameron McIntyre, Ph.D.McIntyre, C.C., and W.M. Grill (2000) Selective microstimulation of central nervous system neurons.Ann. Biomed. Eng. 28:219-233.McIntyre, C.C., and W.M. Grill (2001) Finite element analysis of the current-density and electric fieldgenerated by metal microelectrodes. Ann. Biomed. Eng. 29:227-235.McIntyre, C.C., Richardson, A.G., and W.M. Grill (2002) Modeling the excitability of mammalian nervefibers: influence of afterpotentials on the recovery cycle. J. Neurophysiol. 87:995-1006.McIntyre, C.C., and W.M. Grill (2002) Extracellular stimulation of central neurons: influence of stimuluswaveform and frequency on neuronal output. J. Neurophysiol. 88:1592-1604.McIntyre, C.C., and N.V. Thakor (2002) Uncovering the mechanisms of deep brain stimulation for Parkinson'sdisease through functional imaging, neural recording, and neural modeling. Crit. Rev. Biomed.Eng. 30:249-281.31

NEURAL CONTROLGuang H. Yue, Ph.D.THE YUELABORATORYPROJECT STAFFJing-Zhi Liu, Ph.D.,Wlodzimierz (Vlodek)Siemionow, Ph.D.SENIOR RESEARCH ENGINEERSYin Fang, Ph.D.Vinoth K. Ranganathan, M.S.RESEARCH TECHNOLOGISTJohn Boros, M.S.GRADUATE STUDENTSHaibin Huang, M.S.Beth Lewandowski, B.S.Zuyao Shan, M.S.Bin Yao, M.S.Luduan Zhang, M.S.COLLABORATORSRobert W. Brown, Ph.D. 1Leonard H. Calabrese, D.O. 2Peter J. Evans, M.D., Ph.D. 3Joan E. Fox, Ph.D. 4Mark L. Latash, Ph.D. 5Zong-Ming Li, Ph.D. 6Steven I. Reger, Ph.D. 7Vinod Sahgal, M.D. 7Maria Siemionow, Ph.D., M.D. 81Case Western Reserve Univ.,Cleveland, OH2Dept. of Rheumatic andImmunologic Disease, CCF3Dept. of Orthopaedic Surgery,CCF4Dept. of Molecular Cardiology,CCF5Pennsylvania State Univ.,University Park, PA6Univ. of Pittsburgh, Pittsburgh,PA7Dept. of Physical Medicine andRehabilitation, CCF8Dept. of Plastic and ReconstructiveSurgery, CCFMechanisms of control of humanvoluntary movements have been studiedextensively over the past severaldecades. Although much has been revealedregarding control strategies in the peripheralneuromuscular system, little is known concerning(1) how the brain, the center of any neuromuscularoperation, controls a voluntary motoraction, and (2) how the central nervous system(CNS), including the brain, adapts to variousacute and chronic perturbations, such as fatigue,immobilization, training, aging, microgravity,injury or disease. A better understanding ofthese questions will lead to more effectivetreatment of movement-disorders. Ourlaboratory focuses on investigating issues relatedto these questions.Cortical control of finger movementsHow the brain controls our fingers is oftremendous interest to many disciplines. We areconducting a number of projects to investigatethis question. One project involves studyingforce-sharing patterns among individual fingers,using healthy subjects and different groups ofpatients, while brain signals are recorded.Another study attempts to improve hand/fingerfunction in older adults through training whilemonitoring brain adaptability.Plasticity of neural command for maximalvoluntary contraction (MVC)Is the command from brain to muscle forMVC fixed? If the answer is yes, then musclestrength enhancements can only be achieved byenlarging muscle mass and/or improving musclecoordination; if no, then muscle strength can beimproved by increasing neural commands fromthe brain through training the neuromuscularsystem or even the neural system alone. Thispossibility (training the neural system to improvemuscle strength) has great potential in neuromus-The Department of Biomedical EngineeringMovement Disorder Treatment Optionsto be Expanded with Insight into NeuralControl of Motor Actioncular rehabilitation because it provides opportunitiesfor improving motor function (strength) inpatients who cannot perform forceful musclecontraction training. Our data show that significantstrength gain can be achieved by training theCNS alone.Neural mechanisms of muscle fatigueIncreased fatigability occurs in every patientwith muscle weakness, regardless of whether theweakness is due to a central or peripheral neurologicaldisorder. The underlying mechanisms arenot well understood, and there is a need inneurology and rehabilitation to study fatigabilitysystematically. The behavior of the peripheralneuromuscular system during muscle fatigue hasbeen studied extensively, but the role of the CNSin muscle fatigue is largely unknown. We believethat without a good understanding of mechanismsof fatigue in health, an assessment of mechanismscontributing to increased fatigability in neurologicaldisorders is difficult. Our goal is to determinethe mechanisms underlying increased fatigability inpatients with neurological disorders.Neural mechanisms underlying motorfunctionrecovery in stroke patientsMotor-function recovery after stroke is aprocess of re-learning lost motor skills. Althoughnumerous studies have been performed involvingmotor performance in stroke patients, little isknown about the neural mechanisms that mediatethe re-acquisition of motor skills. This projectuses functional magnetic resonance imaging andmovement-related cortical potential techniques todetermine brain-function mechanisms underlyingmotor-function recovery by examining the patternof brain activation at various stages of therecovery process. This information is importantfor designing rehabilitative treatments and forreducing health care costs by stopping unnecessarytreatment at the earliest appropriate stage.Yue, G.H., Liu, J.Z., Siemionow, V., Ranganathan, V.K., Ng, T.C., and V. Sahgal (2000) Brain activationduring human finger extension and flexion movements. Brain Res. 856:291-300.Liu, J.Z., Dai, T.H., Elster, T.H., Sahgal, V., Brown, R.W., and G.H. Yue (2000) Simultaneous measurementof human joint force, surface electromyograms, and functional MRI-measured brain activation. J.Neurosci. Methods 101:49-57.Dai, T.H., Liu, J.Z., Sahgal, V., Brown, R.W., and G.H. Yue (2001) Relationship between muscle outputand functional MRI-measured brain activation. Exp. Brain Res. 140:290-300.Fang, Y., Siemionow, V., Sahgal, V., Xiong, F., and G.H. Yue (2001) Greater movement-related corticalpotential during eccentric verses concentric muscle contractions. J. Neurophysiol. 86:1764-1772.Liu, J.Z., Dai, T.H., Sahgal, V., Brown, R.W., and G.H. Yue (2002) Nonlinear cortical modulation ofmuscle fatigue: a functional MRI study. Brain Res. 957:320-329.32

ORTHOPAEDICBIOLOGY ANDBIOENGINEERINGThe Department of Biomedical EngineeringExtracellular Matrix and Its Remodelingby MetalloproteasesTHE APTELABORATORYFELLOWSJ. Michael Engle, Ph.D.Robert Somerville, Ph.D.SENIOR TECHNOLOGISTWeiping (Lauren) Wang, M.S.RESEARCH TECHNICIANSKatherine Jungers, B.S.Samantha Oblander, B.S.Extracellular proteases are essential fordevelopment and play a major role in thepathogenesis of diseases such as cancerand arthritis. Remodeling alters tissue architecture,and at the cellular level, proteolysis affectscell behavior and cell fate through processing ofgrowth factors, receptors, cytokines, andadhesion molecules.The laboratory works on two families ofmolecules—the matrix metalloproteases (MMPs)and a novel family of ADAMTSenzymes. Specific ongoing studies are asfollows:1. We are determining thebiological role of novel ADAMTSproteases previously discovered by usthrough the generation of knockoutmice and analysis of human diseases.Two specific functions being addressedare the processing of the aminopropeptideof fibrillar collagens andproteolysis of large aggregatingproteoglycans such as aggrecan andversican. These projects have relevancefor the Ehlers-Danlos syndrome,arthritis and cancer.2. We are investigating thestructural biochemistry of specificdomains within the ADAMTS enzymesas well as in a novel family ofnonproteolytic ADAMTS-like moleculesthat we have recently discovered. This is beingdone by expression and characterization ofrecombinant enzymes.3. We previously generated a line oftransgenic mice with targeted deletion of theMMP-14 gene. These mice have a number ofanomalies, which are presently being characterizedby various morphological and biochemicalapproaches. In particular, we are determining themechanisms of abnormal skeletogenesis andangiogenesis in these mice.Hurskainen, T.L., Hirohata, S., Seldin M.F., and S.S.Apte (1999) ADAM-TS5,ADAM-TS6 and ADAM-TS7, novel members of a new family of zinc metalloproteases(ADAM-TS, A Disintegrin And Metalloprotease domain with ThromboSpondin typeI motifs). General features and genomic distribution of the ADAM-TS family. J.Biol. Chem. 274:25555-25563.Zhou, Z., Apte, S.S., Wang, J., Rauser, R., Baaklini, G., Soininen, R., and K.Tryggvason (2000) Defective skeletal growth, angiogenesis and premature death inMMP-14 deficient mice. Proc. Natl. Acad. Sci. USA 97:4052-4057.Fernandes, R.J., Hirohata H., Engle, J.M., Colige, A., Cohn, D.H., Eyre D.R., andS.S. Apte (2001) Procollagen II amino propeptide processing by ADAMTS-3: insightson dermatosparaxis. J. Biol. Chem. 276:31502-31509.Hirohata, S., Wang, L.W., Miyagi, M., Yan, L., Seldin, M.F., Keene, D.R., Crabb,J.W., and S.S. Apte (2002) Punctin, a novel ADAMTS-like molecule (ADAMTSL-1),in extracellular matrix. J. Biol. Chem. 277:12182-12189.34

The Department of Biomedical EngineeringRegulation of Growth Plate Chondrocytesby Nuclear Hormone ReceptorsORTHOPAEDICBIOLOGY ANDBIOENGINEERINGChildhood obesity, a rapidly growingconcern, now threatens one in fourchildren with long-term health problems.Recent advances in the understanding ofadipogenesis and lipid metabolism at themolecular level have revealed the existence of afamily of nuclear hormone receptors that linknutritional signals to the control of geneexpression, and are induced or activated inresponse to a high-fat diet. These molecules,termed peroxisome proliferator-activatedreceptors (PPARs), are also expressed in boneand cartilage and interfere with thyroid hormonereceptor (TR)-mediated gene transcription incells in which PPARs and TRs are co-expressed.Given that peroxisomal function isrequired for normal endochondral ossificationand that thyroid hormone plays a central role inregulating skeletal maturation at growth plate, itis reasonable to ask if crossstalk exists betweenTR- and PPARmediatedtranscriptionalregulation in growthplate chondrocytes.Crosstalk between theTR and PPAR signalingpathways in growthplate cells may havedirect clinical implicationsregarding theetiology of an obesityrelatedhip disease inchildren, slipped capitalfemoral epiphysis. Inour laboratory, we aretesting the hypothesisthat PPARs areinducible repressors ofTR-mediated genetranscription in growthplate chondrocytes. Thespecific aims of thiswork are: (1) todocument the expressionof PPAR isoforms in the growth plate and toestablish if PPARs are co-expressed with TRs inindividual growth plate chondrocytes; (2) todetermine if exposure of growth platechondrocytes to PPAR activators results ininduction of PPAR expression and activation ofPPAR-mediated gene transcription in these cells;(3) to characterize the molecular interactions ofPPARs with TRs and retinoid X receptors ingrowth plate chondrocytes and describe theeffect of these interactions on TR-mediated genetranscription; and (4) to define the effects ofPPAR activation on thyroid hormone-inducedgrowth arrest and terminal differentiation ofgrowth plate cells.We are also pursuing translational researchopportunities focused on the growth plate. Wehave recently developed a rat model of physealbar formation and are using this model to developnovel strategies for regeneration of physealcartilage to prevent growth abnormalitiesfollowing physeal injury. Another area of stronginterest is the investigation of the molecularmechanisms involved in the Heuter-Volkmanphenomenon, in which growth plate cartilageslows down its growth in response to compressiveforces and speeds up its growth in response totensile loading. We are developing an instrumentedsurgical staple that will be inserted acrossthe rat proximal tibial physis to provide real-timemeasurements of the progressive compressiveforces generated by the physis growing against thestaple.These projects provide several opportunitiesfor training of residents, fellows, doctoral, orpostdoctoralstudents at manylevels of scientificinquiry. From basicmolecular andcellular biologytechniques to animalsurgery and tissueengineering, traineeswill emerge fromtheir experience inour laboratory witha strong backgroundin performing basicscience andtranslational researchprojects and willgain an appreciationfor what is requiredto be a successfulclinician-scientist.R. Tracy Ballock, M.D.THE BALLOCKLABORATORYINVESTIGATORYvonne Shao, Ph.D.COLLABORATORSRobert E. Guldberg, Ph.D. 1Brian Johnstone, Ph.D. 2Jean F. Welter, M.D., Ph.D. 2Matthew C. Stewart, D.V.M., Ph.D. 21Dept. of Mechanical Engineering,Georgia Inst. of Technol.,Atlanta, GA2Dept. of Orthopaedics, CaseWestern Reserve Univ.,Cleveland, OH1Dept. of Mechanical Engineering, Georgia Inst.of Technol., Atlanta, GA2CDept. of Orthopaedics, Case Western ReserveUniv., Cleveland, OHBallock, R., Mita, B.C., Zhou, X., Chen, D.H., and L.M. Mink (1999) Expression ofthyroid hormone receptor isoforms in rat growth plate cartilage in vivo. J. BoneMiner. Res. 14:1550-1556.Ballock, R.T., Zhou, X., Mink, L.M., Chen, D.H., Mita, B.C., and M.C. Stewart (2000)Expression of cyclin-dependent kinase inhibitors in epiphyseal chondrocytes inducedto terminally differentiate with thyroid hormone. Endocrinology 141:4552-4557.Ballock, R.T., Zhou, X., Mink, L.M., Chen, D.H., and B.C. Mita (2001) Both retinoicacid and 1,25(OH)2 vitamin D3 inhibit thyroid hormone-induced terminal differentiationof growth plate chondrocytes. J. Orthop. Res. 19:43-49.Marcelino, J., Sciortino, C.M., Romero, M.F., Ulatowski, L.M., Ballock, R.T., Economides,A.N., Eimon, P.M., Harland, R.M., and M.L. Warman (2001) Human disease-causingNOG missense mutations: effects on noggin secretion, dimerformation, and bone morphogenetic protein binding. Proc. Natl. Acad. Sci. U.S.A.98:11353-11358.35

ORTHOPAEDICBIOLOGY ANDBIOENGINEERINGTHE HASCALLLABORATORYPROJECT SCIENTISTSAnthony Calabro, Ph.D.Csaba Fulop, Ph.D.Aimin Wang, Ph.D.POSTDOCTORAL FELLOWSMark Lauer, Ph.D.Durba Mukhopadhyay, Ph.D.COLLABORATORSAnthony Day, D. Phil. 1Carol de la Motte, Ph.D. 2Edward Maytin, M.D. 3Judy Mack, Ph.D. 3Antonietta Salustri, Ph.D. 4Scott Strong, M.D. 2Markku Tammi, M.D. 5Raija Tammi, M.D. 51MRC Immunochemistry Unit,Univ. of Oxford, Oxford, UK.2Dept. of Colorectal Surg. andImmunol. Res., CCF3Dept. of BiomedicalEngineering, CCF4Dept. of Cell Biol., 2 nd Univ. ofRome, Rome, Italy5Dept. of Anatomy, Univ. ofKuopio, Kuopio, FinlandOur laboratory focuses on the structure,function and metabolism of proteoglycansand hyaluronan in connective tissues.Proteoglycans are specialized proteins containingone or more covalently bound glycosaminoglycanchains: chondroitin/dermatan sulfate, keratan sulfate,heparin/heparan sulfate, or a combination of types.Glycosaminoglycan chains comprisepolymers of repeating disaccharide motifs that(except for hyaluronan) contain sulfoestersubstituents along their carbohydrate backbones.These highly negatively charged glycosaminoglycansendow the parent proteoglycans with a widerange of structures and functions. A briefdescription of proteoglycans currently under studyfollows.The major proteoglycan of cartilages isaggrecan; its core protein is ~200,000 Da molecularweight, with >150 chondroitin sulfate and keratansulfate chains attached. The mature proteoglycan(>2 million Da molecular weight) is the majorcomponent of cartilage extracellular matrix andenables this tissue to resist compressive load withminimal deformation.Biosynthesis and catabolism of aggrecan areclosely regulated, interdependent processesessential for normal tissue function, both duringlong-bone development on cartilage templates andin maintenance of articular cartilages that absorbshock on the ends of mature long bones. Abnormalbiomechanical load on cartilages, e.g., afterinjury to the knee’s cruciate ligaments, can adverselyalter aggrecan metabolism in the tissue and causematrix degeneration that eventually leads to clinicalosteoarthritis. Our goal is to use model chondrocyteor cartilage tissues to study processes thatregulate the amount and fine structure of aggrecansynthesized in normal and pathological situationsand to determine how aggrecan synthesis andcatabolism are regulated.The macromolecule hyaluronan is notsynthesized on a core protein, but rather at or nearthe cell surface, with the growing polymer beingThe Department of Biomedical EngineeringHyaluronan and Proteoglycan BiochemistryForm the Basis of Tissue EngineeringResearch Programextruded into the extracellular space. Hyaluronanconsists of repeating disaccharides of glucuronicacid and N-acetylglucosamine and reaches molecularweights >10 million Da.Hyaluronan is nearly ubiquitous throughoutthe vertebrates and is also synthesized by somebacteria. It serves as a scaffold for the extracellularmatrix in many connective tissues and has a vitalrole in tissue morphogenesis. Despite its prevalenceand deceptively simple structure, details of itssynthesis and metabolic regulation remainenigmatic.We have studied hyaluronan’s role in severalsystems. In cartilage, hyaluronan forms a filamentouslattice structure in the matrix to which aggrecanmolecules are anchored. Its metabolism is closelycorrelated with that of aggrecan and involves cellsurfacehyaluronan receptors as part of catabolism.In the final stages preceding ovulation, the oocytedirects the adjacent cumulus cell population tosynthesize and organize a similar hyaluronan lattice,surrounding itself with an extracellular matrix thatplays a vital role in subsequent fertilization. Inkeratinizing epithelia (e.g., skin or oral mucosa), thecells synthesize hyaluronan, which forms a matrixaround the basal and the differentiating suprabasalcells. These epithelia continuously regenerate andundergo terminal differentiation in the keratinizinglayers; thus hyaluronan must also be catabolizedcompletely within the tissue. Hyaluronan producedby colon or lung smooth muscle cells in responseto viral stimuli is organized in macrostructures thatengage and activate mononuclear leukocytes. Thismechanism is central in the pathogenesis ofinflammatory bowel disease and asthma. Thesecells, as well as mesangeal cells, synthesize similarstructures in response to elevated glucose concentrationsas in uncontrolled diabetes, suggesting thatabnormal hyaluronan matrices synthesized inresponse to elevated glucose are also involved invascular and diabetic pathologies. We are studyingthe mechanisms of both biosynthesis and catabolismof hyaluronan in these systems.Calabro, A., Oken, M.M., Hascall, V.C., and A.M. Masellis (2002) Characterization of hyaluronan synthaseexpression and hyaluronan synthesis in bone marrow mesenchymal progenitor cells: predominantexpression of HAS1 mRNA and up-regulated hyaluronan synthesis in bone marrow cells derived frommultiple myeloma patients. Blood 100:2578-2585.Vincent C. Hascall, Ph.D.See the website“Science ofHyaluronan”http://www.glycoforum.gr.jpKnepper, M.A., Saidel, G.M., Hascall, V.C., and T. Dwyer (2003) Concentration of solutes in the renal innermedulla: interstitial hyaluronan as a mechano-osmotic transducer. Am. J. Physiol. Renal Physiol.284:F433-F446.Fulop, C., Szanto, S., Mukhapadhyay, D., Bardos, T., Kamath, R., Day, A., Salustri, A., Hascall, V.C.,Glant, T., and K. Mikecz (2003) Impaired cumulus mucification and female sterility in tumor necrosisfactor-induced protein-6-deficient mice. Development. 130:2253-2261.Mack, J.A., Abramson, S.R., Ben, Y., Coffin, J.C., Rothrock, J.K., Maytin, E.V., Hascall, V.C., Largman,C., and E.J. (2003) Hoxb13 knockout adult skin exhibits high levels of hyaluronan and enhancedwound healing. FASEB J. 2003 May 20 [Epub ahead of print].de la Motte, C.A., Hascall, V.C., Drazba, J., Bandyopadhyaya, S.K., and S. Strong (2003) Mononuclearleukocytes bind to specific hyaluronan structures on colon mucosal smooth muscle cells treated withpolyinosinic acid:polycytidylic acid. Inter-alpha-trypsin inhibitor is crucial to structure and function. Am.J. Pathol. 163:000-000 (in press).36

Insights into Etiology and Innovative TreatmentModalities for Osteoporosis, Fracture Healing,Osteolysis and OsteonecrosisOsteocytes, osteoclasts, osteoblasts andpluripotent cells within bone marrowform a functional syncytium linking cellsdeep within bone tissue to cells on bone surfacesand/or within close proximity to the vascularsystem. This cellular network permits transmissionof chemical, electrical and mechanical signalsbetween cells that have the machinery to remodelbone tissue (osteocytes, osteoclasts,osteoblasts) and those withthe capacity to affect the populationof bone remodeling cells(pluripotent cells and monocytes inthe marrow, circulating blood) aswell as to invoke a systemicresponse. Remodeling eventsappear highly “choreographed,”but the signaling and timing ofinteractions between osteocytes,osteoclasts and osteoblasts are notclear. A primary focus of theMusculoskeletal MechanobiologyGroup is to understand theseinteractions. Specifically, we aimto uncover mechanisms underlyingprocesses of growth, adaptation, and repair ofmusculoskeletal tissues, in particular, bone.Interstitial fluid flow is a likely mechanismfor mechanochemical transduction in bone. Wehave developed innovative methods to studymechanical load-induced fluid flow and masstransport through tissue, using fluorescent tracersof different molecular weights. Applying thesemethods ex vivo in sheep, in vivo in rat, and in vitrousing bone explants, we have proven thatmechanical loading drives fluid flow throughbone. We have also shown that fluid displacementsresulting from mechanical loading enhancemolecular transport from the blood supply to theosteocytes, thus playing an important role inosteocyte viability. This finding has importantclinical implications for healing bones. We haveThe Department of Biomedical EngineeringMelissa L. Knothe Tate, Ph.D.also developed theoretical computer models topredict flow patterns under simulated conditions.By comparing these predictions with actualexperimental results, we have begun to explicatethe relationship between mechanical loadingparameters and fluid dynamics in bone. To betterunderstand molecular transport processes throughthe relatively impermeable tissue of bone, wehave devoted a major effort tostudying the spaces through whichextravascular fluid flows. We areexploiting insights gained fromthese studies to develop drugdeliverysystems for skeletaltissues and for new bioactiveendoprostheses designed tooptimize osseointegration. Inaddition, we are applying thisknowledge to optimize functionof tissue-engineered bone.Furthermore, new prophylactictreatment modalities to preventosteopenia due to osteoporosisand disuse are under study.In sum, we are achieving aglobal picture of fluid flow and mass transport inbone that has tremendous implications for cellviability and integrity, as well as the governanceof functional adaptation and repair within bonetissue. Furthermore, we can link spatial information(e.g., local architecture) to distribution offlow and to osteocyte signaling. We are correlatingthis information with the distribution ofcytokines through the tissue, thereby establishinga biophysical basis for mechanotransduction andthe biology underlying processes associated withadaptation and repair. These concepts haveprovided the foundation for a new theory ofbone (re)modeling that enhances our understandingof the clinical implications of fluid flow andmass transport for bone in health and disease.Knapp, H.F., Reilly, G.C., Stemmer, A., Niederer, P., and M.L. Knothe Tate (2002) Development of preparationmethods for and insights obtained from Atomic Force Microscopy of fluid spaces in corticalbone. Scanning 24:25-33.Knothe Tate, M.L., Tami, A.E.G., Bauer, T.W., and U. Knothe (2002) Micropathoanatomy of osteoporosis- indications for a cellular basis of bone disease. Adv. Osteoporotic Fracture Manage. 2:9-14.Tami, A.E., Nasser, P., Verborgt, O., Schaffler, M.B., and M.L. Knothe Tate (2002) The role of interstitialfluid flow in the remodeling response to fatigue loading: a theoretical and experimental study. J. BoneMiner. Res. 17:2030-2037.Steck, R., Niederer, P., and M.L. Knothe Tate (2003) A finite element analysis for the prediction ofload-induced fluid flow and mechanochemical transduction in bone. J. Theoretical Biol. 220:249-259.Steck, R., Gatzka, C., Schneider, E., Niederer, P., and M.L. Knothe Tate (2003) Measurement of bonesurface strains on the sheep metacarpus in vivo and ex vivo. Veterinary Comparative Orthop. Traumatol.16:1-9.ORTHOPAEDICBIOLOGY ANDBIOENGINEERINGTHE KNOTHE TATELABORATORYPOSTDOCTORAL FELLOWSSanjay Mishra, Ph.D.Roland Steck, Ph.D.RESEARCH FELLOWPavel Netrebko, M.D.GRADUATE STUDENTSJosée Adamson, M.S.Andrea Tami, Dipl. Masch.-Ing.UNDERGRADUATE STUDENTSMegan Dines 1Scott Koncal 1Ravi Patel 2Jaime Streem 3Christine Tiberio 11Cornell University, Ithaca, NY2Case Western Reserve Univ.,Cleveland, OH3Univ. of California at LosAngelesHIGH SCHOOL STUDENTSMegan ColeyTeleschia LawMarissa SchafferCOLLABORATORSJ. Iwan Alexander, Ph.D. 1Harihara Baskaran, Ph.D. 2Thomas W. Bauer, M.D., Ph.D. 3,4Dwight Davy, Ph.D. 1Steven Eppell, Ph.D. 5Miklos Gratzl, Ph.D. 2Ulrich Hopfer, M.D., Ph.D. 6Joseph Iannotti, M.D., Ph.D. 3Ulf Knothe, M.D., Dr. Med. 3Ronald J. Midura, Ph.D. 7DeVon Griffin, Ph.D. 81Dept. of Mech. and AerospaceEngineering, Case WesternReserve University, Cleveland,OH2Dept. of Chem. Eng., CWRU3Dept. of Orthopaedic Surgery, CCF4Dept. of Anatomic Pathology, CCF5Dept. of Biomed. Eng., CWRU6Dept. of Electrophysiology,CWRU7Dept. of Biomed. Eng., CCF8NASA Glenn Research Center,Cleveland, OHMelissa L. Knothe Tate, Ph.D.37

ORTHOPAEDICBIOLOGY ANDBIOENGINEERINGThe Department of Biomedical EngineeringGenetic Basis of Cell Fate Determinationand DifferentiationTHE LEFEBVRELABORATORYPOSTDOCTORAL FELLOWSSrijeet Mitra, Ph.D.Patrick Smits, Ph.D.Lai Wang, MD, Ph.D.RESEARCH TECHNICIANSPeter Dy, B.S.Michael Patrick, B.S.COLLABORATORSMatthew Warman, M.D. 1Clemencia Colmenares, Ph.D. 2Paul Fox, Ph.D. 31Dept. of Genetics, CaseWestern Reserve Univ.,Cleveland, OH2Dept of Cancer Biol., CCF3Dept of Cell Biol., CCFDeptofCelBiology, Cleveland Clinic FoundationOur main objectives are to identify andcharacterize genetic mechanisms thatvertebrates use to generate the largepanel of their highly different and specialized celltypes. Commitment and differentiation of stemcells towards specific lineages are multi-stepprocesses involving various types of regulatorymolecules. Our laboratory focuses on thedevelopment of the skeleton and hematopoieticsystem and on transcription factors belonging tothe Sox family that are involved in theseprocesses. We use molecular biology, cell biology,and mouse genetic engineeringapproaches.Sox transcriptionfactors are a family of 20proteins in mice and humans.Their common feature is anSry-related HMG boxdomain. Upon binding tospecific DNA sequences, thisdomain induces a strong bendof the DNA helix, a propertythat may allow Sox proteinsto facilitate the assembly ofcell-specific transcriptionalcomplexes. In addition tothe HMG box domain,several Sox proteins harbor atransactivation or atransrepression domain. Theprecise molecular roles ofSox transcription factorsremain largely unknown.Each Sox gene has a uniquespatial and temporalexpression pattern in vivo, such that each isexpressed in one or a few distinct cell types.Studies of mouse and human mutants haveuncovered critical roles for a handful of Soxgenes in determining cell fate and differentiationin specific lineages, but the roles of most SoxLefebvre, V., Li, P., and B. de Crombrugghe (1998) A new long form of Sox5 (L-Sox5), Sox6 and Sox9 are co-expressed in chondrogenesis and cooperatively activatethe type II collagen gene. EMBO J. 17:5718-5733.Smits, P., Li, P., Mandel, J., Zhang, Z., Deng, J.M., Behringer, R.R., de Crombrugghe,B., and V. Lefebvre (2001) The transcription factors L-Sox5 and Sox6 are essentialfor cartilage formation. Developmental Cell 1:277-290.de Crombrugghe, B., Lefebvre, V., and K. Nakashima (2001) Regulatory mechanismsin the pathways of cartilage and bone formation. Curr. Opin. Cell Biol.13:721-727.Lefebvre, V. (2002). Toward understanding the functions of the two highly relatedSox5 and Sox6 genes. J. Bone Miner. Metab. 20:121-130.Smits, P., and V. Lefebvre (2003) L-Sox5 and Sox6 are required for notochord extracellularmatrix sheath formation, notochord cell survival and development of thenucleus pulposus of intervertebral discs (2003). Development 130, 1135-1148.Véronique Lefebvre, Ph.D.genes remain to be uncovered. Our laboratoryworks to uncover new molecular and cellularroles of Sox factors.We currently pursue the study of Sox5 andSox6. These two highly identical genes are coexpressedin chondrocytes and notochord cells.We recently showed that chondrocytes are unableto overtly differentiate in mouse embryos lackingboth Sox5 and Sox6. The cells express all majorcartilage extracellular matrix genes at reduced orundetectable level, hardly proliferate, and matureaberrantly. Cartilage elements remain rudimentaryand are precociously replacedby bone. Embryos die beforebirth with a very severeskeletal dysplasia. Morerecently, we found that Sox5and Sox6 are also required innotochord cells (Smits andLefebvre, 2003). Theycontrol notochord extracellularmatrix formation and cellsurvival, and the transformationof the notochord intothe nucleus pulposus cores ofintervertebral discs. Hence,Sox5 and Sox6 control twocell types that have majorroles in the development ofthe vertebrate skeleton.Current projects are tofurther define the roles ofSox5 and Sox6 in vivo. Weare aiming at identifying thegenes directly targeted by theSox5 and Sox6 proteinproducts, and at determiningthe mode of action of these proteins on targetgenes. We are collaborating with Dr. MatthewWarman to identify human diseases caused bymutations in SOX5 or SOX6 and with Drs.Clemencia Colmenares and Paul Fox to characterizenon-skeletal phenotypes in Sox5 and Sox6mutant mice. New projects are being developedto study the roles of other Sox genes in variousother aspects of skeleton development, and todetermine the roles of Sox genes in the specificationof hematopoietic stem cells toward distinctblood cell lineages.38

Cellular and Molecular Mechanisms ofWound Healing in Orthopaedic SoftTissue and OsteoarthritisOur primary interest is the cellularand molecular processes in thehealing of wounds in the joint tissues.While a substantive literature documents theimportance of the knee joint meniscus, relativelylittle is known about the cell biology of thistissue. The meniscus is especially interesting inthe context of wound healing as it can repairwounds, whereas articular cartilage, a verysimilar tissue, does not.The Cell and Matrix Biology of theNormal MeniscusAt least three distinct populations of cellscan be recognized in the meniscus. Most of theinner nonvascularized portion is populated bycells that are round or oval with a pericellularmatrix of type VI collagen. We have termedthese cells “fibrochondrocytes.” The outermeniscus has fibroblast-like cells with longcytoplasmic processes that interconnect withsimilar cells through gap junctions. Elongatedcells that lack cytoplasmic extensions populatethe superficial zones of the meniscus. Our in vivoand in vitro studies suggest that these cells initiatethe wound-healing process in the meniscus.The meniscus is a fibrocartilage with anextracellular matrix composed mainly of type Icollagen, the typical collagen of fibrous tissues,and small amounts of type II collagen, thegenetic type of collagen found in hyalinearticular cartilage. We have established thatthese two collagens are found together in ahighly organized fibrillar meshwork.The Department of Biomedical EngineeringRepair Mechanisms in the MeniscusWe have developed in vivo and in vitromodels for studying the response of meniscal cellsto wounds in the tissue. The fibrochondrocytesand fibroblast-like cells of the normal meniscusare in a quiescent state with minimal expression ofthe fibrillar collagen genes. With wounding,however, the mRNA levels for type I and type VIcollagen and other matrix proteins are dramaticallyincreased, as assessed by RNase protection assay.The cells in the superficial region undergo divisionand express an alpha smooth muscle isoform. Thecrevice of the wound becomes populated by cellsthat appear to come from the superficial zone.Interestingly, the cells of the meniscus can migrateinto acellular areas created by apoptosis ofresident cells, a phenomenon that apparently doesnot occur in articular cartilage. With time, anintegration of tissues on either side of the woundoccurs.Tissue Engineering of MeniscusOur studies show that the meniscus has itsown distinctive healing process. We are exploringthe use of different macromolecules that, wheninserted into wounds, should promote the healingprocess.Type VI Collagen in the PericellularMatrices of Connective Tissue CellsThe physical and chemical stimuli to a cellmust traverse any pericellular coating it has. Somecells, like chondrocytes and fibrochondrocytes, aresurrounded by a distinct pericellular matrix oftype VI collagen. We can study the structural andfunctional aspects of this pericellular matrix andhow it controls signaling from the matrix to thecell.Kambic, H.E., Futani, H., and C.A. McDevitt (2000) Cell, matrix changes and alpha-smooth muscle actinexpression in repair of the canine meniscus. Wound Repair Regen. 8:554-561.Arnoczky, S.P., and C.A. McDevitt (2000) The meniscus: structure, repair, and replacement. In: Buckwalter,J.A., Einhorn, T.A., and S.R. Simon SR, eds. Orthopaedic Basic Science. 2nd ed. Park Ridge,IL: American Academy of Orthopaedic Surgeons, pp. 531-545.Wildey, G.M., Billetz, A.C., Matyas, J.R., Adams, M.E., and C.A. McDevitt (2001) Absolute concentrationsof mRNA for type I and type VI collagen in the canine meniscus in normal and ACL-deficientknee joints obtained by RNase protection assay. J. Orthop. Res. 19:650-658.McDevitt, C.A., Mukherjee, S., Kambic, H., and R. Parker (2002) Emerging concepts of the cell biologyof the meniscus. Curr. Opin. Orthop. 13:345-350.Kambic, H., and C. McDevitt (2002) Distribution and spatial relationship of collagen type I and type IIin the canine meniscus. Poster 0902, 48th Annual Meeting of the Orthopaedic Research Society, February10-13, 2001, Dallas, TX.Mukherjee, S., and C.A. McDevitt (2003) The superficial zone cells initiate a wound healing process incanine meniscus in vitro [poster]. Trans. Orthop. Res. Soc. vol. 28.Kim, J.H., Billetz, A., Iannotti, J., and C.A. McDevitt (2003) Isolation of a new cell-pericellular matrixstructure from the biceps tendon: longitudinal chains of type VI collagen surround linear arrays of cells[poster]. Trans. Orthop. Res. Soc. vol. 28.ORTHOPAEDICBIOLOGY ANDBIOENGINEERINGTHE MCDEVITTLABORATORYPOSTDOCTORAL FELLOWSSarmistha Mukherjee, Ph.D.Cassius Iyad Ochoa Chaar, M.D.Manojkumar Valiyaveetil, Ph.D.Manojkumar Valiiyaveettil, Ph.D.GRADUATE STUDENTCarlumandarlo Zaramo, B.S.TECHNOLOGISTKristin Rundo, B.S.Cahir A. McDevitt, Ph.D.COLLABORATORSJack Andrish, M.D. 1Brian L. Davis, Ph.D. 2David R. Eyre, Ph.D. 3Joseph P. Iannotti, M.D., Ph.D. 1John R. Matyas, Ph.D. 4Ronald J. Midura, Ph.D. 2John S. Mort, Ph.D. 5Richard D. Parker, M.D. 1Kimerly A. Powell, Ph.D. 2John Sandy, Ph.D. 61Dept. of Orthopaedic Surgery,CCF2Dept. of BiomedicalEngineering, CCF3Dept. of Orthopedics, Univ. ofWashington, Seattle4Dept. of Anatomy andPathology, McCaig Ctr. forJoint Injury and Arthritis Res.,Univ. of Calgary, Alb.,Canada5Shriners Hospital, Montreal,PQ, Canada6Shriners Hospital, Tampa, FLCahir A. McDevitt, Ph.D.39

CONNECTIVETISSUEBIOLOGYTHE MIDURALABORATORYINVESTIGATORSFotini Adamidou, M.D.Michael Ibiwoye, M.D., Ph.D.Sharon Midura, B.S.Matthew Schnoke, B.S.Xiaowei Su, Ph.D.COLLABORATORSJeffrey P. Gorski, Ph.D. 1Mark D. Grabiner, Ph.D. 2David J. McQuillan, Ph.D. 31Dept. of Mol. Biol. &Biochem., Univ. of Missouri,Kansas City2Dept. of Kinesiology, Univ. ofIllinois, Chicago3LifeCell Corp., Branchburg,NJRonald J. Midura, Ph.D.Bone is a dynamic organ system exhibitingcontinual formation, resorption andreformation of resorbed tissue (remodeling).These processes are dependent on themetabolic activity of its constituent cells:osteoblasts form bone,osteocytes maintain bone,while osteoclasts resorbbone.Nearly all bonediseases, or pathologies infracture healing of bone,manifest aberrations in bonematrix production and/orthe mineralization of thismatrix. These cellularactivities in bone are tightlyregulated by cytokines andhormones both in normal andpathologic states. Parathyroidhormone (PTH) is amajor regulator of boneformation and remodeling.Its endocrine function in vivois to maintain Ca 2+ levels inthe blood by reabsorbing Ca 2+from the kidneys andreleasing Ca 2+ from bones. Italso manifests somatotrophiceffects on bone formationwhen used as a therapeuticagent and is one of the mostpromising prospects for theprevention of bone loss (osteopenia) and thetreatment of osteoporosis.Our current research focuses on theregulation of osteoblast function by PTH. OurThe Department of Biomedical EngineeringMolecular Mechanisms of BoneFormation and Osteoporosisinitial approaches have been to analyze the effectsof PTH on an osteoblast’s ability to synthesizespecific macromolecules found in bone matrix,properly assemble them in the extracellularenvironment, and mineralize this matrix. Usingseveral osteoblasticmodels (from cell linesto primary tissue), wehave discovered thatPTH can dramaticallyaffect an osteoblast’sability to produce selectmatrix macromolecules,alter its ability toassemble thesemacromolecules into anextracellular matrix,and regulate itsmineralization of thismatrix. Our objectivesare to define themolecular mechanismsoperating in thesebiological responses ofosteoblasts to PTH.This research will yieldincreased knowledge ofan osteoblast’sfunctions in bonehomeostasis. Ultimately,it will provide abetter understanding ofbone development andpathology and may offer new strategies using PTHto augment bone healing, prevent osteopenia, andtreat osteoporosis.Ronald J. Midura, Ph.D.Bosch, P.P., Stevens, J.W., Noonan, K.J., Buckwalter, J.A., and R.J. Midura (2003) Expression ofCD44 in human neoplastic and normal hyaline cartilage. Iowa Orthop. J. 22:47-54.Muschler, G.F., and R.J. Midura (2002) Connective tissue progenitors: practical concepts for clinical applications.Clin. Orthop. (395):66-80.Plaas, A.H., West, L.A., and R.J. Midura (2001) Keratan sulfate disaccharide composition determinedby FACE analysis of keratanase II and endo-beta-galactosidase digestion products. Glycobiology11:779-790.Wang, A., Martin, J.A., Lembke, L.A., and R.J. Midura (2000) Reversible suppression of in vitro biomineralizationby activation of protein kinase A. J. Biol. Chem. 275:11082-11091.Byzova, T.V., Kim, W.. Midura, R.J., and E.F. Plow (2000) Activation of integrinalpha(V)beta(3) regulates cell adhesion and migration to bone sialoprotein.Exp. Cell Res. 254:299-308.40

The Department of Biomedical EngineeringBone Biology, Skeletal Reconstruction,Aging and OsteoporosisCONNECTIVETISSUEBIOLOGYOur laboratory is focused on advancingunderstanding the field of boneformation and repair. This has particularrelevance to age-associated skeletal changes suchas osteoporosis. Our work includes developmentof more effective, less invasive methods fortreatment of fractures and deformities of theskeleton through advanced clinical procedures.This increasingly draws us into work directed atunderstanding early events in osteoblasticdifferentiation. We integrate and draw ondisciplines of cell and molecular biology, growthfactor expression and action, cell matrixinteraction, image processing, and biomechanics.Osteoblastic Stem CellsNormal bone marrow is populated byundifferentiated stem cells, which are recruitedthroughout life in the processes of bone growth,repair, and remodeling. These cells are critical tobone formation. One aspect of our work focuseson understanding changes which occur in theserare cells during normal aging and additionalchanges which result from hormonal changes atmenopause, medications (e.g., corticosteroids)habits (e.g. smoking) and from selected diseases(e.g. Diabetes and Sickle Cell Anemia).We have also devised innovative andminimally invasive methods for harvest and rapidcollection of bone stem cells, which will facilitatethe use of these cells for bone tissue engineeringand other therapeutic applications.Bone RegenerationRegeneration of new bone segments andlimb lengthening can be accomplished using thebiologic process known as distraction osteogenesis.Distraction osteogenesis involves theestablish of a vascular healing fracture callus andthen stretching the callus using controlledmechanical distraction. This distraction results ina prolonged proliferation of osteoblasticprogenitors in the callus (the growth phase ofbone formation) generating new bone tissue inthe area where the bone is pulled apart. We arecurrently developing fully implantable devicesthat use the process of distraction osteogenesis toregenerate bone segments and lengthen limbswithout cumbersome and painful frames.Development and Testing of EffectiveMaterials for Bone GraftingCurrently, most clinical bone graftprocedures involve the harvest of bone from onesite and transplantation to another. This subjectspatients to additional surgery, blood loss, andpain. A number of synthetic materials (includingcalcium phosphate ceramics, purified collagenpreparations, and some polymers) have been usedsuccessfully to heal small bone defects in animals.Similarly, several growth factors appear to have asignificant positive effect on bone healing.However, the formulation of an optimal compositeof available materials (matrix, proteins, and cells)for clinical use requires evaluation of thesecomposites in a model that is appropriate in bothsize and anatomy to model the clinical setting. Manymaterials work well in small defects in rodent bones,but fail in larger animals.Since one-half of all bone grafting materialsis used for spinal fusions, our laboratory hasdeveloped a segmental canine posterior spinalfusion model that allows efficient and sensitiveevaluation of composite graft materials for spinalfusion. This model has documented the failure ofsome composite materials that were underconsideration for clinical application. Wehave also shown that a polymeric materialdelivering a recombinant human bonemorphogenetic protein (rhBMP-2) inmicrogram quantities results in fusionproperties equal to autogenous cancellousbone.We are currently a clinical test sitein a prospective FDA-approvedmulticenter trial of osteogenic protein-1(BMP-7) for treatment of tibial nonunion.The development of safe andeffective synthetic bone graft materialswill have a dramatic impact in reducingthe morbidity of bone grafting proceduresin the near future.THE MUSCHLERLABORATORYThe Muschler LaboratoryYoichi Matsukura, M.D., Ph.D.Chizu Nakamoto, Ph.D.Cynthia Boehm, B.S.George F. Muschler, M.D.Muschler GF, Kotschi H: Bone Transport and Lengthening System. U.S. Patent#5,429,638. Issued July 4, 1995.Hyodo A, Kotschi H, Kambic H, Muschler G. Bone transport using intramedullaryfixation and a single flexible traction cable. Clin Orthop 325:256-268, 1996.Muschler GF, Boehm C, Easley K: Aspiration to obtain osteoblastic progenitorscells from human bone marrow: The influence of aspiration volume. J Bone JointSurg 79A: 1699-1709, 1997.Majors A, Boehm C, Nitto H, Midura R, Muschler G: Characterization of humanbone marrow stromal cells with respect to osteoblastic differentiation. J OrthopRes 15:546-457, 1997.Muschler GF: Method of preparing a composite bone graft. U.S. Patent #5,824,084issued: Oct. 20, 1998.Muschler GF, Lane JM. Principles of Bone Fusion. In: Principles and Techniques ofSpinal Surgery. Rothman and Simeone eds. (New York, W.B. Saunders, Co.,1999).Muschler GF, Boehm CA, Nitto H: Age and gender related changes in the numberand prevalence of osteoblastic progenitors in human bone marrow (submitted).41

The Department of Biomedical EngineeringBME Staff, Project Scientists with Independent FundingAnthony Calabro, Ph.D.Osteoarthritis is characterized by the gradual and irreversible loss of cartilage surfaces, which eventuallydisable patient mobility. Osteoarthritis affects an estimated 20.7 million Americans, and is responsiblefor ~7 million physician visits a year. Osteoarthritis presents a complicated treatment problem inorthopedic surgery, because of the limited capacity of cartilage to repair itself. The focus of the Calabrolaboratory is on the development of synthetic cartilage substitutes for use in cartilage repair. These novelbiomaterials are formed through an enzyme-selective, biocompatible cross-linking chemistry with thescaffold material obtained from those natural occurring macromolecules within native cartilage that areprimarily responsible for cartilage’s biomechanical properties.Kathe Derwin, Ph.D.My research focuses on tendon mechanobiology and tissue engineering. We have developed an in vitrobioreactor system to study the cellular mechanisms involved in tendon remodeling and injury in responseto mechanical loading conditions. We are also investigating the use of natural extracellular matrices asscaffolds for tendon tissue engineering. The in vitro bioreactor system is used to investigate cell-seedingand mechanical conditioning of these biomaterials for in vivo implantation in a canine rotator cuffinjury model. Further, we are studying the relationship between muscle pathology and tendon-musclefunction in our chronic canine rotator cuff model.Scott Colles, Ph.D.Our research in the laboratory of Linda Graham, M.D., focuses on the role glutathione peroxidase andlipid oxidation products in the development of vascular disease. Lipid oxidation products are thought tobe major factors in the development of various vascular diseases including atherosclerosis. Glutathioneperoxidase is an antioxidant enzyme involved in preventing cellular injury by lipid oxidation products.We have developed mouse models that lack the LDL receptor and have altered expression of glutathioneperoxidase and lipid oxidation products in vascular disease.Susan D’Andrea, Ph.D.My research is focused on the understanding of human locomotor system and the development ofbiomedical solutions to problems related to the foot and ankle. This involves determining a correlationbetween bone structure and the response to the forces acting on the skeleton. Establishing the spectraof bone strains experienced during normal daily activities can directly relate to the evaluation of agerelated bone loss, post-menopausal and disuse osteoporosis as well as fracture healing. Additionally, mywork concentrates on the evaluation of an exercise as a countermeasure to physiological adaptations ofmicrogravity. In particular, we are developing an instrumented treadmill to challenge the posturalcontrol system of astronauts in microgravity.Kiyotaka Fukamachi, M.D., Ph.D.The primary research interest of the Cardiovascular Dynamics Lab is cardiovascular physiology withmajor emphasis on cardiovascular dynamics relating to cardiac devices and surgical interventions used totreat heart failure. We are developing devices that improve cardiac function in patients with dilatedcardiomyopathy or repair mitral regurgitation in beating hearts; advanced heart valves; and novel genetherapies. We are also investigating heart assist devices such as left ventricular assist devices and animplantable total artificial heart. Our lab works in close collaboration with the Departments ofThoracic and Cardiovascular Surgery and Cardiology so that our research addresses clinical needs.42Aimin Wang, Ph.D.The proliferation of vascular smooth muscle cells and glomerular mesangial cells is a prominent earlyhistological feature of many vascular and glomerular diseases in humans and experimental animals. Heparansulfates (HSs) and heparin are inhibitors of smooth muscle cell and mesangial cell growth in both experimentaldisease models and in cell culture, and HSs have been proposed as endogenous defending moleculesto protect resident cells from activation and injury by functioning as autocrine/paracrine growth inhibitors.My research interests are concentrated on the precise structural determinants of HSs/heparin for thegrowth-inhibitory activity, and their beneficial effects in diabetic nephropathy.P. Stephen Williams, Ph.D.Research in this laboratory is currently focused on a novel technique for cell sorting using immunospecificmagnetic labeling. Labeled cells are selectively removed from unlabeled cells by their migration across thethickness of an annular flow channel mounted in a quadrupole magnetic field. I am working on theoptimization of sorting conditions. The influence of small geometrical imperfections on sorting efficiencyis being studied using computational fluid dynamics. Sorting conditions may be adjusted to compensate foraccepted construction tolerances. We are also developing a technique called magnetic field-flow fractionationfor characterizing magnetic particulate species such as immunospecific magnetic labels.

CancerBiology

DEPARTMENT OFCANCER BIOLOGYCHAIRMANBryan R.G. Williams, Ph.D.,Hon. FRSNZThe Betsy de Windt ChairSTAFFFirouz Daneshgari, M.D.Serpil C. Erzurum, M.D.Warren D. (Skip) Heston, Ph.D.Robert H. Silverman, Ph.D.ASSOCIATE STAFFAlexandru Almasan, Ph.D.Graham Casey, Ph.D.Clemencia Colmenares, Ph.D.Jawhar Rawwas, M.D.Michael Vogelbaum, M.D., Ph.D.Yan Xu, Ph.D.Taolin Yi, M.D., Ph.D.ASSISTANT STAFFSipra Banerjee, Ph.D.Joseph DiDonato, Ph.D.S. Jaharul Haque, Ph.D.Nywana Sizemore, Ph.D.STAFF SCIENTISTC. Thomas Powell, Ph.D.PROJECT SCIENTISTSMarina Antoch, Ph.D.Nandan Bhattacharyya, Ph.D.Suzy Comhair, Ph.D.Beihua Dong, Ph.D.Arundhati Ghosh, Ph.D.Pratima Karnik, Ph.D.Xavier Lee, Ph.D.Liming Wang, Ph.D.RESEARCH ASSOCIATESPatricia Stanhope-Baker, Ph.D.Malathi Krishnamurthy, Ph.D.Nilesh Maiti, Ph.D.Suparna Mazumder, Ph.D.Ting Shi, Ph.D.Roger Slee, Ph.D.Rongzhi Wu, Ph.D.Ying Xiang, Ph.D.Yi-Jin Xiao, Ph.D.Weiling Xu, Ph.D.Maryam Zamanian-Daryoush, Ph.D.Xiaoxian Zhao, Ph.D.The Department of Cancer BiologyCancer Biology Investigators ResearchMolecular Events Regulating Cell Division,Differentiation, or ApoptosisThe unifying theme of the Cancer Biologylaboratories is the study of the molecularbiology and genetics of cancer. We wish todefine the basic molecular events controllingcellular responses to external cues that signal cellgrowth, division, differentiation or death. We alsoinvestigate the mechanisms responsible for celltransformation and oncogenesis, particularly asthey relate to human cancers. By applyingmolecular genetic techniques to the study ofcommon human malignancies, we hope to identifymolecular markers that may prove useful in theearly diagnosis or prognosis of cancer and alsoprovide approaches to the development of noveltherapies. By studying the events found inpathological processes, we should also shed lighton normal cellular regulatory processes.The Department of Cancer Biology isdirected by Bryan R.G.Williams, Ph.D., holder ofthe Betsy de Windt Chair ofCancer Biology, and ishoused on the fourth andfifth floors of the LernerResearch Institute. Thedepartment comprises 16laboratories, headed by fiveStaff, seven Associate Staff,four Assistant Staffmembers, and one StaffScientist. The department ispresently supported by over$6.5 million in totalexternal grant funding fromthe NIH annually. Anadditional $1.3 million isderived from other federaland nonfederal sources.Recently, departmentmembers have made asignificant contribution toour understanding of thegenetic basis of prostatecancer. Prostate cancer issecond only to lung canceras the cause of cancerdeaths in men, claimingabout 31,500 American meneach year out of anestimated 200,000 newcases. While the chance ofgetting prostate cancer inone’s lifetime is about 1 in6, in some families the riskis much higher. Hereditaryforms of prostate cancer are due to mutations incertain genes that predispose affected individualsto early-onset and aggressive forms of the disease.The RNASEL gene, cloned in Dr. RobertSilverman’s laboratory, now appears to be a strongcandidate for the prototype of the hereditaryprostate cancer genes, HPC1. The RNASEL genecodes for an enzyme called RNase L, involved inthe protection of cells against viral infections byinterferons (IFNs). RNase L is believed tocounteract prostate cancer by causing the tumorscells to die prematurely. The connection betweenRNase L and prostate cancer was extremelydifficult to prove, but two new studies involvingDr. Silverman and Dr. Graham Casey provide thatevidence. The discovery that RNase L is a strongcandidate for HPC1 was an international effortby Drs. John Carpten and Jeffrey Trent of theNational Human Genome Research Institute, Dr.Continued on Page 45Bryan R.G. Williams, Ph.D., Hon. FRSNZ44

The Department of Cancer BiologyContinued from Page 44William Isaacs of Johns Hopkins University, Dr.Silverman of the Cleveland Clinic, and othercollaborators (Nature Genetics 2002;30:181-4).Subsequently, Drs. Casey and Silverman(Cleveland Clinic) with Dr. John Witte (CaseWestern Reserve University) reported that asingle amino acid change in RNase L is implicatedin as many as 13% of unselected prostatecancer cases (Nature Genetics 2002;32:581-3).These findings suggest the involvement of RNaseL in both hereditary and unselected cases ofprostate cancer. This important development inprostate cancer research could provide anopportunity to screen men for mutations thatcause early-onset disease, thereby allowing earlytreatment, or form the basis for the developmentof a new drug or gene therapy for prostatecancer. A small molecule called 2-5A causesRNase L to become active in cells and leads tothe death of prostate cancer cells. Our hope isthat these studies will lead to a better understandingof how prostate cancer develops.In the NIH-funded George M. O’BrienUrology Research Center, Dr. Warren (Skip)Heston has described prostate-specific membraneantigen (PSMA) as a potential target in prostatecancer patients. PSMA is a type-2 membraneprotein expressed in the prostate and highlyexpressed in metastatic or poorly differentiatedadenocarcinomas. Moreover, PSMA expression isupregulated by androgen deprivation. New radioimmunoconjugateforms of anti-PSMA monoclonalantibody are being used to diagnoseprostate cancer metastasis and recurrence. Therehave also been promising results from Phase Iand II trials using PSMA as a therapeutic target(Urol. Oncol. 2002;7:7-12). Recently, PSMAexpression in endothelial cells of tumorassociatedneovasculature has been described,suggesting a role in tumor angiogenesis anddefining a new target for therapeutic approachesfor all solid tumors.Another important discovery was recentlymade in collaboration with scientists at theNational Institutes of Health. Dr. NicholasRestifo’s group in the National Cancer Institute’sSurgery branch have produced a naked DNAvaccine encoding an alphavirus replicon (selfreplicatingmRNA) and the self/tumor antigentyrosinase-related protein-1. In collaborationwith Drs. Williams and Silverman, his groupshowed that, unlike conventional DNA vaccines,this vaccine can break tolerance and provideimmunity to melanoma. The vaccine mediatesproduction of double-stranded RNA (dsRNA),leading to the activation of the dsRNAdependentprotein kinase R (PKR). This dsRNAproduction was shown to be critical to vaccinefunction because the vaccine’s immunogenicityand anti-tumor activity are blocked in micedeficient for RNase L. This novel study showsthat alphaviral replicon-encoding DNA vaccinesactivate innate immune pathways known to driveantiviral immune responses and suggests strategiesfor improving the efficacy of immunization withnaked DNA by manipulating these pathways.The adenylate uridylate-rich elements(AREs) mediate the rapid turnover of mRNAsencoding proteins that regulate cellular growthand response to exogenous agents such asmicrobes and inflammatory and environmentalstimuli. However, the full repertoire of AREcontainingmRNAs is unknown. Dr. Williams andDr. Khalid Khabar (Adjunct Staff in thisdepartment; King Faisal Hospital ResearchCenter, Riyadh, Saudi Arabia) have explored thedistribution of AREs in human mRNA sequencesand arrived at a computationally derived AREpattern that is the basis of a database containingnonredundant, full-length ARE-mRNAs (NucleicAcids Res. 2001;29:246-54; 2003;31:421-3). TheARE-mRNA database (ARED) reveals that AREmRNAsencode a wide repertoire of functionallydiverse proteins that belong to different biologicalprocesses and are important in several diseasestates. This database has been used to design acustom cDNA array of the entire spectrum ofARE-containing genes checked for p38-dependentregulation of ARE-mediated mRNA turnover. Inaddition, the half-lives of 470 AU-rich mRNAswere determined and a feedback loop identifiedthat regulates macrophage signaling via p38 MAPkinase-dependent transcript stabilization (Mol.Cell. Biol. 2003;23:425-36).Findings from Dr. Clemencia Colmenares’laboratory have suggested that the Skiprotooncogene is involved in neural tubedevelopment and muscle differentiation. Inagreement with this result, the group has foundthat Ski-/- mice display a cranial neural tubedefect that results in exencephaly and a markedreduction in skeletal muscle mass. They haveshown that the penetrance and expressivity of thephenotype changes when the null mutation isbackcrossed into the C57BL6/J background, withthe principal change involving a switch from aneural tube defect to midline facial clefting(Nature Genetics 2002;30:106-9). This phenotypeis found in individuals diagnosed with 1p36deletion syndrome, a disorder caused by monosomyof the short arm of human chromosome 1p.Dept. website:http://www.lerner.ccf.org/cancerbio/Continued on Page 4645

The Department of Cancer Biology46Dept. website:http://www.lerner.ccf.org/cancerbio/Continued from Page 45In fact, Dr Colmenares and colleagues havefound that human SKI is located at distal 1p36.3and is deleted in all the individuals tested whohave this syndrome. Thus, SKI may contribute tosome phenotypes common in the 1p36 deletionsyndrome, particularly to facial clefting. Ski isalso implicated in regulation of the transcriptionfactor Glioblastoma-3 (Gli3). Dr. Colmenaresand collaborators from Japan reported that Skibinds to Gli3 and recruits the histone deacetylasecomplex (Genes & Development 2002;16:2843-8).A Ski mutation enhanced the digit abnormalitiescaused by the Gli3 gene mutation. Thus, Ski playsan important role in pattern formation byregulating the Gli3 activity.Dr. Alex Almasan’s laboratory hasestablished a critical role for the inactivation ofcyclin E/Cdk2 by caspase-mediated cleavageduring apoptosis (Mol. Cell. Biol. 2002;22:2398-409). Cyclin E/Cdk2 is a critical regulator ofcell-cycle progression from G(1) to S in mammaliancells and has an established role in oncogenesis.This proapoptotic mechanism can beinduced by gene-toxic agents such as ionizingradiation in hematopoietic tumor cell lines and isaccompanied by the expression of a novel p18-cyclin E. Caspase-mediated cyclin E cleavageeliminated interaction with Cdk2 and inactivatedthe associated kinase activity. Apoptosis andgeneration of p18-cyclin E were significantlyinhibited by overexpressing the cleavage-resistantcyclin E mutant, indicating a functional role forcaspase-dependent proteolysis of cyclin E inapoptosis of hematopoietic tumor cells.The laboratory of Dr. Sipra Banerjee isuncovering the role of DNA polymerase beta(polβ) in cancer development. She has describedspecific deletions of DNA polβ in differentcancers and has found that the mutant form ofthe enzyme (polβ∆) forms a dominant-negativecomplex in DNA repair. The defective enzymemay facilitate accumulation of mutations inresponse to environmental stimuli, leading to theexpression of a mutator phenotype in differenttumors (Gene Expression 2002;10:115-23).Infection, inflammation and cancer areintimately linked. The laboratory of Dr. JoeDiDonato is studying the role of the IκB kinases(IKKs) activated in response to differentinflammatory stimuli. He described new rolesdiscovered for IKKα, in epidermal differentiationand in B-cell maturation (Science STKE 2001 –http:stke.sciencemag.org/). In epidermaldifferentiation, IKKα regulates the productionof a secreted differentiation factor through apathway that is independent of its role inactivation of NF-κB. In B-cell maturation,conventional NF-κB signal-induced activation ofIKKα results in phosphorylation of p100precursor proteins and increased proteolyticprocessing and constitutive NF-κB activation.The laboratory of Dr. Serpil Erzurum isstudying the role of antioxidants and reactiveoxygen (ROS) and nitrogen species (RNS) in thepathogenesis of lung diseases. The lungs are wellequipped with antioxidant defenses, andaugmentation of these defenses through genetherapy is being studied as a means of preventingcell injury to patients in whom these defenses arecompromised by diseases such as cancer. Thislaboratory has shown that chronically hypoxichuman populations resident at high altitudes inTibet and Bolivia have very high levels of exhalednitric oxide (NO). This fact likely explains thegreater vasodilation and blood flow in theseindividuals (Nature 2001;414:411-2). ROS areimportant in the initiation and promotion of cellsto neoplastic growth; exposure to cigarettesmoke, the primary risk factor in lung cancerdevelopment, leads to high levels of ROS withinthe human airway. Dr Erzurum’s group hasshown that alterations in antioxidant activities,attributable to increased manganese SOD anddecreased catalase protein, and in mRNAexpression occur in lung tumors (Cancer Res.2001;61:8578-85). Since parallel changes inantioxidant activities, protein, and mRNAexpression were noted in lung carcinoma cell linesexposed to proinflammatory cytokines, such astumor necrosis factor-alpha (TNF-α), interleukin(IL)-1beta (IL-1β), and IFN-gamma (IFN-γ),inflammation in the lung may create an intracellularenvironment favorable to DNA damage andthe promotion of cancer.Dr. Jaharul Haque has investigatedaberrant cytokine signaling in malignant gliomas.His laboratory has shown that glioblastomamultiforme (GBM) cells fail to support Jak-Statsignaling in response to both IL-4 and IL-13. Thisfact is in part due to the expression of IL-13Rα2,which acts as a decoy receptor for IL-13. Themechanism of inhibition was mediated via aphysical interaction between the intracellulardomain of IL-13 Rα2 and the cytoplasmicdomain of the IL-4Rα chain (Cancer Res.2002;62:1103-9). The Haque group, in collaborationwith Dr. Michael Vogelbaum (CCF’s BrainTumor Institute), has recently reported that GBMtumors and cell lines contain high levels ofconstitutively activated Stat3 compared withnormal human astrocytes, white matter, andnormal tissue adjacent to tumor. The persistentactivation of Stat3 is, in part, attributable to anautocrine action of IL-6. This activation can beinhibited by the Jak inhibitor AG490 or expressionof a dominant-negative mutant Stat3Continued on Page 47

The Department of Cancer BiologyContinued from Page 46protein. Thus, constitutive activation of Stat3contributes to the pathogenesis of glioblastomaby promoting both proliferation and survival ofGBM cells. Therefore, targeting Stat3 signalingmay provide a potential therapeutic interventionfor GBM (Oncogene 2002;21:8404-13).Dr. Taolin Yi’s group has been investigatingthe role of the tyrosine phosphatase SHP-1in signal transduction and leukemiogenesis. Anexciting new development is the discovery of asmall molecule inhibitor of this phosphatase,sodium stibogluconate, which in preclinicalstudies (in collaboration with Dr. Ernest Borden,Taussig Cancer Center, Drug Discovery andDevelopment) is showing promise as anantitumor agent, either alone or in combinationwith other therapeutics (J. Immunol.2002;169:5978-85). The Yi laboratory has alsoidentified the prolactin (PRL) family ofoncogenic phosphatases as targets for developinganticancer therapeutics. They have shown thatpentamidine, an anti-protozoa drug with anunknown mechanism of action, is an inhibitor ofPRLs, both in cell cultures and of humanmelanoma tumors grown in nude mice. Theseobservations suggest the potential of pentamidinein anticancer therapies and may provide abasis for developing novel protein tyrosinephosphatase-targeted therapeutics (Mol. CancerTher. 2002;1:1255-64).In recent years, lysophospholipids havebeen recognized as important cell signalingmolecules. The laboratory of Dr. Yan Xu hasbeen at the forefront of these advances. Hergroup has identified the cell surface receptor forthe cell membrane and serum lipidlysophosphatidylcholine (LPC) as G2A, alymphocyte-expressed G-protein-coupledreceptor whose genetic ablation results in thedevelopment of autoimmunity. Ovarian cancerG-protein-coupled receptor 1 (OGR1, orGPR68), and G-protein-coupled receptor 4(GPR4) have also been identified as receptors forsphingosylphosphorylcholine and LPC (BiochimBiophys Acta 2002;1582:81-8).Dr. Michael Vogelbaum is interested inmechanisms of apoptosis induction in glioblastoma.He is also collaborating with Drs.Silverman and Haque to identify novel strategiesfor therapeutic intervention in this brain tumortype.Protein kinase C regulation is the subjectof research in two laboratories. Dr. FirouzDaneshgari, a urologist with a research interest inthe regulation of protein kinase C signaling in theurothelium, is working with Dr. Thomas Powell,who is investigating the role of protein kinase Cisoforms in the induction of apoptosis.Dr. Jawhar Rawwas is a pediatric hematologistand oncologist who is interested in themechanisms of resistance to retinoic acid in thechildhood cancer neuroblastoma, including therole of cellular retinoic acid binding proteins andtheir possible interactions with N-myc. Dr.Rawwas is a member of the NeuroblastomaBiology Subcommittee of the Children’s OncologyGroup.The newest member of the Cancer Biologydepartment is Dr. Nywana Sizemore, who hasbeen recruited to the Section of ColorectalCancer Research headed by Dr. Casey. Herresearch interests encompass the role of aberrantsignal transduction by AKT and IKK in controllinggene expression, oncogenesis, and apoptosis incolorectal cancer. Dr. Sizemore discovered a rolefor the phosphatidylinositol 3' kinase (PI3K)/AKT cell survival pathway in the positiveregulation of NFκB. She also showed that thetumor suppressor PTEN negatively regulatesNFκB activity. She has also found that IKKregulates the activation of α-catenin, a criticalfactor regulating colorectal epithelial celltranscription.The Department of Cancer Biology has anactive training program at the graduate studentand postdoctoral levels. Several staff membershave joint appointments at Case Western ReserveUniversity, Cleveland State University and KentState University. Presently, 13 graduate studentsare completing their thesis research in thedepartment, and several rotation and summerstudents spend time throughout the year invarious department laboratories. Three studentsgraduated with a Ph.D. degree during the pastyear. Postdoctoral training is an importantactivity in the department, and we presently haveover 51 fellows representing an internationalcadre of trainees. The training program includes aseminar series, journal club, and meetings with theTaussig Cancer Center Translational Therapeuticsgroup. Trainees are also encouraged to attendother relevant weekly sessions at the LRI seminarsand the annual LRI retreat. Funds are availableto enable trainees to attend national and internationalmeetings to present their work, and thedepartment has an excellent record in obtainingcompetitive travel awards to relevant conferences.47

THE ALMASANLABORATORYVISITING SCHOLARQuan Chen, Ph.D.Inst. of Zoology, Chinese Acad.of Sciences, BeijingPOSTDOCTORAL FELLOWSJohn Hissong, M.D., Ph.D.Suparna Mazumder, Ph.D.Subrata Ray, Ph.D.GRADUATE STUDENTSMeredith Crosby, A.B.Erica DuPree, M.S.Marcela Oancea, B.S.Dragos Plesca, M.S.COLLABORATORSGuy M. Chisolm, Ph.D. 1Daniel Lindner, M.D., Ph.D. 2Roger M. Macklis, M.D. 3Lily Ng, Ph.D. 4Gordan Srkalovic, M.D., Ph.D. 51Dept. of Cell Biology, CCF2Drug Development, TaussigCancer Center, CCF3Dept. of Radiation Oncology,CCF4Dept. of Chemistry, ClevelandState Univ., Cleveland, OH5Dept. of Hematology andMedical Oncology, CCFResponse of Tumor Cells to Genotoxic Stress:Mechanisms of Apoptosis andCell-Cycle ControlThe overall goal of our laboratory is to gaina better understanding of how mammaliancells respond to genotoxic stress elicited byionizing radiation (IR) and chemotherapeuticagents. These agents, commonly used in thetreatment of cancer, are thought to ultimatelykill tumor cells by triggeringapoptosis. Cytochrome c,caspases, and Bcl-2-familyproteins represent the basicregulators of apoptosis. We areinterested in the precisemechanism by which theseproteins interact to regulate thecommitment to cell death.ApoptosisWe have shown that IRinducedapoptosis is dependenton the activation of multiplecaspases, which proteolyticallycleave cellular proteins,including Bcl-2 and cyclin E. Wehave proposed a model forIR-induced apoptosis, activatedby cytochrome c through: (i) aninitial release into the cytoplasm of low levels ofcytochrome c, sufficient to induce caspaseactivation, and (ii) a late-stage depletion ofmitochondrial cytochrome c and loss ofmitochondrial functions. We have identifiedBcl-2 as a mitochondrial caspase target whichthus converts an anti-apoptotic protein to apro-apoptotic one. We are interested in the roleof Bcl-2 and the related Bcl-2 homology domain3 (BH3: Bik, PUMA, Noxa) and multi-domain(Bax, Bak) apoptotic proteins in the control ofcytochrome c release and the commitment toapoptosis. We have also found that the deathChen, Q., Gong, B., Mahmoud-Ahmed, A.S., Zhou, A., Hsi, E.D., Hussein, M., and A.Almasan (2001) Apo2L/TRAIL and Bcl-2-related proteins regulate type I interferoninducedapoptosis in multiple myeloma. Blood 98:2183-2192.Mazumder, S., Gong, B., Chen, Q., Drazba, J.A., Buchsbaum, J.C., and A. Almasan(2002) Proteolytic cleavage of cyclin E leads to inactivation of associated kinaseactivity and amplification of apoptosis in hematopoietic cells. Mol. Cell. Biol. 22:2398-2409.Chen, Q., Chai, Y.C., Mazumder, S., Jiang, C., Macklis, R.M., Chisolm, G.M., and A.Almasan (2003) The late increase in intracellular free radical oxygen species duringapoptosis is associated with cytochrome c release, caspase activation, and mitochondrialdysfunction. Cell Death Differ. 10:323-334.Almasan, A., and A. Ashkenazi (2003) Apo2L/TRAIL: apoptosis signaling, biology, andpotential for cancer therapy. Cytokine Growth Factor Rev. 14:337-348.Ray, S., and A. Almasan (2003) Apoptosis induction in prostate cancer cells and xenograftsby combined treatment with apo2L/TRAIL and CPT-11. Cancer Res. 63:4713-4723.The Department of Cancer BiologyAlexandru Almasan, Ph.D.ligand Apo2 ligand/TNF-related apoptosisinducingligand (Apo2L/TRAIL) is activated byIR or interferons and is responsible for cell deathin T-cell or multiple myeloma-derived tumorsthrough a receptor-mediated pathway. Currenttesting of this unique tumor-specific apoptoticactivator, alone or in combinationwith other therapeutics, invitro and in mouse xenografts,should pave the way for its usein clinical trials.Cyclin ECell-cycle control iscritical for all cellular functions,and its deregulation leads toapoptosis. The cell-cycle arrestor apoptosis induced by DNAdamagingagents such as IRlargely results from activationof the tumor suppressor p53,which transcriptionally inducesmany genes, including inhibitorsof the cyclin E/cyclindependentkinase (CDK)complexes. This in turnprevents phosphorylation of the retinoblastomatumor supressor protein (pRb), thus sequesteringthe E2F transcription activators, which controlexpression of S-phase genes. Cyclin E representsan essential regulator of pRb phosphorylation, aswell as of DNA replication. We have extendedour previous studies on the role of pRb inapoptosis to that of cyclin E. We found that,when overexpressed, cyclin E greatly acceleratesIR-induced apoptosis. We believe that cyclin Eexpression could affect cell death at two distinctlevels. First, cyclin E expression and associatedkinase activity increase following irradiation.Second, most cyclin E is proteolytically cleaved inall human tumor hematopoietic lineages. We haveidentified the site of cyclin E cleavage and foundthat a cyclin E proteolytic fragment is a potentinducer of apoptosis. We are determining itssubcellular localization and interactions withcellular proteins to address the role it plays inregulating apoptosis.To further elucidate the signals to apoptosisand cell-cycle control and discover novelradiation-induced genes, we are using microarraytechnologies. The knowledge gained from theabove studies may lead to a better understandingof signals leading to cell death followingtreatment with genotoxic agents, and ultimatelyimprove cancer therapy by enhancing theelimination of malignant cells.48

The Department of Cancer BiologyRole of DNA Polymerase β,a DNA Repair Gene, in OncogenesisThe focus of this laboratory is to elucidatewhether DNA polymerase β (polβ)contributes to carcinogenesis. DNA polβ isthe major contributor to gap-filling synthesis atAP sites of damaged DNA involved in the baseexcisionrepair pathway. The polβ gene, mappedon chromosome 8p12, is essential for embryonicviability and neurogenesisof mice. Additionalreports provideevidence that polβ playsa role in DNA replication,recombination,meiosis, drug-resistantphenotype and apoptosis.Reports from thislaboratory provided thefirst evidence for an 87-bp deletion encodingamino acid residues208-236 in the codingsequence of polβ inhuman breast carcinomasand fibroadenomasof breast, colorectal andlung carcinomas. A 36-kDa truncated polβprotein (polβ∆) is alsoexpressed in thesetumors.We have recently identified mutations in thegenomic sequence of polβ of primary ovariantumors. Furthermore, several truncated proteins areexpressed in cell lysates of ovarian tumors. Theseresults demonstrate that indeed mutations occur inthe polβ gene of human tumors. It is noteworthythat alterations in the coding sequence of the polβgene and expressions of polβ∆ protein have beenrevealed in low-malignant-potential borderlineovarian tumors.We hypothesize that cells expressing defectivepolβ would have an adverse biological effecton cells, leading to mutagenesis and carcinogenesis.To address this possibility, we established threestable cell lines: 16.3∆P expressing polβ∆ and 39-kDa wild-type (WT) polβ proteins, 19.4∆P expressingthe polβ∆ protein only, and 19.4WT expressingthe polβ WT protein. Possible hypersensitivityof cells expressing polβ∆ was examinedwhen DNA is damaged by MNU, a mutagen andcarcinogen. Hypersensitivity of cells was evaluatedby growth characteristics, susceptibility to morphologicaltransformation, anchorage independency andtumorigenic potential in the nude mouse. Transformationfrequency induced by MNU exhibited in16.3∆P cells was markedly higher than in 16.3 cellsexpressing the WT polβ∆ protein. Similarly, thetransformation frequency was enhanced in 19.4∆Pcells treated with MNU vs. 19.4WT cells exposedto MNU. Transformed cells formed colonies in softagar. These results clearly demonstrate that cellsSipra Banerjee, Ph.D.expressing polβ∆ are susceptible to transformation,especially to carcinogens.To test in vivo which cells are more susceptibleto carcinogenesis, those expressing polβ∆ or WTpolβ, we established 4 transgenic mouse lines thatexpress polβ∆ under the control of a WAP promoterin breast epithelial cells. To determine potentialdifferences in tumorigenicsusceptibility betweentransgenic and nontransgenicanimals, mice weretreated with MNU. Mammarytumors developedearlier in transgenic thanin nontransgenic mice.The polβ∆ expressedin tumor cellsinhibits the biochemicalfunctions of WT polβ,acting as a dominantnegativemutant. Moreover,we showed thatXRCCI, a repair protein, isdirectly involved in thedominant suppressiveactivity of polβ∆, leadingto the genomic instabilitycharacteristic of tumorcells. Notably, we found,by CD analysis, that polβprotein showed conformational instability. To findnovel proteins that might interact with polβ∆, wescreened a HeLa cDNA library using polβ∆ as baitin the yeast two-hybrid system. Two positiveclones with the EST sequence of a hypotheticalprotein, MGC5306, and a ubiquitously expressedtranscript were identified. By RT-PCR, we successfullyamplified the full coding sequence ofMGC5306 from the brain tumor cell line U373and breast cancer cell line MDA468. Characterizationof MGC5306 and study of the relationshipbetween its expression and tumorigenesis areunder way.THE S. BANERJEELABORATORYPROJECT SCIENTISTSNandan Bhattacharyya, Ph.D.Liming Wang, M.D., Ph.D.CLINICAL FELLOWSPedro Escobar, M.D.Effrosyni Mahali, M.D.POSTDOCTORAL FELLOWSSusmita Chakraborty, Ph.D.Neelam Yadav, Ph.D.COLLABORATORSJerome L. Belinson, M.D. 1Rathin Bose, Ph.D. 2Peter J. Mazzone, M.D. 3Joan B. Sweasy, Ph.D. 41Dept. of Gynecology andObstetrics, CCF2Dept. of Chemistry, Kent StateUniversity, Kent, OH3Dept. of Pulmonary andCritical Care Medicine, CCF4Dept. of Therapeutic Radiol.and Genetics, Yale Sch. ofMed., New Haven, CTBhattacharyya, N., and S. Banerjee (1997) A variant of DNA polymerase β acts as a dominantnegative mutant. Proc. Natl., Acad. Sci. USA 94:10324-10329.Chen, H.-C., Bhattacharyya, N., Wang, L., Recupero, A.J., Harter, M.L., and S. Banerjee(2000) Defective DNA repair genes in a primary culture of human renal cell carcinoma. J.Cancer Res. Clinic. Oncol. 126:185-190.Bhattacharyya, N., Banerjee, T., Patel, U., and S. Banerjee (2001) Impaired repair activity of atruncated DNA polymerase β protein. Life Sci. 69:271-280.Bhattacharyya, N., and S. Banerjee (2001) A novel role of XRCCI in the functions of a DNApolymerase β variant. Biochemistry 40: 9005-9013.Bhattacharyya, N., Chen, H,-C., Wang, L., and S. Banerjee (2002) Heterogeneity in expressionof DNA polymerase β and DNA repair activity in human tumor cell lines. Gene Expression10:115-123.Bhattacharyya, N., and S. Banerjee (2003) Analysis of alterations in a base-excision repair genein lung cancer. Methods Mol. Med. 74:413-438.49

Graham Casey, Ph.D.THE CASEYLABORATORYPOSTDOCTORAL FELLOWSMine Cicek, Ph.D.Muzaffer Cicek, Ph.D.Anthony Curran, M.S.Phillippa Neville, Ph.D.LEAD TECHNOLOGISTSarah Plummer, B.S.SENIOR RESEARCHTECHNOLOGISTLisa Krumroy, B.S.RESEARCH TECHNOLOGISTAndrea Moreira, B.S.GRADUATE STUDENTFrederick Schumacher, B.S.COLLABORATORSJames M. Church, M.D. 1Robert W. Haile, Ph.D. 2Eric A. Klein, M.D. 3Noralane M. (Laney) Lindor, M.D. 4Sanford D. Markowitz, Ph.D. 5Nywana Sizemore, Ph.D .6Danny R. Welch, Ph.D. 7John S. Witte, Ph.D. 51Dept. of Colorectal Surgery,CCF2Univ. of Southern California,Los Angeles, CA3Urological Inst., CCF4Mayo Clinic, Rochester, MN5Case Western Reserve Univ.,Cleveland, OH6Dept. of Cancer Biol., CCF7Jake Gittlen Cancer Res. Inst.,Coll. of Med., PennsylvaniaState Univ. Hershey, PAThe overall objectives of our laboratoryare to discern molecular genetic mechanisms of cancer development andmetastasis. Areas of interest include theidentification and characterization of tumorsuppressor genes, gene expression profiling, andcancer genetics-epidemiology studies.An important goal of cancer genetics isthe identification of individuals at increasedrisk for developing the disease. We haverecently mapped a hereditary prostate cancergene to chromosomes 16q23 through wholegenomelinkage analyses and loss-of-heterozygositystudies. Furthermore we have determinedthat tumor aggressiveness may also inpart be genetic, as we have identified two loci,on chromosome 19q13.1 and 7q32-q33, thatalso appear to harbor hereditary prostate cancergenes associated with more aggressive cancer atdiagnosis. We are using a range of molecularapproaches to identify and characterize thesegenes.We are also interested in determining therole of low-penetrance gene mutations in risk ofprostate cancer. We recently determined that asingle common variant of the RNASEL genecalled R462Q is associated with up to 13% ofprostate cancers, making it one of the mostfrequent genetic alterations in any of the commoncancers. Nearly 60% of men in the studypossessed at least one copy of the R462Q variant.Men who inherited only one copy of the varianthad a 50% increased risk of prostate cancer,whereas men who inherited two copies had atwo-fold increased risk of prostate cancer. Thismeans that although the effect of carrying theR462Q variant may be relatively small for anindividual, the effect on men’s health overall isThe Department of Cancer BiologyAnalysis IdentifiesGenetic Alterations ofCommon Human Malignanciesvery large due to the frequency of the R462Q variantin the population.We have also found a correlation between somevariants and more aggressive forms of prostate cancer.We have determined that a variant in the cytochromeP450 gene CYP3A4 is associated with more aggressiveprostate cancer in a case-only study of African-American prostate cancer patients. This result hasparticular relevance as African-American men are notonly more prone to prostate cancer but also todeveloping more aggressive forms of the disease.Mutations in the BRCA1 gene are responsiblefor up to 5% of breast cancers. BRCA1 mutationcarriers appear to have a different prognosis fromnon-BRCA1 breast cancer patients. We havedetermined that breast tumorigenesis in BRCA1mutation carriers occurs by a distinct molecularmechanism from that of age-matched nonfamilialcases. Furthermore, our data suggest that age atdiagnosis, possibly related to menopausal status, maybe an important factor in the expression of specificproteins in breast tumors of BRCA1 mutationcarriers. These results may lead to a better understandingof the underlying mechanisms involved inBRCA1-related tumorigenesis.Metastasis, the spread of the primary tumorto a distant site, is the main cause of death fromcancer. We have applied somatic cell approaches toidentify genes associated with metastasis usingAffymetrix GeneChip arrays. We have been usingthe MDA MB 435 metastasis model system. Acomparison of altered gene expression in metastaticand nonmetastatic cells has yielded a number ofcandidate genes that may function as positive(metastatic oncogenes) or negative (metastasissuppressor genes) regulators of metastasis. Thesecandidate genes are being rigorously examined toconfirm their role in the metastatic process.Paris, P.L., Kupelian, P.A., Hall, J.M., Williams, T.L., Levin, H., Klein, E.A., Casey, G., and J.S. Witte(1999) Association between a CYP3A4 genetic variant and clinical presentation in African-Americanprostate cancer patients. Cancer Epidemiol. Biomarkers Prev. 8:901-905.Paris, P.L., Witte, J.S., Kupelian, P.A. Levin, H., Klein, E.A., Catalona, W.J., and G. Casey (2000)Identification and fine mapping of a region showing a high frequency of allelic imbalance on chromosome16q23.2 that corresponds to a prostate cancer susceptibility locus. Cancer Res. 60:3645-3649.Vaziri, S.A., Krumroy, L.M., Elson, P., Budd, G.T., Darlington, G., Myles, J., Tubbs, R.R., and G. Casey(2001) Breast tumor immunophenotype of BRCA1 mutation carriers is influenced by age at diagnosis.Clin. Cancer Res. 7:1937-1945.Neville, P.J. Conti, D.V. Paris, P.L., Levin, H., Catalona, W.J., Suarez, B.K., Witte, J.S., and G. Casey(2002) Prostate cancer aggressiveness locus on chromosome 7q32-q33 identified by linkage and allelicimbalance studies. Neoplasia 4:424-431.Casey, G., Neville, P.J., Plummer, S.J., Xiang, Y., Krumroy, L.M., Klein, E.A., Catalona, W.J., Nupponen,N., Carpten, J.D., Trent, J.M., Silverman, R.H., and J.S. Witte (2002) RNASEL Arg462Glnvariant is implicated in up to 13% of prostate cancer cases. Nature Genet. 32:581-583.50

Our laboratory is interested in understandingthe mechanisms that controlthe balance between proliferation anddifferentiation during development. Our focusis on the ski oncogene family, whose twomembers, ski and sno, encode transcriptionalregulators that can affect both oncogenictransformation andcellular differentiation.We use gene targetingand transgenictechniques to study thedevelopmentalprocesses that requirethe function of ski andsno in mice, as modelsfor human development.Both ski and snohave been shown tofunction as coactivatorsor corepressors,modulatingtranscription byinteracting with severaldifferent transcriptionfactor complexes.Those interacting withski include members ofthe Nuclear Factor I(NFI) family, theretinoic acid receptor,and the nuclearhormone co-regulators NCoR and skip/NCoA62, and members of the Smad genefamily. Their ability to interact with multipletranscription factors, coupled with theirubiquitous expression, suggests that ski and snoare involved in regulating the expression of alarge, diverse group of genes. Therefore, wehave undertaken a genetic approach, introducingmutations into ski and sno genes to uncoverthe developmental pathways that require theirfunctions.A long-standing area of interest in thelaboratory has been the role of the ski genefamily in skeletal muscle differentiation. Bothski and sno have the surprising property ofinducing, simultaneously, both myogenicdetermination and oncogenic transformation.In vivo, overexpression of ski in skeletalmuscles of transgenic mice induces hypertrophyof specific fiber types. In our ski-deficientmice, skeletal muscle development is defective,as shown by a dramatic reduction in skeletalmuscle mass and fiber organization. We arecurrently using these mutant mice to identifyspecific stages during skeletal muscle differentiationthat require ski expression.The Department of Cancer BiologyProto-Oncogenes ski, sno:Global Regulators of Differentiationand DevelopmentA new focus for our laboratory is the studyof craniofacial defects in ski-deficient mice. Wehave found that ski -/- mice show either a neuraltube defect or median facial clefting and that thepenetrance of these two phenotypes is completelydependent on genetic background. Theseresults suggest that mutations in ski may berelevant to humanfacial clefting, whichis one of the mostcommon birthdefects. These dataled us to correctlypredict the involvementof ski in ahuman geneticsyndrome, monsomy1p36, in which theobserved featuresinclude not only facialclefting, but alsoother phenotypes incommon with ski-nullmice, such as musclehypotonia, openfontanels, and adepressed nasalbridge. Our continuingefforts involvemapping of modifierClemencia Colmenares, Ph.D.genes, and searchingfor mutations in skiamong other familieswith facial clefting defects.Because sno has the same activities as skiwhen over-expressed in vitro, and both genes areexpressed ubiquitously, it seemed likely that theywould have overlapping activities. Our analysesof double-mutant mice lacking both ski and snosuggest that, although there is some functionalredundancy, there are also gene-specific functionsthat lead to distinct phenotypes.THE COLMENARESLABORATORYTECHNOLOGISTBinh To, B.S.Yuanhua LiuGRADUATE STUDENTYishi Chen, M.S.COLLABORATORSHeidi Heilstedt, M.D. 1Shunsuke Ishii, Ph.D. 2Sonia Pearson-White, Ph.D. 3Lisa Shaffer, M.D. 1Edward Stavnezer, Ph.D. 41Dept. of Molecular and HumanGenetics, Baylor Coll. ofMed., Houston, TX2RIKEN Tsukuba Life ScienceCtr., Tsukuba, Japan3Univ. of Virginia, Charlottesville4Case Western Reserve Univ.,Cleveland, OHBerk, M., Desai, S. Y., Heyman, H. C., and C. Colmenares (1997) Mice lacking theski proto-oncogene have defects in neurulation, craniofacial, patterning, and skeletalmuscle development. Genes Dev. 11:2029-2039.Shinagawa, T., Nomura, T., Colmenares, C., Ohira, M., Nakagawara, A., and S. Ishii(2001) Increased susceptibility to tumorigenesis of ski-deficient heterozygous mice.Oncogene 20:8100-8108.Colmenares, C., Heilstedt, H.A., Shaffer, L.G., Schwartz, S., Berk, M., Murray, J.C.,and E. Stavnezer (2002) Loss of the SKI proto-oncogene in individuals affected with1p36 deletion syndrome is predicted by strain-dependent defects in Ski-/- mice. Nat.Genet. 30:106-109.Dai, P., Shinagawa, T., Nomura, T., Harada, J., Kaul, S.C., Wadhwa, R., Khan, M.M.,Akimaru, H., Sasaki, H., Colmenares, C., and S. Ishii (2002) Ski is involved intranscriptional regulation by the repressor and full-length forms of Gli3. Genes Dev.16:2843-2848.51

THE DANESHGARILABORATORYPOSTDOCTORAL FELLOWGuiming Liu, M.D., Ph.D.The Department of Cancer BiologyBladder Disease in Diabetes Arises fromChanges in Tissue, Accumulation ofAdvanced Glycosylation EndproductsSENIOR RESEARCH TECHNOLOGISTLateef Saffore, M.S.COLLABORATORSC. Thomas Powell, Ph.D. 1Frank Bockowitz, M.D., Ph.D. 21Dept. of Cancer Biology, CCF2Dept. of Physiology, CaseWestern Reserve Univ.,Cleveland, OHThe main focus of our laboratory is onbladder physiology and the pathophysiologyof bladder diseases. The initial modelof bladder pathology being investigated in ourlaboratory is diabetes mellitus.Up to 83% of the patients with eitherinsulin-dependent (type 1) or noninsulindependent(type 2) diabetes suffer fromcomplications in the lower urinary tract. Thesecomplications are manifestedas voiding dysfunction,evidenced by increasedbladder capacity withdecreased contractility, andinfection, evidenced byrecurrent and complicatedinfections.The traditional view onthe pathophysiology ofvoiding dysfunction indiabetic cystopathy has beenthat it arises from autonomicneuropathy. Based onaccumulated data from theliterature and from ourlaboratory, our alternativehypothesis is that the mainpathologic condition indiabetic cystopathy arises fromchanges occurring underhyperglycemic conditions inthe bladder tissue itself. Basedon this hypothesis, theaccumulation of advanced glycosylationendproducts (AGEs) could cause alterations inthe phenotype of diabetic bladder and impairedcontractility of the bladder.Our laboratory has shown that, in theimpaired contractility caused by diabetes,alterations in the translocation and temporaldistribution of certain isoenzymes of proteinkinase C (PKC) occur, with a marked change inDaneshgari, F. (2001) Valsalva leak point pressure: steps toward standardization. Curr.Urol. Rep. 2:388-391.Lee, R.S., DeAntoni, E., and F. Daneshgari (2002) Compliance with recommendationsof the urodynamic society for standards of efficacy for evaluation of treatmentoutcomes in urinary incontinence. Neurourol. Urodyn. 21:482-485.Daneshgari, F., Washington, M., Babekir, N., El-Azab, A., Hoogwerf, B., Barber, M.,Rackley, R., and S. Vasavada (2002) Lower urinary tract complications of diabetesmellitus in adult population: A cross sectional study. International Continence SocietyAnnual Meeting. Germany, September 2002.Daneshgari, F., Saffore, L., and S. Choudhary (2002) Temporal profile of proteinkinase C isozymes in the urothelium and Detrusor of rat. 22 nd Annual meeting ofAmerican Urogynecological Society. San Francisco, October 2002.Daneshgari, F., Wyne, K., Ferruchi, P., and S. Choudhary (2002) Animal models ofdiabetes for studies of lower urinary tract complications. J. Urol. (submitted).Firouz Daneshgari, M.D.isoenzyme BII, in particular. Our current effortfocuses on the effects of the accumulation ofAGEs on PKC function in bladder tissue.During 2002, we started translation of theabove work to clinical investigation. WithInstitutional Review Board approval, we havestarted recruiting patients with diabetes into twoparallel clinical studies. In one study, the prevalenceof diabetic complication is assessed byseveral questionnaires,uroflowmetry and post-voidresidual. The patients withevidence of diabeticcystopathy undergo multichannelurodynamic studies,cystoscopy and biopsy oftheir bladder. Contractilitystudies and identification ofPKC isozymes and AGEs aresubsequently performed. Inthe other arm of the study, asample of bladder ofpatients with end-stagediabetes who undergo renaltransplantation is obtained.Similar samples fromoutpatient patients,contractility studies andidentification of PKCisozymes and AGEs areperformed. The dataemerging from thesetranslational studies are veryencouraging.A very exciting development in ourlaboratory during 2002 was an invitation (via aU01 supplement grant from the National Instituteof Diabetes and Digestive and Kidney Diseases)to Dr. Daneshgari to join the Animal Models ofDiabetes Consortium of NIH as the chair of asubcommittee to characterize diabetic uropathy.The aim of this consortium is to create animalmodels of diabetes by varioius knock-out andtransgene technologies.During 2002, the work in our laboratorywas recognized by two awards: a Young InvestigatorAward of the National Kidney Foundationand a Grant-in-Aid from the Diabetes Associationof the Greater Cleveland, awarded to Dr.Daneshgari in July.52

The Department of Cancer BiologyEnteric Pathogen RegulatesNF-κB Activationof Inflammatory AgentsPathogenic stimulation of the nuclearfactor-κB transcription factor (NF-κB)family of proteins leads to the activationof numerous proinflammatory agents. Expressionof cytokines, chemokines, cellular adhesionproteins and immunoreceptor genes that arerequired for inflammation is dysregulated innearly all cases of chronic and debilitatingdiseases. In our laboratory, we contend thatpathogens are responsible for disrupted signaltransduction pathways, which result in errantimmune response gene expression.Our hypothesis states that pathogenicstimuli commandeer and use normal cellularsignal transduction pathways. Those pathogensusurp the normal control points in the flow ofinformation from the extracellular environmentand the cell membrane to the nucleus.Dysregulated gene expression of inflammatorymediators results in aberrant expression ofinflammatory response genes.Intestinal epithelial cells serve as our modelsystem. We are studying the regulation of the NFκBtranscription factor in response to invasion ofthese cells by pathogenic enteric bacteria. We areusing a variety of biochemical and reverse genetictechniques to precisely interfere with discretesteps in the activation pathway(s) used byproinflammatory cytokines. We are comparinghow this interference affects the signalingpathways activated by the pathogens.Identification of key control nodes alongthe pathogenic signaling pathways will aid in thedevelopment of pharmaceutical compounds thatcan block pathogenic signaling but leave the cell’snormal signal transduction pathways largelyunaffected. Such pharmaceuticals will beextremely useful in the treatment of manychronic inflammatory diseases, such as inflammatorybowel disease, rheumatoid arthritis andnumerous autoimmune and neurodegenerativediseases. Recently, we have identified a patternrecognition receptor on intestinal epithelial cellsthat recognizes bacterial flagellin protein. Therecognition of flagellin by Toll-like receptor 5leads (TLR5) to NF-κB activation and proinflammatorygene expression. Current projectsin the laboratory focus on understanding themolecular interactions and signaling properties ofTLR5 and other TLR family members and theirrelationship to inflammation. Recently, with Dr.Amiya K. Banerjee’s laboratory in CCF’s Sectionof Virology, we have identified a TLR-mediatedpathway of NF-κB activation that recognizesproteins from the medically important humanparainfluenza virus (HPIV3). HPIV3 is a majorcause of lung-related morbidity and disease innewborns, young infants and immunologicallysuppressed individuals. Activation of NF-κB viathis pathway causes the virus to be mildlycytotoxic, whereas blocking the activation ofNF-κB turns the virus infection into a virulentone. In addition, this pathway identifies a novelNF-κB-mediated anti-viral innate host defensesystem.Additional projects in the laboratory focuson identifying structural features of the I-κBkinase (IKK) protein complex that are associatedwith its inactive and active states. IKK is thecentral regulator of NF-κB activation in cells inresponse to a wide variety of cell stimuli. Otherstudies are designed to identify amino acidresidues within the IKK complex that are triggerpoints for IKK activation by other upstreamkinases. Once the structural features of theactive IKK complex and identification of keyactivation amino acid residues and upstreamkinases have been identified, they will serve asexcellent potential drug targets for therapeuticintervention.THE DIDONATOLABORATORYPROJECT SCIENTISTLouis Liou, Ph.D., M.D.Urological Inst., CCFRESEARCH ASSOCIATETing Shi, Ph.D.POSTDOCTORAL FELLOWSNiladri Kar, Ph.D.Thomas Tallant, Ph.D.Provash Sadhukhan, Ph.D.GRADUATE STUDENTJoseph Lupica, B.A.Joseph A. DiDonato, Ph.D.DiDonato, J.A., Hayakawa, M., Rothwarf, D.M., Zandi, E., and M. Karin (1997) A cytokine-responsive IκBkinase that activates the transcription factor NF-κB. Nature 388:548-554.Zamanian-Daryoush, M., Mogensen, T.H., DiDonato, J.A., and B.R. Williams (2000) NF-κB activation bydouble-stranded-RNA-activated protein kinase (PKR) is mediated through NF-κB-inducing kinase and IκBkinase. Molec. Cell. Biol. 20:1278-1290.Purcell, N.H., Yu, C., He, D., Xiang, J., Paran, N., DiDonato, J.A., Yamaoka, S., Shaul, Y., and A. Lin(2001) Activation of NF-κB by hepatitis B virus X protein through an IκB kinase-independent mechanism.Am. J. Physiol. Gastrointest. Liver Physiol. 280:G669-G677.DiDonato, J.A. (2001) IKK alpha on center stage. Science STKE [Electronic Resource: Signal TransductionKnowledge Environment]. 2001(97):PE1.Rani, M.R., Asthagiri, A.R., Singh, A., Sizemore, N., Sathe, S.S., Li, X., DiDonato, J.D., Stark, G.R., andR.M. Ransohoff (2001) A role for NF-κB in the induction of β-R1 by interferon-β. J. Biol. Chem.276:44365-44368.53

THE ERZURUMLABORATORYRESEARCH ASSOCIATESSuzy Comhair, Ph.D.Weiling Xu, M.D.CLINICAL FELLOWSTobias Piekert, M.D.Roberto Machado, M.D.VISITING SCIENTISTArnaud Chambellan, M.D.INSERM, Nantes, FrancePOSTDOCTORAL FELLOWSSudakshina Ghosh, Ph.D.Shuo Zheng, Ph.D.SENIOR TECHNOLOGISTTannishia Goggans, B.S.RESEARCH COORDINATORJacqueline Pyle, R.N.TECHNOLOGISTAllison Janocha, B.S.TECHNICAL ASSOCIATEDaniel LaskowskiGRADUATE STUDENTFares Masri54Inducible Nitric Oxide Synthase (NOS2)Expression Key to Airway InflammationOur laboratory studies the molecularmechanisms that initiate and maintainairway inflammation. In this context, wehave focused on the role of antioxidants andreactive oxygen (ROS) and nitrogen species(RNS) in the pathogenesis of lung diseases.Recently, the lungs have been shown toproduce significant amounts of RNS, i.e., nitricoxide (NO). NO is present in exhaled air ofnormal individuals and increased in that of asthmaticindividuals. Although excessive NO may bean injurious oxidantspecies, NO is endogenouslyproduced inhealthy human lungs,suggesting it has aphysiologic role.Three isoforms ofnitric oxide synthase(NOS), the enzymesresponsible for endogenousNO production, are foundin cells: inducible NOS(NOS2) and two isoformsof constitutive NOS(NOS1 and 3). Expressionof NOS2 has traditionallybeen found in cellsstimulated by inflammatorycytokines such as interleukin-1β (IL-1β), tumornecrosis factor-α (TNF-α), and interferon-γ(IFN-γ). However, having identified and clonedthe NOS isoform from freshly obtained healthyhuman airway epithelium, we found that thiscontinuously expressed airway NOS is NOS2.Ours was the first conclusive demonstration ofcontinuous expression of the NOS2 gene innormal, non-inflamed tissues and suggests uniqueKaneko, F.T., Arroliga, A.C., Dweik, R.A., Comhair, S.A., Laskowski, D., Oppedisano, R.,Thomassen, M.J., and S.C. Erzurum (1998) Biochemical reaction products of nitric oxide asquantitative markers of primary pulmonary hypertension. Am. J. Respir. Crit. Care Med.158:917-923.Comhair, S.A., Bhathena, P.R., Dweik, R.A., Kavuru, M., and S.C. Erzurum (2000) Rapid lossof superoxide dismutase activity during antigen-induced asthmatic response. Lancet 355:624.Beall, C.M., Laskowski, D., Strohl, K.P., Soria, R., Villena, M., Vargas, E., Alarcon, A.M.,Gonzales, C., and S.C. Erzurum (2001) Pulmonary nitric oxide in mountain dwellers. Nature414:411-412.Chung-man Ho, J. [Ho, J.C.] Zheng, S., Comhair, S.A., Farver, C., and S.C. Erzurum (2001)Differential expression of manganese superoxide dismutase and catalase in lung cancer.Cancer Res. 61:8578-8585.Zheng, S., De, B.P., Choudhary, S., Comhair, S.A., Goggans, T., Slee, R., Williams, B.R.,Pilewski, J., Haque, S.J., and S.C. Erzurum (2003) Impaired innate host defense causessusceptibility to respiratory virus infections in cystic fibrosis. Immunity 18:619-630.Xu, W., Comhair, S.A., Zheng, S., Chu, S.C., Marks-Konczalik, J., Moss, J., Haque, S.J., andS.C. Erzurum (2003) STAT-1 and c-Fos interaction in nitric oxide synthase-2 gene activation. Am.J. Physiol. Lung Cell. Mol. Physiol. 285:L137-L148.The Department of Cancer Biologymechanisms for regulation of NOS2 in airwayepithelial cells.However, increased expression of theNOS2 gene may contribute to the pathogenesis ofinflammatory airway diseases, i.e., asthma andbronchitis. We found that asthmatic epitheliumexpresses 15-fold higher levels of NOS2 thannormal airway epithelium. Interestingly, the upregulationof NOS2 may be rel-ated to the level ofROS present in the airway. For example, uponexposure of cells to increasing O 2, increasedamounts of NO are produceddue to increased amounts ofNOS2 expression. In contrast,decreased NO may also lead tolung disease. We recently demonstratedthat individuals withprimary pulmonary hypertensionhave decreased NO levelscompared with healthy controls.Thus, airway epithelialNOS2 may mediate pulmonaryvascular response by generatingNO, pharmacologically definedas a potent vasodilator. Furthermore,we recently identifiedthat loss of NO synthesisSerpil C. Erzurum, M.D. in cystic fibrosis (CF) contributesto the susceptibility of CFneonates to viral infection. Weare now studying the source, regulation and role ofNO in the lung; our long-term goal is, by determininghow NO is mechanistically involved in lungdiseases, to design effective therapies.Evidence has emerged in recent years suggestinga role for increased ROS and RNS in lungdiseases, specifically in the airway inflammationcharacteristic of chronic bronchitis and asthma.However, the lungs are well equipped with antioxidantdefenses, and in some experimental systems,antioxidants increase in response to ROS tominimize inflammation and injury. We have shownthat augmentation of antioxidant defenses throughgene therapy prevents cell injury. We investigatedthe response of antioxidants to increased ROS inlung. Our work has identified alterations in theantioxidants glutathione (GSH), Cu-Zn superoxidedismutase (SOD) and glutathione peroxidase(GPx) in cigarette-smoke-exposed lungs, lung cancer,asthma and pulmonary hypertension. SOD degradessuperoxide and exists in three forms, includingintracellular manganese SOD and Cu-Zn SODand an extracellular SOD. GPx removes hydrogenperoxide and organic hydroperoxides by oxidizingGSH, a water-soluble, low-molecular-weight tripeptide(L-γ-glutamyl-L-cysteinyl glycine) that isabundant in lung epithelial lining fluid. Currentwork is focused on identifying the consequences ofloss of antioxidant defenses and increase of ROSandRNS-mediated modifications of proteins inlung diseases.

The Department of Cancer BiologyCytokine-Mediated Cell Signalingin Cancer and InflammationOur long-term objective is to understandhomeostatic control of cytokinemediatedcell signaling in health anddisease. Currently our major focus is to define (i)the role and regulation of Stat3 signaling in thesurvival of glioblastoma cells, (ii) the cellular andmolecular mechanisms that negatively regulate IL-4-mediated signal transduction and subsequentgene expression in allergicinflammation, and (iii) signalingmechanisms underlying the growthand survival of mast cells.Cytokine Signaling in GlioblastomaCellsGlioblastoma multiforme(GBM) is the most common andmalignant form of primary braintumors, with an average survivalof less than one year. The latenttranscription factor Stat3, whenactivated by IL-6-family cytokinesand other growth factors, inducesthe expression of genes that areresponsible for suppression ofapoptosis in a variety of humancancer cells. We found that Stat3 ispersistently activated by theautocrine action of IL-6 in GBM tumor tissuesand GBM cell lines. Inhibition of Stat3 activationinduces apoptosis in these cells. We haveundertaken biochemical and molecular geneticapproaches in both tissue culture system andanimal models to investigate the molecular basesof Stat3-mediated survival of GBM cells. Inaddition, we have addressed the role of IL-13Rα2, a high-affinity non-signaling transmembraneIL-13-binding protein, in the regulation ofJak-Stat signaling in GBM cells.Cytokine Signaling in Allergic InflammationNegative Regulation of IL-4 Signaling: IL-4, apleiotropic cytokine secreted by activated Tlymphocytes, basophils and mast cells, plays a keyrole in the pathogenesis of a variety of inflammatorydisorders, including allergic asthma and otherallergic diseases. Major biological actions of IL-4in the inflammatory cells are mediated throughthe activation of the Jak-Stat pathway. Signaltransduction through this pathway can benegatively controlled at multiple levels by anumber of molecules and by different means. Wechose to focus on the roles of protein tyrosinephosphatases (PTPs) and of proteins in thesuppressors of cytokine signaling (SOCS) familyin the negative regulation of IL-4-dependent Jak-Stat signaling. Our recent work suggested thatPTP activity is a major and primary negativeregulator of the cytokine-activated Jak-Statsignaling pathway, and importantly, more thanone PTP activity is involved in this process. Wehave identified Shp-1 as a negative regulator ofIL-4-mediated cell signaling. Identification ofShp-1 substrate(s), additional PTPs and theirsubstrates in these signaling pathways is underactive investigation. To understand the SOCSmediatednegative feedback mechanisms, we havebegun to characterize the induction profile ofSOCS genes in cytokine-stimulated cells and todefine the structural basis of SOCS-mediatedregulation of the Jak-Statsignaling pathways.Signaling Mast CellGrowth and Survival. Mastcells are critical effectors ininnate immune responses andplay key roles in thepathogenesis of multipleinflammatory disorders,including asthma. Stem cellfactor (SCF) signalingthrough c-Kit is essential forproliferation and survival ofhuman mast cells. However,the precise molecularmechanisms underlying c-Kit-mediated mast cellsurvival remain unclear. TheSaikh Jaharul Haque, Ph.D.long-term goal of thisproject is to (i) identify and characterizeintracellular signaling transduction pathways thatlead to the expression and activation of prosurvivalproteins in mast cells, and (ii) target theidentified pathways in a mouse asthma model toinduce the spontaneous apoptosis of airway mastcells.THE HAQUELABORATORYRESEARCH ASSOCIATENilesh Maiti, Ph.D.PHYSICIAN SCIENTISTFred H. Hsieh, M.D.POSTDOCTORAL FELLOWSShaik Ohidar Rahaman, Ph.D.Pankaj Sharma, Ph.D.TECHNICAL ASSISTANTPhyllis C. Harbor, B.S.COLLABORATORSMark A. Aronica, M.D. 1, 2Gene H. Barnett, M.D. 3Serpil C. Erzurum, M.D. 1,4Abhijit Guha, M.D., FRCS(C) 6George R. Stark, Ph.D. 7Michael Vogelbaum, M.D., Ph.D. 3, 4Bryan R.G. Williams, Ph.D. 4Taolin Yi, Ph.D. 41Dept. of Pulmonary and CriticalCare Medicine, CCF2Dept. of Immunology, CCF3Dept. of Neuro. Surgery, CCF4Dept. of Cell. Path. St.George’sHospital Medical School, London,UK5Dept. of Cancer Biology, CCF6Dept. of Neurosurg. and Surg.Oncol., Univ. of Toronto, Toronto,Ont., Canada7Dept. of Molecular Biology, CCFGoh, K. C., Haque, S.J. and B.R.G. Williams (1999) p38 MAP kinase is required forSTAT1 serine phosphorylation and transcriptional activation induced by interferons.EMBO J. 18:5601-5608.Haque, S.J., Harbor, P.C., and B.R.G. Williams (2000) Identification of criticalresidues required for suppressor of cytokine signaling (SOCS)-specific regulationof IL-4 signaling. J. Biol Chem. 275:26500-26506.Rahaman S.O., Sharma P., Harbor P.C., Aman M.J., Vogelbaum M.A., and S.J.Haque (2002) IL-13Ra2, a decoy receptor for IL-13, acts as an inhibitor of IL-4-dependent signal transduction in glioblastoma cells. Cancer Res. 62:1103-1109.Rahaman S.O., Harbor P.C., Chernova, O., Barnett, G.H., Vogelbaum, M.A., andS.J. Haque (2002) Inhibition of constitutively active Stat3 suppresses proliferationand induces apoptosis in glioblastoma multiforme cells. Oncogene 21:8404-8413.Fox., S.W., Haque, S.J., Lovibond, A.C., and T.J. Chambers (2003) The possiblerole of transforming growth factor-β (TGF-β)-induced SOCS expression in osteoclast/macrophagelineage commitment in-vitro. J. Immunol. 170:3679-3687.Zheng, S., De, B.P., Choudhary, S., Comhair, S.A.A., Goggans, T., Slee, R.,Williams, B.R.G., Pileswski, J., Haque, S.J., and S.C. Erzurum (2003) Impairedinnate host defense causes susceptibility to respiratory virus infections in cysticfibrosis. Immunity 18:619-630.Xu, W., Comhair, S.A.A., Zheng, S., Chu, S.C., Marks-Konczalik, J., Moss, J.,Haque, S.J., and S.C. Erzurum (2003) Stat1 and c-Fos interaction in nitric oxidesynthase 2 gene activation. Am. J. Physiol. Lung Cell Mol. Physiol. 258:L137-L148.55

THE HESTONLABORATORYSTAFF SCIENTISTC. Thomas Powell, Ph.D.PROJECT SCIENTISTArundhati Ghosh, Ph.D.POSTDOCTORAL FELLOWXinning Wang, Ph.D.RESEARCH FELLOWMasifumi Oyama, M.D.RESEARCH TECHNOLOGISTKelly Harsch, B.S.COLLABORATORSNeil Bander, M.D. 1Carlos Cordon-Cardo, M.D., Ph.D. 2Robert Dreicer, M.D. 3Yuman Fong, M.D. 2Eric Klein, M.D. 4Peter Molloy, Ph.D. 5Joe Neal, Ph.D., 6Dianne Perez, Ph.D. 7Victor Reuter, M.D. 2Barbara Slusher, Ph.D. 81Weill Med. College, CornellUniv., New York, NY2Memorial Sloan-Kettering CancerCtr., New York, NY3Dept. of Hematol./Oncol., CCF4Glickman Urol. Inst., CCF5CSIRO, Sydney, Australia6Georgetown Univ., Washington,DCCSIRO, Sydney, Australia7Dept. of Mol. Cardiol., CCF8Guilford Pharmaceuticals, Baltimore,MDOur laboratory cloned the gene encoding aunique protein that is highly expressed inprostate cancers, which we designatedprotein prostate-specific membrane antigen (PSMA).Surprisingly, PSMA is also highly expressed in theneovasculature of solid tumors, but not expressed inthat of normal tissues. PSMAhas modest homology with thetransferrin receptor, yet it doesnot bind transferrin, nor is itinvolved in cellular irontransport by a transferrin. Wediscovered that PSMA has anenzymatic function as a folatehydrolase. We also discoveredtwo alternative spliced formsof PSMA: PSMA’ is a cytosolicprotein, and the mRNAencoding PSMA’ predominatesin normal prostate cells,whereas PSMA is the predominantform encoded in cancercells and encodes for a type-twoThe Department of Cancer BiologyTHE GEORGE M. O’BRIEN UROLOGY RESEARCH CENTERPSMA May Be Key to Targeted Deliveryof Drugs to Tumorsmembrane protein. In normalcells, given PSMA’s folatehydrolase activity, the humanprostate may be an organ at riskfor developing “localized folatedeficiency.” A cell that is folatedeficient is a cell at high risk to develop cancer, andin the human male, the cells with the highestpropensity for developing cancer are those of theprostate.Because PSMA predominates in prostatecancer and is a type-two membrane protein, we areexploring means to target the extracellular domainfor treatment of prostate cancer. We have foundthat the membrane form of PSMA is expressed inthe tumor-associated neovasculature in all other solidSkip Heston, Ph.D.Director,The George M. O’BrienUrology Research Centertumors. Thus, this form of PSMA becomes a broadspectrumtarget for all tumors. We are developingprodrug strategies based on the folate hydrolase/glutamate carboxypeptidase activity of PSMA.Because PSMA can be induced to internalize, we areusing phage display technology to identify ligands withhigh affinity for PSMA that willinduce internalization to providefor additional therapeutictargeting agents.We have also producedhuman PSMA-expressingtransgenic mice that will serveas a model for targeting thehuman protein in immunocompetentanimals. In mice,the murine homolog of PSMAis expressed in the testes,kidney, and brain. We havegenerated PSMA-knockoutmice to understand thefunctional role of PSMA inthese tissues. In brain, PSMAis considered to be responsiblefor the hydrolysis of N-acetylaspar-tylglutamate(NAAG), a neurotransmitter.PSMA hydrolysis of NAAGreleases the neurotransmitterglutamate. In abnormal situations such asischemia, neurotoxic levels of glutamate areproduced by the hydrolysis of released NAAG,resulting in large areas of neuronal death.Inhibitors of murine PSMA attenuate the damage.Likewise, in the PSMA-knockout animals, thedamage associated with a stroke is significantlyless than in the wild-type animal. Thus, theseknockout animals model the role of this proteinContinued on Page 57Uchida, A.. O’Keefe, D.S., Bacich, D.J., Molloy, P.L., and W.D. Heston (2001) In vivo suicide genetherapy model using a newly discovered prostate-specific membrane antigen promoter/enhancer: apotential alternative approach to androgen deprivation therapy. Urology 58(2 Suppl 1):132-139.Thomas, J., Gupta, M., Grasso, Y., Reddy, C.A., Heston, W.D., Zippe, C., Dreicer, R., Kupelian, P.A.,Brainard, J., Levin, H.S., and E.A. Klein (2002) Preoperative combined nested reverse transcriptasepolymerase chain reaction for prostate-specific antigen and prostate-specific membrane antigen does notcorrelate with pathologic stage or biochemical failure in patients with localized prostate cancer undergoingradical prostatectomy. J. Clin. Oncol. 20:3213-3218.Bacich, D.J., Ramadan, E., O’Keefe, D.S., Bukhari, N., Wegorzewska, I., Ojeifo, O., Olszewski, R.,Wrenn, C.C., Bzdega, T., Wroblewska, B., Heston, W.D., and J.H. Neale (2002) Deletion of the glutamatecarboxypeptidase II gene in mice reveals a second enzyme activity that hydrolyzes N-acetylaspartylglutamate.J. Neurochem. 83:20-29.Cozzi, P.J., Burke, P.B., Bhargav, A., Heston, W.D., Huryk, B., Scardino, P., and Y. Fong (2002)Oncolytic viral gene therapy for prostate cancer using two attenuated, replication-competent, geneticallyengineered herpes simplex viruses. Prostate 53:95-100.Chang, S.S., and W.D. Heston (2002) The clinical role of prostate-specific membrane antigen (PSMA).Urol. Oncol. 7:7-12.56

The Department of Cancer BiologyContinued from Page 56in neuronal function, and studying them will aidour understanding of such disorders as stroke,pain, diabetic neuropathy, epilepsy, andamyotropic lateral sclerosis, as well asAlzheimer’s, Huntington’s, and Parkinson’sdisease.We have identified an enhancer region thatis responsible for the strong expression of PSMAin prostate cancer. We are using this enhancerregion for gene therapy approaches such asprodrug strategies using the nontoxic prodrug 5fluorocytosine (5FC). 5FC is converted by theenzyme cytosine deaminase to 5 fluorouracil(5FU), which is toxic to dividing cells. When wetransfect cells with expression vectors containingcytosine deaminase driven by the PSMApromoter/enhancer, following expression andtreatment with 5FC, we kill PSMA-expressingcancer cells but not non-PSMA-expressing tumorcells. This expression is enhanced with hormonedeprivation and is thus likely to provide a usefulstrategy, even when the prostate cancer patienthas been treated with hormones. We havegenerated mice in which green fluorescent protein(GFP) expression is being driven by the humanPSMA promoter/enhancer. Expression isobserved in the brain, kidney and testes, but onlyto a lesser extent in the prostate. The PSMApromoter/enhancer GFP reporter system will helpus model factors that effect tissue-specificexpression in vivo and identify factors responsiblefor the extremely high expression of humanPSMA in prostate.Prostate Cancer Program participants: The Prostate Cancer Program, comprised of a team of investigatorsfrom the Cleveland Clinic Glickman Urological Institute and the Lerner Research Institute, earned the first annualCleveland Clinic “Research Program of the Year” Award during 2002. The award honored the program that bestfulfilled the mission of the Programmatic Research Teams – to promote excellence through fully integrated scientific andeducational initiatives resulting from the collaborative efforts of basic and clinical researchers. Investigators contributingto the award winning prostate cancer research program are, from left, Howard Levin, M.D., Co-Director Skip Heston,Ph.D., Yan Xu, Ph.D., Robert Silverman, Ph.D., Graham Casey, Ph.D., Co-Director Eric Klein, M.D., AndreiGudkov, Ph.D., Raymond Tubbs, D.O., Jay Ciezki,. M.D., and Arul Mahadevan, M.D. Participants not picturedinclude Robert Dreicer, M.D., Tatiana Byzova, Ph.D., Ed Plow, Ph.D,.and Jennifer Brainard, M.D.57

The Department of Cancer BiologyIdentification of N-myc Regulated GenesOpens Paths for Targeted Therapyof NeuroblastomasTHE RAWWASLABORATORYLEAD TECHNOLOGISTKarrie Trevarthen, B.S.Neuroblastoma is the most commonextracranial solid tumor of childhood,accounting for 8-10% of all tumors inchildren. It is a heterogeneous disease thatdisplays a spectrum of clinical behaviorsdepending on the biology of the tumor and theage of presentation. Older children usuallypresent with advanced stage progressive diseasethat has poor outcome. According to the NationalCancer Institute statistics, the five-year survivalof US children with neuroblastoma above 5 yearsof age was still around 40% during the period1985-1994. Surgery, intensive chemotherapy,radiation therapy and stem cell transplant appearto be insufficient or too toxic to achieve thedesired goal of significant improvement inoutcome. Therefore, understanding the mechanismsof neuroblastoma proliferation anddifferentiation and the genes involved is a keyelement in understanding disease developmentand the factors that determine the clinical courseand patient outcome.Patients with aggressive advanced stageneuroblastoma usually have a high rate of N-myconcogene amplification. N-myc is a nuclearphosphoprotein transcription factor that bindsDNA and affects gene regulation. Its in vitrooverexpression results in malignant transformationand increased proliferation and suppressionof differentiation. Overexpression of N-myccauses neuroblastoma in transgenic mice. We havepreviously identified nucleotide diphosphatekinase-A as a gene associated with advanced stageneuroblastoma tumors. We have also correlatedN-myc amplification with high levels of CellularRetinoic Acid Binding Protein-II in neuroblastomatumors using 2-dimensional PAGE. Morerecently, we have utilized cDNA microarraytechnology to examine global patterns of geneexpression between different types of tissues andcell lines (ex. N-myc amplified tumor or cell linevs. normal N-myc copy number tumor or cell line).My lab is interested in identifying the genesthat are directly or indirectly activated by N-mycand to understand their potential role in thedevelopment and progression of neuroblastoma.Dissection of the biologic pathways involved willhelp identify novel targets for therapeuticintervention in neuroblastoma patients.Jawhar Rawwas, M.D.Chang, C.L., Zhu, X.X., Thoraval, D.H., Ungar, D., Rawwas, J., Hora, N., Strahler, J.R., Hanash, S.M.,and E. Radany (1994) Nm23-H1 mutation in neuroblastoma. Nature 370:335-336.Shulkin, B.L., Wieland, D.M., Baro, M.E., Ungar, D.R., Mitchell, D.S., Dole, M.G., Rawwas, J.B., Castle,V.P., Sisson, J.C., and R.J. Hutchinson (1996) PET hydroxyephedrine imaging of neuroblastoma. J. Nucl.Med. 37:16-21.Chen, C.L., Rawwas, J., Sorrell, A., Eddy, L., and F.M. Uckun (2001) Bioavailability and pharmacokineticfeatures of etoposide in childhood acute lymphoblastic leukemia patients. Leuk. Lymphoma 42:317-327.58

The Department of Cancer BiologyTumor Suppressor andAntiviral Roles ofInterferon-Induced ProteinsThe relationship between innate immunity andtumor suppression is the focus of thislaboratory. Understanding the molecularmechanisms of interferon (IFN) action against cancercells and viruses is the long-range goal. The IFNs are afamily of pleiotropic cytokines responsible forproviding innate immunityagainst a wide-range of virusesand other microbial pathogens.Moreover, IFNs regulate cellproliferation, apoptosis andimmune responses, propertiesthat underlie their uses in thetreatment of cancer. IFNs alterpatterns of gene expression incells by binding to specific cellsurface receptors and activatingJAK/STAT pathways. TheIFN-stimulated genes (ISGs)encode proteins that mediatethe biological effects of IFNs.Amongst the mosthighly inducible proteins area family of 2-5A-synthetasesthat are activated by doublestrandedRNA (dsRNA) toproduce 2',5'-oligoadenylates(2-5A) of the generalformula [pppA(2'p5'A)n,n>2]. The function of 2-5A is to activate RNaseL, an 83-kDa protein that we cloned in 1993.Recent progress from the laboratory has contributedto our understanding of the basic eventsunderlying the tumor suppressor and antiviralactivities of the 2-5A system. The absence ofRNase L in mice leads to enhanced susceptibilityto virus infections and to a defect in apoptosis(programmed cell death). Therefore, the 2-5Asystem probably eliminates virus-infected cellsthrough the induction of apoptosis.In addition, we recently showed thatRNase L participates in non-viral apoptoticpathways. Interactions of RNase L with otherstress response proteins and viral strategies forcounteracting the 2-5A system are underinvestigation. In addition, the RNA targets ofRNase L are being determined using genemicroarrays. In a recent collaboration with theNational Human Genome Research Institute andJohns Hopkins University, RNase L was mappedto a major, human cancer susceptibility gene,HPC1 [Carpten J., Nature Genet. (2002)30:181-184]. These findings were extended in a collaborationwith Dr. Graham Casey (Cleveland Clinic)[Casey et al., Nature Genet. (2002), 32:581-583].The presence of both germline mutations inRNASEL segregating with disease within HPCaffectedfamilies and loss of heterozygosity(LOH) in tumor tissues suggest a novel role forthe regulated endoribonuclease in the pathogenesisof prostate cancer. The association ofmutations in RNASEL with prostate cancer casesfurther suggests a relationship between innateimmunity and tumor suppression.In addition, we are studying the tumorsuppressor function of RNase Lby monitoring expression indifferent types of humancancers and by breeding RNaseL-/- mice with mice that havedeficiencies in other tumorsuppressor genes. The goals areto probe the fundamental roleof RNase L in tumor biologyand to develop novel diagnosticsand experimental therapeuticsfor cancer. Our hypothesis isthat RNase L functions as atumor suppressor for prostatecancer through the induction ofapoptosis.By using DNAmicroarray technology, we haverecently identified many novelinterferon-regulated genes (inRobert H. Silverman, Ph.D. collaboration with Dr. BryanWilliams, Cleveland Clinic). Oneof these genes encodesphospholipid scramblase, a calcium-dependentprotein linked to the transbilayer movement ofphospholipids. The role of scramblase induction inthe antiviral and anticancer activity of IFNs is acurrent topic of investigation. We have found thatphospholipid scramblase inhibits viral replication andfunctions as a suppressor of human ovariancarcinoma in a mouse model. Therefore, an emergingtheme is one of host defense genes that have dualroles in the suppression of tumor and viral growth.THE SILVERMANLABORATORYPROJECT STAFFBeihua Dong, M.D.RESEARCH ASSOCIATESMalathi Krishnamurthy, Ph.D.Ying Xiang, Ph.D.Zhan Yin, Ph.D.POSTDOCTORAL FELLOWJunko Murakami, M.D., Ph.D.GRADUATE STUDENTSGeqiang Li, B.Sc.Chandar Thakur, B.Sc.LEAD TECHNOLOGISTSJayashree Paranjape, M.S.SENIOR TECHNOLOGISTGreggory Wroblewski, B.Sc.COLLABORATORSJoseph Carpten, Ph.D. 1Graham Casey, Ph.D. 2W. Isaacs, Ph.D. 3Eric Klein, M.D. 4Peter J. Sims, M.D., Ph.D. 5Paul F. Torrence, Ph.D. 6Jeffrey Trent, Ph.D. 1Bryan R.G. Williams, Ph.D. 2Aimin Zhou, Ph.D. 71National Human Genome Res.Inst., Baltimore, MD2Dept. of Cancer Biology, CCF3Brady Urological Inst., JohnsHopkins Medical Inst., Baltimore,MD4Glickman Urological Institute,CCF5Scripps Research Inst., La Jolla,CA6Northern Arizona Univ., Flagstaff,AZ7Cleveland State Univ. Cleveland,OHSilverman, R.H., Halloum, A., Zhou, A., Dong, B., Al-Zoghaibi, F., Kushner, D., Zhou,Q., Zhao, J., Wiedmer, T., and P.J. Sims (2002) Suppression of ovarian carcinoma cellgrowth in vivo by the interferon-inducible plasma membrane protein, phospholipidscramblase 1. Cancer Res. 62:397-402.Carpten, J., et al. (2002) Germline mutations in the ribonuclease L gene in familiesshowing linkage with HPC1. Nat. Genet. 30:181-184.Casey, G., Neville, P.J., Plummer, S.J., Xiang, Y., Krumroy, L.M., Klein, E.A.,Catalona, W.J., Nupponen, N., Carpten, J.D., Trent, J.M., Silverman, R.H., and J.S.Witte (2002) RNASEL Arg462Gln variant is implicated in up to 13% of prostate cancercases. Nat. Genet. 32:581-583Xiang, Y., Condit, R.C., Vijaysri, S., Jacobs, B., Williams, B.R., and Silverman, R.H.Blockade of interferon induction and action by the E3L double-stranded RNA bindingproteins of vaccinia virus. J. Virol. 76:5251-5259.Silverman, R.H. (2003) Implication for RNase L in prostate cancer biology. Biochemistry42:1805-1812.59

THE SIZEMORELABORATORYRESEARCH FELLOWSAnju Agarwal, Ph.D.Kingshuk Das, M.D.RESEARCH ASSOCIATEKwok Peng Ng., Ph.D.SENIOR RESEARCH TECHNOLOGISTNatalia Lerner, B.S.COLLABORATORSGrahan Casey, Ph.D. 1George R. Stark, Ph.D. 2Bryan R.G. Williams, Ph.D. 11Dept. of Cancer Biology, CCF2Dept. of Molec. Biology, CCFThe Department of Cancer BiologyRole and Signaling Mechanism of thePI3K/AKT/ IκB Kinase Pathway in CancerThe overall focus of our laboratory is theinvestigation of the role of aberrant signaltransduction by AKT and IκB kinase incontrolling gene expression, oncogenesis, andapoptosis in colorectal and other cancers.Our group was the first to discover a roleof the phosphatidylinositol 3’ kinase (PI3K)/AKT cell survival pathway in the positiveregulation and the tumor suppressor PTEN in thenegative regulation of the activity of the antiapoptotictranscription factor NFκB. NFκBactivates gene products that protect cells fromapoptosis and inhibit cell death. We alsoestablished that IκB kinase (IKK) mediates thephosphorylation of NFκB in response toactivated AKT. We haverecently discovered thatIKK also regulates theactivation of anotherimportant transcriptionfactor, β-catenin. BothNFκB and β-catenin areimportant in cancerdevelopment by promotingcellular transformation,proliferation, and resistanceto apoptosis. The focus ofour current studies is onthe role and signalingmechanisms of AκT andPTEN in controlling theactivity of IkK towardsthese transcription factors,as well as the roles these transcription factors playin controlling tumorigenesis and apoptosis.Additional ongoing studies are focusing on therole of autocrine growth factor/cytokinesignaling and the identification of novel IKKsubstrates involved in the evolution of cancer.Currently, the laboratory’s efforts arespecifically focused on these goals:• To define the role and molecularmechanism of AKT in regulation of the IKKactivity towards the nuclear factor kappa B(NFκB) and β-catenin transcription factors.• To define the functional and structuralrequirements of IKK to modulate the NFκB andβ-catenin transcriptionfactors.•To identify changesin the phosphorylation andtranscriptional activity ofNFκB and β-catenininduced by IKK.•To identify NFκBandβ-catenin-dependentgenes involved in colorectalcancer development andprogression.•To identify novelmolecular targets of IKKaction.Nywana Sizemore, Ph.D.Sizemore, N., Gangarosa, L.M., Graves-Deal, R., Oldham, S.M., Der, C.J., and R.J. Coffey (1997) ARaf-independent EGF receptor autocrine loop is necessary for Ras-transformation of rat intestinalepithelial cells. J. Biol. Chem. 272:18926-18931.Sizemore, N., Cox, A.D., Barnard, J.A., Oldham, S.M., Reynolds, E.R., Der, C.J., and R.J. Coffey(1999) Pharmacological inhibition of Ras-transformed epithelial cell growth is linked to down-regulation ofepidermal growth factor-related peptides. Gastroenterology 117:567-576.Sizemore, N., Leung, S., and G.R. Stark (1999) Activation of phosphatidylinositol 3-kinase in response tointerleukin-1 leads to phosphorylation and activation of the NFκB p65/RelA subunit. Mol. Cell Biol.19:4798-4805.Rani, M.R., Hibbert, L., Sizemore, N., Stark, G.R., and R.M. Ransohoff (2002) Requirement ofphosphoinositide 3-kinase and Akt for interferon-beta-mediated induction of the beta-R1 (SCYB11) gene.J. Biol. Chem. 277:38456-38461.Sizemore, N., Lerner, N., Dombrowski, N., Sakurai, H., and G.R. Stark (2002) Distinct roles of the IκBkinase α and β subunits in liberating nuclear factor κB (NFκB) from IκB and in phosphorylating the p65subunit of NFκB. J. Biol. Chem. 277:3863-3869.60

Traditional approaches to the treatment ofbrain tumors focus on the hypothesis thattumors arise and grow as a consequence ofdisordered regulation of proliferation. It hasbecome apparent that tumor growth depends notonly on the rate of cellular proliferation, but alsoon the rate of cell death. Apoptosis is a“physiological” form of cell death, which isactively controlled by a number of signaltransduction pathways that act to regulate theactions of a unique set of genes and geneproducts. In a number of organ systems,dysregulation of apoptosis contributes directly totumor development,progression, and resistance tochemotherapy. Recentstudies have helped todevelop an increasedunderstanding of specificproteins responsible for theregulation of apoptosis. Thisknowledge may allowdevelopment of clinicaltherapies directed at alteringlevels of expression ofspecific pro-apoptoticproteins, which maypotentiate effects ofchemotherapeutic agents.The most importantgenes/gene productsidentified in the control ofDNA damage-inducedapoptosis include p53,members of the bcl-2 genefamily, the caspases, andcytochrome c, amongstothers. Previous work hasThe Department of Cancer BiologyMolecular Genetic Approaches UnravelRegulation of Apoptotic Pathwaysin Malignant Brain TumorsMichael A. Vogelbaum, M.D., Ph.D.Center for Molecular Geneticsdemonstrated that more than 50% of malignantgliomas have mutations of p53, thereby increasingthe resistance of these tumors to DNA damageinducedapoptosis. Restoration of wild-type p53to gliomas having a mutant, but not a wild-type,p53 genotype results in spontaneous apoptosis. Incontrast, overexpression of bax, a pro-apoptoticmember of the bcl-2 gene family, in gliomas havinga wild-type, but not mutant, p53 genotype alsoresults in spontaneous apoptosis. Finally, gliomaswith a wild-type p53 genotype that are subjectedto a DNA-damaging stimulus fail to transactivatebax (a known target of P53) and fail to undergoapoptosis.We have generatedthe following hypothesis toaccount for this set ofobservations: DNAdamage-induced apoptosisin glioma cell lines requiresactivation of wild-typeP53, which results in bothbax transactivation andactivation of a separatesignal transductionpathway (which we havetermed the “death signal”transduction pathway) thatis distinct from itstransactivation of bax.The major focus of mylaboratory is to establishthe validity of thishypothesis in malignantglioma cell lines and inprimary cultures of tumorstaken from the operatingroom.THE VOGELBAUMLABORATORYPOSTDOCTORAL FELLOWUtpal Datta, Ph.D.TECHNICAL ASSISTANTSDmitry LeontievChunbiao LiCOLLABORATORSGene Barnett, M.D. 1S. Jaharul Haque, Ph.D. 2Bryan R.G. Williams, Ph.D. 21Brain Tumor Institute, CCF2Dept. of Cancer Biology, CCFVogelbaum, M.A., Tong, J.X., Higashikubo, R., Gutmann, D.H., and K.M. Rich (1998) Transfection of C6 glioma cells with the bax gene andincreased sensitivity to treatment with cytosine arabinoside. J. Neurosurg. 88:99-105.Vogelbaum, M.A., Tong, J.X., Perugu, R., Gutmann, D.H, and K.M. Rich (1999) Overexpression of Bax in human glioma cell lines. J.Neurosurg. 91:483-9.Rahaman, S.O., Sharma, P., Harbor, P.C., Aman, M.J., Vogelbaum, M.A., and S.J. Haque (2001) IL-13Rα2, a decoy receptor for IL-13, actsas an inhibitor of IL-4 dependent signal transduction in glioblastoma cells. Cancer Res. 62:1103-1109.Prayson, R.A., Castilla, E.A., Vogelbaum, M.A., and G.H. Barnett (2002) Cyclooxygenase-2 (COX-2) expression by immunohistochemistry inglioblastoma multiforme. Ann. Diagn. Pathol. 6:148-153.Rahaman, S.O., Harbor, P.C., Chernova, O., Barnett, G.H., Vogelbaum, M.A., and S.J. Haque (2002) Inhibition of contitutively active Stat3suppresses proliferation and induces apoptosis in glioblastoma multiforme cells. Oncogene 21:8404-8413.61

THE WILLIAMSLABORATORYPROJECT SCIENTISTSMarina Antoch, Ph.D.Pratima Karnik, Ph.D.Xavier Lee, Ph.D.RESEARCH ASSOCIATESRoger Slee, Ph.D.Patricia Stanhope-Baker, Ph.D.Maryam Zamanian-Daryoush, Ph.D.POSTDOCTORAL FELLOWSJoao Marques, Ph.D.Ikenna Okereke, M.D.Anthony Sadler, Ph.D.Aristobolo Silva, Ph.D.Mark Whitmore, Ph.D.LEAD TECHNOLOGISTPatricia Kessler, M.S.TECHNOLOGISTSJeanna M. Guenther, B.S.Die Wang, B.S.GRADUATE STUDENTSMichelle Holko, B.S.Wenliang Li, B.S.Carol Ann Sledz, B.S.COLLABORATORSRobert Silverman, Ph.D. 1Jun Qin, Ph.D. 21Dept. of Cancer Biology, CCF2Center for Structural Biology,CCFThe Department of Cancer BiologySignaling Innate Immunity andTumor SuppressionThe major theme of my laboratory isinvestigation of the molecular mechanismscontrolling cellular responses to extracellularstimuli. In particular, our work focuses on the rolethat potential tumor suppressor genes may play inregulating cell growth and differentiation andapoptosis. We use genetic approaches to understandingthe mechanisms of action of interferons(IFNs), the potent cell-growth-regulating cytokines,and characterizing molecular genetic events involvedin Wilms tumorigenesis.Molecular Mechanisms of Interferon ActionMammalian cells use complex, overlappingsignal transduction pathways to sense environmentalchanges. When cells are subjected to viral challenge,double-stranded RNA (dsRNA), produced as partof the viral replicative cycle, stimulates cellulardefense mechanisms, resulting in the production ofinterferon and the development of an antiviralstate. We are investigating the hypothesis thatspecific signaling pathways are activated in thiscellular response. Signaling by IFNs and othercytokines activates a cascade of kinase activities andprotein-protein interactions. We have identified thestress-activated kinase p38 mitogen-activatedprotein (MAP) kinase as a key player in the IFNresponse. PKR, an IFN-induced protein kinase thatis autophosphorylated when activated by dsRNA, isan essential component of the cellular responses toextracellular stimuli. Using genetic and biochemicalapproaches, we have characterized the dsRNAbindingdomains (dsRBD) of PKR and identifiedsingle amino acid residues that are essential fordsRNA binding. In collaboration with Dr. Jun Qin(Center for Structural Biology), we solved thestructure of the dsRBD and provided a model forthe activation of this kinase by dsRNA. Furtherstructural studies, using X-ray crystallography, areunder way.PKR is able to act as a signal transducer notonly for dsRNA but also for growth regulatorycytokines. Formation of the transcription factorNFκB is induced by dsRNA, IFN or tumor necrosisfactor via a PKR-dependent pathway. In cellsderived from mice in which the PKR gene has beenhomozygously deleted, the response of NFκBdependentgenes is deficient. PKR, a stressresponsivekinase, is required to activate p38 MAPkinase by a number of cellular stress stimuli,including those mediated by pattern recognition Tollreceptors. In PKR-deleted cells, there is a deficiencyin signaling via transcription factor Stat3 in responseto platelet-derived growth factor. PKR is alsosubject to functional regulation during the cell cycleand regulates cellular responses to apoptotic stimuli.In collaboration with Dr. R.H. Silverman,we have deleted the known antiviral genes (PKRand RNASEL) from the germ line of mice anddiscovered that IFN is still able to offer someprotection against viral infection. To elucidate thepathways involved in this response, we have usedgene chip technology and identified novel IFNregulatedgenes. These data, including functionalcategories, can be accessed at: http://www.lerner.ccf.org/labs/williams/. The roleof these genes in IFN-responses is presently beinginvestigated.Tumor Suppressor GenesTumor suppressor genes are implicated in theinitiation/progression of a number of cancers.Wilms tumor (WT), a pediatric nephroblastoma, isassociated with the deletion or mutation of theWT1 gene residing at band p13 on chromosome 11.The expression pattern of WT, supported bymutational analyses in human disease and mousemodels, suggests a role in development of thegenitourinary system, in tumors originating fromthese tissues, and in the etiology of some leukemiasand possibly breast cancer. We are investigating theupstream regulators and downstream targets ofWT1 using custom cDNA arrays produced in ourlaboratory to better understand its role in developmentand cancer.Leitner, W.W., Hwang, L.N., DeVeer, M.J., Zhou, A., Silverman, R.H., Williams, B.R., Dubensky, T.W.,Ying, H., and N.P. Restifo (2003) Alphavirus-based DNA vaccine breaks immunological tolerance byactivating innate antiviral pathways. Nat. Med. 9:33-39.Frevel, M.A., Bakheet, T., Silva, A.M., Hissong, J.G., Khabar, K.S., and B.R. Williams (2003) p38Mitogen-activated protein kinase-dependent and -independent signaling of mRNA stability of AU-richelement-containing transcripts. Mol. Cell. Biol. 23:425-436.Tebo, J., Der, S., Frevel, M., Khabar, K.S., Williams, B.R., and T.A. Hamilton (2003) Heterogeneity inControl of mRNA stability by AU rich elements. J. Biol. Chem. 2003 Jan 28 [epub ahead of print].Bryan R.G. Williams, Ph.D.,Hon. FRSNZKhabar, K.S., Siddiqui, Y.M., Al-Zoghaibi, F., Al-Haj, L., Dhalla, M., Zhou, A., Dong, B., Whitmore, M.,Paranjape, J., Al-Ahdal, M., Al-Mohanna, F., Williams, B.R., and R.H. Silverman (2003) RNase Lmediates transient control of interferon response through modulation of the double-stranded RNAdependent protein kinase PKR. J. Biol. Chem. 2003 Feb 11 [epub ahead of print].Espert, L., Degols, G., Gongora, C., Blondel, D., Williams, B.R., Silverman, R.H., and N. Mechti (2003)ISG20, a new interferon-induced RNase specific for single-stranded RNA, defines an alternative antiviralpathway against RNA genomic viruses. J. Biol. Chem. 2003 Feb 19278:16151-16158.62

The Department of Cancer BiologyRole, Signaling Mechanisms of BioactiveLysophospholipids/Receptors in CancerTHE XU LABORATORYVISITING PROFESSORKwan-sik Kim, M.D., Ph.D.My main research interests center onunderstanding, at the molecular level,the mechanism of development ofovarian, breast, and prostate cancers. Theseinclude revealing the molecular signalingmechanisms of cancer development andidentifying effective diagnostic and prognosticbiomarkers and novel therapeutic targets forthese diseases.We have pioneered research in the role ofsignaling lipid molecules in ovarian cancer. Weidentified the first lipid growth factor in ovariancancer cells and demonstrated that lysophosphatidicacid (LPA) is a potential marker for theearly detection of ovarian cancer. We havedeveloped a highly effective method to analyzelysophospholipids in body fluids and have shownthat certain bioactive lysophospholipids areelevated in blood, ascites, and peritonealwashings from patients with ovarian cancer.These findings suggest that these lipid moleculesare likely to be pathologically relevant to ovariancancer.We have investigated the role and signalingmechanisms of sphingosine-1-phosphate (S1P),sphingosylphosphorylcholine (SPC), LPA, andother lysophospholipids in ovarian cancerdevelopment. In addition, we have successfullyemployed the Clontech PCR-select cDNAsubtraction kit, Affymetrix GeneChip and cDNAarrays in identification of genes up- ordownregulated by LPA, S1P, and SPC.Lysophospholipids and other smallbioactive molecules function through G-proteincoupledreceptors (GPCRs). There is increasingevidence that abnormalities in the structure andfunction of GPCRs are responsible for manydiseases, including cancers. Molecular cloningand characterization of novel GPCRs willcontribute to a better understanding of thenormal functioning and signaling mechanisms ofthese receptors, as well as the role that thesereceptors play in cancer. GPCRs hold enormouspromise for therapeutic drug discovery, sinceabout 50% of all existing pharmaceuticals aretargeted towards these receptors. To identifyGPCRs in ovarian cancer cells, we have cloned anovel GPCR gene (ovarian cancer G-proteincoupledreceptor 1, or OGR1) from HEY ovariancancer cells by degenerate oligonucleotide PCRamplification (Nature Cell Biol. 2:261, 2000). Wehave shown that SPC is a high-affinity ligand forOGR1 through calcium mobilization, ligandbinding, receptor internalization, MAP kinase,and cell proliferation assays. LPC is known toplay an important role in human systemicautoimmune disease and atherosclerosis. G2A isan orphan GPCR, expressed predominantly inlymphocytes. Genetic ablation of G2A functionin mice results in the development of autoimmunityassociated with hyperproliferative responsesof T lymphocytes to antigen receptor stimulation.We have shown recently that G2A is a highaffinityreceptor for LPC (Science 293:618, 2001;a commentary describes the importance of ourfinding). More recently, we have identified anOGR1-related GPCR, GPR4, as not only anotherhigh-affinity receptor for SPC, but also a receptorfor LPC (J. Biol. Chem. 276:41325, 2001).Calcium mobilization, ligand binding, receptorinternalization, MAP kinase activation, cellproliferation, and cell migration assays wereconducted to establish the ligand-receptorrelationship.We will continue to: 1) investigate the roleand signaling mechanisms of LPA, LPC, and SPCand their receptors in cancer and other diseases; 2)determine the structure-function relationship ofthe OGR1-subfamily receptors and establishmolecular models of these receptors for drugdevelopment; and 3) determine the physiologicaland pathological roles and functions of OGR1-subfamily receptors using knockout-mousemodels.INVESTIGATORSYing Jiang, Ph.D.Juan Ren, M.D., Ph.D.Saubhik Sengupta, Ph.D.Lisam Shanjukumar Singh, Ph.D.Zeneng Wang, Ph.D.RESEARCH ASSOCIATESYijian Xiao, Ph.D.Xiaoxian Zhao, Ph.D.LEAD TECHNOLOGISTMichael Berk, M.S.RESEARCH TECHNOLOGISTRussel TippsGRADUATE STUDENTSJanice Battistuta, B.S.Alexander Zaslavsky, B.S.Yan Xu, Ph.D.Kabarowski, J.H., Zhu, K., Le, L.Q., Witte, O.N., and Y. Xu (2001) Lysophosphatidyl-choline as a ligand for the immunoregulatory receptor G2A. Science 293:702-705.Zhu, K., Baudhuin, L.M., Hong, G., Williams, F.S., Cristina, K.L., Kabarowski, J.H., Witte, O.N., and Y. Xu (2001) Sphingosylphosphorylcholine and lysophosphatidylcholineare ligands for the G protein-coupled receptor GPR4. J. Biol. Chem. 276:41325-41335.Baudhuin, L.M., Cristina, K.L., Lu, J., and Y. Xu (2002) Akt activation induced by lysophosphatidic acid and sphingosine-1-phosphate requires both mitogen-activatedprotein kinase kinase and p38 mitogen-activated protein kinase and is cell-line specific. Mol. Pharmacol. 62:660-671.Xu, Y., Xiao, Y.J., Zhu, K., Baudhuin, L.M., Lu, J., Hong, G., Kim, K.S., Cristina, K.L., Song, L.S, Williams, F., Elson, P., Markman, M., and J. Belinson (2003)Unfolding the pathophysiological role of bioactive lysophospholipids. Curr. Drug Targets Immune Endocr. Metabol. Disord. 3:23-32.Xu, Y. (2002) Sphingosylphosphorylcholine and lysophosphatidylcholine: G protein coupled receptors and receptor-mediated signal transduction. Biochim. Biophys.Acta 1582:81-88.Lauber, K., Bohn, E., Kröber, S.M., Xiao, Y., Blumenthal, S.G., Lindemann, R.K., Marini, P., Baksh, S., Schulze-Osthoff, K., Xu, Y., et al. (2003) Apoptotic cellsinduce migration of phagocytes via caspase-3 mediated release of a lipid attraction signal. Cell 113:717-730.Sengupta, S., Xiao, Y., and Y. Xu (2003) A novel laminin-induced LPA autocrine loop in the migration of ovarian cancer cells. FASEB J. June 3, 10.1096/fj.02-0963.63

THE YI LABORATORYPOSTDOCTORAL FELLOWSKekke Fan, Ph.D.Jing Li, M.D.TECHNICIANSMingli CaoKundu SumanThe Department of Cancer BiologyPathogenic Role of PTPases in Cancer andAnti-Cancer Activities of PTPase InhibitorsProtein tyrosine phosphatases (PTPases) arekey switches in many important eukaryoticcellular signaling pathways and are tumorsuppressers or anti-oncogenes. Consistent with itsnegative role in signaling, PTPase SHP-1deficiency has been found by us and others incertain human hematopoietic malignancies(polycythemia vera, lymphoma and acuteleukemia) and could be a contributing pathogenicfactor. Certain PTPases may also function asoncogenes. Increased expression of PTPase-alphaand PRLs was associated with late-stage humancolon cancer and caused cell transformation invitro. Amplification of HePTP was detected insome cases of human acute myeloid leukemia.We hypothesize that SHP-1 interacts withand dephosphorylates distinct substrates thatmediate signals of activation,proliferation and differentiation inhematopoietic cells. We have detectedseveral novel SHP-1 substratesinvolved in the growth control ofhematopoietic cells by affinitypurification and mutational analysis.Further characterization of thesesubstrates and determination of theirrole in mitogenic signaling are pivotalin elucidating the mechanisms thatcontrol normal proliferation ofhematopoietic cells. Such efforts will alsoprovide novel insights about the pathogenicprocess of SHP-1 deficiencies in causinghematopoietic diseases.As PTPases play a pivotal role in intracellularsignaling, their inhibitors are expected to havesignificant clinical activities and are targets ofextensive research efforts. Our recent studiesdemonstrated, for the first time, that sodiumstibogluconate is a potent PTPase inhibitor andhas marked anti-cancer activity as a single agentor, more strikingly, in combination with IFNalpha,which is currently used in anti-cancertherapies. Our ongoing studies will furtherexploit its potential as a novel anti-cancer drugand characterize the biologic activities of otherPTPase inhibitors.Taolin Yi, Ph.D.Kant, A.M., De, P., Peng, X., Yi, T., Rawlings, D.J., Kim, J.S., and D.L. Durden (2002) SHP-1 regulatesFcgamma receptor-mediated phagocytosis and the activation of RAC. Blood 100:1852-1859.Yang, W., Tabrizi, M., and T. Yi (2002) A bipartite NLS at the SHP-1 C-terminus mediates cytokineinducedSHP-1 nuclear localization in cell growth control. Blood Cells, Molecules, and Diseases 28:63-74.Joliat, M.J., Lang, P.A., Lyons, B.L., Burzenski, L., Lynes, M.A., Yi, T., Sundberg, J.P., and L.D. Shultz(2002) Absence of CD5 dramatically reduces progression of pulmonary inflammatory lesions in SHP-1protein-tyrosine phosphatase-deficient ‘viable motheaten’ mice. J. Autoimmun. 18:105-117.Pathak, M.K., Hu, X., and T. Yi (2002) Effects of sodium stibogluconate on differentiation and proliferationof human myeloid leukemia cell lines in vitro. Leukemia 16:2285-2291.Yi, T., Pathak, M.K., Lindner, D.J., Ketterer, M.E., Farver, C., and E.C. Borden (2002) Anticanceractivity of sodium stibogluconate in synergy with IFNs. J. Immunol. 169:5978-5985.Pathak, M.K., Dhawan, D., Lindner, D.J., Borden, E.C., Farver, C., and T. Yi (2002) Pentamidine is aninhibitor of PRL phosphatases with anticancer activity. Mol. Cancer Ther. 1:1255-1264.64

CellBiology

DEPARTMENT OFCELL BIOLOGYINTERIM CHAIRMANGuy M. Chisolm, III, Ph.D.STAFFMartha K. Cathcart, Ph.D.Paul E. DiCorleto, Ph.D.Donna M. Driscoll, Ph.D.Paul L. Fox, Ph.D.Stanley L. Hazen, M.D., Ph.D.Philip H. Howe, Ph.D.Donald W. Jacobsen, Ph.D.Jonathan Smith, Ph.D.ASSOCIATE STAFFJosephine C. Adams, Ph.D.Michael T. Kinter, Ph.D.Richard E. Morton, Ph.D.Alan Wolfman, Ph.D.ASSISTANT STAFFMarc S. Penn, M.D., Ph.D.Thomas Weimbs, Ph.D.The Department of Cell Biology is home toa group of scientists investigating cellularevents and underlying mechanisms relatedto normal function and a broad spectrum ofdiseases. Major areas of interest include vascularcell and molecular biology, inflammation, renalcell biology and cancer. Despite this diversity intopic areas, there are extensive interactions andcollaborations among the department’s 15laboratories, as evidenced by numerous coauthoredpublications and grant applications.Collaborations are also common outside thedepartment with other researchers and physicianscientists at both CCF and other institutions. Anenvironment of cooperation and collegiality isone of the hallmarks and greatest strengths ofthe department. Overall, the department issupported by ~$7 million dollars of totalexternal grant revenue annually, the majorityfrom the NIH.The Department of Cell BiologyMultidisciplinary Scientists Ask How CellDysfunction Contributes to Disease ProcessesBiology of Renal and Cancer CellsResearch in the department probes whycancer cells lose their ability to regulate growthand why epithelial cell polarity is lost as aconsequence of cell transformation. Topicsinclude:• Transforming growth factor-beta signal transductionpathways leading to growth inhibitionand apoptosis• Interactions between multiple signaling proteinsassociated with the various members ofthe p21 ras oncoprotein family• The role of Ras isoforms in regulating theapoptotic set point• Role of the N-ethylmaleimide-sensitive factor-attachmentprotein receptor (SNARE)membrane fusion machinery in cytokinesisand exocytosis in epithelial cells• Cell biology and signaling mechanisms involvedin polycystic kidney diseasePROJECT SCIENTISTSBarbara A. Hocevar, Ph.D.Seng Hui Low, Ph.D.Barsanjit Mazumder, Ph.D.Jayasri Nanduri, Ph.D.Marie Odile Parat-Salvado, Ph.D.Evgeny A. Podrez, M.D., Ph.D.Gary M. Wildey, Ph.D.Bo Xu, Ph.D.RESEARCH ASSOCIATESSmarajit Bandyopadhyay, Ph.D.Marie-Luise Brennan, Ph.D.Sara G. Carlson, Ph.D.Kevin Carnevale, M.D.Unni Chandrasekharan, Ph.D.Michael Greenberg, Ph.D.Thomas E. Patterson, Ph.D.Janice C. Wolfman, Ph.D.Renliang Zhang, Ph.D.JOINT APPOINTMENTSBela Anand-Apte, M.B.B.S., Ph.D. 1Jay Ciezki, M.D. 2John W. Crabb, Ph.D. 1Joe Hollyfield, Ph.D. 1Byron J. Hoogwerf, M.D. 3Damir Janigro, Ph.D. 4Alan Marmorstein, Ph.D. 1Jonathan Sears, Ph.D. 1Suyu Shu, Ph.D. 5Maria Siemionow, M.D., Ph.D. 6Mary Jane Thomassen, Ph.D. 8Eric J. Topol, M.D. 71Ophthalmology Research, CFF2Radiation Oncology, CFF3Endocrinology, CFF4Neurological Surgery, CFFContinued on Page 6766Vascular Cell Biology and InflammationIn these programs, we investigate themolecular events that occur in atherosclerosis andother inflammatory diseases. Topics include:• Transcriptional regulation during activationof the endothelium• Thrombin-induced signaling pathways in theendothelium• Regulation of endothelial cell migration• Ceruloplasmin, iron metabolism and vasculardisease• Proteomics• Cell responses to oxidative stress• Mechanisms of oxidant stress in vivo• Synthesis and regulation of selenoproteins• Mechanisms of mRNA editing• Lipoprotein remodeling and factorsregulating cellular cholesterol deposition• Oxysterol-induced apoptosis• Regulation of tissue factor expression• Mechanism of ABCA1-mediated cholesterolefflux from macrophages• Regulation of monocyte chemotaxis• Genetics of atherosclerosis susceptibility inmice• Molecular basis of homocysteine-inducedvascular injury• Vascular biochemistry of vitamin B 12:transport, processing and coenzyme function• Nitric oxide biochemistry• Leukocyte activation, signaling, and role inhost defense and tissue injury• Gene therapy approaches to myocardialdamage• Thrombospondins and other extracellularmatrix proteins in cell adhesion andmigration• Cytoskeleton and signaling molecules inregulation of cell-matrix contactsEducational OpportunitiesThe Department of Cell Biology has anactive training program at both the pre- andpostdoctoral levels. Most members of thedepartment have faculty appointments at CaseContinued on Page 67Guy M. Chisolm, III, Ph.D.Interim Chairman

The Department of Cell BiologyThe joint LRI Department of Cell Biology and Case Western Reserve University Cell Biology graduateprogram brought fine minds together to exchange understanding about cells and their molecules at SawmillCreek Resort during April 2002.Continued from Page 66Western Reserve University, Cleveland StateUniversity, Kent State University or Ohio StateUniversity. Our faculty members have close tieswith CWRU’s Cell Biology Program faculty andare heavily involved in developing the newCleveland Clinic Lerner College of Medicine ofCWRU. Currently, about 15 doctoral students areperforming their thesis research in the department.In addition, many undergraduate studentsfrom John Carroll University and other neighboringinstitutions perform research projects in ourlaboratories. Postdoctoral fellows represent anintegral part of the research effort in everylaboratory in the department. Currently, over 40postdoctoral researchers are employed in thedepartment. Our fellows and students aresupported through multiple external sources,including fellowships from the NIH and theAmerican Heart Association, as well as an NIHNational Research Service Award, “TrainingProgram in Vascular Cell Biology,” which is jointlysponsored by the Department of MolecularCardiology.Our training program includes severalseminar series and journal clubs in which fellowsand graduate students present their work andcomment on that of others. In addition, traineesare encouraged to attend seminars of interest atCCF and at neighboring institutions, and all areinvited to attend annual departmental andinstitute retreats held at area resorts. Fellows andstudents are provided resources to invite twoseminar speakers per year to the department.They also have the opportunity to participate inroundtable discussions over lunch with thedepartment’s many invited speakers followingtheir presentations. We strongly encourage ourstudents and fellows to attend national meetingsand present their research.Dept. website: http://www.lerner.ccf.org/cellbio/Continued from Page 665Ctr. for Surgery Research, CFF6Prim w/Plastic and ReconstructiveSurgery, CFF7Cardiovascular Medicine, CFF8Pulmonary and Critical Care, CFFADJUNCT APPOINTMENTSStan A. Duraj, Ph.D. 9Joseph D. Fontes, Ph.D. 9Sandra Harris-Hooker, Ph.D. 10Diana Kunze, Ph.D. 11Lily Ng, Ph.D. 9Gerald M. Saidel, Ph.D. 12William P. Schilling, Ph.D. 11Crystal M. Weyman, Ph.D. 39Cleveland State University10Morehouse School of Medicine11MetroHealth Medical Center12Case Western Reserve University67

The Department of Cell BiologyRole of Cell Protrusionsin Cell Migration MechanismsTHE ADAMSLABORATORYPOSTDOCTORAL FELLOWRitu Chakravarti, Ph.D.RESEARCH TECHNICIANRaymond MonkGRADUATE STUDENTSoren PragSTUDENTJamie CannonThe research interest of my lab is in theresponses of cells to extracellular matrix.ECM is fundamental to cell interactions intissue organization and, by the formation ofadhesive contacts with cells, regulates cellfunction through effects on the cytoskeleton, oncell signaling processes and on gene expression.Matrix adhesion is thus crucial to normal cellinteractions. Changes in the expression orfunction of adhesion molecules have causal rolesin numerous genetic and acquired human diseases.We have focused on the responses to cellsto thrombospondin-1 (TSP-1), an ECM componentthat is highly associated with tissueremodeling and is also an inhibitor of angiogenesis.Cell adhesion to TSP1 induces cells to formprotrusive matrix-contacts based on fascin spikes.We have established molecular mechanisms thatare needed for assembly of fascin protrusions. Weare now investigating the role of fascin protrusionsin cell migration and invasion and thesignaling mechanisms by which fascin protrusionsare integrated with contractile focal adhesions.We are also defining the role of a novel intracellularprotein, muskelin, that affects cell spreadingon TSP1. Muskelin is a member of the kelchrepeat superfamily of proteins, many of whichact as components of large protein complexes.Understanding these cellular processes shouldidentify new candidate targets for translationalapplications.COLLABORATORSMartha K. Cathcart, Ph.D. 1Melissa L. Knothe Tate, Ph.D. 2Tony Ng, Ph.D. 3Marc S. Penn, M.D., Ph.D. 1Martin A. Schwartz, Ph.D. 41Dept .of Cell Biology, CCF2Dept. of BiomedicalEngineering, CCF3Cancer-Research UK, London4Univ. of Virginia,CharlottesvilleJosephine Adams, Ph.D.Adams, J.C., Kureishy, N., and A.L. Taylor (2001) A role for syndecan-1 in coupling fascin spike formationby thrombospondin-1. J. Cell Biol. 152:1169-1182.Anilkumar, N., Annis, D., Mosher, D.F., and J.C. Adams (2002) The trimeric assembly of thrombospondin-1or thrombospondi-2 is necessary for cell spreading and fascin spike organisation. J. Cell Sci. 115:2357-2366.Kureishy, N., Sapountzi, V., Prag, S., Anilkumar, N., and J.C. Adams (2002) Fascins, and their roles in cellstructure and function. Bioessays 24:350-361.Adams, J.C. (2002) Characterisation of a Drosophila melanogaster orthologue of muskelin. GENE 297:69-78.Jawhari, A.U., Buda, A., Jenkins, M., Shehzad, K., Sarraf, M., Noda, M., Farthing, M.J.G., Pignatelli, M. andAdams, J.C. (2003) Fascin, an actin-bundling protein, modulates colonic epithelial cell invasiveness anddifferentiation in vitro. Am. J. Path. 162:69-80.Adams, J.C., Monk, R., Taylor, A., Ozbek, S., Fascetti, N., Baumgartner, S. and Engel, J. (2003)Characterisation of Drosophila thrombospondin defines an early origin of pentameric thrombospondins. J.Mol. Biol. 325: 479-494.68

Novel Pathways RegulatingMonocyte Inflammatory ActivitiesThe focus of the research conducted in ourlaboratory is to define the mechanismsresponsible for regulating the oxidation oflipids during the activation of human monocytesand to study the role of lipids in regulatingmonocyte chemotaxis into sites of inflammation.These events likely contribute to inordinate lipidaccumulation in atherosclerosis and may mediatetissue injury in pathologic settings.Our work has defined several essentialsteps in the potentially pathologic process ofmonocyte-mediated lipid oxidation. Amongthese, we have demonstrated a critical role for thehighly reactive oxygen radical superoxide anion.Additionally, we have identified an apparentrequirement for an enzyme in the lipoxygenasefamily, enzymes that catalyze highly specificoxidation of lipids.In the course of these studies, we haveidentified a requisite role for both calcium influxand calcium release from intracellular stores; yetcalcium is not the sole required stimulus forinducing the oxidizing events. We are presentlyinvestigating the relationship between the roles ofcalcium and other signal transduction pathways inregulating monocyte-mediated lipid oxidation, inparticular, the involvement of various isoformsof protein kinase C (PKC) and phospholipase A 2(PLA 2).In this regard, we found, through the useof pharmacologic inhibitors and antisenseoligonucleotides, that PKC activity is essential foractivated monocytes to oxidize LDL lipids. Wetherefore designed studies to identify theparticular isoenzymes of PKC that participate inthis process. Isoenzymes of the cPKC family(including PKCα, PKCβI and PKCβII) wereshown to be required, and recently we have foundthat PKCα is the isoenzyme required formonocyte-mediated LDL lipid oxidation.The phospholipase A 2family of enzymesincludes an enzyme called cytosolic PLA 2(cPLA 2),believed to function in signal transduction. Inmonocytes, the activity of this enzyme isregulated by calcium. We have also examined theparticipation of this enzyme in the signaltransduction processes required for monocyteproduction of superoxide anion and oxidation ofLDL lipids. Our studies have implicated thisenzymatic pathway as an essential one and havefurther shown that cPLA 2activity is regulated byPKCα activity, thus linking these two pathways.We are pursuing studies to determine themechanisms for enzymatic regulation of thephosphorylation and translocation of thecomponents of the enzyme complex responsiblefor producing superoxide anion.The Department of Cell BiologyIn related studies, we are monitoringlipoxygenase (LO) activity during monocyteactivation and investigating the possible involvementof each of the above enzymatic pathwayson regulating LO activity and LO expression,particularly 15-LO. We were the first todemonstrate the detection of the products of thisenzyme in human atherosclerotic tissue. We areespecially interested in understanding thecytokine-mediated induction of expression ofthis enzyme. To this end, we have recentlyidentified the receptor components and immediatesignal transducing kinases involved in themonocyte response to IL-13, a cytokine that is auniquely potent inducer of 15-LO expression.Our understanding of the regulation of expressionof this enzyme may prove important forlimiting atherogenesis.Other studies in the lab are focused onunderstanding the role of phospholipases andtheir products in regulating monocyte chemotaxisin response to MCP-1. This chemokine plays acentral role in bringing monocytes into the vesselwall in atherosclerosis, and if this process isblocked, atherosclerosis can be markedlyinhibited. We have found two lipid products ofphospholipases that are required for MCP-1-induced monocyte chemotaxis and are pursuingstudies to understand how they control monocytemovement.In summary, we are studying a variety ofregulatory pathways and defining their contributionsto modulating the activity of monocytes ininflammatory responses. Our findings will suggestnew approaches for inhibiting these processes andlimiting the progression of atherosclerosis andinflammation.THE CATHCARTLABORATORYPROJECT SCIENTISTBo Xu, Ph.D.RESEARCH ASSOCIATEKevin Carnevale, M.D.POSTDOCTORAL FELLOWAshish Bhattacharjee, Ph.D.STUDENTSRyan FiccoTsung-Fu YuTECHNICIANClaudine Horton, M.S.Martha K. Cathcart, Ph.D.Cathcart, M.K., and V.A. Folcik (2000) Lipoxygenases and atherosclerosis: protection versus pathogenesis.Free Radical Biol. Med. 28:1726-1734.Carnevale, K., and M.K. Cathcart (2001) Calcium independent phospholipase A 2is required forhuman monocyte chemotaxis to monocyte chemoattractant protein 1. J. Immunol. 167:3414-3421.Roy, B., Bhattacharjee, A., Xu, B., Ford, D., Maizel, A.L., and M.K. Cathcart. (2002) IL-13 signaltransduction in human monocytes: phosphorylation of receptor components, association with Jaks,and phosphorylation/activation of Stats. J. Leuko. Biol. 72:580-9.Bey E.A., and M.K. Cathcart. (2002) Antisense oligodeoxyribonucleotides: a better way to inhibitmonocyte superoxide anion production? Methods Enzymol. 353:421-34.Zhao X., Bey, E.A., Wientjes, F.B., and M.K. Cathcart. (2002) Cytosolic phospholipase A 2(cPLA 2)regulation of human monocyte NADPH oxidase activity. cPLA 2affects translocation but not phosphorylationof p67(phox) and p47(phox). J. Biol. Chem. 277:25385-92.Xu, B., Bhattacharjee, A., Roy, B., Xu, H.M., Anthony, D., Frank, D.A., Feldman, G.M., and M.K.Cathcart (2003) Interleukin-13 induction of 15-lipoxygenase gene expression requires p38 mitogenactivatedprotein kinase-mediated serine 727 phosphorylation of Stat1 and Stat3. Mol. Cell. Biol.23:3918-3928.Carnevale, K.A., and M.K. Cathcart (2003) Protein kinase Cb is required for human monocytechemotaxis to MCP-1. J. Biol. Chem. 278:25317-25322.69

THE CHISOLMLABORATORYRESEARCH ASSOCIATESara G. Carlson, Ph.D.POSTDOCTORAL FELLOWSudesh Agrawal, Ph.D.TECHNOLOGISTSRichard A. Cole, B.S.Charles A. Kaul, B.S.STUDENTSShu-Ling Liang, M.S.Cleveland State Univ., Cleveland,OHTina ChoudhriJohn Carroll Univ., UniversityHts., OHCOLLABORATORSMunna L. Agarwal, Ph.D. 1Alex Almasan, Ph.D. 2Martha K. Cathcart, Ph.D. 3Paul E. DiCorleto, Ph.D. 3Donna M. Driscoll, Ph.D. 3Stanley L. Hazen, M.D., Ph.D. 3,4Roger M. Macklis, M.D. 5Marc S. Penn, M.D., Ph.D. 3,4George R. Stark, Ph.D. 1Yan Xu, Ph.D. 21Dept. of Molecular Biology, CCF2Dept of Cancer Biology, CCF3Dept. of Cell Biology, CCF4Dept of Cardiovascular Medicine,CCF5Dept of Radiation Oncology,CCFLow-density lipoprotein (LDL) is theprincipal cholesterol-carrying molecularcomplex in normal human plasma, andLDL levels correlate strongly with risk ofatherosclerosis. For twodecades, our laboratory hasbeen evolving and testing atheory that the oxidativemodification of lipoproteinspromotes the development ofatherosclerotic lesions.Alterations in cellfunction induced by oxidizedLDL in vitro mimic events inlesion development observedin vivo. Our research focuses oncellular changes brought aboutby oxidized LDL that aredistinct from those resultingfrom exposure of cells tounaltered LDL. We haveidentified constituents ofoxidized LDL that arebioactive and probedmechanisms by which theseconstituents change particularcell functions.Oxidized Lipoproteins and Cell InjuryWe have shown that oxidized LDL injurescells in culture, that the cytotoxicity is independentof the mode of LDL oxidation, that cellsare significantly more susceptible during the S-phase of the cell cycle, and that the delivery ofthe toxin does not require lipoprotein receptors.Lipoproteins oxidized in vivo, such as thoseisolated from diabetic rats or human lesions, arealso cytotoxic to cells in culture. Infusion ofoxidized LDL injures vascular endothelial cells invivo and impairs their function.The Department of Cell BiologyLipoprotein Oxidation, LipoproteinRegulation of Vascular Cell Function:Roles in Apoptosis, Thrombosis,Inflammation and AtherosclerosisGuy M. Chisolm, III, Ph.D.We have identified multiple cytotoxinsborne by oxidized LDL that accumulate invascular lesions and we have studied cell deathmechanisms. For example, a hydroperoxide ofcholesterol kills cells byperoxidation of cell lipids; itsderivative oxysterols kill byapoptosis. With Dr. DonnaDriscoll, we are studying theregulation of antioxidantselenoenzymes that can reducetoxic lipid peroxides and protectcells from apoptosis. With Drs.Munna Agarwal, Martha Cathcartand George Stark, we arestudying Stat1-dependent cellsignaling pathways of apoptosisinduced by oxysterols. These arebeing explored in cell culture andin vivo in genetically altered mice.Prothrombotic Actions ofOxidized LDLWith Drs. Marc Penn andPaul DiCorleto, we have shownthat LDL and oxidized LDL, aswell as various of their lipids,can induce the gene or enhancethe activity of the clotting cascade initiator,tissue factor, on the surfaces of vascular smoothmuscle cells (SMCs). These effects may beimportant contributors to lesion development,since tissue factor induction could lead toincreased local production of thrombin, andthrombin not only enhances coagulation, but isalso mitogenic for SMCs. We are studying theregulation by specific oxidized LDL constituentsof tissue factor gene expression, proteinproduction and cell surface tissue factor activity.Penn, M.S., Cui, M.Z., Winokur, A.L., Bethea, J., Hamilton, T.A., DiCorleto, P.E., and G.M. Chisolm (2000) Smooth muscle cell surface tissuefactor pathway activation by oxidized low-density lipoprotein requires cellular lipid peroxidation. Blood 96:3056-3063.Chisolm, G.M., and D. Steinberg (2000) The oxidative modification hypothesis of atherogenesis: an overview. Free Radical Biol. Med. 28:1815-1826.Agrawal, S., Agarwal, M.L., Chatterjee-Kishore, M., Stark, G.R., and G.M. Chisolm (2002) Stat1-dependent, p53-independent expression ofp21(waf1) modulates oxysterol-induced apoptosis. Mol. Cell. Biol. 22:1981-1992.Hazen, S.L., and G.M. Chisolm (2002) Oxidized phosphatidylcholines: pattern recognition ligands for multiple pathways of the innate immuneresponse. Proc. Natl. Acad. Sci. USA 99:12515-12517.Chen, Q., Chai, Y.C., Mazumder, S., Jiang, C., Macklis, R.M., Chisolm, G.M., and A. Almasan (2003) The late increase in free radical oxygenspecies during apoptosis is associated with cytochrome c release, caspase activation and mitochondrial dysfunction. Cell Death Differ. 10:323-334.Cui, M.Z., Zhao, G., Winokur, A.L., Laag, E., Bydash, J.R., Penn, M.S., Chisolm, G.M., and X. Xu (2003) Lysophosphatidic acid induction oftissue factor expression in aortic smooth muscle cells. Arterioscler. Thromb. Vasc. Biol. 23:224-230.70

The central objective of the research in thislaboratory is to understand the role of geneexpression by vascular endothelial cells(ECs) in the physiological maintenance of vesselwall homeostasis and in the pathogenesis ofvascular diseases. The working hypothesis is thataberrant expression of embryonic or “pathological”genes in the endothelium of adults leads toatherosclerotic plaque development and otherdiseases of large blood vessels. Two specificresearch areas are under investigation. The firstfocuses on the regulation of expression ofleukocyte adhesion molecules, including E-selectinand VCAM-1, by the endothelium in response tothrombin, TNF-alpha and cytokines. The secondarea is the regulation of expression of the plateletderivedgrowth factor (PDGF) A and B chain genesby ECs. In both pursuits, we are defining thecascade of intracellular signaling events that lead tothe expression of these genes, the transcriptionalfactors and DNA elements responsible for theirinduction, and post-transcriptional events thatcontribute to the expression of active proteins.Endothelial Cell Expression of LeukocyteAdhesion MoleculesMonocyte binding to the endothelium is arequisite first step in the emigration of thisleukocyte from the circulation to tissue duringinflammation and wound healing. The overallobjective of this project is to understand themonocyte/endothelium interaction and to definethe intracellular signaling pathways and agonistinducedgenes that are involved in the regulationof monocyte binding to ECs. We have beenstudying thrombin- and TNF-induced signalingpathways and transcription factors that areresponsible for induction of the leukocyteadhesion molecules. In addition, we are studyingthe relative importance of the two TNF-alphareceptors in the induction of leukocyte adhesionin vitro and in vivo using mice that are null for oneor both of the receptor genes. We have recentlyobserved that TNFR2 appears to perform a criticalrole in TNF-induced firm adhesion to postcapillary venules in a mouse cremaster musclemodel.The Department of Cell BiologyEndothelial Cell Gene Expression Linkedto Vascular DiseaseThrombin-induced PDGF Production by theEndotheliumOur major objective in this project is toidentify and characterize regulatory pathwaysinvolved in the expression of the polypeptidemitogen PDGF by ECs in response to the proteasethrombin. We have recently identified a novel modeof transcription factor activation using this modelsystem. We first identified a thrombin responseelement in the PDGF B chain promoter and a novelthrombin-dependent trans-acting factor that boundto this element to cause increased transcription. Wepurified this factor, and amino acid sequenceinformation identified it as a member of the Y-boxfamily of transcription factors. Y-box-bindingproteins are known to act constitutively to positivelyor negatively regulate the expression of severalcellular and viral genes; however, activation of thisclass of transcription factors in response to cellstimulation had not previously been reported. Wehave shown that the Y-box protein is bound tomRNA in the cytosol under basal conditions, but inresponse to thrombin stimulation, it is cleaved andtranslocated to the nucleus, where it induces PDGFexpression. The cleaved protein has a DNA-bindingsequence distinct from the full-length Y-box protein,which is present in the nucleus constitutively.We have used micro-array gene-screeningtechnique to identify seven novel thrombinresponsivegenes that were verifiable by Northernblot analysis in human umbilical vein ECs. Amongthem CL-100, a.k.a. MAP kinase phosphatase-1, a dualspecificityphosphatase, was found to be inducedstrongly, but transiently by thrombin-- mRNApeaked at 1 h with a t 1/2of 45 min. CL-100induction by thrombin was protease-activatedreceptor-1 (PAR-1)-mediated, protein synthesisindependent and at the transcriptional level. Srckinase and p42/p44 ERK activity are critical for theinduction of CL-100 by thrombin, whereas PKCactivation is not required. CL-100 inhibition by aspecific anti-sense oligonucleotide inhibitedthrombin-induced PDGF-A and -B genes, whileVCAM-1 and E-selectin expression levels were upregulatedfurther. These results suggest an importantregulatory role of CL-100 in thrombin-inducedsignaling and gene expression in EC.THE DICORLETOLABORATORYRESEARCH ASSOCIATESSmarajit Bandyopadhyay, Ph.D.Unni Chandrasekharan, Ph.D.POSTDOCTORAL FELLOWSZahid Ashraf, Ph.D.Lin Yang, M.D.TECHNICIANLori Mavrakis, B.A.STUDENTSCorttrell KinneyPam DaherCOLLABORATORSGuy Chisolm, Ph.D. 1Stephen Ellis, M.D. 2Stanley Hazen, M.D., Ph.D. 1Marc Penn, M.D., Ph.D. 1Maria Siemionow, M.D., Ph.D. 31Dept. of Cell Biology, CCF2Dept. of CardiovascularMedicine, CCF3Dept. of Plastic and ReconstructiveSurgery, CCFPaul E. DiCorleto, Ph.D.Patel, C., Sharangpani, R. Bandyopadhyay, S., and P.E. DiCorleto (1999) Endothelial cells express a novel, TNF-alpha-regulated variant of HOXA9. J.Biol. Chem. 274: 1415-1422.Stenina, O.I., Poptic, E.J., and P.E. DiCorleto (2000) Thrombin activates a Y box-binding protein (DNA-binding protein B) in endothelial cells. J. Clin.Invest. 106:579-587.Mao, C.D., Hoang, P., and P.E. DiCorleto (2001) Lithium inhibits cell cycle progression and induces stabilization of p53 in bovine aortic endothelialcells. J. Biol. Chem. 276:26180-8. [Stenina, O.I., Shaneyfelt, K. , and P.E. DiCorleto (2001) Thrombin induces the release of the Y-box protein dpbB from mRNA: a new mode oftranscription factor activation. Proc. Natl. Acad. Sci. USA 98:7277-82.Byzova, T.V., Goldman, C.K., Jankau, J., Chen, J., Cabrera, G., Achen, M.G., Stacker, S.A., Carnevale, K.A., Siemionow, M., Deitcher, S.R.,and P.E. DiCorleto, (2002) Adenovirus encoding vascular endothelial growth factor-D induces tissue-specific vascular patterns in vivo. Blood99:4434-42.71

THE DRISCOLLLABORATORYPOSTDOCTORAL FELLOWSLaurent Chavatte, Ph.D.Carri Gerber, Ph.D.Mithu Majumder, Ph.D.Lisa Middleton, Ph.D.TECHNICAL ASSOCIATEAnne Relich, B.S.COLLABORATORGuy M. Chisolm, Ph.D.Dept. of Cell Biology, CCF72Donna M. Driscoll, Ph.D.Posttranslational Regulation of KeyProteins Involved in AtherosclerosisOur research focuses on two pathwaysthat expand the genetic diversity of thegenome: mRNA editing andselenoprotein biosynthesis. In both projects, thegenes we are studying encode proteins that playcritical roles in the development of atherosclerosis.One of the most unexpected developmentsin molecular biology was the discovery ofmRNA editing. In base modification editing,single nucleotides in the coding region of atranscript are deaminated to generate alternativemRNAs that encode proteins with differentbiological functions. The editing ofapolipoprotein-B (apo-B) mRNA involves thedeamination of C6666 to U, which converts aglutamine codon (CAA) to an in-frame stopcodon (UAA). The full-length andtruncated apo-B proteins have distinctfunctions in lipoprotein metabolismand atherosclerosis susceptibility. Thegoals of our research are to identifyand characterize the trans-acting factorsthat catalyze the editing of apo-BmRNA and determine whether othermRNAs are edited by this samemechanism. We defined a minimalholoenzyme that edits apo-B mRNA invitro. This complex is composed of acatalytic subunit, apobec-1, and anRNA-binding subunit, apobec-1complementation factor (ACF). Tounderstand how the editing enzymerecognizes its target mRNA, weinvestigated the structure and functionof ACF, which contains three copiesof an RNA recognition motif (RRM).Our results suggest that the threeindividual RRMs in ACF contributedifferently to RNA binding. Thepresence of three nonequivalent RRMsin ACF may allow this protein tointeract with several different mRNAsequences. This hypothesis is supported by ourfinding that ACF is widely expressed in tissuesthat lack apo-B mRNA, which suggests that it isinvolved in other mRNA editing or mRNAprocessing events. We are currently developingexperimental approaches to identify novelmRNA targets of ACF.The laboratory’s second project isinvestigation of the biosynthesis ofselenoproteins. Selenium is an essential traceelement that is incorporated into proteins asselenocysteine (Sec), the 21st amino acid. Manyselenoproteins catalyze redox reactions andcontain a Sec residue at their active site. Thesynthesis of selenoproteins, which is encoded bya UGA codon, necessitates the reprogramming oftranslation, since UGA is normally read as aThe Department of Cell Biologytranslational stop codon. Our aim is to understandhow the ribosome copes with a codon thathas a dual function. In eukaryotes, the recodingof UGA as Sec requires a Sec insertion sequence(SECIS) element in the 3' untranslated region ofthe mRNA. We purified, cloned, and characterizedSECIS-binding protein 2 (SBP2), a novelRNA-binding protein that binds to a non-Watson-Crick base-pair quartet in the SECIS element. Wealso developed the first system for translatingselenoprotein mRNAs in vitro and showed thatSBP2 is an essential and limiting factor in thispathway. SBP2 contains a putative RNA-bindingdomain found in ribosomal proteins and atranslation termination agent, eukaryotic releasefactor-1 (eRF1). SBP2 mRNA is widely expressed.However, SBP2 protein levels varydramatically between tissues; in some cell types,the protein is undetectable. Our recent resultssuggest that SBP2 expression is regulated at thetranslational level. Ongoing projects in thelaboratory include: analyzing the structure,function and regulation of SBP2; identifyingother factors involved in Sec incorporation; andinvestigating the translational regulation of thispathway in vivo.Copeland, P.R., Fletcher, J.E., Carlson, B.A.,Hatfield, D.L., and D.M. Driscoll (2000) A novelRNA binding protein, SBP2, is required for thetranslation of mammalian selenoprotein mRNAs.EMBO J. 19:306-314.Mehta, A., Kinter, M.T., Sherman, N.E., andD.M. Driscoll (2000) Molecular cloning ofapobec-1 complementation factor, a novel RNAbindingprotein involved in the editing ofapolipoprotein B mRNA. Mol. Cell. Biol. 20:1846-1854.Copeland, P.R., Stepanik, V.A., and D.M.Driscoll (2001) Insight into mammalianselenocysteine insertion: domain structure andribosome binding properties of Sec insertionsequence binding protein 2. Mol. Cell. Biol.21:1491-1498.Fletcher, J.E., Copeland, P.R., Driscoll, D.M.,and A. Krol (2001) The selenocysteineincorporation machinery: interactions betweenthe SECIS RNA and the SECIS-binding proteinSBP2. RNA 7:1442-1453.Mehta, A., and D.M. Driscoll. (2002) Identificationof domains in apobec-1 complementationfactor required for RNA binding andapolipoprotein-B mRNA editing. RNA 8:69-82.Copeland, P.R., and D.M. Driscoll (2002)Purification and analysis of selenocysteineinsertion sequence-binding protein 2. MethodsEnzymol. 347:40-49.Driscoll, D.M., and P.R. Copeland (2003)Mechanism and regulation of selenoproteinsynthesis. Annu. Rev. Nutr. 2003 Jan 8 [epubahead of print].

Maintenance of blood-vessel-wallfunction depends on the equilibriumbetween inflammatory or injuriousprocesses that may damage vascular cells andimpede their function and healing processes thataccelerate the recovery of the blood vessel frominjury. We are investigating specific aspects ofboth processes, since both are likely to influencethe behavior of blood vessels under normal andpathological conditions, especially duringatherogenesis.Endothelial Cell MotilityEndothelial cell (EC)movement is a critical,initiating event in theformation of new bloodvessels and the repair ofinjured vessels. Our laboratoryis interested in the regulationof EC motility by lipids andlipoproteins, specifically thefunction of the plasmamembrane in cell motility. Wehave recently shown that themicroviscosity of the plasmamembrane is a criticaldeterminant in regulating ECmovement. Using multiplephysiological agents, a simplebiphasic dependency wasobserved in which moderate increases inmembrane microviscosity stimulated ECmigration; however, increases in membranemicroviscosity beyond an optimal thresholdinhibited migration. Surprisingly, two angiogenicgrowth factors, vascular endothelial growthfactor and basic fibroblast growth factor, alsoincreased EC membrane microviscosity. We arenow investigating the biochemical mechanisms bywhich angiogenic growth factors alter membranemicroviscosity, e.g., caveolin-mediated cholesteroltranslocation. We are also investigating themechanisms by which membranes influence cellmovement; our attention is focused on interactionsof membranes with cytoskeletal proteinsand with rac and other small G-proteins thatregulate motility. In a related collaborativeproject with Dr. Linda Graham, we are studyingthe effects of oxidized lipids and lipoproteins onthe healing of synthetic vascular grafts in vivo.Structure, Function of Human CeruloplasminA second major project involves studies ofthe function of ceruloplasmin, an acute-phasereactant protein secreted by the liver and byactivated macrophages. Ceruloplasmin contains 7copper atoms and carries 95% of the serumcopper. Evidence from our laboratory indicatesthat ceruloplasmin has a potent pro-oxidantactivity, catalyzed by a single copper atom, thatThe Department of Cell BiologyEndothelial Cell Motility, CeruloplasminFunction Impact Vessel Wall Healthdramatically increases the rate of oxidation oflow-density lipoprotein. We are actively pursuingthe role of ceruloplasmin in cell-mediatedoxidative processes in vitro and in atheroscleroticlesions. A principal approach involves the use oftransgenic animal models of atherosclerosis. Weare also investigating the molecular mechanisms,at both the transcriptional and posttranscriptionallevels, that regulate ceruloplasmin synthesis.The recent discovery of “aceruloplasminemia”as a humangenetic disorder leading topathologic accumulationof iron has led us toinvestigate the specificrole of ceruloplasmin iniron homeostasis. We haveshown that ceruloplasminpromotes iron flux intocells of erythroid originand also that ceruloplasminproduction istranscriptionally regulatedby hypoxia-induciblefactor-1. These findingssuggest that ceruloplasminhas an important role inerythropoiesis, particularlyduring stress. We arePaul L. Fox, Ph.D.investigating the role ofceruloplasmin in iron metabolism in mice lackingthe ceruloplasmin gene and in a mouse model ofchronic renal failure.THE P. FOXLABORATORYPROJECT SCIENTISTBarsanjit Mazumder, Ph.D.POSTDOCTORAL FELLOWSPrabar Ghosh, Ph.D.Rupak Mukhopadhyay, Ph.D.Vasudevan Seshadri, Ph.D.Nicholas Tripoulas, Ph.D.RESEARCH TECHNOLOGISTSAlena Nikolskaya, M.S.Angela Serrani, B.S.GRADUATE STUDENTSSrujana Cherukiri, B.S.Paul Pavicic, B.S.Prabha Sampath, M.S.Joydeep Sarkar, B.A.Amit Vasanji, B.S.Ke Ya, M.S.COLLABORATORSLinda Graham, M.D. 1Alan Lichtin, M.D. 2Saul Nurko, M.D. 31Depts. of Vascular Medicineand Biomedical Engineering,CCF2Dept. of Hematology/MedicalOncology, CCF3Dept. of Nephrology/Hypertension, CCFMukhopadhyay, C.K., Attieh, Z.K., and P.L Fox (1998) Role of ceruloplasmin in cellulariron uptake. Science 279:714-717.Salvado, M.O., and P.L. Fox (2001) Palmitoylation of caveolin-1 in endothelial cells ispost-translational but irreversible. J. Biol. Chem. 276:15776-15782.Mazumder, B., Seshadri, V., Imataka, H., Sonenberg, N., and P.L. Fox. (2001)Translational silencing of ceruloplasmin requires the essential elements of mRNAcircularization: poly(A) tail, poly(A)-binding protein, and eukaryotic translation initiationfactor 4G. Mol. Cell. Biol. 21:6440-6449.Seshadri, V., Fox, P.L., and C.K. Mukhopadhyay (2002) Dual role of insulin intranscriptional regulation of the acute phase reactant ceruloplasmin. J. Biol. Chem.277:27903-27911.Ghosh, P.K., Vasanji, A., Murugesan, G., Eppell, S.J., Graham, L.M., and P.L. Fox(2002) Membrane microviscosity regulates endothelial cell motility. Nat. Cell Biol.4:894-900.Fox, P.L. (2003) The copper-iron chronicles: the story of an intimate relationship.Biometals 16:9-40.Mazumder, B., Seshadri, V., and P.L. Fox (2003) Translational control by the 3'-UTR:the ends specify the means. Trends Biochem. Sci. 28:91-98.Sampath, P., Mazumder, B., Seshadri, V., and P.L. Fox (2003) Transcript-selectivetranslational silencing by gamma interferon is directed by a novel structural element inthe ceruloplasmin mRNA 3' untranslated region. Mol. Cell. Biol. 23:1509-1519.73

THE HAZENLABORATORYRESEARCH ASSOCIATESMichael Greenberg, Ph.D.Marie-Luise Brennan, Ph.D.Renliang Zhang, M.D., Ph.D.RESEARCH FELLOWSRon Aviles, M.D.Waddah Maskoun, M.D.Mehdi Shishehbor, D.O.POSTDOCTORAL FELLOWSNagella Nukuna, Ph.D.Lian Shan, Ph.D.LEAD TECHNICIANDave SchmittTECHNICIANSXiaoming Fu, M.S.Shirley MannSteven MaximukDragos MihaitaMeghan SmithSTUDENTSPaula FintonCheryl MolendaLaura NarineWei SongCOLLABORATORSGuy Chisolm, Ph.D. 1Raed Dweik, M.D. 2Stephen Ellis, M.D. 3Serpil Erzurum, M.D. 2Gary Francis, M.D. 3Paul Fox, Ph.D. 1Mani Kavuru, M.D. 2Michael Lauer, M.D. 3Patrick McCarthy, M.D. 4Steven Nissen, M.D. 3Marc Penn, M.D.,Ph.D. 1,3Eugene Podrez, M.D., Ph.D. 1Robert G. Salomon, Ph.D. 5Dennis Stuehr, Ph.D. 6James Thomas, M.D. 3Eric Topol, M.D. 3Jay Yadav, M.D. 3James Young, M.D. 31Dept. of Cell Biology, CCF2Dept. of Pulmonary andCritical Care Medicine, CCF3Dept. of CardiovascularMedicine, CCF4Dept. of Thoracic andCardiovascular Surgery, CCF5Dept. of Chemistry, CaseWestern Reserve Univ.,Cleveland, OH6Dept. of Immunology, CCFThe overall goals of my laboratory are tounderstand the mechanisms throughwhich phagocytic cells promote protein,lipid and DNA oxidative damage as part of theirnormal function and in chronic inflammatorydiseases. Three major research programscurrently focus on the role of oxidative damagein the pathogenesis of disease. We employ amultidisciplinary approach, combining clinical,animal model, cellular and molecular biologicalstudies with those that rely heavily on chemicaland analytical methods (e.g., mass spectrometry,nuclear magnetic resonance, high-performanceliquid chromatography, and electron paramagneticresonance).Myeloperoxidase, Oxidant Stress andAtherogenesisAtherosclerosis is a chronic inflammatoryprocess in which oxidative damage within theartery wall is implicated in the pathogenesis ofthe disease. Mononuclearphagocytes,an inflammatory cellcapable of generatinga variety of oxidizingspecies, are earlycomponents ofarterial lesions.Myeloperoxidase(MPO) is anabundant hemeprotein secreted fromactivated phagocytesand is present inhuman atheroscleroticlesions. Wehave shown thatMPO is one pathwayfor protein andThe Department of Cell BiologyFree Radicals and Reactive OxidantsCause Inflammatory Injury in DiseaseStanley Hazen, M.D., Ph.D.lipoprotein oxidation in vivo. Multiple distinctproducts of MPO are enriched in humanatherosclerotic lesions and low-density lipoproteins(LDLs) recovered from human atheroma.In recent clinical studies we demonstrated thatMPO levels serve as an independent predictor ofcardiovascular risk. Current research efforts areaimed at examining the mechanisms of howMPO participates in atherogenesis through use ofgenetic, biochemical, analytical, cell biologicaland clinical studies.Eosinophil Peroxidase and AsthmaEosinophils play an essential role in vivo,destroying pathogenic microorganisms, parasitesand tumor cells. To perform these functions,they have evolved enzymatic mechanisms togenerate an arsenal of reactive oxidant species;however, their potent oxidants also have greatpotential to harm healthy tissue. Oxidativeproducts of eosinophil activation are implicatedin the genesis of tissue injury inasthma. Eosinophil peroxidase(EPO), an abundant hemeprotein secreted duringeosinophil activation, usesH 2O 2to generate potentcytotoxic oxidants.We have identifiedseveral pathways for EPOdependentoxidative damage ofcellular proteins and lipids thatmight contribute to the originsof cellular injury in theinflammatory response inasthma. The potential roles ofthese pathways in human andmurine models of asthma arebeing examined.Brennan, M.L., Wu, W., Fu, X., Shen, Z., Song, W., Frost, H., Vadseth, C., Narine, L., Lenkiewicz, E.,Borchers, M.T., Lusis, A.J., Lee, J.J., Lee, N.A., Abu-Soud, H.M., Ischiropoulos, H., and S.L. Hazen (2002)A tale of two controversies: defining both the role of peroxidases in nitrotyrosine formation in vivo usingeosinophil peroxidase and myeloperoxidase-deficient mice, and the nature of peroxidase-generatedreactive nitrogen species. J. Biol. Chem. 277:17415-17427.Podrez, E.A., Poliakov, E., Shen, Z., Zhang, R., Deng, Y., Sun, M., Finton, P.J., Shan, L., Gugiu, B., Fox,P.L., Hoff, H.F., Salomon, R.G., and S.L. Hazen (2002) Identification of a novel family of oxidizedphospholipids that serve as ligands for the macrophage scavenger receptor CD36. J. Biol. Chem.277:38503-38516.Podrez, E.A., Poliakov, E., Shen, Z., Zhang, R., Deng, Y., Sun, M., Finton, P.J., Shan, L., Febbraio, M.,Hajjar, D.P., Silverstein, R.L., Hoff, H.F., Salomon, R.G., and S.L. Hazen (2002) A novel family ofatherogenic oxidized phospholipids promotes macrophage foam cell formation via the scavenger receptorCD36 and is enriched in atherosclerotic lesions. J. Biol. Chem. 277:38517-38523.Hazen, S.L., and G.M. Chisolm (2002) Oxidized phosphatidylcholines: Pattern recognition ligands formultiple pathways of the innate immune response. Proc. Natl. Acad. Sci. USA 99:12515-12517.Zhang, R., Brennan, M.L., Shen, Z., MacPherson, J.C., Schmitt, D., Molenda, C.E., and S.L. Hazen (2002)Myeloperoxidase functions as a major enzymatic catalyst for initiation of lipid peroxidation at sites ofinflammation. J. Biol. Chem. 277:46116-46122.74

The Department of Cell BiologyTGF-β Signaling PathwaysTHE HOWELABORATORYThe central focus of the work in mylaboratory is to determine the molecularsignaling pathway used by transforminggrowth factor β (TGFβ). We have recentlyidentified an adaptor molecule termed disabled-2(Dab2) as a mediator of TGFβ signaling. Dab2serves as a link between activated cell surfaceTGFβ receptors and their downstream signalingmediators, the Smad proteins. Currently, twospecific aspects of Dab2 are under investigation.We have recently determined that Dab2 binds toseveral key regulatory molecules, Dvl-3 and axin,that function in Wnt-mediated signalingtransduction. We have shown that Dab2 servesas a negative regulator of the Wnt signalingpathway and results in modulation of nuclear β-catenin levels. We are currently investigating, at amolecular level, the mechanism through whichthe binding of Dab2 to Dvl-3 and axin ultimatelyregulates β-catenin levels. In a related project, weare examining the role of Dab2 in mediatingTGFβ-induced epithelial to mesenchymal transdifferentiation(EMT) in mouse mammaryepithelial cells. We have observed that duringTGFβ-induced EMT in these cells, there is aconcomitant upregulation of the p96 form ofDab2 and a downregulation in the p67 form ofDab2. This effect of TGFβ on the respectiveDab2 proteins is due to its effects on alternativepre-mRNA splicing. We are therefore examiningwhether Dab2 alternative splicing regulates thetransdifferentiation of epithelium into mesenchyme.Our second major area of interest focuseson determining the molecular mechanisms bywhich TGFβ aids in maintenance of selftolerance through its induction of apoptosis in Blymphocytes. We have obtained preliminaryresults demonstrating that TGFβ-inducedapoptosis in B lymphocytes is mediated throughthe induction of the pro-apoptotic Bcl-2 familymember Bim. It appears that TGFβ-mediatedinduction of Bim protein is regulated at both thetranscriptional and post-transcriptional levels. Weare investigating the signal transduction pathwaysthat mediate post-transcriptional effects on Bimprotein and the 5′ promoter region of Bim forTGFβ-regulated transactivation.INVESTIGATORSBarbara A. Hocevar, Ph.D.Gary L. Wildey, Ph.D.POSTDOCTORAL FELLOWSCeline Prunier, Ph.D.Xiaojun Qi, Ph.D.TECHNICAL ASSISTANTJessica RennoldsCOLLABORATORSJonathan Cooper, Ph.D. 1Edward B. Leof, Ph.D. 2Xiangxi (Mike) Xu, Ph.D. 31Fred Hutchinson Cancer Fndn.,Seattle, WA2Dept of Biochemistry, MayoClinic, Rochester, MN3Dept. of Biochemistry, EmoryUniv., Atlanta, GAPhilip H. Howe, Ph.D.Hocevar, B.A., Brown, T.L., and P.H. Howe (1999) TGF-β- induces fibronectin synthesis through a c-Jun N-terminal kinasedependent,Smad4-independent pathway. EMBO J. 18:1345-1356.Brown, T.L., Patil, S., Cianci, C.D., Morrow, J.S., and P.H. Howe (1999) Transforming growth factor β induces caspase 3-independent cleavage of α II-spectrin (α-fodrin) coincident with apoptosis. J. Biol. Chem. 274:23256-23262.Patil, S., Wildey, G.M., Brown, T.L., Choy, L., Derynck, .R, and P.H. Howe (2000) Smad7 is induced by CD40 and protectsWEHI 231 B-lymphocytes from transforming growth factor-β-induced growth inhibition and apoptosis. J. Biol. Chem.275:38363-38370.Hocevar, B.A., Smine, A., Xu, X.X., and P.H. Howe (2001) The adaptor molecule Disabled-2 links the transforming growthfactor β receptors to the Smad pathway. EMBO J. 20:2789-2801.Wildey, G.M., Patil, S., and P.H. Howe (2003) Smad3 potentiates transforming growth factor β (TGFβ)-induced apoptosisand expression of the BH3-only protein Bim in WEHI 231 B lymphocytes. J. Biol. Chem. 278:18069-18077.Hocevar, B.A., Mou, F., Rennolds, J.L., Morris, S.M., Cooper, J.A., and P.H. Howe (2003) Regulation of the Wnt signalingpathway by disabled-2 (Dab2). EMBO J. 22:3084-3094.Prunier, C., Pessah, M., Ferrand, N., Howe, P.H., and A. Atfi (2003) The oncoprotein Ski acts as an antagonist of TGFβsignaling by suppressing Smad2 phosphorylation. J. Biol. Chem. (in press).75

The Department of Cell BiologyHyperhomocysteinemia: Mechanisms ofVascular DamageTHE JACOBSENLABORATORYPOSTDOCTORAL FELLOWShantanu Sengupta, Ph.D.TECHNICAL ASSOCIATEPatricia M. DiBello, M.S.GRADUATE STUDENTSBeatrix BüdyEumelia V. TipaDept. of Chemistry, ClevelandState Univ.,Cleveland, OHCOLLABORATORSMichael Kinter, Ph.D. 1Ralph O’Brien, Ph.D. 2Byron Hoogwerf, M.D. 3Vincent Dennis, M.D. 4Marc Pohl, M.D. 4Rick Austin, Ph.D. 5Warren Kruger, Ph.D. 61Dept. of Cell Biology, CCF2Dept. of Biostatistics and Epidemiology,CCF3Dept. of Endocrinology, CCF4Dept. of Nephrology and Hypertension,CCF5McMaster Univ., Hamilton,Ont., Canada6Fox Chase Cancer Ctr., Philadelphia,PAElevated blood homocysteine(hyperhomocysteinemia) is a strongindependent risk factor for cardiovasculardisease. Approximately 40% of patientsdiagnosed with coronary artery disease at theCleveland Clinic Foundation havehyperhomocysteinemia. Our laboratory is studyingthe vascular biochemistry of homocysteine andmechanisms of homocysteine-induced endothelialdysfunction.We determined that human aortic endothelialcells have a limited capacity to metabolize homocysteine.By direct enzyme assay and western blottingand northern blotting, we demonstrated that the firstenzyme of thetranssulfuration pathway,cystathionine β-synthase, isnot expressed. We hypothesizedthat because of itslimited capacity to metabolizehomocysteine, the vascularendothelium may beparticularly vulnerable to highconcentrations of homocysteine.Continuous exposure toelevated homocysteine in thecirculation ofhyperhomocysteinemicsubjects causes endothelialcell dysfunction.In atherogenesis,monocytes are recruited tosites of vascular injury, wherethey transmigrate to the intimal space, transform intomacrophages, and engorge lipids. What role doeshomocysteine play in atherogenesis? We have shownthat in cultured human aortic endothelial cells,homocysteine induces expression of monocytechemoattractant protein-1 (MCP-1) and interleukin 8(IL-8), chemokines for the recruitment of monocytesPoddar, R., Sivasubramanian, N., DiBello, P.M., Robinson, K., and D.W. Jacobsen(2001) Homocysteine induces expression and secretion of MCP-1 and IL-8 in humanaortic endothelial cells: implications for vascular disease. Circulation 103:2717-2723.Sengupta, S., Chen, H., Togawa, T., DiBello, P.M., Majors, A.K., Büdy, B., Ketterer,M.E., and D.W. Jacobsen (2001) Albumin thiolate anion is an intermediate in theformation of albumin-bound homocysteine. J. Biol. Chem. 276:30111- 30117.Sengupta S., Wehbe, C., Majors, A.K., Ketterer, M.E., DiBello, P.M., and D.W. Jacobsen(2001) Relative roles of albumin and ceruloplasmin in the formation of homocystine,homocysteine-cysteine-mixed disulfide, and cystine in circulation. J. Biol. Chem.276:46896-46904.Majors, A.K., Sengupta, S., Willard, B., Kinter, M.T., Pyeritz, R.E., and D.W. Jacobsen(2002) Homocysteine binds to human plasma fibronectin and inhibits its interaction withfibrin. Arterioscler. Thromb. Vasc. Biol. 22:1354-1359.Hossain, G.S., Van Thienen, J.V., Werstuck, G.H., Zhou, J., Sood, S.K., Dickhout, J.G.,De Koning, A.B., Tang, D., Wu, D., Falk, E., Poddar, R., Jacobsen, D.W., Zhang, K.,Kaufman, R.J., and R.C. Austin (2003) TDAG51 is induced by homocysteine, promotesdetachment-mediated programmed cell death and contributes to the development ofatherosclerosis in hyperhomocys-teinemia. J. Biol. Chem. 2003 278:30317-30327.Donald W. Jacobsen, Ph.D.,F.A.H.A.and neutrophils, respectively. In addition to theinduction of mRNA for MCP-1 and IL-8, homocysteinealso triggers the release of MCP-1 and IL-8protein. Induction and release is mediated by 5-50 µML-homocysteine (the D enantiomer is inactive). L-Cysteine is inactive, suggesting that the mechanism isnot due to a general thiol effect involving thegeneration of reactive oxygen species. Studies areunder way to elucidate the mechanisms of homocysteine-inducedchemokine expression and release.Using in vitro model systems, we have studiedthe vascular biochemistry of homocysteine andcysteine in circulation. In our model, we propose thatcysteine undergoes autooxidation to cystine in areaction catalyzed by ceruloplasmin.Albumin-Cys34 thiolate anion,secreted into circulation from theliver, attacks cystine to formalbumin-S-S-cysteine. Homocysteinethiolate anion then attacks thecysteine sulfur of albumin-S-Scysteineto form homocysteinecysteinemixed disulfide andalbumin-Cys34 thiolate anion. About20% of the homocysteine enteringcirculation undergoes autooxidationto homocystine, but this reaction iscatalyzed by albumin-bound copper,not ceruloplasmin copper. Thealbumin thiolate anion can then reactwith either the mixed disulfide orhomocystine to form albumin-S-Shomocysteine,which accounts forup to 80% of circulating plasma total homocysteine.This process, which we term “molecular targeting”by homocysteine, may explain the adverse effects thathomocysteine has on the vascular endothelium.Vitamin B 12and folate are B-complexmicronutrients that drive homocysteine metabolism.Subjects with B 12and/or folate deficiency arehyperhomocysteinemic. B 12and folate serve ascoenzyme and substrate, respectively, for methioninesynthase, the enzyme that remethylates homocysteineback to methionine. We know very little about howthese micronutrients are transported and processedby vascular cells. We believe that the vascularendothelium plays a major role in B 12homeostasis bytranscytosis of the B 12-transcobalamin complex.Transcobalamin is a serum B 12-binding protein thatdelivers B 12to cells throughout the body. Ourlaboratory studies B 12and folate transport andmetabolism in vascular cells and tissues. We recentlyestablished that cultured human aortic endothelialcells express 12-15,000 transcobalamin receptors percell, an observation that supports our endothelial cellB 12transcytosis hypothesis. We are currently studyingintracellular B 12processing and mitochondrialtransport by cultured human aortic endothelial andsmooth muscle cells.76

Oxidative stress is believed to contributeto tissue injury in a number of humandiseases including atherosclerosis,neurodegenerative disorders, asthma, and aging.In atherosclerosis, for example, the productionof reactive oxygen and reactive nitrogen speciesappears to play a role in thevariety of processes thatinjure the arterial wall.Specifically, these reactivespecies have been found toparticipate in a cascade ofevents that could contributeto the progression of thedisease, including modificationof low-density lipoprotein(LDL), initiation of lipidloading into macrophages andsmooth muscle cells, cellproliferation and migration,and damage to the endothelium.The research activitiesin my laboratory are focusedon attaining a better understandingof the molecularmechanisms of oxidant injurythrough two lines ofinvestigation: characterizingthe anti-oxidant defensesystems in cells, and characterizingthe sites and chemicalstructure of oxidant damage to proteins.A unique aspect of these experiments isthe utilization of mass spectrometric methods ofprotein characterization and sequencing.The first area of investigation in thelaboratory uses a proteomic approach (2Delectrophoresis and tandem mass spectrometry)to map and identify the proteins that areoverexpressed and underexpressed in cell linesthat are resistant to oxidative injury. The generalhypothesis being tested in this work is that thesechanges in protein expression are responsible forthe resistant phenotype. As the differentiallyexpressed proteins are identified, subsequentexperiments will be designed and carried out totest their role in the injury process. Suchexperiments will use combinations of transfection,to increase protein expression, and antisenseoligonucleotide treatment, to inhibit proteinexpression, in combination with various in vitroassays of oxidant injury. The goal of this work isto discover previously unidentified and unstudiedThe Department of Cell BiologyProteomics and Mass Spectrometry TargetCharacterization of Anti-Oxidant DefenseMechanisms, Oxidative Protein Damage Sitesproteins that help cells resist the damaging effectsof oxidative stresses. In the longer term, we canthen begin to devise new methods to utilize theseproteins to intervene in diseases such as atherosclerosis.The second area of investigation in thelaboratory uses tandem massspectrometry to characterizethe site and structure ofoxidative modifications toproteins. One theory ofhow oxidative stress affectscells is that key proteinsbecome modified in amanner that alters theirfunction. The exact natureof these modifications,however, is not wellunderstood. It is envisionedthat the specific structuresthat are detected andcharacterized will providenew information about theoxidation reactions leadingto those modifications.Further, we expect that asour understanding of thesite and structure ofoxidative modifications isadvanced, it will be possibleto identify new molecularmarkers to monitor the effects oxidative stress invivo. This work includes the development ofnovel, site-specific quantitative methods foroxidized proteins.Michael T. Kinter, Ph.D.THE KINTERLABORATORYPOSTDOCTORAL FELLOWSJ. Andrew Keightley, Ph.D.Belinda B. Willard, Ph.D.STUDENTSJames ConwayLemin ZhengKinter, M., and N.E. Sherman (2000) Protein Sequencing and Identification UsingTandem Mass Spectrometry. John Wiley & Sons, Inc. New York, NY.Willard, B.B., and M. Kinter (2001) Effects of the position of internal histidine residueson the collision-induced fragmentation of triply protonated tryptic peptides. J. Am.Soc. Mass Spectrom. 12:1262-1271.Ruse, C.I., Willard, B., Jin, J.P., Haas, T., Kinter, M., and M. Bond (2002) Quantitativedynamics of site-specific protein phosphorylation determined using liquid chromatographyelectrospray ionization mass spectrometry. Anal. Chem. 74:1658-1664.Chikamori, K., Grabowski, D.R., Kinter, M., Willard, B.B., Yadav, S., Aebersold, R.H.,Bukowski, R.M., Hickson, I.D., Andersen, A.H., Ganapathi, R., and M.K. Ganapathi(2003) Phosphorylation of serine 1106 in the catalytic domain of topoisomerase IIalpha regulates enzymatic activity and drug sensitivity. J. Biol. Chem. 278:12696-12702.Willard, B.B., Keightley, J.A., Ruse, C.I., Bond, M., and M. Kinter (2003) Site-specificquantitation of protein nitration using liquid chromatography-tandem mass spectrometry.Anal. Chem. 75:2370-2376.77

The Department of Cell BiologyRole of CETP and LTIP in Plasma HDL,LDL Concentrations ExaminedTHE MORTONLABORATORYPOSTDOCTORAL FELLOWSYu-Bin He, M.D.Sung-Koo Kang, Ph.D.Lahoucine Izem, Ph.D.Viktor Paromov, Ph.D.TECHNICAL ASSISTANTDiane J. Greene, B.S.COLLABORATORSDonna M. Driscoll, Ph.D. 1Eder Quintão , C.R., M.D. 21Dept. of Cell Biology, CCF2Univ. of São Paulo, São Paulo,Brazil78It is increasingly appreciated that the lipidconstituents of human lipoproteins are notpassive components, but are rapidly transferredbetween lipoproteins and other membranestructures. Whether this transferoccurs by simple diffusionthrough the aqueous space orwhether it is protein-mediateddepends on the nature of thelipid itself.The transfer of the apolarcomponents of lipoproteins,cholesteryl ester and triglyceride,depends on the action of aspecific plasma protein,cholesteryl ester transfer protein(CETP). CETP can facilitate thenet transfer of triglyceride andcholesteryl ester betweenlipoproteins, thus playing animportant role in defining thelipid composition of plasmalipoproteins.Our laboratory’s majorfocus is investigating the role of CETP in theintra-/extravascular metabolism of plasmalipoproteins and lipids. Our current interests takeseveral directions. First, we are investigating themechanism of the transfer process itself. We havemade significant progress in defining the kineticsof binding between CETP and plasma lipoproteins.We have shown that lipid transfer requiresformation of a CETP-lipoprotein complex andthat all plasma lipoproteins bind CETP withsimilar affinities but markedly different capacities.By reconstitution techniques, we are studyinghow lipoprotein composition affects the bindingevent and the association of CETP with differentWang, X., Driscoll, D.M., and R.E. Morton (1999) Molecular cloning and expression of lipidtransfer inhibitor protein reveals its identity with apolipoprotein F. J. Biol. Chem. 274:1814-20.Morton, R.E. (1999) Cholesteryl ester transfer protein and its plasma regulator: lipid transferinhibitor protein. Curr. Opin. Lipidol. 10:321-27.Greene, D.J., Skeggs, J.W., and R.E. Morton (2001) Elevated triglyceride content diminishesthe capacity of high density lipoprotein to deliver cholesteryl esters via the scavengerreceptor class B type I (SR-BI). J. Biol Chem. 276:4804-4811.Izem, L., and R.E. Morton (2001) Cholesteryl ester transfer protein biosynthesis and cellularcholesterol homeostasis are tightly interconnected. J. Biol Chem. 276:26534-26541.Morton, R.E., Nunes, V., Izem, L., and E.C. Quintão (2001) Markedly elevated lipid transferinhibitor protein in hypercholesterolemic subjects is mitigated by plasma triglyceride levels.Arterioscler. Thromb. Vasc. Biol. 21:1642-1649.Skeggs, J.W., and R.E. Morton (2002) LDL and HDL enriched in triglyceride promoteabnormal cholesterol transport. J. Lipid Res. 43:1264-1274.Morton, R.E., D.J. Greene (2003) The surface cholesteryl ester content of donor andacceptor particles regulates CETP: a liposome-based approach to assess thesubstrate properties of lipoproteins. J. Lipid Res. 44:1364-1372.Richard E. Morton, Ph.D.lipoprotein classes in vivo. Ultimately, we hope todefine the “binding site” of CETP and todelineate the mechanism of lipid transfer.We are investigating regulation of CETPactivity by the physicochemicalproperties of its lipoproteinsubstrates and regulation of thecirculating CETP by other plasmaproteins. The initial approachfocuses on how modification oflipoprotein composition, inducedby dietary factors or metabolicaberrations, alters the rate anddirectionality of lipid transfer. Wefound that the capacity of CETPto facilitate the mass transfer ofcholesteryl ester correlatespositively with the unesterifiedcholesterol content of plasmalipoproteins, demonstrating thatunesterified cholesterol stimulatesthe metabolic pathways thatdeliver tissue cholesterol to theliver, where it can be excreted.Recently, we have begun characterizing anovel protein in human plasma, designated LTIP,which suppresses CETP activity in vitro. LTIP isnot simply a general suppressor of CETP activity,but preferentially inhibits CETP-mediated lipidtransfers involving low-density lipoprotein. Thisresults in a reduced capacity of the apolar lipidswithin the low-density lipoprotein pool toequilibrate with those in other lipoproteins.Therefore, LTIP plays a key role in defining thelipid transfer events that CETP can mediate inplasma. In general, LTIP appears to promote apattern of lipid transfers that are considered to beanti-atherogenic. We have purified LTIP andcloned its cDNA. We are now performing detailedkinetic and functional studies to define the roleof LTIP in determining the composition andconcentration of individual lipoprotein classesand subclasses.We are also defining the capacity of CETPto alter the accumulation or deposition of lipidswithin cells. These studies are an extension of ourobservations that CETP can promote the netremoval of cholesteryl esters from lipid-loadedmacrophages in culture and that CETP expressionis essential for normal cellular cholesterolhomeostasis, suggesting novel roles for CETP inextravascular lipid metabolism.Collectively, these studies should not onlyyield important information concerning thefunction and regulation of CETP, but shouldprovide additional useful insights into themechanisms underlying the formation ofputatively atherogenic lipoproteins and thecellular deposition of lipids leading to foam cellformation.

The Department of Cell BiologyMolecular Mechanisms of VentricularRemodeling and Regeneration FollowingMyocardial InfarctionThe major focus of my laboratory is thestudy of left ventricular remodeling anddysfunction following myocardialischemia, and mechanisms of myocardialregeneration. The prevalence of congestive heartfailure in the American population is continuallyincreasing, and is now~10% in people over 65years of age.To understand howthe left ventricle respondsto ischemia, we arecharacterizing (1) generegulation and (2) proteaseactivation during earlymyocardial infarction(MI). Specifically, we arefocusing on the role ofleukocyte-generatedoxidants on left ventricular(LV) dilation followingMI. We have recentlydemonstrated thatmyeloperoxidase (MPO)mediated oxidation ofplasminogen activatorinhibitor-1 (PAI-1) plays acentral role in determing left ventricular sizefollowing MI. Ongoing studies are now focusingon the role of other mediators of leukocytegenerated oxidants on LV function following MI,as well as the role of single nucleotide polymorphismsin the genes encoding MPO and/or PAI-1on LV function in clinicalpopulations.Another focus ofthe laboratory is todetermine the molecularmechanisms responsiblefor stem cell homing to,and stem cell differentiationin, injured myocardium.Our goal is to reestablishthese signalingsystems in models ofchronic congestive heartfailure in order toregenerate cardiacfunction. We believe thatthis approach will lead tovaluable discoveries andpotential therapies thatcan be utilized to treatclinical populations.THE PENNLABORATORYRESEARCH FELLOWSDavid Lee, M.D.Samuel Unzek, M.D.Kai Wang, M.D., Ph.D.Xiaorong Zhou, M.D.Zhongmin Zhou, M.D.TECHNOLOGISTSFarhad Forudi, B.S.Matthew Kiedrowski, B.S.COLLABORATORSGuy M. Chisolm, Ph.D. 1 .Paul E. DiCorleto, Ph.D. 1Stanley L. Hazen, M.D., Ph.D. 1Patrick M. McCarthy, M.D. 2Edward F. Plow, Ph.D. 3Eric J. Topol, M.D. 4Marc S. Penn, M.D., Ph.D.Penn, M.S., Francis, G.S., Young, J.B., McCarthy, P.M., and E.J. Topol (2002)Autologous cell transplantation and the treatment of myocardial damage. Prog.Cardiovasc. Dis. 45:21-32.1Dept. of Cell Biology, CCF2Dept. of Thoracic andCardiovascular Surgery, CCF3Dept. of Molecular Cardiology,CCF4Dept. of CardiovascularMedicine, CCFAskari, A.T., and M.S. Penn (2002) Targeted gene therapy for the treatment of cardiacdysfunction. Semin. Thorac. Cardiovasc. Surg. 14:167-177.Yen, M.H., Pilkington, G., Starling, R.C., Ratliff, N.B., McCarthy, P.M., Young, J.B.,Chisolm G.M., and M.S. Penn (2002) Increased tissue factor expression predictsdevelopment of cardiac allograft vasculopathy. Circulation 104: 992-997.Askari, A.T., Brennan, M.L., Zhou, X., Drinko, J., Morehead, A., Thomas, J.D., Topol,E.J., Hazen, S.L., and M.S. Penn (2003) Myeloperoxidase and plasminogen activatorinhibitor 1 play a central role in ventricular remodeling after myocardial infarction. J.Exp. Med. 197:615-624.Askari, A.T., and M.S. Penn (2003) Cell therapy for the treatment of ischemic heartdisease: Approaching a new frontier. In: E.J. Topol, ed. Textbook of InterventionalCardiology. Philadelphia: W.B. Saunders, Chapter 52, pp. 1053-1061, 2003.Shishehbor, M.H., Aviles, R.J., Brennan, M.L., Fu, X., Goormastic, M., Pearce, G.L.,Gokce, N., Keaney, J.F. Jr., Penn, M.S., Sprecher, D.L., Vita, J.A., and S.L. Hazen(2003) Association of nitrotyrosine levels with cardiovascular disease and modulationby statin therapy. JAMA 289:1675-1680.Askari, A.T., et al. (2003) Stromal cell-derived factor-1 mediates stem cell homing andtissue regeneration in ischemic cardiomyopathy. Lancet (In press).79

THE J. SMITHLABORATORYTECHNOLOGISTGreg Brubaker, M.A.TECHNICIANMegan Settle, B.A.POSTDOCTORAL FELLOWSWilfried LeGoff, Ph.D.Daoquan Peng, Ph.D.COLLABORATORJan L. Breslow, M.D.The Rockefeller Univ.,New York, NYJonathan D. Smith, Ph.D.Our laboratory works on a diverse groupof subjects, all related by the importantroles of cholesterol and apolipoproteinE. Our approaches include cell biology andmouse genetics to explore the relevance of thesefactors to common human diseases.Macrophage Cholesterol EffluxCholesterol-loaded macrophage foam cellsare the earliest cellular lesion in atherosclerosis.We aim to determine how macrophages ridthemselves of excess cholesterol in the reversecholesterol transport pathway. Using a macrophagecell line, we have shown that cAMPanalogues induce a pathway for cholesterol andphospholipid efflux to apolipoproteins that thenform nascent high-density lipoprotein (HDL).This pathway is deficient in humans with the raregenetic disorder Tangier disease, due to mutationsin the ABCA1 gene. Our research indicates thatABCA1-mediated lipid efflux depends onextracellular calcium ions and involves endocytosisand resecretion of the apolipoprotein ligands.One theory of ABCA1 action is that it translocatesphosphatidylserine from the inner to theouter leaflet of the plasma membrane and thatthis cell-surface phosphatidylserine is responsiblefor apolipoprotein binding to the cells. However,our research indicates that cell-surfacephosphatidylserine is not sufficient to mediatelipid efflux and that there is no competition foreither cell binding or lipid efflux betweenapolipoproteins and the phosphatidylserinebindingprotein annexin V. We are investigatingother proteins involved in this pathway in anattempt to delineate the mechanism of lipidefflux to the apolipoprotein acceptors.Takahashi, Y., and J.D. Smith (1999) Cholesterol efflux to apolipoprotein AI involvesendocytosis and resecretion in a calcium-dependent pathway. Proc. Nat. Acad. Sci.USA 96:11358-11363.Smith, J.D., Waelde, C., Horwitz, A., and P. Zheng (2002) Evaluation of the role ofphosphatidylserine translocase activity in ABCA1 mediated lipid efflux. J. Biol. Chem.277:17797-17803.Dansky, H.M., Barlow, C.B., Lominska, C., Sikes, J.L., Kao, C., Weinsaft, J.,Cybulsky, M.I., and J.D. Smith (2001) Adhesion of monocytes to arterial endotheliumand initiation of atherosclerosis are critically dependent on VCAM-1 gene dosage.Arterioscler. Thromb. Vasc. Biol. 21:1662-1667.Dansky. H.M., Shu, P., Donavan. M., Montagno, J., Nagle, D.L., Smutko, J.S., Roy,N., Whiteing, S., Barrios, J., McBride, T.J., Smith, J.D., Duyk, G., Breslow, J.L., andJ.K. Moore (2002) A phenotype-sensitizing Apoe deficient genetic background revealsnovel atherosclerosis predisposition loci in the mouse. Genetics 160:1599-1608.Smith, J.D., James, D., Dansky, H.M., Wittkowski, K.M., Moore, K.J., and J.L.Breslow (2002) In silico quantitative trait locus map for atherosclerosis susceptibilityin apolipoprotein E-deficient mice. Arterioscler. Thromb. Vasc. Biol. 23:117-122.The Department of Cell BiologyMechanism of Macrophage CholesterolEfflux and Identification of Genes thatAlter Susceptibility of Mouse Models toAtherosclerosis and Alzheimer DiseaseGenes that Modify Atherosclerosis andAlzheimer SusceptibilityMice are very resistant to atherosclerosis,perhaps in part because of their low levels oflow-density lipoprotein (LDL, bad cholesterol)and high levels of HDL (good cholesterol). Weuse apoE-deficient mice that develop hypercholesterolemiaand atherosclerosis even on a low-fatchow diet. Using both candidate gene andgenetic approaches in this model, we areidentifying genes that modify the animals’susceptibility to atherosclerosis. We have shownthat mice deficient in both apoE and themacrophage cytokine MCSF have about a 90%reduction in lesion area, despite having about 2.5-fold elevated plasma cholesterol levels comparedwith their apoE-deficient littermates. Thisfinding showed the important role that monocyte-macrophagenumber and/or function plays inthis model of atherosclerosis and in lipidmetabolism.We then showed, using the RAG-1knockout model, that mature T and B cells playonly a minor role in regulating lesion size inapoE-deficient mice. We also showed, bybreeding apoE-deficient mice to mice withseverely reduced levels of a mutant VCAM-1protein, that the adhesion molecule VCAM-1plays an important role in atherosclerosis. We arenow using a gene-mapping positional cloningstrategy to identify atherosclerosis susceptibilitygenes in mice. By breeding apoE deficiency ontodifferent inbred mouse strains, we identifiedstrains with large differences in lesion size. Thesestrains were intercrossed, and a large cohort ofthe F 2generation was subjected to lesionmeasurement and genome scans. After quantitativetrait locus (QTL) mapping analysis, threemajor loci (on chromosomes 10, 14, and 19) werefound to be associated with lesion size in the F 2cohort.We have recently used a new method tomap QTL in silico based upon the mouse SNPdatabase and have confirmed the loci onchromosomes 10 and 14. I have identified anadditional three novel potential atherosclerosissusceptibility loci. Next, we will determine theidentity of these disease-modifying genes. We areperforming similar studies using a mouse modelfor Alzheimer disease-like pathology. By breedingonto different mouse strains, we have foundstrain effects on the levels of the β-amyloidpeptide and its precursor protein. We are usingsimilar genetic methods to map and identify theresponsible gene(s).80

The Department of Cell BiologyGM-CSF/Surfactant, NO/CytokineRegulation Central to Pulmonary DiseaseOur research program focuses on understand-ing the role and regulatory aspectsof inflammatory cytokines in pulmonarydisease. Our access to the Cleveland Clinic’sextensive patient population gives us theopportunity to study cellular and molecularprocesses involved in the development of humanpulmonary diseases. Granulocyte macrophagecolonystimulating factor (GM-CSF) regulationof surfactant homeostasisand nitric oxide (NO)regulation of cytokines aretwo areas of current interest.Pulmonary alveolarproteinosis (PAP) is a rarelung disease characterized bythe accumulation ofsurfactant material within thealveoli. GM-CSF-deficientmice develop a PAP-likesyndrome that can becorrected by exogenous GM-CSF, suggesting this factor’spivotal role for GM-CSF innormal lung homeostasis andMary Jane Thomassen, Ph.D.clearance of surfactant. Administration ofexogenous GM-CSF ameliorates lung disease in asubset of PAP patients, providing support for aGM-CSF role in human PAP. Monocytes andalveolar macrophages from PAP patients produceGM-CSF and respond to GM-CSF, indicating nointrinsic defects in the cells’ ability to produceGM-CSF or in the GM-CSF receptor. Further, allPAP patients tested have antibodies against GM-CSF in both BAL and serum. Interleukin-10 (IL-10), a pleiotropic cytokine that stimulatesantibody production, is also a potent inhibitor ofGM-CSF production from alveolar macrophages.PAP patients have less detectable GM-CSF inbronchoalveolar lavage fluids but higher levels ofIL-10 than healthy controls. IL-10 polymorphismshave been associated with increased IL-10in some autoimmune diseases. A polymorphismin the GM-CSF gene of a PAP patient has beendescribed, although the functional significancehas not been studied. Based on these data, wehypothesize that in PAP, the availability of GM-CSF is decreased by anti-GM-CSF antibodies andheightened IL-10; these events may be associatedwith polymorphisms in the IL-10/GM-CSFgenes. The long-term objective of these studiesis to delineate the role of anti-GM-CSFantibodies and IL-10 in decreasing the availabilityof GM-CSF, which is pivotal in the pathophysiologyof alveolar proteinosis. These studies,coupled with data from the GM-CSF clinicaltrial, will provide novel insights into the basicmechanisms underlying human PAP.NO is an important regulatory moleculeimplicated in both pro- and anti-inflammatoryprocesses in the lung. Abnormalities in airway NOlevels are associated with such disease states asasthma and primary pulmonary hypertension (PPH).Interestingly, alterations in inflammatory cytokineshave been reported for both of these diseases.We hypothesized that NO might be involvedin cytokine regulation by alveolar macrophages. Wehave demonstrated that NO decreases inflammatorycytokine production from human alveolar macrophages.NO may regulate cytokine gene expressionthrough effects on the transcriptionfactor nuclear factor-κB (NF-κB), whichcontrols the expression of manyinflammatory cytokine and growthfactor genes. Thus, we investigatedwhether NO affects NF-κB activation inhuman alveolar macrophages in vitro andin vivo.To study the mechanism of NOeffects on NF-κB activation, alveolarmacrophages were stimulated withlipopolysaccharide (LPS) ± a NOgeneratingcompound (DETANONOate). Results indicated that NOdecreased NF-κB activation in a dosedependentmanner. NO also preventedthe degradation of the inhibitory protein I-κB.Interference with I-κB degradation resulting infailure of NF-κB activation may be one mechanismby which NO affects cytokine gene expression.In vivo investigations were performed usingfreshly isolated alveolar macrophages from healthy(control) individuals and those with asthma and withPPH. In healthy individuals, NF-κB activation isdetected at low levels. Asthma patients with highairway NO levels showed minimal NF-κB activationwhereas asthmatics with low NO levels showedsignificantly greater NF-κB activation. In those withPPH, patients with low NO had high NF-κBactivation. These in vivo results, together with the invitro observations, support an inverse relationshipbetween NO and NF-κB activation.THE THOMASSENLABORATORYSTAFFCarol Farver, M.D.PROJECT SCIENTISTSTracey L. Bonfield, Ph.D.Baisakhi Raychaudhuri, Ph.D.POSTDOCTORAL FELLOWSDaniel A. Culver, M.D.TECHNICAL ASSOCIATESSusamma AbrahamSujata BurgessAnagha MalurADJUNCT STAFFBarbara P. Barna, Ph.D.COLLABORATORSAlejandro C. Arroliga, M.D. 1Kevin K. Brown, M.D. 2Raed Dweik, M.D. 1Serpil Erzurum, M.D. 1Stan Hazen,M.D, Ph.D. 3Mani S. Kavuru, M.D. 1Alton L. Melton, M.D. 4Herbert P. Wiedemann, M.D. 1Taolin Yi, Ph.D. 51Dept. of Pulmonary and CriticalCare Medicine, CCF2National Jewish Medical Center,Denver, CO3Dept. of Cell Biology, CCF4Dept. of Medical Subspecialties/PediatricAllergy, CCF5Dept. of Cancer Biology, CCFRaychaudhuri, B., Dweik, R., Connors, M.J., Buhrow, L., Malur, A., Drazba, J., Arroliga,A., Erzurum, S.C., Kavuru, M.S., and M.J. Thomassen (1999) Nitric oxideblocks nuclear factor-κB activation in alveolar macrophages. Am. J. Respir. CellMol. Biol. 21:311-316.Raychaudhuri, B., Fisher, C.J., Buhrow, L., Malur, A., Connors, M.J., Kavuru,M.S., and M.J. Thomassen (2000) Interleukin-10 (IL-10) mediated inhibition of inflammatorycytokine production by human alveolar macrophages. Cytokine12:1348-1355.Thomassen, M.J., Raychaudhuri, B., Malur,A., and M.S. Kavuru (2000) Pulmonaryalveolar proteinosis is a disease of decreased availability of GM-CSF rather thanan intrinsic cellular defect. Clin. Immunol. 95:85-92.Thomassen, M.J., and M.S. Kavuru (2001) Human alveolar macrophages and monocytesas a source and target for nitric oxide. Int. Immunopharmacol. 1:1479-1490.81

THE WEIMBSLABORATORYPROJECT SCIENTISTSSeng Hui Low, Ph.D.Jayasri Nanduri, Ph.D.POSTDOCTORAL FELLOWXin Li, M.D., Ph.D.GRADUATE STUDENTSNikunj Sharma, MSShivakumar Vasanth, MSSENIOR TECHNOLOGISTBonnie Gorzelle, MSTECHNICIANSClaire Larson, BSMark DeGennaro, BSRESEARCH STUDENTSMin HeJunko IwasakiThomas Weimbs, Ph.D.We are interested in the molecularmechanisms of the establishment andmaintenance of epithelial cell polarity.Epithelial cells, e.g., kidney tubule cells, havetwo distinct plasma membrane (PM) domains,the apical and basolateral domains. The proteinand lipid compositions of these two PM domainsare very different from each other, which isessential for the proper function of epithelialtissues. Apical and basolateral PM proteins aretargeted to their respective domains by highlyspecific vesicular transport. The final step in eachpathway is the fusion of the transport vesicleswith the PM. A ubiquitous membrane fusionmachinery, the “SNARE machinery,” has beenidentified to be involved in most, if not all,intracellular vesicle fusion events. Soluble N-ethylmaleimide-sensitive factor attachmentprotein receptors (SNAREs) are membraneproteins localized on the vesicle membrane (v-SNAREs) and target membrane (t-SNAREs).Membrane fusion requires the binding of amatching combination of v- and t-SNAREs. Wehave previously characterized in detail thesubcellular localization and function of differentSNARE isoforms in polarized Madin Darbycanine kidney (MDCK) cells, a model cell line forthe study of membrane traffic in epithelial cells.We are currently focusing on several interrelatedquestions.SNARES/Cell PolarityWe are investigating whether the mutuallyexclusive localization of the t-SNAREs syntaxin3 and 4 on the apical and basolateral plasmamembrane domains, respectively, is necessary forcell polarity. We are identifying the targetingsignals on these syntaxins, investigating howLi, X., Low, S.H., Miura, M., and T. Weimbs (2002) SNARE expression and localizationin renal epithelial cells suggest mechanism for variability of trafficking phenotypes.Am. J. Physiol. Renal Physiol. 283:F1111-F1122.Low, S.H., Marmorstein, L.Y, Miura, M., Li, X., Kudo, N., Marmorstein, A.D., and T.Weimbs (2002) Retinal pigment epithelial cells exhibit unique expression andlocalization of plasma membrane syntaxins which may contribute to their traffickingphenotype. J. Cell Sci. 115:4545-4553 (journal cover article).Kreitzer, G., Schmoranzer, J., Low, S.H., Li, X., Gan, Y., Weimbs, T., Simon, S.M.,and E. Rodriguez-Boulan (2003) Three-dimensional analysis of post-Golgi carrierexocytosis in epithelial cells. Nat. Cell Biol. 5:126-136.Sherief, M.H., Low, S.H., Miura, M., Kudo, N., Novick, A.C., and T. Weimbs (2003)Matrix metalloproteinase activity in urine of patients with renal cell carcinoma leads todegradation of extracellular matrix proteins: possible use as a screening assay. J.Urol. 169:1530-1534.Low, S.H., Li, X., Miura, M., Kudo, N., Quinones, B., and T. Weimbs (2003) Syntaxin 2and endobrevin are required for the terminal step of cytokinesis in mammalian cells.Dev. Cell 4:753-759.Weimbs, T., Low, S.H., Li, X., and G. Kreitzer (2003) SNAREs and epithelial cells.Methods 30:191-197.The Department of Cell BiologyMembrane Trafficking Trackedin Polarized Epithelial Cellsthese SNAREs themselves reach the apical andbasolateral PM domain, and testing whetherdisruption of their targeting leads to defects inepithelial cell polarity.SNARES/Cell DivisionWe are exploring the role of SNAREs incell division (cytokinesis). We have identified, forthe first time, that two SNARE proteins localizespecifically to the so-called midbody, a narrowcytoplasmic bridge that connects the twoprospective daughter cells. Only after cleavage ofthis midbody is cytokinesis complete. Inhibitionof these SNAREs leads to failure of midbodycleavage and results in binucleated cells. This isthe first demonstration of a requirement forSNARE-mediated membrane fusion during thefinal step of the cell cycle and identifies newpotential molecular targets for tumor therapy.Polycystin-1 in Kidney DiseasePolycystin-1 is the protein affected in~85% of the cases of autosomal-dominantpolycystic kidney disease (PKD). Although it iswell established that renal epithelial cells in PKDexhibit defects in polarized protein targeting, cellproliferation and apoptosis, the function ofpolycystin-1 remains unknown. We are using anovel in vitro cell culture approach in which wecan interfere with polycystin-1 function byoverexpression of dominant-negative inhibitorsto identify the downstream signaling pathwaysinvolved in the pathogenesis of PKD. This workwill ultimately allow clinicians and researchers todevise strategies for pharmacological treatment ofthis disease.Renal Cell Carcinoma MarkersThe aim of this project is to identifyurinary protein markers for the early detection ofrenal cell carcinoma (RCC), a highly malignantcancer for which such early detection is currentlynot possible. We have discovered that the urineof RCC patients contains highly elevated levels ofmatrix metalloproteinase activity that leads to thedegradation of normally excreted extracellularmatrix proteins. Urinalysis has allowed us todetect RCC, even at the earliest stages, with asensitivity close to 100%. We are currentlydeveloping these detection assays for possiblefuture commercial use, which will allow widespreadapplication.82

The Department of Cell BiologyRas Knockouts Aidin Defining OncogenicContribution of Normalc-RasAlan Wolfman, Ph.D.We are testing the hypothesis that thedifferent Ras isoforms (N-, Ha-, K(A),and K(B)-Ras) perform uniquefunctions. Using N and K(i)-Ras knockout celllines, we will test whether normal c-Ras isoformsare required for the transformation by specificoncogenic Ras proteins. Previous data from ourlaboratory support the hypothesis that transformationby oncogenic Ha-Ras, at protein levelsresembling those found in human tumors, requiresthe function of c-N-Ras. Oncogenic Ha-ras putsforth signals through an unidentified target tosecrete an EGF receptor ligand, which thenactivates c-N-Ras, Raf-1 and the MAP kinasecascade. This is the first report of Ras isoformcooperativity.Using cell lines established from N-Rasknockout mice, we have observed that c-N-Rasplays a pivotal role in regulating the cellularapoptotic set point. In the absence of c-N-Ras,mouse fibroblasts are hypersensitive to allapoptotic agents tested, including serumwithdrawal.In contrast, cells lacking c-K-Rasexpression are insensitive to the induction ofapoptosis. Together, these data suggest thatcellular Ras isoforms provide steady-state(“tonic”) signaling that regulates cells’ sensitivityto apoptotic agents. We are investigating themechanisms through which each of the cellularRas isoforms regulated the apoptotic mechanisms.In a second project, we are testing thehypothesis that c-N-Ras is distributed amongcellular organelles in discrete signaling modules.In quiescent cells we find all the plasma membraneassociated c-N-Ras complexed to Raf-1 andPKCe. We have also demonstrated that c-N-Rasis associated with membrane compartments otherthan the plasma membrane, such as the mitochondria.We are currently examining the role of c-N-Ras within each organelle and whether the c-N-Ras in specific organelles is associated with aunique Ras binding partner that might be requiredfor organelle-specific functions.Our laboratory is also investigating thesignaling systems that might be regulated bypulsed electromagnetic fields (PEMFs) that havea clinical outcome on patients having non-unionbone fractures. Using primary osteoblasts and anFDA-approved device that generates PEMFs, weare currently determining the initial biologicaloutcomes that are PEMF dependent. Using thisbiological outcome in cultured cells, we will thenexamine the signaling systems that are activatedupon PEMF treatment of primary osteoblasts.THE WOLFMANLABORATORYRESEARCH ASSOCIATESThomas PattersonSarah PlanchonPOSTDOCTORAL FELLOWSJinhui LiaoJanice WolfmanWolfman, J.C., and A. Wolfman (2000) Endogenous c-N-Ras provides a steady-state anti-apoptotic signal.J. Biol. Chem. 275:19315-19323.Hamilton, H. Liao, J., Cathcart, M., and A. Wolfman (2001) Constitutive association of c-N-Ras with Raf-1and protein kinase Ce in latent signaling modules. J. Biol. Chem. 276:29079-29090.Karasarides, M., Anande-Apte, B., and A. Wolfman (2001) A direct interaction between oncogenic Ha-Rasand phosphatidylinositol-3 kinase is not required for Ha-Ras-dependent transformation of epithelial cells. J.Biol. Chem. 276:39755-39764.Wolfman, J.C., Palmby, T., Der, C.J., and A. Wolfman (2002) Cellular N-Ras promotes cell survival bydownregulation of Jun N-terminal protein kinase and p38. Mol. Cell Biol. 22:1589-1606.83

The Department of Cell BiologyCell Biology Project Scientists with Independent FundingBarsanjit Mazumder Ph.D.The primary goal of my research has been to elucidate the posttranscriptionalcontrol of gene expression in monocytes and macrophages.These control mechanisms may have an important role in theendogenous cellular strategy to resolve inflammation by terminating theexpression of certain inflammatory gene products. Our studies showthat Interferon-gamma can induce ceruloplasmin an inflammatoryprotein in monocyte but the induction of ceruloplasmin is subject to aunique translational silencing. Recently we have identified a novelmechanism for the translational silencing, where Ribosomal proteinL13a was found to be the molecular switch of the mechanism. Studiesare ongoing to identify more target genes for this mechanism.Seng Hui Low, Ph.D.My research focuses on the mechanics and regulation ofthe terminal step of cell division. Cytokinesis is completedby the abscission of the midbody, a cytoplasmic bridgethat connects the two prospective daughter cells. We haveidentified two members of the SNARE membrane fusionmachinery, syntaxin 2 and endobrevin, that localize to themidbody during cytokinesis and whose function is requiredfor midbody abscission. Inhibition of these proteins leadsto failure of cell division. Understanding this fundamentalmechanism is especially important because it is requiredfor the proliferation of cancer cells, and may provide newdrug targets for cancer therapy.Eugene A. Podrez, M.D., Ph.D.I have a broad interest in the field of cell biology ofatherosclerosis. Recently, my research was focused onidentifying mechanisms of the recognition of modifiedlipoproteins by macrophages. We described a novelpathway for converting LDL into a proatherogenic formthat involves oxidative modification of LDL by monocytegeneratedreactive nitrogen species. We demonstrated thatmacrophage scavenger receptor CD36 is responsible for theformation of foam cells in vitro and in vivo. Finally, wehave identified a novel family of oxidized cholineglycerophospholipids that serve as ligands for CD36.Currently, my focus is on the elucidation of the biologicalrole of novel ligands for CD36 in atherosclerosis andthrombosis.84

Immunology

DEPARTMENTOF IMMUNOLOGYCHAIRMANThomas A. Hamilton, Ph.D.STAFFRobert L. Fairchild, Ph.D.James H. Finke, Ph.D.Andrew Larner, M.D., Ph.D.Dennis J. Stuehr, Ph.D.Vincent Tuohy, Ph.D.ASSOCIATE STAFFCharles L. Bevins, M.D., Ph.D.Peter Heeger, M.D.ASSISTANT STAFFMark Aronica, M.D.Xiaoxia Li, Ph.D.PROJECT SCIENTISTSKulwant Aulak, Ph.D.Sudip Bandyopadhyay, Ph.D.Roopa Biswas, Ph.D.Alana Majors, Ph.D.Charles S. Tannenbaum, Ph.D.Julie M. Tebo, Ph.D.Anna Valujskikh, Ph.D.Chin-Chuan Wei, Ph.D.RESEARCH ASSOCIATESAnton Gorbachev, Ph.D.Zhengfan Jiang, Ph.DZhigiang Wang, Ph.D.Koustubh Panda, Ph.D.Continued on Page 87The Department of Immunology has six fullStaff members (Drs. Thomas Hamilton, JamesFinke, Robert Fairchild, Andrew Larner,Dennis Stuehr, and Vincent Tuohy), two AssociateStaff (Drs. Charles Bevins and Peter Heeger) and twoAssistant Staff (Drs. Xiaoxia Li and MayumiNaramura). In addition, eight investigators holdprimary departmental appointments in clinicaldivisions. Of these, three (Mark Aronica [Pulmonaryand Critical Care Medicine], Scott Strong [ColorectalSurgery] and Anthony Stallion [Pediatric Surgery])have active laboratory research programs housed inthe Department of Immunology. The remaining five(Drs. Ashok Agarwal [Urological Institute], RonaldBukowski [Taussig Cancer Center, Hematology-Oncology], Peter Cohen [Center for SurgeryResearch], Victor Perez [Cole Eye Institute] andRaymond Tubbs and Belinda Yen-Lieberman [bothof Clinical Pathology]) participate in substantialThe Department of ImmunologyInteractive Programs Aim to DefineInteractions Between Adaptive andInnate Immune Systemscollaborative interactions with colleagues holdingprimary appointments in Immunology and therebyserve as important liaisons between the laboratorybench and the clinical setting.Research programs within the department arepursued within two broad themes. The first includesstudies of T-cell development and function at thecellular and molecular levels, using a variety ofdisease models. Areas of focus include the recruitmentand function of specific T cells at sites of organtransplantation, the development and evolution ofspecific T-cell populations in autoimmune disease,and the suppression of T-cell responses in cancerpatients.The second theme centers on the regulationand function of the innate immune system. Specificprograms include the regulation of cytokine signalingand gene expression, the control of inflammatoryresponses in a range of disease models, the biochemistryand enzymology of nitric oxidesynthases, and the identification andcharacterization of mammalian antimicrobialpeptide genes.These broad themes provideremarkable opportunities forinteraction among the individuallaboratories and together represent aconcept that integrates the departmentas a whole: the interface betweeninnate and acquired immunity and theresulting complexity of immunemediatedinflammation. This conceptappears to be highly relevant in aspectrum of clinically importantentities that have either acute orchronic inflammation as a keycausative or symptomatic feature ofpathogenesis.Continued on Page 87Thomas A. Hamilton, Ph.D.86

The Department of ImmunologyContinued from Page 86Recent highlights include the followingobservations and discoveries:• Reprogramming the autoimmune responseby genetic modification so that an inflammatorydisease-inducing response may be shifted to an antiinflammatorydisease-inhibiting response (Tuohylaboratory).• The latent activity of human defensins inthe intestinal mucosa is unmasked via post-translationalprocessing of pro-defensins by action of aserine protease; definition of critical in vivo roles ofepithelial defensins in mammalian host defense(Bevins laboratory).• Identification of the role fortetrahydrobiopterin in nitric oxide synthesis throughthe regulation of oxygen activation in the enzyme(Stuehr laboratory).• Cell type specific regulation of functionallysimilar chemokines provides distinct patterns ofchemokine expression in vivo at sites of inflammation(Hamilton laboratory).• The importance of Tyk2 kinase inmediating regulation of innate immunity in responseto type I interferons (Larner laboratory).• Human renal tumors promote apopotosisin T cells through the action of gangliosides andreactive oxygen species generation, thus promotingimmune dysfunction in cancer patients (Finkelaboratory).• IL-1 receptor-associated kinase (IRAK)serves as a scaffold for assembly of the signalingcomplex during the activation of NFκB (Li laboratory).• The innate immune system regulatesantigen-primed T-cell infiltration of allografts andsites of contact hypersensitivity in the skin (Fairchildlaboratory).• Indirectly primed CD8+ T cells are aprominent component of the allogeneic T-cellrepertoire following skin graft rejection in mice(Heeger laboratory).Continued from Page 86JOINT APPOINTMENTSAshok Agarwal, M.D. 1Mark Aronica, M.D. 2Ronald M. Bukowski, M.D. 3Peter Cohen, M.D. 4Victor Perez, M.D. 5Gregory Plautz, M.D. 4Maria Siemienow, M.D., Ph.D. 6Anthony Stallion, M.D. 7Scott A. Strong, M.D. 8Raymond Tubbs, D.O. 9Belinda Yen-Lieberman, Ph.D. 91Andrology Lab, Glickman UrologicalInst., CCF2Dept. of Pulm./Crit. Care Med.,CCF3Taussig Cancer Ctr., CCF4Ctr. for Surg. Res., CCF5Cole Eye Inst., CCF6Dept. of Plastic/ReconstructiveSurgery, CCF7Dept. of Pediatrics, CCF8Dept. of Colorectal Surg, CCF9Dept. of Clin. Pathology, CCFDept. website: http://www.lerner.ccf.org/immuno/ADJUNCT APPOINTMENTSValentin Gogonea, Ph.D. 1Michael Lamm, M.D. 2Donald Lindmark, Ph.D. 3Tobili Y. Sam-Yellowe, Ph.D. 31Dept. of Chemistry, Penn. StateUniv., University Park, PA2Dept. of Pathology, CaseWestern Reserve Univ.,Cleveland, OH3Dept. of Biol., and Environ’l.Sci., Cleveland State Univ.,Cleveland, OH87

THE ARONICALABORATORYSENIOR RESEARCH TECHNOLOGISTShadi SwaidaniCOLLABORATORSMark R. Boothby, M.D., Ph.D. 1R. Stokes Peebles, M.D. 21Dept. of Microbiology andImmunology, Vanderbilt Univ.,Nashville, TN2Dept. of Allergy, Pulmonary andCritical Care Med., VanderbiltUniv., Nashville, TNAn individual’s susceptibility to allergy, asthmaand autoimmune disease is influenced byresponses of the white blood cells toactivation. In particular, the regulation of the subsetsof T lymphocytes called Th1 and Th2 cells isimportant in these processes.Type 1 T-cell effectors activatemacrophages and are important inthe development of delayed-typehypersensitivity (DTH), in partthrough the production of IFN-γand TGF-β. In contrast, type 2effector cells serve as mediatorsof the humoral response andinduce IgE- and eosinophilmediatedreactions through theproduction of IL-4, IL-5 and IL-13. Type 2 responses are thoughtto be important in limiting orinhibiting type 1 inflammatoryresponses and may play a role insuppressing autoimmune diseases.A wealth of data from mice andhumans has led to the recognitionof T cells and cytokines characteristicof type 2 T cells as crucialpathogenic components in allergicdiseases. So many known featuresof allergic processes can beproduced by Th2-dependentcytokines that asthma has come tobe viewed as a Th2 disease. In contrast to this simplemodel, recent data suggest a role for Th1 cells as well.Transcription Factors and Regulation of Type 1and Type 2 T-Cell ResponsesWe have shown that inhibition of NF-κ/Reltargeted specifically to the T lineage selectivelyAronica, M.A., Mora, A.L., Mitchell, D.B., Finn, P.W., Johnson, J.E., Sheller, J.R., andM.R. Boothby (1999) Preferential role for NF-kB/Rel signaling in the type 1 but nottype 2 T cell-dependent immune response in vivo. J. Immunol. 163:5116-5124.Aronica, M.A., Goenka, S., and M. Boothby (2000) IL-4-dependent induction of BCL-2and BCL-X(L)IN activated T lymphocytes through a STAT6- and pi 3-kinase-independentpathway. Cytokine 12:578-587.Boothby, M., Mora, A.L., Aronica, M.A., Youn, J., Sheller, J.R., Goenka, S., and L.Stephenson (2001) IL-4 signaling, gene transcription regulation, and the control ofeffector T cells. Immunol. Res. 23:179-191.Boothby, M., and M.A. Aronica (2002) Transcription regulation, allergic responses, andasthma. Allergy Immunol. Clin. N. Am. (In press),Corn, R.A., Aronica, M.A., Zhang, F., et al. (2003) T cell-intrinsic requirement for NFkBinduction in postdifferentiation IFN-g production and clonal expansion in a Th1response. J. Immunol. 171:1816-1824.The Department of ImmunologyRole of T Lymphocytes and T-Cell Memory inthe Development of Airway Inflammationand AsthmaMark A. Aronica, M.D.impairs development of Th1 responses only, withnormal development of Th2 responses, as measuredby cytokine production and antigen-specific IgEproduction. Despite evidence of an adequate Th2response, there is only a modest influx of eosinophilsin a model of allergic pulmonaryinflammation (asthma), providingadditional support that Th1 cells needto collaborate with Th2 cells in thedevelopment of asthma. Usingtransgenic and knockout mice withimpaired Th1 function, we hope tobetter delineate the role of Th1 cellsin the development of asthma.Susceptibility to Asthma: A Linkwith the Development ofAntigen-Experienced/Memory-Phenotype CellsDespite the known importanceof immunologic memory inprotection from viral and bacterialpathogens, little is known about therole of memory in allergy. Therefore,we have developed a model toinvestigate whether memory T-cellresponses are important for thedevelopment of allergic diseases.To investigate the role ofmemory T cells in the developmentof allergic pulmonary inflammationand airway hyperresponsiveness (AHR), we havedeveloped a novel mouse model. In this system, T-cell receptor (TCR)-transgenic CD4 + effector cells aregenerated in vitro and then transferred into naïverecipients. After waiting until the effectors have beendemonstrated to resume quiescence and haveacquired memory status, recipient mice are exposedto aeroallergen. Using this system, we have shownthat antigen-specific memory Th2 cells are sufficientto lead to the development of AHR and allergicpulmonary inflammation. Interestingly, if Th1 cellsare transferred and resume quiescence, they are notrecruited to the lung upon antigen exposure. Usingthis model, we hope to better understand mechanismsregulating the trafficking of Th1 and Th2 memorycells.88

The Department of ImmunologyDefensins Pose a First DefenseAgainst PathogensThe recent isolation and characterization ofantimicrobial peptides have unveiled apreviously unrecognized component of animalhost defense. These peptidesexhibit a broad spectrum ofantimicrobial activity in relationto bacteria, fungi and certainviruses. The peptides have beenisolated from diverse speciesthroughout the plant and animalkingdoms.Recent work from ourlaboratory has revealed thatthese peptides are abundantlyexpressed at wet mucosalsurfaces of humans and othermammals. Our long-range goalis to understand how theseepithelial antimicrobial peptidescontribute to mucosal hostdefense and to characterize thepathophysiology that resultsfrom altered expression of thesepeptides.Although the epithelia ofmammalian mucosal tissues were once regarded as aprimarily passive barrier to noxious agents, our recentwork supports a model: Induction of antimicrobialpeptide expression by bacterial and pro-inflammatorystimuli constitutes part of an active, early hostdefense response of challenged epithelial cells.Charles L. Bevins, M.D., Ph.D.A major interest of this laboratory isantimicrobial peptides of the mammalian intestine.Previously, we identified two human defensinsexpressed at high levels in thePaneth cells of the smallintestine. Work in our laboratoryis better defining the role ofthese (and similar) entericpeptides in human host defenseand in disease processes,including inflammatory boweldisease (IBD). These investigationswill serve as a basis forbuilding an interactive bridgingprogram, linking basic sciencestudies in the Lerner ResearchInstitute with the resources ofCCF’s Center for InflammatoryBowel Disease.Our current studiesinclude: (i) characterizing theprimary structure and biologicalactivity of the tissue forms ofenteric defensins, (ii) defining thekey regulatory steps for theexpression of these molecules, and (iii) exploringpotential mechanisms of therapeutic modulationof these systems. The investigations includebiochemical and molecular biological approachesand analysis of transgenic models.THE BEVINSLABORATORYFELLOWSScott Howell, Ph.D.Bo Shen, M.D.Aurelie Tasiemski, Ph.DTECHNICAL ASSOCIATEDennis Wilk, B.S.COLLABORATORSJohn Crabb, Ph.D. 1Gill Diamond, Ph.D. 2Judy Drazba, Ph.D. 3Tomas Ganz, M.D., Ph.D. 4Paul McCray, M.D. 5Edith Martin Porter, M.D. 4Nita Salzman, M.D., Ph.D. 6Satya Yadav, Ph.D. 71Cole Eye Inst., CCF2UMDNJ Med. School, Newark,NJ3Imaging Core, CCF4UCLA Med. School, LosAngeles, CA5Univ. of Iowa Coll. of Med.,Iowa City6Med. Coll. of Wisconsin,Madison7Biotechnology Core, CCFJia, H.-P., Wowk, S.A., Schutte, B.C., Lee, S.K., Vivado, A., Tack, B.F., Bevins, C.L., and P.B. McCray, Jr. (2000) Anovel murine β-defensin expressed in tongue, esophagus and trachea. J. Biol. Chem. 275:33314-33320.Diamond, G., Kaiser, V., Rhodes, J., Russell, J., and C. Bevins (2000) Transcriptional regulation of β-defensin geneexpression in tracheal epithelial cells. Infect. Immun. 68:113-119.Ghosh, D., Porter, E., Shen, B., Lee, S.K., Wilk, D., Drazba, J., Yadav, S.P., Crabb, J.W., Ganz, T., and C.L.Bevins (2002) Paneth cell trypsin is the processing enzyme for human defensin-5. Nature Immunol. 3:583-590.Bevins, C.L., and G. Diamond (2002) Mammalian antimicrobial peptides. In: Dutton, C.J., Haxell, M.A., McArthur,H.A.I., and R.G. Wax, eds. Peptide Antibiotics: Discovery, Modes of Action, and Applications. New York: MarcelDekker, pp. 145-192.Ghosh, D., Porter, E., Shen, B., Lee, S.K., Wilk, D., Drazba, J., Yadav, S.P., Crabb, J.W., Ganz, T., and C.L.Bevins (2002) Paneth cell trypsin is the processing enzyme for human defensin-5. Nat. Immunol. 3:583-590.Bevins, C.L. (2003) Antimicrobial peptides as effector molecules of mammalian host defense. Contrib. Microbiol.10:106-48.Salzman, N.H., Ghosh, D., Huttner, K.M., Paterson, Y., and C.L. Bevins (2003) Protection against entericsalmonellosis in transgenic mice expressing a human intestinal defensin. Nature 422:522-526.89

THE FAIRCHILDLABORATORYPOSTDOCTORAL INVESTIGATORSHiroyuki Amano, M.D.Motoo Araki, M.D.Anton Gorbachev, Ph.D.Eun-jie Oh, M.D.Qi-Wei Zhang, M.D., Ph.D.GRADUATE STUDENTTarek El-SawyRESEARCH TECHNOLOGISTSMichael Auerbach, B.S.Danielle Kish, B.S.COLLABORATORSSuneel Apte, M.B.B.S., D. Phil. 1Joshua Farber, Ph.D. 2Stuart Flechner, M.D. 3David Goldfarb, M.D. 3Peter Heeger, M.D. 4Venkatesh Krishnamurthi, M.D. 3Charles Modlin, M.D. 3Andrew Novick, M.D. 3Daniel Remick, M.D. 5Randall Starling, M.D. 6Robert Strieter, M.D. 7Mohamed Yamani, M.D. 6James Young, M.D. 61Dept. of Biomedical Engineering,CCF2NIAID, NIH, Bethesda, MD3Urological Inst., CCF4Dept. of Immunology, CCF5Dept. of Pathology, Univ. ofMichigan, Ann Arbor6Dept. of CardiovascularMedicine, CCF7Div. of Pulmonary and CriticalCare Medicine, UCLA, LosAngeles90Mechanisms of T-Lymphocyte-MediatedInflammation in Skin Diseaseand TransplantationThe focus of this laboratory is to definemechanisms involved in T-cell-mediatedinflammation in skin disease and during therejection of transplanted allografts. Based on resultsfrom our laboratory, we have developed the hypothesisthat T-cell responses are preceded by inflammatoryprocesses that are mediated by innate componentsof the immune response and that the innatecomponents regulate the magnitude and duration ofthe T-cell response.One model we have been using to study theinflammatory T cells involves the priming andactivity of T cells in responseto epidermal application ofhaptens. Challenge of haptensensitizedanimals results in animmune response contacthypersensitivity (CHS), acommon dermatologicalproblem (e.g., poison ivy). Wehave demonstrated the effectorrole of CD8 + T cells and theregulatory role of CD4 + T cellsin this immune response.Recent studies have indicatedthat CD4 + T cells mediate thisregulation by killing off thehapten-presenting dendritic cellsthat carry the hapten from theskin to the draining lymphoidtissues where the T cells are.This killing restricts the windowof time that the effector CD8 +T cells can be primed to the hapten and limits thesize of the reactive T cell compartment.Recruitment of the primed effector CD8 + Tcells to the antigen challenge site is controlled by theproduction of chemoattractant cytokines, orchemokines, such as Groα. This recruitment and theGorbachev, A.V., Heeger, P.S., and R.L. Fairchild (2001) CD4+ and CD8+ T cell primingfor contact hypersensitivity occurs independently of CD40-CD40L interactions. J.Immunol. 166:2323-2332.Baldwin, W.W., Fairchild, R.L., and C.P. Larsen (2001) Innate immune responses totransplants: a significant variable with cadaver donors. Immunity 14:369-376.el-Sawy, T., Fahmy, N.M., and R.L. Fairchild (2002) Chemokines: directing leukocyteinfiltration into allografts. Curr. Opin. Immunol. 14:562-568.Gorbachev, A.V., and R.L. Fairchild (2002) Mechanisms of T cell priming for contacthypersensitivity. Crit. Rev. Immunol. 21:451-472.Kobayashi, H., Novick, A.C., Toma, H., Fairchild (2002) Chronic antagonism of Miginhibits cellular infiltration and promotes survival of class II MHC disparate skinallografts. Transplantation 74:387-395.Fahmy, N.M., Yamani, M.H., Starling, R.C., Ratliff, N.B., Young, J.B., McCarthy, P.M.,Feng, J., Novick, A.C., and R.L. Fairchild (2003) Chemokine and chemokine receptorThe Department of ImmunologyCHS response is blocked by treatment with Groαantibodies. Groα does not directly recruit effectorCD8 + T-cells to the challenge site but recruitsneutrophils that are, in turn, stimulated to produce (asyet) unidentified chemoattractants for the T-cells.Depletion of neutrophils or blocking their functionalso inhibits effector T-cell recruitment into thechallenge site and the CHS response. Althoughprimed T-cell recruitment and the CHS response arenot observed to low doses of antigen challenge,manipulating neutrophil infiltration into the challengesite results in T-cell infiltration and the CHSresponse. These studies emphasizethe critical role of innate immunecomponents on the recruitment andactivities of antigen-specific T cells.These studies have also demonstratedthe ability to manipulate T-cell-mediated immune responsesthrough the innate immune system.The second model used tostudy T-cell-mediated inflammationis allogeneic tissue transplantation.The recruitment of alloantigenspecificT-cells to the graft siteinitiates allograft rejection. Thefactors directing alloantigen-primedT-cells to the allograft duringrejection are poorly defined. Duringrejection of skin and heart allograftsRobert L. Fairchild, Ph.D. in mice, chemokines produced earlyfollowing transplantation mediateneutrophil and macrophagerecruitment to the allograft. As this productionsubsides, chemokines that mediate alloantigenprimedT-cell recruitment are produced. We aretesting the ability of anti-chemokine antibodies todelay or abrogate allograft rejection. In a murineheart allograft model, recipient treatment withantibodies to an IFN-γ-induced chemokine, Mig,inhibits T-cell recruitment into the allograft andacute graft rejection. In situ hybridization studieshave demonstrated that Mig is derived from bothdonor and recipient. Current experiments areutilizing Mig-deficient heart allograft donors and/or recipients to further test the role of thisrecruiting factor in acute and chronic rejection andto define the Mig mediated mechanisms directingalloantigen-primed T cells into allografts. Similar toCHS, antagonism of innate immune components inheart allograft recipients attenuates T-cell graftinfiltration and acute rejection. We are participatingin a study with both the cardiac and renal transplantteams at the Cleveland Clinic to investigatethese aspects of the immune response in clinicaltransplantation. The understanding of thesemechanisms is essential to the design of practicalclinical strategies to maintain allograft acceptancein transplant recipients.

The Department of ImmunologyTumor-induced Apoptosis of T-Cells;A Mechanism of Tumor EvasionTHE FINKELABORATORYRESEARCH SCIENTISTCharles S. Tannenbaum, Ph.D.Depressed T-cell function, resulting inineffective anti-tumor responses, iscommon among cancer patients. Thetumor microenvironment has a deleterious effecton tumor-infiltrating lymphocytes (TILs), althoughthe immunosuppressive effects extend into theperiphery as well. TILs from patients with renal cellcarcinoma (RCC) are impaired in their ability toproliferate and mediate anti-tumor cytotoxicactivity.We hypothesize that tumors evade theimmune system by inhibiting TIL signal transductionand increasing T cells’sensitivity to apoptosis. Thisincreased sensitivity may bea barrier to the developmentof an effectiveimmune response to tumors.This idea is supported byour observation that inapproximately half of RCCtumors, as many as 5-15%of the TILs are apoptotic insitu. Even T cells within thetumor bed that appear viableare actually compromised, asthey are easily induced toundergo activation-inducedcell death (AICD) ifstimulated in vitro. Peripheralblood T cells isolated from RCC patients are alsosusceptible to AICD, suggesting that tumor-derivedproducts can trigger the heightened T-cell susceptibilityto apoptosis and AICD. Indeed, solubleproducts from approximately 50% of explanted, invitro cultured renal tumors directly induce apoptosisof T cells, whereas supernatants from other RCCtumors sensitize T cells to AICD.Our recent findings implicate gangliosideswithin the tumor supernatants as one class ofproducts that can induce both phenotypes in Tcells. Gangliosides isolated from some RCCsupernatants induce apoptosis; those from otherRCC supernatants only sensitize T cells to AICD.Aldehyde products of fatty acid oxidation, likelyresulting from oxidative stress within the tumormicroenvironment, represent another class ofproducts within tumor supernatants that caninduce apoptosis in T cells. Among the mostprominent aldehyde product is 4-hydroxynonenal(HNE). We are testing whether the heterogeneouscapacity of RCC to directly induce apoptosis of Tcells, or sensitize them to AICD, is related to thevariations in the specific products expressed byindividual tumors. We are also testing if specificstructural features of these molecules dictatewhether individual products induce apoptosis orsensitize T cells to AICD. Another major goal is todefine the mechanism by which gangliosides andHNE sensitize and/or induce apoptosis in T cells.James H. Finke, Ph.D.Gangliosides, as well as HNE, can alsoinhibit activation of the transcription factorNFkB. The ability of these products to inhibitNFkB activity may increase the sensitivity of Tcells to apoptosis, since NFkB regulates transcriptionof several key anti-apoptotic genes (Bfl-A1,Bcl-2, XIAP, cIAP1 and cIAP2). Impairment inNFkB activation resulting in reduced expression ofanti-apoptotic genes and increased sensitivity toapoptosis may hinder the development of T-cellimmunity in cancer patients. Studies are under wayto define how tumor-derived products inhibitNFkB activation and how thisaffects the expression of antiapoptoticgenes.We are also testing if thereis cooperation between gangliosidesand death-receptor ligandsexpressed on tumor cells ininitiating T-cell apoptosis. Wefound that in the absence ofcaspase-8 and FADD, Jurkat Tcells are significantly protectedfrom SK-RC-45-mediatedapoptosis. Blocking gangliosidesynthesis with SK-RC-45 alsosignificantly reduced that line’sability to induce T-cell apoptosis.These findings and others suggestthat SK-RC-45-mediatedapoptosis of T cells involves both receptor ligandsand gangliosides expressed by the tumor. Experimentsare in progress to examine the interactionbetween gangliosides and death ligands in promotingT-cell apoptosis.Studies to elucidate the mechanismsresponsible for these abnormalities may facilitatethe future development of new strategies to blockor bypass these signaling defects and restore antitumorT- cell immunity.RESIDENTGeath Al-Atrash, M.D., Ph.D.Dept. of Internal Medicine, CCFPOSTDOCTORAL FELLOWSJustin Albani, M.D.Ithaar Derweesh, M.D.Gira Raval, Ph.D.TECHNICAL ASSOCIATESPatricia A. Rayman, M.S.Cynthia Combs, B.S.Christina Moon, B.S.Amy Richmond, B.S.Mark Thornton, B.S.COLLABORATORSRonald M. Bukowski, M.D. 1Inderbir Gill, M.D. 2Eric D. Hsi, M.D. 3Andrew C. Novick, M.D. 3Walter Storkus, Ph.D. 41Experimental Therapeutics,Taussig Cancer Ctr., CCF2Urological Inst., CCF3Dept. of Clinical Pathology, CCF4Univ. of Pittsburgh, Pittsburgh, PAUzzo, R., Rayman, P., Kolenko, V., Clark, P.E., Cathcart, M.K., Bloom, T., Novick, A.C.,Bukowski, R.M., Hamilton, T. and J.H. Finke (1999) Renal cell carcinoma-derivedgangliosides suppress nuclear factor-kB activation in T cells. J. Clin. Invest. 104:769-776.Finke, J.H., Rayman, P., George, R., Uzzo, R., Tannenbaum, C.S., Kolenko, V., Novick,A.C., and R.M. Bukowski (2001) Tumor induced sensitivity to apoptosis in T cells frompatients with renal cell carcinoma: role of NFkB suppression suppression. Clin. CancerRes. 7(3 suppl.):940s-946s.Tatsumi, T., Kierstead, L.S., Ranieri, E., Gesualdo, L., Schena, F.P., Finke, J.H.,Bukowski, R.M., Mueller-Berghaus, J., Kirkwood, J.M., Kwok, W.W., and W.J. Storkus(2003) Disease-associated bias in T helper type 1 (Th1)/Th2 CD4+ T cell responsesagainst MAGE-6 in HLA-DRB1*0401+ patients with renal cell carcinoma or melanoma. JExp. Med. 196:619-628.Kudo, D., Rayman, P., Horton, C., Cathcart, M., Bukowski, R.M., Thornton, M.,Tannenbaum, C., and J.H. Finke (2003) Gangliosides expressed by the renal cellcarcinoma cell line SK-RC-45 are involved in tumor-induced apoptosis of T cells. CancerRes. 63:1676-1683.Molto L, Rayman P, Paszkiewicz-Kozik E, Thornton M, Reese L, Thomas JC, Das T,Bukowski R, Finke J and Tannenbaum C. The Bcl-2 transgene protects T cells fromRCC-mediated apoptosis. (2003) Clinical Cancer Research, accepted.91

THE HAMILTONLABORATORYPROJECT SCIENTISTSRoopa Biswas, Ph.D.Julie Tebo, Ph.D.POSTDOCTORAL FELLOWSYalei Dai, Ph.D.Shyamasree Datta, Ph.D.TECHNICAL ASSOCIATESAlison ChudykCarol CrooksJennifer MajorMichael NovotnyThe diversity of the inflammatory process stemsfrom multiple sources, including the collectionof cell types that participate, the nature andcomplexity of the inflammatory stimulus, thetransmembrane and intracellular signaling processesthat occur following stimulation, and the largenumber of independently regulated genes whoseexpression is subject to modulation during theprocess. Our research program is focused upondefining the molecularevents that control theexpression of induciblegenes during responsesto multiple forms ofstimulation. Thesestudies focus uponalterations in transcriptionaland posttranscriptionalactivitiesthat can producesignificant changes inlevels of inflammatorygene products.We have focusedattention on theregulation ofchemoattractant cytokine(chemokine) geneexpression in mononuclearphagocytes andother cell types thatparticipate in inflammatoryresponses. Thespecific genes we haveutilized include theinterferon-inducibleprotein (IP)-10 and themonokine induced by interferon-γ (Mig) chemokines,critical determinants of activated T-cell recruitmentto sites of antigen-driven immune response, and theELR-CXC chemokines KC and macrophageinflammatory protein-2 (MIP-2), which targetThe Department of ImmunologyRegulation of Inflammatory ChemokineGene ExpressionThomas A. Hamilton, Ph.D.neutrophils during the early inflammatory response toinjury and infection. In each case, the sequenceregions controlling transcription for each gene havebeen characterized, as have sequences within themRNA bodies that contribute to post-transcriptionalcontrol of mRNA stability.Although the mechanisms that increasechemokine gene expression are important, it is equallynecessary to provide negative regulation of theseinflammation-generatinggenes, as over-expressionhas the potential to producesubstantial tissue injury. Toexamine this process, wehave studied the mechanismsinvolved in suppressionof IFN-β- or LPSinducedgene expression byanti-inflammatory cytokines,including interleukin-4 and-10 and transforminggrowth factor-β. Someinhibitory effects aremediated at the level oftranscription and appear toinvolve the sequestration ofnecessary co-activators,whereas others involvemodulation of mRNAdecay.The goals of currentprojects include (1)determination of mechanismsassociated with IL-4mediated, STAT6 dependentand independent suppressionof inflammatory genetranscription, (2) the definition of mechanismsthrough which mRNA decay is achieved andregulated, and (3) the importance of specifictranscriptional and post-transcriptional mechanismsfor control of inflammatory gene expression in vivo.Hamilton, T.A., Ohmori, Y., and J. Tebo (2002) Regulation of chemokine expression by antiinflammatorycytokines. Immunol. Res. 25:229-245.Endlich, B., Armstrong, D., Brodsky, J., Novotny, M., and T.A. Hamilton (2002) Distinct temporal patterns ofmacrophage-inflammatory protein-2 and KC chemokine gene expression in surgical injury. J. Immunol.168:3586-3594.Toshchakov, V., Jones, B.W., Perera, P.-Y., Thomas, L., Cody, J., Williams, B., Major, J., Hamilton, T.A.,,Fenton, M.J., and S.N. Vogel (2002) TLR4, but not TLR2, mediates IFN-beta-induced STAT1alpha/betadependentgene expression in macrophages Nat. Immunol. 3:392-398.Major, J., Fletcher, J.E., and T.A. Hamilton (2002) IL-4 pretreatment selectively enhances cytokine andchemokine production in lipopolysaccharide-stimulated mouse peritoneal macrophages. J. Immunol. 168:2456-2463.Tebo, J., Der, S., Frevel, M., Khabar, K.S., Williams, B.R., and T.A. Hamilton (2003) Heterogeneity in controlof mRNA stability by AU rich elements. J. Biol. Chem. 278:12085-12093.92

Our laboratory focuses on understanding thecellular and molecular immunologic eventsinvolved in rejection and tolerance ofallogeneic organ grafts. Despite significant progressin the development of medications and therapiescapable of preventing rejection of organ transplants,acute rejection episodes,with their concomitantmorbidities, remain a clinicalproblem. Perhaps moreimportantly, chronic rejectionleads to organ failure in a largeproportion of transplantrecipients 5-10 years posttransplant surgery.Our laboratory usesanimal models to delineate themechanisms involved in acuteand chronic transplant rejectionand to further determine themechanisms that mediatetolerance to transplanted organs.We have an additional interest indeveloping and testing clinicallyrelevant, immune-basedprocedures for identifyinghuman organ transplant recipients at high risk ofincipient organ failure.Studies funded through the NationalInstitutes of Health (NIH) evaluate mechanisms ofallograft rejection in mouse models using skin andheterotopic cardiac allografts, with a particularemphasis on indirect allorecognition by T cells. Wehave demonstrated that T cells responding todonor-derived peptides expressed by recipientantigen-presenting cells can mediate a chronic formof skin graft rejection, despite an inability torecognize any antigen expressed on the graft cellsthemselves. Studies in a model of heart transplantationsuggest that this “indirect pathway” is animportant mediator of graft fibrosis and chronicrejection. Ongoing studies are focused onunderstanding the molecular mechanisms of theseeffects so as to be able to ultimately design specifictherapies for preventing or treating this disease. Aspart of these studies, we have developed andcharacterized a state-of-the-art, computer-assistedELISPOT assay, capable of detecting cytokineproduction by individual alloreactive T lymphocytes.A related project in the laboratory is focusedon understanding mechanisms of induction andmaintenance of allograft tolerance. Immunetolerance is a condition in which the recipientaccepts the donor organ in the absence ofimmunosuppressive medications, has no pathologicimmune reactivity directed towards the transplant,and has an otherwise intact immune system. Wehave developed several well-characterized animalThe Department of ImmunologyCellular and Molecular Mechanismsof Rejection and Toleranceto Allogeneic Transplanted OrgansPeter Heeger, M.D.models in which to study immune tolerance toorgan transplants. Our findings show that theimmunosuppressive cytokine TGF-β is an essentialmediator of tolerance under selected conditions.We have further shown that the ability to inducetolerance to a transplanted organ is in partdependent on the activation state(naïve, effector or memory) of thealloreactive T cells prior to placementof the graft. These latterfindings have important implicationsfor attempts at inducing tolerance inhumans, as human peripheral T-cellrepertoires often contain memoryalloreactive T cells presumablyprimed through previous environmentalexposure to cross-reactiveantigens.Our laboratory is alsoinvolved in several clinically basedstudies, funded through the NIH, inwhich we are testing ELISPOTbasedimmunologic monitoring ofhuman organ transplant recipientsusing peripheral blood samples. Ourinitial findings reveal that thisapproach provides a useful measure of the antidonorimmune response in a recipient and that theresults can provide supplemental predictiveinformation about the risk of clinical rejection posttransplant. Ongoing studies will determine whetherELISPOT-based monitoring can be used clinicallyto predict incipient rejection and to adjust immunosuppressivemedications.THE HEEGERLABORATORYPROJECT SCIENTISTAnna Valujskikh, Ph.D.POSTDOCTORAL FELLOWSYifa Chen, M.D.Chunshui He, M.D.LEAD TECHNOLOGISTEarla Biekert, B.S.TECHNOLOGISTSAlla Gomer, B.S.Jocelyn Riley, B.S.Michael Clemente, B.A.Heeger, P.S., Greenspan, N.S., Kuhlenschmidt, S., Dejelo, C., Hricik, D.E., Schulak, J.A.,and M. Tary-Lehmann (1999) Pretransplant frequency of donor-specific, interferongammaproducing lymphocytes is a manifestation of immunologic memory and correlateswith the risk of post transplant kidney rejection episodes. J. Immunol. 163:2267-2275.Valujskikh, A., Lantz, O., Celli, S., Matzinger, P., and P.S. Heeger (2002) Cross-primedCD8+ T cells mediate graft rejection via a distinct effector pathway. Nature Immunol.3:844-851.Illigens, B.M., Yamada, A., Fedoseyeva, E.V., Anosova, N., Boisgerault, F., Valujskikh,A., Heeger, P.S., Sayegh, M.H., Boehm, B., and G. Benichou G. (2002) The relativecontribution of direct and indirect antigen recognition pathways to the alloresponse andgraft rejection depends upon the nature of the transplant. Hum. Immunol. 63:912-925.Gebauer, B.S., Hricik, D.E., Atallah, A., Bryan, K., Riley, J., Tary-Lehmann, M.,Greenspan, N.S., Dejelo, C., Boehm, B.O., Hering, B.J., and P.S. Heeger (2002) Evolutionof the enzyme linked immunosorbent spot assay for post-transplant alloreactivity as apotentially useful immune monitoring tool. Am. J. Transplant. 2:857-866.Pantenburg, B., Heinzel, F., Das, L., Heeger, P.S., and A. Valujskikh (2002) T cells primedby Leishmania major infection cross-react with alloantigens and alter the course of allograftrejection. J. Immunol. 169:3686-3693.Heeger, P.S. (2003) Allorecognition pathways and transplant rejection: a summary andupdate. Am. J. Transplant. 3:525-533.93

THE LARNERLABORATORYPOSTDOCTORAL FELLOWMaria Navarro, Ph.D.The Department of ImmunologySignaling Cascades that Regulate theBiological Actions of InterferonsGRADUATE STUDENTSRamesh PotlaJinzhong QinMy laboratory is interested in the signalingcascades regulated by interferons and howthese various cascades are responsible forthe biological actions of these cytokines. There arethree main efforts ongoing in the laboratory.One project examines the role of tyrosinekinases and phosphatases that have traditionallybeen associated with signaling through the T-cellreceptor in the antiproliferative actions ofinterferons. Another project examines themechanisms by which interferons stimulateprogrammed cell death. The third project, justinitiated, will examine the signaling cascadesrequired for interferons to downregulate cellularRNAs both transcriptionally andposttranscriptionally.Andrew C. Larner, M.D., Ph.D.Petricoin, E.F. III, Ito, S., Williams, B.L., Audet, S., Stancato, L.F., Gamero, A., Clouse, K., Grimley, P.,Weiss, A., Beeler, J., Finbloom, D.S., Shores, E.W., Abraham, R., and A.C. Larner (1997) Antiproliferativeaction of IFN-alpha requires components of T-cell receptor signaling. Nature 390:629-632.Stancato, L.F., Yu, C.-R., Petricoin, E.F., and A.C. Larner (1998) Activation of Raf-1 by interferon-gammaand oncostatin M requires expression of the Stat1 transcription factor. J. Biol. Chem. 273:18701-18704.Gamero, A.M., and A.C. Larner (2000) Signaling via the T-cell antigen receptor induces phosphorylation ofStat1 on serine 727. J. Biol. Chem. 275:16574-16578.Larner, A.C., and A. Keightley (2000) The Jak/Stat signaling cascade: its role in the biological effects ofinterferons. In: Gudkind, J.S., ed. Signaling Networks and Cell Cycle Control: The Molecular Basis ofCancer and Other Diseases. Totowa, NJ: Humana Press, Inc., pp. 393-409.Dong F, Gutkind, S.J., and A.C. Larner (2001) Granulocyte colony-stimulating factor Induces Erk5 activationwhich is differentially regulated by protein tyrosine kinases and protein kinase C and is involved inthe regulation of cell proliferation and survival. J. Biol. Chem. 276:10811-10816.Gamero, A.M., and A,C. Larner (2001) Vanadate stimulates interferon-mediated apoptosis that is dependenton the Jak/Stat pathway. J. Biol. Chem. 276:13547-13553.94

The Department of ImmunologyGenetic Analysis of IL-1 Signaling andRegulation of NFκB ActivationAprimary interest in my laboratory is thestudy of interleukin-1 (IL-1)-mediatedsignaling. IL-1 is a major pro-inflammatorycytokine with a wide range of biological activitiesin inflammation. It functionsmainly by activating proteinkinase cascades, leading to theactivation of nuclear factorκB(NFκB) and Jun kinase,which in turn phosphorylatesand activates activationtranscription factor (ATF)and activator protein-1 (AP-1). The IL-1 receptor belongsto the IL-1 receptor/Toll-likereceptor (IL-1R/TLR)superfamily. Human TLRshave recently emerged as keycomponents in the generationof immune and inflammatoryresponses due to their abilityto recognize pathogen-associatedmolecules. Tremendousefforts have beendevoted to understanding themolecular mechanisms bywhich IL-1Rs/TLRs mediatesignaling, with the long-termobjective of developing moreeffective anti-inflammatory small-molecule drugs.We have previously taken a genetic approachto studying IL-1-dependent signaling pathways,using random mutagenesis to generate IL-1-unresponsive cell lines lacking specific componentsof the pathways. Employing the siRNA interferenceapproach in cultured human cells, we havealso successfully abolished the expression ofsignaling components of the pathway. The resultingmutant cell lines have provided us with a uniqueopportunity to study the IL-1R-mediated signalingpathway. We are now focusing on the key stepsthat govern the receptor proximal signaling eventsof the IL-1 pathway, including the IL-1-inducedformation and dissociation of the receptor complexand the translocation and activation of transforminggrowth factor-β-activated kinase 1 in responseto IL-1 stimulation.We recently identified a novel member ofthe IL-1R/TLR superfamily, named Sigirr. Sigirr -/-mice show an enhanced inflammatory state uponstimulation with IL-1 and various Toll ligands,including lipopolysaccharide, CpG DNA anddsRNA, suggesting that Sigirr might be a negativeregulator for the IL-1R/TLR-mediated pathway.Upon stimulation with IL-1, Sigirr interacts withIL-1 receptor, IL-1R-associated kinase and TNFreceptor-associated factor-6, suggesting that Sigirrprobably functions through its interaction with thereceptor complex. We are investigating how Sigirrnegatively regulates the IL-1 pathway via itsXiaoxia Li, Ph.D.interaction with the IL-1 receptor complex.We are also interested in the signalingpathways mediated by the TNF receptor (TNFR)superfamily, whose members exert many importantbiological functions,including cell proliferation/differentiation, inflammation,immune responses, andhomeostasis. These TNFRfamily members functionmainly by activating proteinkinases cascades, leading tothe activation of transcriptionfactors, includingNFκB, ATF and AP-1.Although much effort hasbeen made toward understandingthe molecularmechanisms of signaltransduction mediated bythese receptors, manyquestions still remain. Werecently identified a noveladapter molecule, NFκBactivator 1 (Act1), thatfunctions in signalingpathways mediated by asubset of TNFR familymembers, including CD40and lymphotoxin-β receptor. We are particularlyinterested in the CD40-mediated pathway, since itis not only essential for normal immune responses,but is also associated with several pathologicalconditions of autoimmune and chronic inflammatorydiseases. We have generated Act1 -/- mice, anessential tool to investigate the physiologicalfunction of Act1.THE LI LABORATORYPOSTDOCTORAL FELLOWSZhenfang Jiang, Ph.D.Youcun Qian, Ph.D.TECHNICAL ASSISTANTGrace ChiGRADUATE STUDENTSJinzhong QinDave WaldCOLLABORATORSTim Bird, Ph.D. 1Robert Fairchild, Ph.D. 2Peter Heeger, Ph.D. 2Brian L. Kotzin, M.D. 3Kunihiro Mastumoto, M.D. 4Robert Rickert, Ph.D. 5George R. Stark, Ph.D. 6Paul D. Wightman, Ph.D. 71Immunex Corp., Seattle, WA2Dept. of Immunology, CCF3Depts. of Med. and Immunol.,Univ. of Colorado Health Sci.Ctr., Denver, CO4Dept. of Molec. Biology,Nagoya Univ., Nagoya, Japan5Div. of Biology and UCSDCancer Ctr., Univ. of California/SanDiego, La Jolla, CA6Dept. of Molec. Biology, CCF73M Pharmaceuticals, St.Paul, MNQian, Y., Zhao, Z., Jiang, Z., and X. Li (2002) Role of NFκB activator Act1 in CD40-mediated signaling in epithelial cells. Proc. Natl. Acad Sci. USA 99:9386-9391.Jiang, Z., Ninomiya-Tsuji, J., Qian, Y., Matsumoto, K., and X. Li (2002) Interleukin-1 (IL-1)receptor-associated kinase-dependent IL-1-induced signaling complexes phosphorylateTAK1 and TAB2 at the plasma membrane and activate TAK1 in the cytosol. Mol. Cell. Biol.22:7158-7167.Jiang, Z., Johnson, H.J., Nie, H., Qin, J., Bird, T.A., and X. Li (2003) Pellino 1 is requiredfor interleukin-1 (IL-1)-mediated signaling through its interaction with the IL-1 receptorassociatedkinase 4 (IRAK4)-IRAK-tumor necrosis factor receptor-associated factor 6(TRAF6) complex. J. Biol. Chem. 278:10952-10956.Jiang, Z., Zamanian-Daryoush, M., Nie, H., Silva, A.M., Williams, B.R., and X. Li (2003)Poly(dI x dC)-induced Toll-like receptor 3 (TLR3)-mediated activation of NFκB and MAPkinase is through an interleukin-1 receptor-associated kinase (IRAK)-independent pathwayemploying the signaling components TLR3-TRAF6-TAK1-TAB2-PKR. J. Biol. Chem.278:16713-16719.Zhao, Z., Qian, Y., Wald, D., Xia, Y.F., Geng, J.G., and X. Li (2003) IFN regulatory factor-1 is required for the up-regulation of the CD40-NF-κB activator 1 axis during airwayinflammation. J. Immunol. 170:5674-5680.95

THE M. SIEMIONOWLABORATORYRESEARCH FELLOWSYavuz Demir, M.D.Selahattin Ozmen, M.D.Maciej Zielinskii, M.D.POSTDOCTORAL FELLOWSDariusz Izycki, M.D., Ph.D.TECHNOLOGY ASSISTANTSJennifer Mule, B.S.Emilia SierkoMEDICAL STUDENTSKrzysztof Siemionow, M.D.Howard YoungCOLLABORATORSTatiana Byzova, Ph.D.. 1Paul E. DiCorleto, Ph.D. 2Robert L. Fairchild, Ph.D. 3David Hicks, M.D. 4Jaroslaw Maciejewski, MD., Ph.D. 51Dept. of Mole. Cardiology, CCF2Dept. of Cell Biology, CCF3Dept. of Immunology, CCF4Dept. of Anat. Pathology, CCF5Taussig Cancer Center, CCFMaria Siemionow, M.D.,Ph.D., D.Sc.The Microsurgery Laboratory of theDepartment of Plastic and ReconstructiveSurgery has a broad research focus andincludes studies related to the followingcategories:TransplantationThe major focus of our MicrosurgeryLaboratory has been on induction of tolerance incomposite tissue allografts. CTAs comprise acombination of skin, subcutaneous tissue,neurovascular tissue, and mesenchymal tissuesuch as bone, muscle, fascia and cartilage (e.g.,the hand, knee joint or larynx). Although“nonvital to life,” these tissues are structurally,functionally and aesthetically important to thosewho specialize in functional restoration ofmusculoskeletal defects. Most attempts atcomposite tissue transplantation have beenunsuccessful, which illustrates the difficultbarrier associated with vascularized allograftscomposed of a variety of tissues. Severalexperimental designs of tolerance induction havebeen reported. Side effects related to theseprotocols limit their use to only carefully selectedapplications.Our recent approach to induce tolerance isbased on the pivotal role of T cells in allograftrejection. To create a window of immunologicalincompetence, we are investigating the possibilityof specifically eliminating alpha-beta-T-cellreceptor-positive cells, which confer the abilityto reject allograft tissues. We achieved significantdepletion of this T-cell population at theend of immunodepleting therapy and observedthe potential for repopulation of the recipient Tcells’ repertoire once the treatment protocol wasstopped. This allowed for over 700 days ofallograft survival without chronic immunosuppression.The functional outcome of CTAs isassessed by our standardized technique, includingOrtak, T., Oke, R., Unsal, M., Carnevale, K., and M. Siemionow (2001) A model ofvascular thymus transplantation in athymic rats. Transplant. Proc. 33:372-374.Meirer, R., Babuccu, O., Unsal, M., Nair, D.R., Gurunluoglu, R., Skugor, B., Meirer, B.,and M. Siemionow (2002) Effect of chronic cyclosporine administration on peripheral nerveregeneration: a dose-response study. Ann. Plastic Surg. 49:96-103.Byzova, T.V., Goldman, C.K., Jankau, J., Chen, J., Cabrera, G., Achen, M.G., Stacker,S.A., Carnevale, K.A.., Siemionow, M., Deitcher, S.R., and P.E. DiCorleto (2002)Adenovirus encoding vascular endothelial growth factor-D induces tissue-specific vascularpatterns in vivo. Blood 99:4434-4442.Siemionow, M., and K. Ozer (2002) Advances in composite tissue allograft transplantationas related to the hand and upper extremity. J. Hand Surg. [Am] 27:565-580.The Department of ImmunologyMicrosurgery Studies Focus onInduction of Transplant Tolerance andReturn of Functionclinical tests and electrophysiological methods.We evaluate the hemodynamics of the rejectingand surviving allografts at the microcirculatorylevel using our intravital microscopy system.Microvascular permeability is monitoredfollowing FITC albumin injection underfluorescence microscopy.Our newest approach is directed towardevaluating the role of bone-marrow-derived stemand proprietor cells in tolerance inductionfollowing vascularized bone-marrow transplantation.These findings will increase our understandingof mechanisms involved in CTA rejection,tolerance induction, development of GVHD andoptimization of clinically applicable treatmentprotocols.MicrocirculationOur work focuses on ischemia-reperfusioninjury and the hemodynamic effects of TNFalpha,VEGF 165 and angiopoietin 1 on thecremaster muscle’s microcirculation. Ourintravital microscopy system measures RBCvelocity, vessel diameters, capillary density andleukocyte endothelial interactions (rolling,sticking, transmigrating leukocytes), andendothelial edema index as we evaluate vascularpermeability. The new cremaster muscletransplantation model was established and istested in TNF-alpha Receptor I, II and I + IIknock-out mouse models.Nerve RegenerationWe are evaluating different surgicaltechniques used for enhancing nerve regeneration.We are studying the effect of Cyclosporine A onnerve degeneration, as well as the effect ofdifferent enhancing factors (such as DHEA andVEGF 165) on nerve regeneration in normal anddiabetic rats, using the standard clinical andfunctional tests, such as somatosensory evokedpotentials, motor evoked potentials, walkingtrack analysis, pin-prick test, and toe spread test.Nerve morphometry of EM sections is assessed byour computerized system, which measures totalnumber of myelinated axons, axon diameters,cross sectional area and myelin thickness.Wound HealingThis category involves the induction ofangiogenesis in abdominal skin flaps and a limbischemia model by the recombinant adenovirusVEGF 165. The effect of pre-operative vs. postoperativeradiation on wound healing is studiedon the rat TRAM flap model.96

Our laboratory studies nitric oxide (NO)biosynthesis in mammals. Discovered in1987, the NO biochemical pathway is nowconsidered to be a widespread means for regulationof cell function and communication. Specific systemsin which the pathway participates are signal transductionin the brain, stroke, control of blood pressureand heart rate, gastric motility, and immunologicdestruction of tumor cells and microbes. Our maininterests are in uncovering the enzyme reactionmechanism, understanding how enzyme structurerelates to function, learning how other cellularproteins can control NOS activity, and the cell biologyof NO as it relates to gene expression and proteinnitration.Reaction Mechanism and Structure/FunctionBiosynthesis of NO is carried out byenzymes named nitric oxide synthases (NOSs). Ourlab works with three distinct NOSs: A cytokineinducedmacrophage isoform and two constitutivelyexpressed NOS from neurons and endothelium.All NOS isoforms catalyze a reaction that iscomplex and biochemically novel: the conversionof L-arginine to nitric oxide and citrulline.We are currently working out the reactionmechanism of NOS through a variety of strategies.These include using chemically modified substrates,intermediates, and cofactors, techniques such asRaman and electron paramagnetic spectroscopy,crystallography, rapid mixing stopped flowtechnology, and single turnover studies to probe theenzyme’s active site chemistry. We also are analyzingimportant domains within the enzyme (forexample, those involved in calmodulin, flavin, orheme binding) by site-directed mutagenesis andcrystallography. Using crystallography, we havealready visualized the active center of the NOS andidentified regions in the protein that enable twoNOS subunits to interact and bind its cofactors andsubstrate.We are studying why a dimeric interaction isrequired to activate the enzyme and have foundthat it allows electron transfer between two NOSsubunits in the active dimer. Ongoing mutagenesisprojects are based on our crystallographic informationand include mapping the surface residues thatThe Department of ImmunologyStructure, Function Key to UnderstandingRegulation of Cellular Nitric Oxideallow for electron transfer between the twosubunits of NOS, identifying catalytically importantresidues in the active site, and residues that areimportant in transducing the effects of boundessential cofactors such as tetrahydrobiopterin.Assembly, Activation, Control of NOSynthasesThe NOS are controlled at multiple levels.We are studying how the active form of theenzyme is assembled within cells. At this point, weknow that intracellular heme availability determineshow much of NOS is assembled into itsactive form and that NO produced by the enzymecan somehow prevent heme incorporation into theprotein. Our understanding of assembly regulationmay help develop a new means to control NOsynthesis during inflammation.Another area of research involves activationof the neuronal and endothelian NOS by calcium.Two steps in the NOS electron transfer sequenceare controlled by the calcium-binding proteincalmodulin. Calmodulin enables electrons to pass tothe iron atom and also greatly speeds the introductionof electrons into the enzyme’s flavin groups.We are studying how calmodulin attachmentchanges the enzyme’s structure to allow thesechanges in electron transfer, using rapid spectroscopictechniques and mutants of calmodulin andNOS.Cell BiologyWe are studying three aspects of NOSregarding cell biology. One project seeks tounderstand how NOS interactions with othercellular proteins such as caveolins or kalirinmodulate the assembly and activity of the NOSenzymes at a molecular level. Another project isfocused on nitration of cellular proteins during NOsynthesis–what components participate, hownitration can be selectively blocked, whethernitration causes change in protein function or geneexpression, and whether enzymes exist that canremove or reduce the nitro group from proteins.A third project is investigating geneexpression changes in response to NO synthesisunder different cellular conditions, keying onenzymes involved in iron uptake, storage, andhomeostasis.THE STUEHRLABORATORYINVESTIGATORSSubrata Adak, Ph.D.Kulwant Aulak, Ph.D.Deborah Durra, B.Sc.Thomas Koeck, Ph.D.David Konas, Ph.D.John McDonald, B.Sc.Koustubh Panda, Ph.D.Manisha Sharma, M.Sc.Zhiquiang Wang, Ph.D.Chin-Chuan Wei, Ph.D.COLLABORATORSTimothy Billiar, M.D. 1Gary Brudvig, Ph.D. 2John Crabb, Ph.D. 3Brian Crane, Ph.D. 4Serpil Erzurum, M.D. 5Elizabeth Getzoff, Ph.D. 6Russ Hille, Ph.D. 7Daniel Mansuy, Ph. D. 8David Meisler, M.D. 4Denis Rousseau, Ph.D. 9John Tainer, Ph.D. 41Univ. of Pittsburgh, Pittsburgh, PA2Yale Univ., New Haven, CT3Cole Eye Inst., CCF4Cornell Univ., Ithaca, NY5Dept. of Pulmonary and CriticalCare Medicine, CCF6Scripps Research Inst., La Jolla,CA7Ohio State Univ., Columbus8Univ. of Paris, France9Albert Einstein Coll. of Med.,Bronx, NYWang, Z.Q., Wei, C.C., and D.J. Stuehr (2002) A conserved tryptophan 457 modulates the kinetics and extentof N-hydroxy-L-arginine oxidation by inducible nitric-oxide synthase. J. Biol. Chem. 277:12830-12837.Adak, S., Aulak, K.S., and D.J. Stuehr (2002) Direct evidence for nitric oxide production by a nitric-oxidesynthase-like protein from Bacillus subtilis. J. Biol. Chem. 277:16167-16171.Dennis J. Stuehr, Ph.D.Panda, K., Rosenfeld, R.J., Ghosh, S., Meade, A.L., Getzoff, E.D., and D.J. Stuehr (2002) Distinct dimerinteraction and regulation in nitric-oxide synthase types I, II, and III. J. Biol. Chem. 277:31020-31030.Wei, C., Wang, Z., Meade, A., McDonald, J., and D.J. Stuehr (2002) Why do nitric oxide synthases usetetrahydrobiopterin? J. Inorg. Biochem. 91:618.Wei, C.C., Wang, Z.Q., Arvai, A.S., Hemann, C., Hille, R., Getzoff, E.D., and D.J. Stuehr (2003) Structure oftetrahydrobiopterin tunes its electron transfer to the heme-dioxy intermediate in nitric oxide synthase.Biochemistry 42:1969-1977.97

The Department of ImmunologyHyaluronan and Associated Proteins AreCritical to Leukocyte Adhesion in IBDImmune ResponseScott A. Strong, M.D.THE STRONGLABORATORYRESEARCH ASSOCIATESSudip Bandyopadhyay, Ph.D.Alana Majors, Ph.D.TECHNOLOGISTCarol de la Motte, Ph.D.COLLABORATORSAnthony Calabro, Ph.D. 1Anthony Day, Ph.D. 2Judith Drazba, Ph.D. 3Csaba Fulop, Ph.D. 1Vincent Hascall, Ph.D. 1Thomas Wight, Ph.D. 41Dept. of Biomedical Engineering,CCF2Oxford University, UK3Imaging Core, CCF4Univ. of Washington, SeattleOur laboratory is a link between theClinic’s Department of ColorectalSurgery and the Department of Immunologyin the Lerner Research Institute As aphysician-directed laboratory, we are primarilyconcerned with the cellular events involved ininflammation, particularly those pertinent to thechronic inflammatory processes related toCrohn’s disease and ulcerative colitis, collectivelyknown as inflammatory bowel disease (IBD).The overriding hypothesis of our laboratory isthat the bi-directional interaction betweenmesenchymal cells and leukocytes within themicroenvironment of the colonic mucosa iscrucial to inflammation and disease progression.The origin of IBD is thought to bemultifactorial. Environmental triggers, includingmicrobiological agents, initiate and perpetuate animmune response in the intestine of geneticallysusceptible individuals, which results in the clinicalmanifestations of Crohn’s disease and ulcerativecolitis.Viruses are thought to be involved in thepathogenesis of IBD because of the clinicalassociation of respiratory virus infections withsubsequent IBD exacerbations. We have recentlydescribed a novel in vitro mechanism by whichviruses may affect the interactions betweenmesenchymal cells and leukocytes.Normally, colonic mucosal tissue contains apopulation of leukocytes, including T- and B-lymphocytes, plasma cells, histiocytes and mastcells, which are scattered in a network of collagenfibers and smooth muscle cell bundles. In IBD, themucosal immune cell population increasesdramatically, and the infiltrate is predominantlycomposed of mononuclear leukocytes. Further, ahyperplastic thickening of the juxtaposed muscularismucosae cells also occurs. This suggests thatinteractions between leukocytes and mesenchymalsmooth muscle cells are important in thede la Motte, C.A., Hascall, V.C., Calabro, A., Yen-Lieberman, B., and S.A. Strong(1999) Mononuclear leukocytes preferentially bind via CD44 to hyaluronan on humanintestinal mucosal smooth muscle cells after virus infection or treatment withpoly I:C. J. Biol. Chem. 274:30747-30755.Strong, S.A., and C.A. de la Motte (1999) Hyaluronan and colon smooth musclecells. In: Hascall VC, Yanagishita M, eds. Science of Hyaluronan Today (web sitepublication, accessible at http://www.glycoforum.gr.jp/).Itoh, J., de la Motte, C.A., Strong, S.A., Levine, A.D., and C. Fiocchi (2001) DecreasedBax expression by mucosal T cells favours resistance to apoptosis inCrohn’s disease. Gut 49:35-41.de la Motte, C.A., Hascall, V.C., Drazba, J., and S.A. Strong (2001) Poly I:C inducesmononuclear leukocyte-adhesive hyaluronan structures on colon smoothmuscle cells: IaI and versican facilitate adhesion. In Kennedy, J.F., Phillips, G.O.,and P.A. Williams, eds. Hyaluronan: Proceedings of an international meeting, Sept.2000, North East Wales, Inst. of Higher Educ., UK. Vol. 1. Cambridge: Woodhead,pp. 381-388.development of IBD. We have recently shownthat colonic mesenchymal cells can proliferate inresponse to leukocyte-derived pro-inflammatorycytokines.In one of our model systems, virusinfection or poly I:C (viral mimic) treatment ofcolon mucosal smooth muscle cells (M-SMCs)increases mononuclear leukocyte adhesion via M-SMC-expressed hyaluronan interacting withleukocyte-expressed CD44. This mechanism isvery different from the cytokine (TNF-α)-induced mechanism of leukocyte adhesion, whichinvolves VCAM-1 on the M-SMCs interactingwith its integrin ligand, VLA-4, on the mononuclearleukocytes. Treatment of M-SMCs withpoly I:C upregulates the production of hyaluronanby M-SMCs, and mononuclear leukocyte adhesionto poly I:C-treated M-SMCs dramatically increases(10- to 20-fold in most experiments). Inaddition, we have shown that leukocyte activationis not required for binding to hyaluronan onvirus-induced M-SMCs. In human colon tissuesections, we observe a large increase in thehyaluronan content of inflamed mucosa ascompared to non-inflamed tissue, even whenderived from the same patient, supporting therelevance of our in vitro results to in vivo observations.Experimentally, we have been trying toreconcile the issue of why a common bodysubstance, hyaluronan, should also play a role in aninflammatory process such as leukocyte recruitment.Our data suggest that in addition to chemicalcomposition, the arrangement of hyaluronan on thecell surface is important; virus-induced M-SMCsform hyaluronan cable structures that are responsiblefor binding leukocytes, whereas patchy “coat”structures are not involved. We are currentlyinvestigating several hyaluronan-binding proteins,their role in structure formation, and how they maydirectly or indirectly dictate leukocyte adhesion.In addition to leukocyte accumulation, we areinvestigating another possible outcome of mononuclearleukocyte adhesion to hyaluronan (i.e.,activation). Under certain circumstances, leukocytescan be activated by ligation of CD44 (the majorhyaluronan receptor) and can produce proinflammatoryproducts. We speculate that leukocytebinding via the virus-induced hyaluronan pathwaywill result in chemokine, cytokine and proteaseproduction that will perpetuate the cascade ofchronic inflammation through leukocyte recruitmentand activation, as well as colon tissue remodeling.98

The Department of ImmunologyMechanisms and Treatment of AutoimmunityTHE TUOHYLABORATORYOur laboratory focus involves (1) understanding the complex self-recognition events thatlead to progression of autoimmune diseaseand (2) developing novel therapeutic strategies thatprevent disease progression. Our laboratory has along-standing history ofresearch on multiplesclerosis (MS) and hasdeveloped a widely usedmouse model calledexperimental autoimmuneencephalomyelitis (EAE)that mimics many of thefeatures of MS. Our studieshave shown that progressionof MS and EAE involves apredictable cascade ofnewly acquired selfrecognitionevents called“epitope spreading,” and ourrecent studies show thattransfer of regulatory Tcells targeting the “epitopespreading cascade” causesabrogation of diseaseprogression. Theseregulatory T cells areproduced by geneticmodification such that,upon engagement with selfantigens,they release anti-inflammatory factorsinstead of disease-inducing inflammatory factors.Thus, another focus of our laboratory involves reprogrammingthe autoimmune response by geneticmodification so that an inflammatory diseaseinducingresponse may be shifted to an antiinflammatorydisease-inhibiting response.Vincent K. Tuohy, Ph.D.Although autoimmune diseases are characteristicallymore prevalent in females, the severity ofautoimmune disease is typically worse in males. Thebasis for this perplexing yet consistently observedmale bias in disease severity is unclear and serves as arecent new focus for investigation inour laboratory. To this end, we havedeveloped a mouse model forexperimental autoimmune myocarditis,in which male mice show farmore pronounced inflammation,fibrosis, and dilated cardiomyopathy(DCM) than females, therebymimicking the skewed male biasobserved in human idiopathic DCM,a disease that accounts for about 25%of all cases of congestive heartfailure in the United States and killsmen at a 2- to 3-fold greaterfrequency than women.Finally, we have recentlyinvestigated the role of inflammatoryT cells in autoimmune sensorineuralhearing loss (ASNHL), a disease thattypically produces a sudden bilateralrapidly progressive loss of hearing.Although autoantibodies andautoreactive T cells have beenimplicated in the etiopathogenesis ofASNHL, several central issuesremain unresolved, including the relative prominenceof B-cell or T-cell autoimmunity in the initiation andprogression of ASNHL, the identity of the putativeinner ear self-antigen(s) that target ASNHL, and thedevelopment and application of contemporaryimmunosuppressive therapies to prevent progressivehearing loss.POSTDOCTORAL FELLOWSAndrea E. Edling, Ph.D.Riitika Jaini, Ph.D.Seiko Kataoko, M.D., Ph.D.C. Arturo Solares, M.D.TECHNICAL ASSOCIATESJustin M. Johnson, B.S.GRADUATE STUDENTSCengiz Zubeyir Altuntas, B.S.Bakhautdin Bakytzhan, B.S.Debasmita Chandra, M.S.Daniel Jane-wit, B.SPavani Kesaraju, B.S.Cara A. McCracken, B.A.COLLABORATORSRobert Fox, M.D. 1Keiko Hirose, M.D. 2Gordon Hughes, M.D. 2Wendy B. Macklin, Ph.D. 3Christine S. Moravec, Ph.D. 4Richard M. Ransohoff, M.D. 31Dept. of Neurology, CCF2Dept.of Otolaryngology andCommunicative Disorders, CCF3Dept. of Neurosciences, CCF4Dept. of CardiovascularMedicine, CCFYu, M., Johnson, J.M., and V.K. Tuohy (1996) A predictable sequential determinant spreading cascadeinvariably accompanies progression of experimental autoimmune encephalomyelitis: A basis for peptidespecifictherapy after onset of clinical disease. J. Exp. Med. 183:1777-1788.Mathisen, P.M, Yu, M., Johnson, J.M., Drazba, J.A., and V.K. Tuohy (1997) Treatment of experimental autoimmuneencephalomyelitis with genetically modified memory T cells. J. Exp. Med. 186:159-164.Tuohy, V.K., Yu, M., Yin, L., Kawczak, J.A., Johnson, J.M., Mathisen, P.M., Weinstock-Guttman, B., and R.P. Kinkel(1998) The epitope spreading cascade during progression of experimental autoimmune encephalomyelitis andmultiple sclerosis. Immunol. Rev. 164:93-100.Tuohy, V.K., Yu, M., Yin, L., Kawczak, J.A., and R.P. Kinkel (1999) Spontaneous regression of primary autoreactivityduring chronic progression of experimental autoimmune encephalomyelitis and multiple sclerosis. J. Exp. Med.189:1033-1042.Klein, L., Klugmann, M., Nave, K.-A., Tuohy, V.K., and B. Kyewski (2000) Shaping of the autoreactive T-cellrepertoire by a splice variant of self protein expressed in thymic epithelial cells. Nat. Med. 6:56-61.Huang, D., Tani, M., Wang, J., Han, Y., He, T.T., Weaver, J., Charo, I.F., Tuohy, V.K., Rollins, B.J., and R.M.Ransohoff (2002) Pertussis toxin-induced reversible encephalopathy dependent on monocyte chemoattractantprotein-1 overexpression in mice. J. Neurosci. 22:10633-10642.Jane-wit, D., Yu, M., Edling, A.E., Kataoka, S., Johnson, J.M., Stull, L.B., Moravec, C.S., and V.K. Tuohy (2002) Anovel class II-binding motif selects peptides that mediate organ-specific autoimmune disease in SWXJ, SJL/J, andSWR/J mice. J. Immunol. 169:6507-6514.99

The Department of ImmunologyImmunology Project Scientistswith Independent FundingKulwant Aulak, Ph.D.The physiology of nitric oxide is complex and its role ininflammation controversial with both anti-inflammatory andpro-inflammatory effects. Tyrosine nitration is a chemicallymediated, post translational modification that results fromexcessive NO production. A pathogenic role of proteinnitration is apparent in numerous vascular and neurologicaldiseases such as sepsis, diabetes, ARDS, Parkinsons disease,ALS and tissue rejection. The focus of our lab is to investigatethe mechanisms and functional consequences of proteinnitration. Our studies will uncover the physiological roles andthe factors that affect nitration and will identify new avenuesfor therapeutic interventions in the disease process.Chin-Chuan Wei, Ph.D.Our Research Interests lie in understanding the molecular basisof protein function, in particular functions of metalloenzymesthat are involvied in biological signal transduction. Our currentproject aims to understand the mechanism of reactive oxgenspecies (ROS) production by non-phagocytic cells. In such cellsROS are generated by NADPH oxidase enzymes (NOX). TheNOX play important roles in mitosis, apotosis, cell migration,hypertrophy, and modification of extracellular matrix. Understandinghow auxiliary cellular components interact with NOXenzymes to control their biochemical reactions presents excitingchallenges. The primary methods we use in our research includebiophysical and biochemical techniques to elucidate the chemicalmechanism and protein-protein interactions at a molecular level.Anna Valujskikh, Ph.D.The focus of our project is the role of donor-specific memory T cellsin allograft rejection and tolerance. Memory T cells exhibit specificproperties that permit efficient control of infections previouslyencountered by the host. At the same time, memory T cells can besignificant hurdle to transplant tolerance induced throughcostimulatory blockade. The goal of this study is to understand howdonor-reactive memory T cells mediate allograft rejection despitecostimulatory blockade. As memory T cells are prevalent in humantransplant recipients, the results obtained from this work couldtranslate into improved therapies for prolongation of humanallograft survival.100

MolecularBiology

DEPARTMENT OFMOLECULAR BIOLOGYCHAIRMANAndrei V. Gudkov, Ph.D.The Thomas Lord ChairSTAFFDonal S. Luse, Ph.D.Richard A. Padgett, Ph.D.Ganes C. Sen, Ph.D.Dennis W. Stacey, Ph.D.George R. Stark, Ph.D.ASSOCIATE STAFFMarian L. (Nikki) Harter, Ph.D.Kurt W. Runge, Ph.D.ASSISTANT STAFFPeter Chumakov, Ph.D.Kwaku Dayie, Ph.D.PROJECT SCIENTISTSMunna Agarwal, Ph.D.Mikhail Chernov, Ph.D.Katerina Gurova, Ph.D.Masahiro Hitomi, Ph.D.Eugene Kandel, Ph.D.Sean Kessler, Ph.D.Elena Komarova, Ph.D.Asoke Mal, Ph.D.Alo Ray, Ph.D.Girish Shukla, Ph.D.William Taylor, Ph.D.RESEARCH ASSOCIATESLyudmila Burdelya, Ph.D.Yang Guo, Ph.D.Julia Kichina, Ph.D.Mahadeb Pal, Ph.D.Subramania Sanker, Ph.D.Saumendra Sarkar, Ph.D.Fulvia Terenzi, Ph.D.Ke Yang, Ph.D.102Targeting the Molecular Basis of DiseaseThe Department of Molecular Biology wasestablished in 1987 with the mission tobuild the programmatic and methodologicalinfrastructure of molecular biology within theResearch Institute and to stimulate research onthe structure, regulation and mechanisms ofactivity of viral and cellular genes. During thistime, the Department has developed into a strongacademic union of nationally recognized expertsrepresenting a broad spectrum of disciplines. Itmaintains the spirit of collegiality and mutualsupport, has a solid technical infrastructure and isefficiently run by an experienced administrativeteam. Ongoing research programs cover thefollowing areas.Understanding the molecular mechanismsof stress-, cytokine-, and oncogene-mediatedThe Department of Molecular BiologyAndrei Gudkov, Ph.D., D.Sci.signal transduction remains a major area ofresearch within the Department. George Stark’sand Ganes Sen’s laboratories have a long-standinginterest in deciphering mechanisms by whichinterferon (IFN)-mediated signaling occur inmammalian cells via activation of various signaltransducers, resulting in changes in gene expression.Stark’s team, in addition, is working onunderstanding the mechanisms of activation ofthe p53 tumor suppressor protein by variousstresses and its effects on cellular growthregulation. Sen’s laboratory is involved indetermining functional characteristics of severalIFN-induced proteins as well as signaling initiatedby double-stranded RNA. Another area ofinterest is the analysis of specific physiologicalroles of different isoforms of angiotensinconvertingenzymes with tissuespecific expression. Dennis Stacey’slaboratory is focused on the role ofoncogene ras-mediated signaling andtopoisomerase II expression in controlof cell cycle and cell response tochemotherapeutic drugs. The groupdevelops and applies unique experimentalapproaches to the analysis ofthe cell cycle allowing revision ofestablished dogmas in this highlycompetitive field. Nikki Harter’slaboratory is working on understandingthe molecular mechanics of cellgrowth and differentiation byoncoprotein E1A and myogenictranscription factor MyoD, bothcontributing to understanding themolecular mechanisms of oncogenesis.The laboratories of two staffmembers are involved in understandingmost basic mechanisms of cellularRNA synthesis and processing. DonalLuse leads a team that studiestranscription by RNA polymerase IIwith specific focus on promoterclearance and regulation of initialsteps of transcript elongation. RichardPadgett primarily focuses his studieson the molecular mechanisms of RNAsplicing, including the chemistry ofthis process, identification of cellularfactors involved in control of RNAsplicing and evolution of splicingmachinery.Kurt Runge continues work onthe replication and maintenance oftelomeres, the physical ends ofeukaryotic chromosomes, usingbudding yeast as a model organism.Besides understanding telomere structure andfunction, his work provides insights into themechanisms of cellular aging, and the control ofContinued on Page 103

The Department of Molecular BiologySECTION ONVIROLOGYContinued from Page 102malignant transformation and is evolvingtowards development of new cancer treatmentapproaches.Recently, the Department was strengthenedby the newly organized laboratory led by ayoung structural biologist. Kwaku T. Dayie is anexpert in biomolecular NMR spectroscopymethods development and application to analysisof RNA-RNA and RNA-protein interactions.This work complements the Department’sexisting strengths in RNA synthesis/processingand signal transduction research.The laboratory of Peter Chumakovconducts active research programs in the p53tumor suppressor field. Chumakov, a pioneer ofthis area of molecular oncology, is pursuing aprogram of development of new anti-cancerp53-targeting pharmaceuticals based on uniquecellular model systems. A series of smallmolecules active against cervical cancer has beenrecently isolated, forming a solid basis for newanticancer drug development. This group is alsoinvolved in identification and functional analysisof new members of the p53 signaling pathway aspotential targets for anticancer treatment.The Department of Molecular BiologySection on Virology is comprised of investigatorswho specialize in and recognize the paramountimportance of investigating the molecular andimmunological bases of the infectious processesof human viruses. Understanding the fundamentalbasis of how viruses cause the disease processstill remains a colossal challenge to virologists.The Section on Virology’s includes three principalinvestigators who are dedicated to studying themechanisms of viral pathogenesis and to recruitvirologists to a interact and foster interdisplinaryresearch with physicians in the clinical departmentswith the Cleveland Clinic Foundation.Dr. Amiya K. Banerjee is the Head of theSection on Virology. Dr. Banerjee’s laboratory isinvolved in understanding the biosyntheticpathways underlying how the genetic material ofpathogenic viruses, e.g., vesicular stomatitis virusand human parainfluenza viruses, is expressedand regulated during their invasion of host cells.The laboratory’s primary goal is to understand themolecular basis of pathogenicity of these twoviruses, with an eye to developing antivirals andvaccines to combat these deadly viruses. Inaddition, research is being carried out to probeinto the molecular basis of pathogenicity ofhuman hepatitis virus C (Dr. Ashim K. Gupta)and to understand the mechanism of antiapoptoticfunctions of viral gene products (Dr.Nickolay Neznanov).Dr. Philip Pellet joined our Department asa senior Staff member early in 2003. Dr. Pellet’sstudies aim to expand the fundamental understandingof the relationships among the variousherpesviruses, the processes by which humanherpesviruses replicate and persist in populationson an evolutionary time scale, and the mechanismsby which these viruses cause disease. As theDirector of Herpesvirus Translational and BasicResearch, Dr. Pellet will lead research that willuse newly gained knowledge about herpesvirusbiology to prevent or treat herpesvirus-associateddisease.The laboratory of Dr. Miguel E. Quiñones-Mateu studies the molecular basis of variability,evolution and antiretroviral resistance of thehuman immunodeficiency virus (HIV). Thesestudies are directed to addressing the basic andclinically related questions with regard to HIVdynamics in AIDS patients undergoingantiretroviral therapy.Finally, the Gudkov laboratory is engagedin a broad research program involving gene anddrug discovery branches, mainly in the field ofcancer. The Gudkov program is aimed atidentification of modulators of apoptosis as anapproach to reduce cancer treatment side effectsand to cure other pathologies resulting from acutestresses. Scientists in the Gudkov laboratory aredeveloping and applying new gene discovery toolsbased on functional genetic technologies.Although the laboratory's program is primarilyfocused on cancer treatment applications thedeveloped methodologies are applicable foridentification of molecular targets in variousdiseases-an opportunity that is being aggressivelyexplored in collaboration with clinical Departmentsof CCF. Drug Screening Core that wasrecently organized in the Department acts as apowerful technological tool for facilitation oftranslational aspects of laboratory research byproviding services in chemical library screening fordrug discovery programs in a variety of fieldsexplored by the scientists from Molecular Biologyand other Departments of LRI.In summary, the Department of MolecularBiology combines basic and translational researchcovering a whole range of problems, fromunderstanding the basics of cell regulation todevelopment of clinically useful pharmaceuticals.Our productive laboratories, led by nationallyrecognized principal investigators, are taking awell-defined course towards strengtheningdisease-oriented translational research programs incollaboration with other divisions of theCleveland Clinic.Dept. website: http://www.lerner.ccf.org/molecbio/SECTION HEADAmiya K. Banerjee, Ph.D.STAFFPhilip E. Pellett, Ph.D.ASSISTANT STAFFMiguel E. Quiñones-Mateu, Ph.D.PROJECT STAFFAshim K. Gupta, Ph.D.Nickolay Neznanov, Ph.D.RESEARCH ASSOCIATESJing Gao, M.D., Ph.D.Manjula Mathur, Ph.D.103

THE GUDKOVLABORATORYPROJECT SCIENTISTSMikhail Chernov, Ph.D.Katerina Gurova, Ph.D.Elena Komarova, Ph.D.Asoke Mal, Ph.D.RESEARCH ASSOCIATESLyudmila Burdelya, Ph.D.Julia Kichina, Ph.D.Roman Kondratov, Ph.D.Jason Tasch, Ph.D.POSTDOCTORAL FELLOWSAnna Khodyakova, Ph.D.Vadim Krivokrysenko, Ph.D.GRADUATE STUDENTSAlexander BoikoJason HillPratibha KhanduryAndrei KomarovInna ShyshynovaAatur SinghiRESEARCH TECHNOLOGISTSKris GoodrichAnna KondratovaVISITING FELLOWSCatherine Burkhart, Ph.D.Chengyuan XueCOLLABORATORSVladimir Botchkarev, M.D.,Ph.D. 1Michael Cole, Ph.D. 2Elena Feinstein, M.D., Ph.D. 3Michelle Haber, Ph.D. 4Alain Jaqcemin-Sablon, Ph.D. 5Hioaki Kiyokawa, Ph.D. 6Murray Norris, Ph.D. 4Varda Rotter, Ph.D. 71Boston Univ., Boston, MA2Princeton Univ., Princeton, NJ3Quark Biotech, Inc., Nes Ziona,Israel4Children’s Cancer InstituteAustralia for Medical Research,Sydney, Australia5Institut Bergonié, Bordeaux,France6Univ. of Illinois at Chicago7Weizmann Inst. of Science,Rehovot, IsraelThe Department of Molecular BiologyIdentifying and TargetingCancer-Related GenesMy laboratory explores functional gene anddrug discovery methodologies fordeveloping new strategies of cancertreatment and diagnostics.Novel Gene Discovery ApproachesWe use functional selection of expressionlibraries for identifying genes encoding prospectivedrug targets. A number of such genes havebeen selected by using the Genetic SuppressorElement (GSE) methodology, including newcandidate tumor suppressor (ING1), drugsensitivity (Falcor) or pro-apoptotic (SIRPα/SHPS-1) genes.One branch of our gene discovery programis devoted to isolating new viral and bacterialanti-apoptotic genes as potential leads to newcellular mechanisms controlling programmed celldeath. Thus, three anti-apoptotic proteins havebeen identified among polypeptides encoded bypoliovirus.Recently, we have developed a newfunctional genetic methodology, the Selection-Subtraction Approach (SSA), which allows adirect functional selection of growth-suppressiveor killing clones from expression libraries. Weconsider the SSA our major tool for genediscovery and are applying it to isolate novelcancer-related genes for future drug targeting.p53 in Cancer Diagnostics and TreatmentOur p53 studies are focused on the role ofthis tumor suppressor gene in response of normaltissues to the stresses associated with cancertreatment. We defined p53 as a determinant ofcancer treatment side effects; the new therapeuticconcept—targeting p53 for therapeutic suppression—wasjustified by isolating a small moleculep53 inhibitor that rescues mice from lethal dosesof gamma irradiation. Furthermore, analysis of amouse model of chemotherapy-induced hair losshas indicated that p53 plays a major role in thiscommon side effect, thus opening another areafor clinical application of p53 inhibitors.We also study the role of p53-dependentapoptosis and growth arrest and the interactionof p53 with other signaling pathways (TNF, Fas,heat shock, etc.) in determining its tumorsuppressor function. Analysis of mechanisms oftissue specificity of the p53 response resulted indevelopment of a new rational approach to newcancer markers encoded by the genes that areunder the negative control of p53. We found thatone of the most important cancer markers,prostate specific antigen (PSA), belongs to thiscategory and initiated a large-scale screening forother candidate markers sharing similar properties.Drug Discovery ProgramOur drug discovery program involvessearching for new p53 inhibitors and testing theirpotential therapeutic applications for reducingcancer treatment side effects and possibly otherpathologies involving p53-inducing stresses. It isbased on the creation of new cell-based readoutsystems and high-throughput screening ofchemicals with the desired biological properties.We are also isolating a new class of smallmolecules acting as modulators of multidrugtransporters that can greatly change the patternof cross-resistance, including the ability toenhance their activity against certain compounds.The molecular mechanisms of activity of newlyisolated compounds are being addressed, as aretherapeutic fields for their practical applications.Komarov, P.G., Komarova, E.A., Kondratov,R.V., Christov-Tselkov, K., Coon, J.S., Chernov,M.V., and A.V. Gudkov (1999) A chemicalinhibitor of p53 that protects mice fromthe side effects of cancer therapy. Science285:1733-1737.Kondratov, R.V., Komarov, P.G., Becker, Y.,Ewenson, A., and A.V. Gudkov (2001) Smallmolecules that dramatically alter multidrug resistancephenotype by modulating the substratespecificity of P-glycoprotein. Proc.Natl. Acad. Sci. USA 98:14078-14083.Gurova, K.V., Roklin, O.W., Krivokrysenko,V.I., Chumakov, P.M., Cohen, M.B., Feinstein,E., and A.V. Gudkov (2002) Expressionof prostate specific antigen (PSA) is negativelyregulated by p53. Oncogene 21:153-157.Zou, X., Ray, D., Aziyu, A., Christov, K.,Boiko, A.D., Gudkov, A.V. and Kiyokawa H.(2002). Cdk4 disruption renders primarymouse cells resistant to oncogenic transformation,leading to Arf/p53-independent senescence.Genes Dev. 16, 2923-2934.Neznanov, N., Neznanova, L., Kondratov,R.V., Burdelya, L., Kandel, E.S., O’Rourke,D.M., Ullrich, A., and A.V. Gudkov (2003)Dominant negative form of signal-regulatoryprotein-a (SIRPa/SHPS-1) inhibits tumor necrosisfactor-mediated apoptosis by activationof NF-kB. J. Biol. Chem. 278:3809-3815.Gudkov, A.V., and E.A. Komarova (2003) Therole of p53 in determining sensitivity to radiotherapy.Nat. Rev. Cancer. 3:117-129.Komarova, E.A., Neznanov, N., Komarov,P.G., Chernov, M.V., Wang, K., and A.V. Gudkov(2003) p53 inhibitor pifithrin alpha cansuppress heat shock and glucocorticoid signalingpathways. J. Biol. Chem. 278:15465-15468.Gurova, K.V., Rokhlin, O.W., Budanov, A.V.,Burdelya, L.G., Chumakov, P.M., Cohen,M.B., and A.V. Gudkov (2003) Cooperation oftwo mutant p53 alleles contributes to Fas resistanceof prostate carcinoma cells. CancerRes. 63:2905-2912.104

We focus on p53-dependent mechanisms ofproliferation control. One direction of ourresearch involves identification and studyof mechanisms that are responsible for programmedp53 activation in response to such faults asabnormalities in cytoskeleton assembly, changes incell contacts, formation of micronuclei, changes incell ploidy, prolonged signaling from activated Ras,etc. The goal of our other research is the understandingof the functional role of missensemutations within the p53 gene in human cancer.We have identified several new gain-offunctionactivities of p53 mutants that contribute tothe malignant phenotype of cancer cells anddetermine increased resistance to cancer therapy.Particularly, we found that certain forms of p53mutant proteins increase resistance of cancer cells to5-FU and other fluoropyrimidine aniticancer drugsthrough specific activation of dUTPase genetranscription. We have been studying factorsaffecting expression of retroviral constructsdepending on the position of integration intorecipient cells, and conditions determining sustainedexpression of a transgene following multiple celldivisions. These studies have yielded several usefulretroviral vectors for tetracycline-regulatedexpression. Several state-of-the-art retrovirus-basedreporter constructs enable us to compare andmonitor activity of transcriptional factors afterintroduction into a wide range of cell types.One of the major goals is development ofapproaches for restoration of disrupted p53pathways in human cancer. The p53 gene is mutatedin almost half of cancer cases. In the other half, thep53 pathway is disrupted by other mechanisms.Many alterations in p53 interfere with induction ofapoptotic programs. The p53 pathway staysstructurally intact in cervical carcinomas, reflectinghigh efficiency that makes further mutationsunnecessary. Human papilloma virus gene productsare putatively excellent targets for discovery of drugsthat reactivate p53-dependent suicidal programs incervical carcinomas. In human tumors harboringmissense mutations within the p53 gene, theapoptotic response to exogenous wild-type p53 isalso well preserved. Therefore, selection of peptidesor small molecules that facilitate correct folding ofthr mutant p53 protein might restore the p53pathway, helping programmed elimination of cancercells. In our recent collaborative work with theKarolinska Institute, Sweden, a small molecule hasbeen selected from a chemical library that inducesapoptosis of tumor cells through reactivation ofmutant p53.To develop a system for high-throughputscreening of chemical libraries for small moleculesthat reactivate the p53 pathway in tumor cells, weintroduced our retrovirus-based reporter constructthat expresses β-galactosidase under control of a p53-responsive promoter into the HPV-positive cervicalcarcinoma cell lines HeLa and SiHa, and into severalThe Department of Molecular BiologyRestoration of Disrupted p53 Pathways inHuman Cancerhuman tumor cell lines bearing different pointmutations within the p53 gene. Testing of theobtained reporter cell lines revealed low backgroundβ-galactosidase expression and high inducibility of thereporter in response to introduction of exogenouswild-type p53. In collaboration with Quark BiotechInc., screening of a 46 k chemical library hasrevealed several distinct classes of small moleculesthat reactivate p53-specific transcriptional activity.Activation of p53 transcriptional activity results ininduction of p53-specific apoptosis and causesreduction of tumorigenicity in nude mice, both as amonotherapy and in combination with γ-radiation.We are currently working on identification of specificmechanisms of action of the identified p53-reactivating drugs.To study mechanisms of p53 inactivation intumors bearing wild-type p53, we are planning toidentify small molecules that reactivate p53 transcriptionalactivity in cell lines in which the cause of thedefect is not known. Identification of specific targetsof selected drugs might shed clues to the mechanismsof the defect. We introduced p53-specific reporterconstructs into several dozen human carcinoma celllines that express wild-type p53. Quantitative analysisof β-galactosidase expression following treatmentwith several types of p53-inducing drugs has revealedseveral cell lines with significantly impaired inductionof the p53-responsive genes. In addition, these samecell lines show greatly reduced induction of the p53-specific reporter after infection with recombinantadenovirus carrying wild-type p53. Some of the celllines have been selected for future screening ofchemical libraries for p53-reactivating compounds.We also study p53-dependent mechanisms thatregulate viability of cells following different stresses.Recently we described the novel hypoxia- and DNAdamage-induciblegene Hi95 that is involved inregulation of differential viability of cells to variousstress conditions through p53-dependent mechanisms.THE CHUMAKOVLABORATORYPOSTDOCTORAL FELLOWSLarissa Agapova, Ph.D.Andrei Budanov, Ph.D.VISITING FELLOWSGalina Ilyinskaya, Ph.D.Dmitry KotchetkovYulia KravchenkoOlga RazorenovaAnna Sablina, Ph.D.COLLABORATORSAndrei Gudkov, Ph.D., D.Sc. 1Alexey Ivanov, Ph.D. 2Pavel Komarov, Ph.D. 3Boris Kopnin, Ph.D., D.Sc 4Arnold Levine, Ph.D. 5Galina Selivanova, Ph.D. 21Dept. Mol. Biol. CCF2Karolinska Institute, Stockholm,Sweden3Quark Biotech, Inc., Cleveland4Russian Acad. of Sci., CancerResearch Center, Moscow,Russia,5Dept of Mol. Biol., PrincetonUniv., Princeton, NJPeter M. Chumakov, M.D.,Ph.D., D.Sc.Alexandrova A., Ivanov A., Chumakov P., Kopnin B., and J. Vasiliev (2000) Changesin p53 expression in mouse fibroblasts can modify motility and extracellular matrix organization.Oncogene 19:5826-5830.Sablina A.A., Chumakov P.M., Levine A.J., and B.P. Kopnin (2001) p53 activation inresponse to microtubule disruption is mediated by integrin-Erk signaling. Oncogene20:899-909.Pugacheva E.N., Ivanov A.V., Kravchenko J.E., Kopnin B.P., Levine A.J., and P.M.Chumakov (2002) Novel gain of function activity of p53 mutants: activation of thedUTPase gene expression leading to resistance to 5-fluorouracil. Oncogene 21:4595-4600.Budanov A.V., Shoshani T., Faerman A., Zelin E., Kamer I., Kalinski H., Gorodin S.,Fishman A., Chajut A., Einat P., Skaliter R., Gudkov A.V., Chumakov P.M., and E.Feinstein (2002) Identification of a novel stress-responsive gene Hi95 involved in regulationof cell viability. Oncogene 21:6017-6031.Bykov V.J., Issaeva N., Shilov A., Hultcrantz M., Pugacheva E., Chumakov P., BergmanJ., Wiman K.G., and G. Selivanova (2002) Restoration of the tumor suppressorfunction to mutant p53 by a low-molecular-weight compound. Nature Med. 8:282-288.105

THE HARTERLABORATORYPOSTDOCTORAL FELLOWMrinal Ghosh, Ph.D.COLLABORATORSJane Flint, Ph.D. 1Saikh Jaharul Haque, Ph.D. 2Tony Hunter, Ph.D. 31Princeton Univ., Princeton, NJ2Dept. of Cancer Biology, CCF3Salk Institute, La Jolla, CAThe Department of Molecular BiologyAdenovirus Holds Keys to DissectingMechanisms of Cell Cycle, ProliferationThe human adenovirus relies largely on themammalian host DNA synthesis apparatusfor its replication in quiescent or terminallydifferentiated cells. Consequently, this virus hasevolved a specific gene (E1A) to induce cellularDNA synthesis in these cells. E1A, however, cancarry on this activity outside the context of thevirus, and in fact, promote DNA synthesis in cellsblocked in G1, either by TGF-β signaling, the useof a ras neutralizing antibody, or a DNAdamaging agent.Our primary focus isto identify the mechanismsby which E1A affects cellcyclepathways and, in turn,understand some of themolecular details underlyingcell proliferation andterminal differentiation.E1A is best knownfor targeting the retinoblastomafamily of proteins(pRb, p107, p130), whichare co-repressors oftranscription. We recentlydiscovered that E1A couldalso inactivate the cyclindependentkinase (cdk)inhibitors p27 and p21.These interactions respectivelypromote TGF-βtreatedcells into S phase andlead to the induction ofDNA synthesis in differentiatedmuscle cells. In effect, these studiesestablished an entirely new way in which E1Acould modulate the cell cycle.We have also discovered that E1A canperturb the mechanistic links between histoneMal, A., Poon, R. Y. C., Howe, P.P., Toyoshima, H., Hunter, T., and M.L. Harter(1996) The E1A oncoprotein disables the CDK inhibitor p27Kip1 in TGF-ß treated cells.Nature 380:262-265.Mal, A., Chattopadhyay, D., Ghosh, M.K., Poon, R.Y. Hunter, T., and M.L. Harter(2000) p21 and retinoblastoma protein control the absence of DNA replication in terminallydifferentiated muscle cells. J. Cell Biol. 149:281-292.Mal, A., Sturniolo M., Schiltz, R.L., Ghosh, M.K., and M.L. Harter (2001) A role for histonedeacetylase HDAC1 in modulating the transcriptional activity of MyoD: Inhibitionof the myogenic program. EMBO J. 20:1739-1753.Chattopadhyay, D., Ghosh, M.K., Mal, A., and M.L. Harter (2001) Inactivation of p21by E1A leads to the induction of apoptosis in DNA-damaged cells. J. Virol. 75:9844-9856.Mal, A., and M. L. Harter (2003) MyoD is functionally linked to the silencing of amuscle-specific regulatory gene prior to skeletal myogenesis. Proc. Natl. Acad. Sci.USA 100:1735-1739.Marian L.(Nikki) Harter, Ph.D.modifications and chromatin function toreactivate E2F-responsive genes that arenecessary for inducing DNA synthesis in quiescentcells. E1A has the ability to target chromatin andthereby interact with a promoter-bound p130,which associates with the histone methylaseSUV39H1. This interaction induces p130’sremoval from the E2F-regulated promoters, andconsequently, this causes the surroundingnucleosomal histones to lose their methylationand become alternativelymodified (phospho-acetylated),an event that leads to theactivation of transcription.These studies provide the firstexample of a viral protein thatcan specifically affect chromatinfunction.The molecularmechanism(s) that are responsiblefor suppressing MyoD’stranscriptional activities inundifferentiated skeletal musclecells have not yet been determined.We have now shownthat MyoD associates with ahistone deacetylase-1 (HDAC1)in these cells and that thisinteraction is responsible forsilencing MyoD-dependenttranscription of muscle specificgenes. We have also shown thatan endogenous MyoD can beacetylated by P/CAF but onlywhen cells have been induced to differentiate.These results provide for a model, whichpostulates that MyoD may be codependent onHDAC1 and P/CAF for temporally controlling itstranscriptional activity before and after thedifferentiation of muscle cells.Most of the genes (e.g., myogenin) that arecentral to the process of skeletal muscledifferentiation remain in a transcriptionally silentor “off ” state until myoblasts are induced todifferentiate. Importantly, we have now shownthat both MyoD and HDAC1 occupy themyogenin promoter in myoblasts, which issurrounded by methylated H3 histones. Aftermyoblasts are induced to differentiate, however,HDAC1 is no longer detected at this promoter,and instead, both MyoD and acetyltransferase P/CAF now occupy this promoter. In addition,enrichment of histone H3 acetylation andphosphorylation is now observed at this promoter.These data suggest that in addition tobeing an activator of differentiation-specificgenes, MyoD can also act as a transcriptionalrepressor in proliferating myoblasts while inpartnership with a histone deacetylase.106

Our laboratory studies transcript initiationand elongation by RNA polymerase II.We are currently focusing on (i) theprocess by which RNA polymerase II beginstranscription and clears the promoter; that is, thetransition from initiation to the transcriptelongation phase of RNA synthesis, (ii) themolecular mechanisms involved in the elongationprocess, and (iii) the effects of chromatinstructure on transcript elongation. All of theseaspects of transcription are important checkpointsin the regulation of gene expression. Theultimate goal of these studies is a more comprehensivepicture of transcription through itsaccurate duplication in test-tube systems.We have recently found that repetitivetemplate sequences in the initially transcribedregion can allow the nascent RNA to slipupstream and reassociate with the template,leading to the synthesis of an RNA longer thanpredicted by the template sequence (Pal andLuse, 2002). This observation has proved to bean important tool in allowing us to understandthe structural transitions within the RNApolymerase that must occur so that the initial,unstable transcription complex can successfullypass into the processive transcript elongationphase (Pal and Luse, submitted; Pal and Luse, inpreparation).Several major projects are under way onpromoter clearance and transcript elongationmechanisms. The first is an extension of ourexperiments (Samkurashvili and Luse, 1998) inwhich we explored the linkage betweentranslocation of RNA polymerase II along thetemplate and transcriptional arrest. We showedthat the polymerase tends to slide back along thetemplate, and in some cases to arrest, until about25 bonds have been made. Surprisingly, the fullymature form of the transcription complex doesnot emerge until the nascent RNA is about 45bases long. We have recently found that thisbehavior is a complex function of bothtranscript length and template sequence (Pal etal., 2001). We have also found that thetransition into the fully processive form of thetranscription complex is reversible (Újvári andLuse, 2002). Dr. Újvári, is currently extendingher recent observations to investigate interactionsbetween the transcript and the proteins ofthe transcription complex that stabilize thepolymerase in the elongation-committed state.We are also studying the sequence requirementsfor arrest by polymerase II (Hawryluk and Luse,submitted). Our results should help to explainthe known tendency of RNA polymerase II topause about 25-40 bases into the transcriptionunit for many eukaryotic genes.The Department of Molecular BiologyRNA Polymerase II Function Evaluated inTranscription Initiation, Checkpoints forTranscript ElongationOur other major project concerns theability of RNA polymerase II to elongate nascentRNAs on nucleosomal templates. We have shownthat nucleosomes assembled from highly purifiedhistones form an essentially absolute barrier toelongation by RNA polymerase II (Chang andLuse, J. Biol. Chem. 1997;272:23427) and thatprotein factors can partially relieve this barrier(Orphanides et al., 1998). We are currentlyfocusing on the effects of nucleosome structureon transcript elongation by RNA polymerase IIand, in a collaborative study with the laboratoryof Vasily Studitsky, on mechanistic aspects of thenucleosomal block to transcript elongation.Donal S. Luse, Ph.D.THE LUSELABORATORYRESEARCH ASSOCIATESMahadeb Pal, Ph.D.Subramaniam Sanker, Ph.D.POSTDOCTORAL FELLOWSLouise Steele, Ph.D.Andrea Újvári, Ph.D.TECHNICAL ASSOCIATESusan W. LuseCOLLABORATORVasily M. Studitsky, Ph.D. 11Dept. of Biochemistry andMolecular Biology, Wayne StateUniv., Sch. of Med., Detroit, MIOrphanides, G., LeRoy, G., Chang, C.-H., Luse, D.S., and D. Reinberg (1998) FACT, a factorthat facilitates transcript elongation through nucleosomes. Cell 92:105-116.Samkurashvili, I.. and D.S. Luse (1998) Structural changes in the RNA polymerase II transcriptioncomplex during the transition from initiation to elongation. Mol. Cell. Biol. 18:5343-5354.Pal, M., McKean, D., and D.S. Luse (2001) Promoter clearance by RNA polymerase II is anextended, multistep process strongly affected by sequence. Mol. Cell. Biol. 21:5815-5825.Pal, M., and D.S. Luse (2002) Strong natural pausing by RNA polymerase II within 10 basesof transcription start may result in repeated slippage and reextension of the nascent RNA.Mol. Cell. Biol. 22:30-40.Újvári, A., Pal, M., and D.S. Luse (2002) RNA polymerase II transcription complexes maybecome arrested if the nascent RNA is shortened to less than 50 nucleotides. J. Biol.Chem. 277:32527-32537.Pal, M., and D.S. Luse (2003) The initiation-elongation transition: lateral mobility of RNA inRNA polymerase II complexes is greatly reduced at +8/+9 and absent by +23. Proc. Natl.Acad. Sci. USA 100:5700-5705.107

THE PADGETTLABORATORYPROJECT SCIENTISTSJayendra Prasad, Ph.D.Girish C. Shukla, Ph.D.POSTDOCTORAL FELLOWKrishna P. Kota, Ph.D.TECHNICAL ASSOCIATESRosemary C. Dietrich, M.S.Kelly EmmettJohn D. FullerCOLLABORATORSChristopher Burge, Ph.D. 1Kwaku Dayie, Ph.D. 21Dept. of Biology, Mass. Inst. ofTechnology, Cambridge, MA2Dept. of Molecular Biology, CCFRemoval of intervening sequences (introns)from the primary RNA transcripts of mostvertebrate genes is a required step in theexpression of genetic information. This premRNAsplicing process requires the formation ofa multicomponent complex known as thespliceosome at the sites of splicing. Thespliceosome is composed of over 200 proteinsand five small nuclear RNAs (snRNAs). ThesesnRNAs are involved in splice site recognitionsteps leading to the formationof the spliceosome.They are also believed to beinvolved in the catalyticsteps of RNA cleavage andligation. The research in mylaboratory is directed atunderstanding the processesinvolved in specifying thesites of splicing within a premRNAand the mechanismof the splicing reactions.A few years ago, weidentified a previouslyunsuspected second class ofintrons within eukaryoticgenes. Subsequent work hasshown that this second classis widely distributed innature and must have beenpresent for at least a billionyears. More recent work hasled us to argue that bothclasses of introns werepresent in one of the earliestancestors of eukaryoticorganisms.In modern organisms, these introns arespliced in a spliceosome composed of foursnRNAs unique to this class of introns and onesnRNA that is common to both classes ofintrons. The spliceosomal proteins are also likelyto contain a mixture of unique and sharedproteins. The splicing mechanisms and many ofThe Department of Molecular BiologyIntrons Removed from RNA byRNA-Based MachinesRichard A. Padgett, Ph.D.the RNA-RNA interactions involved in theformation and function of the spliceosome arestrikingly similar in both classes of introns, whichsuggests that they probably share a similar origin.Over the last several years, we have beenmapping the RNA-RNA interactions involved inthe splicing of this new class of introns usinggenetic and biochemical techniques. These studiesare directed toward understanding the structureand function of the spliceosome and gaininginsights into how theseintrons are identified by thesplicing machinery in theprimary RNA transcripts ofgenes.Recently, we havefocused on the function of asmall RNA element in thespliceosome that may beinvolved in the catalysis ofthe splicing reactions. Wehave shown that this elementcan be functionally replacedby a similar RNA elementfrom a class of self-splicingRNA introns. This findingsuggests that both thespliceosome and these selfsplicingintrons use similarcatalytic mechanisms andsupports the idea thatspliceosomal introns evolvedfrom self-splicing introns.We are pursuing structuralstudies of these RNAelements to further definetheir functions in splicing.We are also extending our studies of theminor class spliceosome to the investigation ofthe proteins involved in the splicing of theseunusual introns. We will determine the overlapof protein factors between the two splicingsystems and identify proteins unique to the newlydiscovered spliceosome.Shukla, G.C., and R.A. Padgett (2001) The intramolecular stem-loop structure of U6 snRNA can functionallyreplace the U6atac snRNA stem-loop. RNA 7:94-105.Dietrich, R.C., Peris, M.J., Seyboldt, A.S., and R.A. Padgett (2001) Role of the 3’ splice site in U12-dependentintron splicing. Mol. Cell. Biol. 21:1942-1952 (additional information in Dietrich et al., Mol. Cell.Biol. 2002;21:3563).Shukla, G.C., and R.A. Padgett (2002) A catalytically active group II intron domain 5 can function in theU12-dependent spliceosome. Mol. Cell 9:1145-1150.Padgett, R.A., and G.C. Shukla (2002) A revised model for U4atac/U6atac snRNA base pairing [letter].RNA 8:125-128.Shukla, G.C., Cole, A.J., Dietrich, R.D., and R.A. Padgett (2002) Domains of human U4atac snRNA requiredfor U12-dependent splicing in vivo. Nucl. Acids Res. 30:4650-4657.108

Telomeres are the nucleoprotein complexesrequired for the stability and completereplication of chromosome ends. Brokenchromosome ends undergo permanent fusions toother DNA molecules in the cell or are degraded,resulting in genomic rearrangements that can leadto cancer. In contrast to broken ends, telomeresare stable and preserve the cell’s genomicintegrity.Telomere DNA consists of tandem arraysof TG-rich sequences: 10-20 kb of TTAGGG inhuman germ cells and 250-400 bp of TG 1-3inbudding yeast, where the TG-rich strand formsthe 3' end of the chromosome. The length ofthese repeats is nearly constant in human germcells and yeast and is probably maintained byregulating the processes of lengthening viatelomerase (a special enzyme that can synthesizetelomere repeats) and shortening caused byincomplete replication or nucleolytic processingof telomeric DNA.How these two processes are regulated tomaintain telomere lengths within a defined rangeis unknown. In human somatic cells, the lengthof the TTAGGG tract decreases as cells divide,leading to cell senescence when telomeresbecome too short. How telomere lengthinformation is transmitted to the cell-cyclemachinery is unknown. Our long-term goal is touse yeast as a model system to understand theseprocesses.Yeast measures telomere length bycounting the number of molecules of the majortelomere binding protein Rap1p; however, howRap1p molecules are counted is unknown. We(Ray and Runge, 1999a,b) developed a workingmodel for telomere length regulation based onour construction of yeast synthetic telomeres andon our work with the telomere length regulatorTEL2. We propose that yeast telomeres form afolded structure with Rap1p, Rif1p and Rif2p tocount Rap1p molecules andblock elongation. Shorttelomeres have too few Rap1pmolecules to form thisstructure, so they are elongated.We (Kota and Runge,1999) have found that theprotein encoded by TEL2(Tel2p) regulates yeast telomerelength in vivo and binds totelomeric TG 1-3in vitro. TEL2 isin the same genetic pathway asTEL1, a homolog of themammalian kinase DNA-PKcsand the yeast DNA damagecheckpoint regulator of the cellcycle, MEC1. The morphologyof cells lacking TEL2 suggeststhat they arrest in a specificThe Department of Molecular BiologyTelomere Length Regulation,Transcriptional Silencing and Cell Agingphase of the cell cycle. Thus, TEL2 may functionto link telomeres to cell-cycle control.We (Roy and Runge, 2000) have recentlydiscovered conditions that cause Sir proteins to beredistributed in the cell in a manner that extendscell life span. Because this redistributioncorrelated with Sir3p phosphorylation, weidentified the Sir3p kinase as Slt2p (a yeast MAPkinase), eliminated Sir3p phosphorylation byeliminating the predicted phosphorylation sites,and showed that lack of Sir3p phosphorylationcauses an increase in cell life span. Because theMAP kinase that phosphorylates Sir3p is activatedby the cell’s decision to grow, the Sir3p systemforms a model for the known phenomenon oforganisms exhibiting slow reproduction andlonger life span understarvation conditions,and more reproudctionand shorter life spanwhen nutrients areplentiful.THE RUNGELABORATORYRESEARCH ASSOCIATESRonald Hector, Ph.D.Alo Ray, Ph.D.Ken richardson, M.D.TECHNICAL ASSOCIATESThihan NyunAnna YakubenkoKurt W. Runge, Ph.D.Kota, R.S., and K.W. Runge (1999) Tel2p, a regulator of yeast telomerre length in vivo, binds to singlestrandedtelomeric DNA in vitro. Chromosoma 108:278-290.Ray, A., and K.W. Runge (1999a) Varying the number of telomere bound proteins does not alter telomerelength in tel1? cells. Proceedings of the National Academy of Sciences USA 96:15044-15049.Ray, A., and K.W. Runge (1999b) The yeast telomere length counting machinery is critically sensitiveto sequences at the telomere/non-telomere junction. Molecular and Cellular Biology 19:31-45.Roy, N., and K.W. Runge (2000) Two paralogs involved in transcriptional silencing that antagonisticallycontrol yeast life span. Curr. Biol. 10:111-114.Ray, A., and K.W. Runge (2001) Yeast telomerase appears to frequently copy the entire template invivo. Nucleic Acids Res. 29:2382-2394.Ray*, A., Hector*, R.E., Roy, N., Song, J.H., Berkner, K.L., and K.W. Runge (2003) Sir3p phosphorylationby the Slt2p pathway effects redistribution of silencing function and shortened lifespan. Nat. Genet.33:522-526 (*co-first authors).109

THE G. SENLABORATORYPROJECT SCIENTISTSSean Kessler, Ph.D.Saumendra Sarkar, Ph.D.RESEARCH ASSOCIATEFulvia Terenzi, Ph.D.POSTDOCTORAL FELLOWSMitali Pandey, Ph.D.Gregory Peters, Ph.D.Kristi Peters, Ph.D.TECHNICAL ASSOCIATESSrabani PalTheresa RoweTom ScheidemantelHeather SmithPaulette ZavackyGRADUATE STUDENTSChristopher ElcoDaniel HuiShoudong LiCOLLABORATORSJun Qin, Ph.D. 1George Stark, Ph.D. 2Bryan Williams, Ph.D. 3Vivien Yee, Ph.D. 41Dept. of Molec. Cardiol., CCF2Dept. of Molec. Biol., CCF3Dept. of Cancer Biol., CCF4Case Western Reserve University,Cleveland OHMy laboratory’s research interests are intwo areas: mechanism of regulation andfunctions of viral stress-inducible genes(VSIGs) and tissue specific functions ofangiotensin-converting enzyme (ACE).Transcription of a group of mammaliangenes is strongly induced by a variety of stimulirelated to virus infection, suchas double-stranded (ds) RNA,interferons (IFNs) and viralproteins. We are identifyingthese genes (VSIGs) bymicroarray analyses. Differentinducing agents appear to usedistinct signaling pathways forinducing the same genes. Todelineate these pathways, weuse mutant cell lines thatrespond to some, but not all,stimuli. Using this approach,distinct pathway-specifictranscription factors andprotein kinases have beenidentified. Moreover, fordsRNA signaling, we haveestablished a critical role ofToll-like receptor 3 andspecific tyrosine residues of itscytoplasmic domain.The most prominent VSIG products arethe members of the P56 family. They containTPR motifs and interact specifically with proteinscontaining PCI motifs including subunits ofproteasomes, COP9/signalosomes and translationinitiation factor eIF3. Binding of P56 to the P48(eIF3e) subunit of eIF3 inhibits its functions,causing an impairment of protein synthesis. Thus,a new IFN, dsRNA and virus-mediated pathwayof translational regulation has been discovered.Identification of other cellular functions of theseproteins is one of our future goals.Sen, G.C. (2001) Viruses and interferons. Annu. Rev. Microbiol. 55:255-281.Peters, K.L., Smith, H.L., Stark, G.R., and G.C. Sen (2002) IRF-3-dependent, NFκBandJNK-independent activation of the 561 and IFN-β genes in response to doublestrandedRNA. Proc. Natl. Acad. Sci. USA 99:6322-6327.Peters, G., Khoo, D., Mohr, I., and G.C. Sen (2002) Inhibition of PACT-mediated activationof PKR by Herpes Simplex Virus Type 1 Us11 protein. J. Virol. 76:11054-11064.Kessler, S.P., Scheidemantel, T.S., Gomos, J.B., Rowe, T.M., and G.C. Sen (2003)Maintenance of normal blood pressure and renal functions are independent effects ofangiotensin-converting enzyme. J. Biol. Chem. 278:21105-21112.Sarkar, S.N., Smith, H.L., Rowe, T.M., and G.C. Sen (2003) Double-stranded RNA-signalingby Toll-like receptor 3 requires specific tyrosine residues in its cytoplasmic domain.J. Biol. Chem. 278:4393-4396.Williams, B.R., and G.C.Sen (2003) A viral on/off switch for interferon. Science300:1100-1101.The Department of Molecular BiologyViral Stress-Inducible Gene Regulationand ACE FunctionsGanes C. Sen, Ph.D.We are interested in identifying cellulardsRNA-binding proteins and determining theirfunctions. The IFN-induced protein kinase PKRand its activator, PACT, are two such proteins.We have identified a small domain of PACT thatis responsible for PKR activation. This domain isdistinct from two other domains that bindstrongly to PKR or dsRNA.We are generating PACT knockoutmice to examine thisprotein’s physiological functions.2-5(A) Synthetases are afamily of enzymes thatpolymerize ATP into a series of2´,5´-linked oligoadenylates.The enzymes are inactive assuch: they require dsRNA fortheir activation. Using acombination of site-directedmutagenesis, chemical crosslinking,mass spectroscopy,fluorescence quenching andbiochemical and enzymaticanalyses, we have identified theacceptor binding, the donorbinding, the catalytic and thedimerization sites of the P69isozyme. Mutations in any ofthese sites inactivate the enzyme, butheterodimers of certain mutants are active,demonstrating that the two subunits of P69participate in cross-cross reactions. Analysis ofthe cellular functions of these enzymes revealed anovel property of the 9-2 small isozyme. Itcontains a Bcl2-homology domain 3 that causescellular apoptosis. This property of the protein isindependent of its enzymatic function. Recently,in collaboration with Dr. Vivien Yee, we haveobtained the crystal structure of another smallisozyme.The most well-studied physiologicalproperty of ACE is its pivotal role in bloodpressure regulation. Recent studies, however,indicate a much broader activity of ACE in renal,immunological and male reproductive functions.To understand the full repertoire of ACE actions,we have used a combination of gene knockoutand tissue-specific transgene expression toconclude that expression of the germinal isozymeof ACE in sperm is sufficient to maintain malefertility, but the somatic isozyme cannotsubstitute for this function of the germinalisozyme. Using a similar approach, we haveshown that regulation of blood pressure andmaintenance of normal kidney functions are twoseparable properties of the somatic isozyme.Further studies are in progress to delineateadditional physiological functions of ACE.110

We use two novel approaches to study thecontrol of cell-cycle progression andcell growth. Instead of synchronizingcells and performing biochemical analyses, as intraditional studies, we study actively growingcultures with time-lapse and quantitative imageanalytical approaches. Our results reveal importantaspects of cell-cycle control not previouslyobserved. Our goal is to extend these approachesand observations to the control of cell-cycleregulateddrug targets and to the analysis ofalterations in tumor cell-growth control.Cyclin D1 and the Cell CycleStudies in quiescent cells following growthstimulation have identified numerous moleculesand their interactions that are required to initiatecell-cycle progression. Unfortunately, loss ofsynchrony within such cultures prevents us fromanalyzing how the cell cycle continues andeventually terminates. Within actively proliferatingcultures, we can define the cell-cycle positionof individual cells, determining the molecularcharacteristics of each in relationship to the cellcycle.Our results reveal a critical, cell-cycleregulatedinteraction between cellular Ras andcyclin D1 proteins that controls the cell’s abilityto continue active proliferation. An importantclue was the fact that cyclin D1 is expressed athigh levels in actively dividing G1- and G2-phasecells, but at extremely low levels in S-phase cells(those actively synthesizing DNA). High levelsof cyclin D1 were strictly dependent uponcellular Ras activity in all cell-cycle phases, butthese levels could be stimulated by Ras activityonly during G2 phase. Thus, growth factorsstimulated by Ras activity induced an increase incyclin D1 levels in G2 phase. These levelsremained high through mitosis andG1 phase to promote the initiationof DNA synthesis; thereafter, cyclinD1 dropped to low levels.These facts form the basis forour model defining cyclin D1 as thecritical switch controlling ongoingcell growth. Cyclin D1 is needed toinitiate DNA synthesis; we haveshown this to be true also in activelycycling cells. Once the cell enters Sphase, however, cyclin D1 isautomatically suppressed to lowlevels. Levels remain low through Sphase, but on entering G2 phase, thecell must make a critical decision. Tocontinue proliferating, it must inducecyclin D1 levels during G2 phase.This requires the activity of growthfactors to stimulate cellular Rasactivity. If cyclin D1 levels areThe Department of Molecular BiologyProliferative Signal Transductionand Cell Cycle Regulationincreased, the cell becomes committed to passthrough not only the upcoming mitosis, but throughthe entire next cell cycle, completing the subsequentmitosis as well. But if conditions are not conducivefor continued cell-cycleprogression, cyclin D1 levelsare not induced during G2phase, and the cell entersquiescence immediatelyfollowing mitosis. CyclinD1 thus performs a switchfunction in the control ofcell-cycle progression. This“switch” automatically shutsoff during S phase, forcingthe cell to turn it on againduring G2 phase if the cell isto continue cycling.Topoisomerase II andDrug ToxicityThe technicalapproach utilized in thecell-cycle studies describedabove has also been appliedto the study of topoisomeraseII alpha, the targetof several anti-cancerdrugs. We have detailedthis molecule’s cell-cycleexpression characteristicsand the cell cycle’s role indetermining the cell’ssensitivity to the anti-topoisomerase II drugetoposide. We are determining the molecularcharacteristics of etoposide’s cell-cycle regulationin normal and tumor cells.THE STACEYLABORATORYINVESTIGATORSYang Guo, Ph.D.Masahiro Hitomi, M.D., D.M.S.Ke Yang, Ph.D.Dennis W. Stacey, Ph.D.Hitomi, M., and D.W. Stacey (1999) Cyclin D1 production in cycling cells depends on Ras in a cellcycle-specificmanner. Curr. Biol. 9:1075-1084.Stacey, D.W., Hitomi, M., and G. Chen (2000) Influence of cell cycle and oncogene activity upontopoisomerase IIalpha expression and drug toxicity. Mol. Cell. Biol. 20:9127-9137.Hitomi, M., and D.W. Stacey (2001) Ras-dependent cell cycle commitment during G2 phase. FEBSLett. 490:123-131.Sa, G., Hitomi, M., Harwalkar, J., Stacey, A.W., Chen, G.C., and D.W. Stacey (2002) Ras is activethroughout the cell cycle, but is able to induce cyclin D1 only during G2 phase. Cell Cycle1:50-58.Guo, Y., Stacey, D.W., and M. Hitomi (2002) Post-transcriptional regulation of cyclin D1 expressionduring G2 phase. Oncogene 21:7545-7556.Stacey, D.W. (2003) Cyclin D1 serves as a cell cycle regulatory switch in actively proliferatingcells. Curr. Opin. Cell Biol. 15:158-63.111

THE STARKLABORATORYPROJECT SCIENTISTSMunna L. Agarwal, Ph.D.Eugene S. Kandel, Ph.D.William R. Taylor, Ph.D.POSTDOCTORAL FELLOWSMukesh Agarwal, Ph.D.Mark W. Jackson, Ph.D.Tao Lu, Ph.D.Jinbo Yang, Ph.D.GRADUATE STUDENTSYulan QingDavid B. ShultzDavid WaldCOLLABORATORSIan M. Kerr, Ph.D. 1Xiaoxia Li, Ph.D. 2Nywana Sizemore, Ph.D. 31Cancer Research UK, London,UK2Dept. of Immunology, CCF3Dept. of Cancer Biology, CCFForward Genetic Analysis ofMammalian Signaling PathwaysMajor objects of our study are theinterferons (IFNs), pathways thatactivate or repress the transcriptionfactor NFκB, stress-induced pathways thatactivate the tumor suppressor protein p53, andpathways that respond to activated p53. We useforward genetics extensively, driving expressionof Herpes thymidine kinase (TK) with signalresponsivepromoters and selecting unresponsivecells with gancyclovir. Chemical or insertionalmutagenesis randomly inactivates a gene requiredfor signaling, resulting in a mutant cell line lackingthe corresponding protein.Re-covery of complementinggenes or the identificationof the insertion siteiden-tifies novel componentsof signaling pathwaysor con-firms the roles ofcomponents previouslyidentified by biochemicalanalyses. The sameselections can also be usedto clone cDNAs en-codingproteins that, whenoverexpressed, activate orinhibit the pathways.Interferons and STATsUse of cell lineslacking STAT1 has revealedthat STAT1-independentsignals are generated inresponse to IFN-γ and IFNβ.Unraveling this newcomplexity in IFN signalingwill help us to understandthe full set of biologicalroles of this importantcytokine. We also study the role of STAT1 andSTAT3 in untreated cells, where they are requiredfor efficient constitutive expression of manyRamana, C.V., Gil, M.P., Han, Y., Ransohoff, R.M., Schreiber, R.H., and G.R. Stark(2001) Stat1-independent regulation of gene expression in response to IFNγ. Proc. Natl.Acad. Sci. USA 98:6674-6679.Li, X., Commane, M., Jiang, Z., and G.R. Stark (2001) IL-1-induced NFκB and c-JunN-terminal kinase (JNK) activation diverge at IL-1 receptor-associated kinase (IRAK).Proc. Natl. Acad. Sci. USA 98:4461-4465.Taylor, W.R., Schönthal, A.H., Galante, J., and G.R. Stark (2001) p130/E2F4 binds toand represses the cdc2 promoter in response to p53. J. Biol. Chem. 276:1998-2006.Agarwal, M.L., Ramana, C.V., Hamilton, M., Taylor, W.R., DePrimo, S.E., Bean, L.J.,Agarwal, A., Agarwal, M.K., Wolfman, A., and G.R. Stark (2001) Regulation of p53 expressionby the Ras-MAP kinase pathway. Oncogene 20:2527-2536.Sizemore, N., Lerner, N., Dombrowski, N., Sakurai, H., and G.R. Stark (2002) Distinctroles of the IκB kinase α and β subunits in liberating nuclear factor-κB (NF-κB) fromIκB and in phosphorylating the p65 subunit of NF-κB. J. Biol. Chem. 277:3863-3869.The Department of Molecular BiologyGeorge R. Stark, Ph.D.Distinguished Scientist,Cleveland Clinic Foundationgenes. Unphosphorylated STATs formheterodimers with other transcription factors,activating the expression of promoters withcomplex DNA elements. We are defining thedetails of these interactions as well as the role ofoverexpressed unphosphorylated STAT3 incancer.IL-1 and NFκBWe have isolated several mutant cell linesunresponsive to IL-1, which activates the phosphorylationof IκB by IκB kinase, leading to IκB degradationand the consequent liberation of NFκB, and thephosphorylation andactivation of NFκB,mediated by receptorassociatedphosphoinositol-3-kinase (PI3K). We arecollaborating with XiaoxiaLi to study these mutants.We have also isolatedmany mutants in which thenormal suppression ofNFκB-dependent signalinghas been disrupted, throughthe loss of negativeregulators. Insertionalmutagenesis will allow us toidentify such regulators.We are also working todefine the biological roleand detailed function ofSIGGR, a novel Toll-likereceptor, in collaborationwith Xiaoxia Li.p53Using p53-responsive promoters, wehave isolated severalmutant cells in which p53signaling is altered. By using retroviral-mediatedinsertional mutagenesis alone, we are nowobtaining dominant mutants in which a retroviralLTR drives overexpression of a full-length ortruncated protein that inhibits p53-dependenttranscription, or an antisense RNA.Biochemical and cell-cycle analyses havehelped to reveal the complexity of p53 action. Inaddition to its well-known ability to arrest cells inG1 in response to stress, p53 can also arrest cellsat other points in the cell cycle. We are studyingthe detailed mechanisms of p53-mediated arrestwithin the S phase at the G2/M boundary.112

The Department of Molecular BiologyViral Pathogenicity Examined inRNA VirusesOur laboratory’s long-term goal is tounderstand the molecular basis of thepathogenicity of viruses of the negativestrandRNA (nsRNA) family, using vesicularstomatitis virus (VSV) and human parainfluenzavirus type 3 (HPIV-3) as the prototype viruses. Athorough understanding of the mode of viraltranscription and replication is fundamental todeveloping reagents to combat them. Ouremphasis is on establishing the functions of keyviral proteins, such as L (the RNA polymerase),P (the transcription factor), and N (the nucleocapsidprotein), encapsidating the genome RNA.The virus ribonucleoprotein (RNP) complexcontaining these polypeptides transcribes thegenome RNA in vitro as well as in vivo, by which itinitiates infection within the infected cells. Wealso study host-virus interaction and havediscovered several host proteins that play criticalroles in the gene expression of these viruses.Vesicular Stomatitis VirusUsing recombinant expression vectors, wehave expressed, in biologically active form, viralpolypeptides that constitute the transcribingRNP (i.e., L, N and P proteins) in prokaryoticand eukaryotic cells. Several important discoverieshave been made, especially with respect to thesubunit composition of the L protein and theputative replicase complex. We have shown thatL protein expressed in insect cells associates withthe cellular translation elongation factor EF-1 a/b/g subunits for its activity. In this respect, VSVRNA polymerase bears a striking similarity tobacteriophage Qb replicase, which requires thebacterial homologue of the translation elongationfactors Ts and Tu. We have shown that thehost cell capping enzyme specifically interactswith the viral RNA polymerase (L) and mediatesthe capping of viral mRNAs, whereas themethyltransferase activity that methylates thecapped structure is encoded by the L protein.These unique findings may help unravel the rolesof these cellular proteins in VSV RNA polymerasefunction. Using a reverse-genetics systemto study transcription and replication of speciallyconstructed mini-genome or defective interfering(DI) particles using cDNAs encoding P proteinsand wild-type L and N proteins, we havedemonstrated that although the transcriptioncomplex is composed of L-P 2-3, the replicaseappears to be a tripartite complex between L (N-P) that initiates the replication reaction. Understandingthe structure and function of thereplicase complex is key to gaining insight into thereplicative pathways in the VSV life cycle.Human parainfluenza virusThe transcription complex of HPIV-3consists of L, P, and N proteins in which the Nprotein encapsidates the genome RNA. We haveshown that specific interaction and polymerizationof actin on the RNP complex leads to theactivation of transcription in vitro, and that thevirus replicates in the cytoskeletal network, whereactin plays a critical role in the replication process.Continuing studies have led to discovery of twoadditional cellular proteins that specificallyinteract with cis-acting viral RNAs, including thekey glycolytic enzyme glyceraldehyde 3-phosphatedehydrogenase (GAPDH) and the nuclearautoantigen La protein. Understanding, in detail,the molecular basis of the interplay of viruses andcellular proteins will further our knowledge ofthe host’s role in promoting virus replication. Wehave shown that HPIV-3 entry and budding is bidirectional,although the apical pole is greatlypreferred. We are identifying and characterizingthe receptor(s) involved in the entry process ofHPIV-3. We have further shown that HPIV-3upregulates MHC class I and II expression onrespiratory epithelial cells without involvementof the STAT1 and class II transactivator (CIITA)pathways. These results suggest that HPIV-3 playsan important role in infection-related immunityand pathogenesis.A full-length cDNA clone of the HPIV-3genome (called pHPIV-3) was constructed, andrecombinant, infectious HPIV-3 was generated bytransfecting pHPIV-3 and support plasmidsencoding the HPIV-3 NP, P, and L proteins intoHeLa cells infected with a recombinant vacciniavirus expressing T7 RNA polymerase. We havemapped the replication and transcriptionpromoters on the genome RNA using a uniqueHPIV-3 mini-genome system. Availability of aninfectious clone for HPIV-3 and the mini-genomesystem will enhance our understanding of themolecular biology of HPIV-3 gene expression andmay help develop an effective vaccine against thisimportant human pathogen.VIROLOGYTHE A.K. BANERJEELABORATORYPROJECT STAFFSantanu Bose, Ph.D.Manjula Mathur, Ph.D.RESEARCH ASSOCIATESSantanu Bose, Ph.D.Achut Malur, Ph.D.POSTDOCTORAL FELLOWSMausumi BasuPatricia Bates, Ph.D.Shaji Daniel, Ph.D.Yuwen Huo, Ph.D.Jared LeMaster, Ph.D.Kaustubha Qanungo, Ph.D.TECHNOLOGISTDouglas Younger, III, B.S.COLLABORATORSAsit K. Pattnaik, Ph.D. 1Richard Ransohoff, M.D. 2Focco van den Akker, Ph.D. 31Dept. of Microbiology andImmunology, Univ. of Miami, FL2Dept. of Neurosciences, CCF3Case Western ReserveUniversity, Cleveland, OHMathur, M., and A.K. Banerjee (2002) Novel binding of GTP to the phosphoprotein (P) of vesicular stomatitisvirus. Gene Exp. 10:193-200.Gupta, A.K., Mathur, M., and A.K. Banerjee (2003) Unique capping activity of the recombinant RNApolymerase (L) of vesicular stomatitis virus: association of cellular capping enzyme with the L protein.Biochem. Biophys. Res. Commun. 293:264-268.Bose, S., and A.K. Banerjee (2002) Role of heparan sulfate in human parainfluenza virus type 3 infection.Virology 298:73-83.Gupta, A.K., Shaji, D., Banerjee AK. Identification of a novel tripartite complex involved in replication ofvesicular stomatitis virus genome RNA. J. Virol. 77:732-738.Amiya K. Banerjee, Ph.D.Head, Section of Virology113

THE PELLETTLABORATORYRESEARCH ASSOCIATEFu-Zhang Wang, Ph.D.POSTDOCTORAL FELLOWSSubhendu Das, Ph.D.Olena Skomorovska-Prokvolit, Ph.D.SENIOR RESEARCHTECHNOLOGISTGeorge CompitelloCOLLABORATORSVIROLOGYRobin Avery, M.D. 1Belinda Yen-Liberman, Ph.D. 21Dept. of Infectious Diseases,CCF2Dept. of Pathology andLaboratory Medicine, CCFHerpesviruses: Molecular Marvelsand Potent PathogensHerpesviruses constitute a virus family ofgreat genetic and biologic diversity. Theherpesviruses that infect humans includeherpes simplex virus types 1 and 2 (whichpredominantly cause oral and genital disease,respectively), the virus that causes chicken poxand shingles (varicella-zoster virus), most casesof infectious mononucleosis (Epstein-Barrvirus), roseola infantum (Human herpesvirus 6B),and Kaposi’s sarcoma (Kaposi’s sarcomaassociatedherpesvirus, a.k.a. Human herpesvirus8). Of the human herpesviruses,the one with perhapsthe greatest clinical impact ishuman cytomegalovirus,which is a leading cause ofcongenitally acquired mentalretardation and deafness,causes some infectiousmononucleosis, is a majorpathogen in immunocompromisedpatients, and mayhave a role in the developmentof atherosclerosis andother occlusive vasculardiseases. An importantshared biologic property ofall herpesviruses is latency,the process by which theseviruses persist in their host ina generally quiescent mannerfor the life of the host. Inimmunocompromised hosts,such as organ transplant recipients who havebeen chemically immunosuppressed to preventrejection of the transplanted organ, theseformerly quiescent viruses can each emerge fromlatency and cause significant disease that is oftendifficult to treat.The genetic complexity and diversity ofCannon, M.J., Dollard, S.C., Smith, D.K., Klein, R.S., Schuman, P., Rich, J.D., Vlahov,D., Pellett, P.E. for the HIV Epidemiology Research Study Group (2001) Blood-borne andsexual transmission of human herpesvirus 8 in women with or at risk for human immunodeficiencyvirus infection. N. Engl. J. Med. 344:637-643.Krug, L.T., Inoue, N., and P.E. Pellett (2001) Sequence requirements for interaction of humanherpesvirus 7 origin binding protein with the origin of lytic replication. J. Virol. 75:3925-3936.Cannon, M.J., Dollard, S.C., Black, J.B., Edlin, B.R., Hannah, C., Hogan, S.E., Patel,M.M., Jaffe, H.W., Offermann, M.K., Spira, T.J., Pellett, P.E., and C.J. Gunthel (2003)Risk factors for Kaposi’s sarcoma in men seropositive for both Human herpesvirus 8 andhuman immunodeficiency virus. AIDS 17:215-222.Stover, C.T., Smith, D.K., Schmid, D.S., Pellett, P.E., Stewart, J.A., Klein, R.S., Mayer,K., Vlahov, D., Schuman, , P. and M.J. Cannon, for the HIV Epidemiology ResearchStudy Group (2003) Prevalence of and risk factors for viral infections among HIV-infectedand high-risk-uninfected women. J. Infect. Dis. 187:1388-1396.Cannon, M.J., Laney, A.S., and P.E. Pellett (2003) Human herpesvirus 8: current issues.Clin. Infect. Dis. 37:82-87.The Department of Molecular BiologyPhilip E. Pellett, Ph.D.herpesviruses, coupled with their complex hostbiology, make these viruses engaging objects ofscientific study, providing fascinating and importantintersections with almost every area ofmolecular biology, cellular biology, immunology,pathogenesis, and epidemiology.The purposes of this laboratory (establishedin 2003) are (i) to conduct basic researchthat adds to our fundamental understanding ofthe relationships among the various herpesviruses,the processes by which human herpesvirusesreplicate and persist inpopulations on an evolutionarytime scale, and themechanisms by which theseviruses cause disease, and (ii)to use this knowledge toprevent or treat herpesvirusassociateddisease.Initiation of HerpesvirusDNA ReplicationOur studies ofherpesvirus replicationinclude structure-functionanalysis of the origin-bindingprotein (OBP), a protein thatis con-served among manymembers of the herpesvirusfamily. OBP binds to theorigin of lytic replication andserves as a nucleatingscaffold for assem-bly of thereplication fork and as an active parti-cipant inpreparing the origin sequence for repli-cationinitiation. We have mapped the structuraldomains responsible for the sequence-specificDNA binding activity of OBP, defined sequencesit can bind to, and found that the protein interactswith one face of the DNA double helix. In subsequentwork, we will identify important conservedfeatures of the structure of the proteinand identify means by which the protein-DNAinteraction can be blocked, so as to inhibit viralreplication.Human Cytomegalovirus Molecular andCellular BiologyWe are initiating a research programdirected at understanding the role of humancytomegalovirus in disease processes, such asinflammation and immune diversion, and themanner in which certain virally encoded proteinsand viral genetic variation contribute to this.Herpesvirus Translational ResearchThe translational research component of ourprogram is directed at improving patient out-comeswith respect to herpesvirus-related disease. Thiseffort will involve a multidisciplinary collaborationbetween basic laboratory scientists, clinical virologists,infectious and vascular disease specialists,epidemiologists, and biostatisticians.114

The AIDS pandemic has been recognizedas one of the most important healththreats of the coming century. Therapeuticinterventions, host immuneresponse, and vacci-nations haveall failed to control the humanimmunodeficiency virus (HIV)epidemic because the virusevolves so rapidly. Our researchis focused on HIV-1 evolution toanswer both basic and clinicalresearch questions about viraldynamics and development ofnew antiretroviral strategies.Clinical Significance ofHIV-1 FitnessMultidrug-resistant (MDR)variants of HIV, with reducedsusceptibility to antiretroviralcompounds from two or more classesof drugs, are now commonly found in treated patients. Inthe absence of drug pressure, the most fit virus would beexpected to be the wild-type virus. But in the presence ofdrug-selective pressure, resistant virus is the most fit virus.However, despite the multitude of in vitro data, we still donot know how this relatively reduced viral fitness couldeventually affect the clinical outcome of patients withMDR virus. Using growth competition experiments andTaqMan ® real-time PCR technology, models of the impactof antiretroviral therapy on HIV-1 fitness and diseaseprogression are being developed.Anti-HIV-1 Mechanisms of CombinationTherapy with IFN-α and Protein TyrosinePhosphatase InhibitorsAlthough advances in HIV/AIDS therapeuticshave caused a decrease in both AIDS incidence anddeath in the U.S. and Europe, treatments withantiretroviral com-binations, host immune response,and vaccinations have all failed to control the HIV type 1(HIV-1) epidemic due to rapid evolution of the virus.Therefore, the need for therapeutic alternatives in thetreatment of HIV infec-tions has become clear.Interferons (IFNs) are part of the natural humandefensive response directed against virus infections,including HIV. Multiple studies have shown thatexogenous IFN type I inhibits HIV-1 replication in vitro.The Department of Molecular BiologyHIV: Fitness and Evolution StudiesLeading to New Antiretroviral TherapiesMiguel E. Quiñones-Mateu,Ph.D.Clinical studies of exogenous IFNs taken alone or incombinations with antiretroviral drugs have shownsome success, especially for earlier HIV-1 primaryinfections. However, the role of IFNsin antiretroviral therapy and AIDSpathogenesis is still unknown. On theother hand, it has been reported thatinhibition of protein tyrosinephosphatases (PTPs, enzymes thatcontrol a diverse array of cellularproces-ses) by sodium stibogluconate(SSG, a drug used for the treatment ofleishmaniasis) enhances the in vitro signalingof IFN type I, perhaps increasingits antiviral effect. We are evaluatingthe in vitro and in vivo anti-HIV-1activity of IFN type I in combinationwith SSG and other PTP inhibitors, toboost the effects of IFNs in antiviraland/or immunotherapeutic treatments.Lethal Mutagenesis as a NewAntiretroviral TherapyTreatments with combinations of antiretroviralmedications do not completely inhibit HIV replication,eventually leading to treatment failure. Thus, there is asubstantial need for the availability of novel agents,which target different sites involved in the virus lifecycle. RNA viruses (such as HIV-1) replicate andevolve as complex mutant distributions termed viralquasi-species, which are powered by error-pronereplication and high mutation frequency. However,maintaining such a high mutation frequency isdangerous for the virus. An increase in the averageerror rate above a critical threshold during viralreplication should result in the loss of geneticinformation in a process that has been referred to asviolation of the error threshold or entry into errorcatastrophe. If an RNA virus quasi-species goesbeyond this mutation limit, the population will nolonger be viable. Interestingly, it has been pre-dictedthat RNA viruses’ high mutation frequencies are closeto this limit and can be forced into error catas-tropheby a moderate increase in mutation rate. This newconcept is opening new avenues for understanding viralinfections, and it should allow an assessment of thepossibilities of lethal mutagenesis as an antiviralstrategy against HIV.VIROLOGYTHE QUIÑONES-MATEU LABORATORYPOSTDOCTORAL FELLOWSJan Weber, Ph.D.Hector Rangel, Ph.D.RESEARCH TECHNICIANSBikram Chakraborty, B.S.Patti Kaiser, M.S.GRADUATE STUDENTMichael Marotta, B.S.COLLABORATORSEric J. Arts, Ph.D. 1Esteban Domingo, Ph.D. 2Jose A. Este, Ph.D. 3Michael M. Lederman, M.D. 1Miguel A. Martinez, Ph.D. 3Robert H. Silverman, Ph.D. 4Vicente Soriano, M.D. 5Cheryl A. Stoddart, Ph.D. 6Zahra Toossi, M.D. 1Guido Vanham, M.D., Ph.D. 7Taolin Yi, Ph.D. 41Case Western Reserve Univ.,Cleveland, OH2Centro de Biologia Molecular“Severo Ochoa,” UniversidadAutonoma deMadrid, Spain3Fundacio irsiCaixa, Barcelona,Spain4Dept. of Cancer Biology, CCF5Instituto de Salud Carlos III,Madrid, Spain6Gladstone Inst. of Virology andImmunology, Univ. of Californiaat San Francisco7Inst. of Tropical Medicine,Antwerp, BelgiumQuiñones-Mateu, M.E., Albright, J.L., Mas, A., Soriano, V., and E.J. Arts (1998) Analysis of pol gene heterogeneity, viral quasispecies, and drugresistance in individuals infected with group O strains of human immunodeficiency virus type 1. J. Virol. 72:9002-9015.Quiñones-Mateu, M.E., Ball, S.C., Marozsan, A.J., Torre, V.S., Albright, J.L., Vanham, G., van der Groen, G., Colebunders, R.L., and E.J. Arts(2000) A dual infection/competition assay shows a correlation between ex vivo HIV-1 fitness and disease progression. J. Virol. 74:9222-9233.Quiñones-Mateu, M.E., Gao, Y., Ball, S.C., Marozsan, A., Abraha, A., and E.J. Arts (2002) In vitro intersubtype recombinants of human immunodeficiencyvirus type 1: comparison to recent and circulating in vivo recombinant forms. J. Virol. 76:9600-9613.Quiñones-Mateu, M.E., Tadele, M., Parera, M., Mas, A., Weber, J., Rangel, H.R., Chakraborty, B., Clotet, B., Domingo, E., Menendez-Arias, L., andM.A. Martinez (2002) Insertions in the reverse transcriptase increase both drug resistance and viral fitness in a human immunodeficiency virustype 1 isolate harboring the multi-nucleoside reverse transcriptase inhibitor resistance 69 insertion complex mutation. J. Virol. 76:10546-10552.Ball, S.C., Abraha, A., Collins, K.R., Marozsan, A.J., Baird, H., Quiñones-Mateu, M.E., Penn-Nicholson, A., Murray, M., Richard, N., Lobritz, M.,Zimmerman, P.A., Kawamura, T., Blauvelt, A., and E.J. Arts (2003) Comparing the ex vivo fitness of CCR5-tropic human immunodeficiency virustype 1 isolates of subtypes B and C. J. Virol. 77:1021-1038.115

The Department of Molecular BiologyGlobal effect of tumorsuppressor gene p53 onradiation response ofmammalian tissues. Wild type(left) and p53-deficient (right) micewere treated with 10Gy of total bodygamma radiation and cell proliferationin tissues was determined in situ by14C-thymidine incorporation followedby autoradiography of whole-bodysections. While p53-wild type animalsshowed dramatic reduction in DNAreplication in all tissues, p53-nullmice maintained the same rate ofDNA synthesis as non-irradiatedanimals. This result reflects the keyrole of p53 in genotoxic stressresponse in mammalian organism.(From Komarova et al., 2000.Oncogene 19, 3791-3798).116

MolecularCardiology

DEPARTMENT OFMOLECULARCARDIOLOGYThe Department of Molecular CardiologyMolecular Cardiology: Basic Research onCardiovascular DiseasesCHAIRMANEdward F. Plow, Ph.D.The Robert C. Tarazi, M.D.,ChairVICE-CHAIRMANEric J. Topol, M.D.Dept. of CardiovascularMedicine, CCFSTAFFJoan E. B. Fox, Ph.D.Subha Sen, Ph.D.ASSOCIATE STAFFKathleen Berkner, Ph.D.Sadashiva Karnik, Ph.D.Kunio Misono, Ph.D.Dianne M. Perez, Ph.D.Jun Qin, Ph.D.ASSISTANT STAFFTatiana Byzova, Ph.D.Indira Sen, Ph.D.Qing Wang, Ph.D.STAFF SCIENTISTTatiana Ugarova, Ph.D.PROJECT SCIENTISTSJoel Dopp, Ph.D.Tom Haas, Ph.D.Timothy O’Toole, Ph.D.Shaoqi Rao, Ph.D.Olga Stenina, Ph.D.RESEARCH ASSOCIATESKasia Bialkowska, Ph.D.Sudhiranjan Gupta, Ph.D.Rajkumar Kadaba, PH.D.Supriya Patil, Ph.D.Elzbieta Pluskota, Ph.D.Mary Ruehr, Ph.D.Mary Russell, Ph.D.Gong Qing Shen, Ph.D.Olga Vinogradova, Ph.D.Yanwu Yang, Ph.D.JOINT APPOINTMENTFetnat M. Fouad-Tarazi, M.D.Kandice Kottke-Marchant, M.D., Ph.D.Michael Lincoff, M.D.Todor Mazgalev, M.D.Christine Moravec, Ph.D.Joseph V. Nally, Jr. M.D.Norman B. Ratliff, M.D.Patrick Tchou, M.D.David Van Wagoner, Ph.D.118The major goals of the Department ofMolecular Cardiology are:• Continuation of the long-standingtradition at the Cleveland Clinic Foundation forresearch excellence in the area of cardiovascularbiology.• Development of cutting-edge researchprograms to address significant physiological andpathophysiological mechanisms that occur in theheart and the vasculature.• Maintenance of an active dialogue withclinicians by addressing medically importantproblems.• Provide educational opportunities forstudents and fellows to receive research trainingin cardiovascular biology.To realize these goals, faculty memberswithin the department lead active researchprograms that deal with a broad range ofcardiovascular problems. The types of analysesrange from basic studies of protein structure, tothe dissection of molecular and cellular mechanisms,to investigations conducted in animalmodels and in humans. Currently, the departmentis composed of 20 staff members (Full StaffEdward F. Plow, Ph.D.through the Project Scientist level). Researchprograms in the Department of MolecularCardiology are organized into six major areas ofinterest: hypertension, heart failure, thrombosis,vascular biology, and structural biology andcardiovascular genetics.HypertensionResearch in the hypertension area seeks tocontinue the tradition of research excellence inthis area established by Cleveland Clinic investigators,such as Irvine Page, Merlin Bumpus andRobert Tarazi. Studies include the analyses of thestructure and function of the angiotensin I-converting enzyme (ACE), which controlsformation of the regulatory peptide angiotensin II(Dr. I. Sen). Angiotensin II is a focus of theresearch effort in the laboratory of Dr. Karnik,who seeks to understand (a) the mechanisms bywhich this peptide regulates blood pressurethrough its function as a ligand for specificreceptors and (b) the signaling events and cellularresponses initiated by recognition of this ligandby these receptors. The angiotensin receptorsbelong to the large family of seven transmembrane-spanningreceptors. Dr. Perez’s laboratoryis involved in in vitro and in vivo studies to analyzethe basis for ligand recognition, activation and thefunctional consequences ofoccupancy of the adrenergicmembers of this receptor family.Heart FailureThe second major researchemphasis in the department isheart failure. Heart failure hasbecome a major health problem inthe United States, reachingepidemic proportions. Threelaboratories of senior investigators(Drs. S. Sen, Misono andBond) have dedicated researchefforts to meet this challenge. Dr.S. Sen’s laboratory seeks toidentify the molecular playersinvolved in the initiation andprogression of heart failure and todevelop strategies to reverse thetransition from hypertrophy toheart failure. Dr. Bond’slaboratory investigates thesignaling pathways and mechanismsassociated with thesimulation major receptor systemsinvolved not only in the normalphysiologic response of the heartbut also in pathogenic mechanismsof heart failure. Dr. Misono’slaboratory is characterizing atrialContinued on Page 119

The Department of Molecular CardiologyContinued from Page 118natriuretic factor and its receptor at a structurallevel. This ligand-receptor system is believed toplay a central role in the heart failure response.ThrombosisThrombosis remains the leading cause ofdeath in the United States. A better understandingof the molecular mechanisms of thrombosis,hemostasis and fibrinolysis represents a third area ofresearch, emphasized within several laboratories inthe department. The laboratory of Dr. Plow isinterested in the function of the components ofthe plasminogen system, the molecular pathwaythat is responsible for the dissolution of blood clotsand a system that also influences cell migration. Hisstudies use mice in which the genes for variouscomponents of the plasminogen system have beeninactivated, and these knockout mice provide ameans to dissect the role of the plasminogen systemin physiologic and pathophysiologic events.Essential to the function of blood coagulationproteins is their post-translational modification byγ-carboxylation. Dr. Berkner’s lab seeks to definethe regulation of this event and to characterize theresponsible γ-carboxylase. The laboratories of Drs.J. Fox and Plow have major research effortsfocused on the role of platelets in thrombusformation. The receptors that mediate plateletadhesion and aggregation, events that are central tothrombus formation, are under analysis. Dr.Byzova’s laboratory investigates how particularsignaling molecules and events control plateletadhesive responses. Her studies utilize transgenicmouse models to dissect the contributions of thesesignaling molecules.Vascular BiologyVascular biology, the department’s fourtharea of emphasis, is broadly studied throughout theLerner Research Institute. Cell adhesion, intracellularsignaling events and pathways and regulation ofthe cytoskeleton represent specific cellular processesthat contribute to complex physiologic andpathophysiologic responses, such as angiogenesis,atherosclerosis and restenosis. Cell adhesionmechanisms are major areas of emphasis within thelaboratories of Drs. J. Fox and Plow. Studies ofligand binding to integrin adhesion receptors andactivation of these adhesion receptors are majortopics of investigation. Dr. Qin, a structuralbiologist (see below), uses NMR to study thestructural basis for integrin activation and celladhesion. A major focus of research in Dr. Byzova’slaboratory is angiogenesis; in vitro and in vivo modelsare utilized to study how growth factors induce thisresponse. Together, these laboratories seek to gaininsights into the processes underlying atherosclerosis,restenosis, and therapies for these diseases.Downstream signaling events and cytoskeletallinkages triggered by activation and occupancy ofthese receptors are areas of intense investigation.Genetic Basis of Cardiovascular DiseaseThe genetic basis of cardiovascular diseasehas evolved into a major theme of research in theDepartment. Dr. Wang’s laboratory is seeking toidentify mutations in genes that cause cardiacarrhythmias and vascular abnormalities in humansand then develops animal and cellular models todetermine how these mutations lead to the abnormalphenotypes. Dr. Topol has organized largemulticenter studies to identify allelic variantsinvolved in premature coronary artery disease andmyocardial infarction. These studies have spurredcollaborations with Dr Wang to find the genesleading to these cardiovascular diseases and withDrs. Plow and Byzova to determine why a singlenucleotide polymorphism in a particular family ofproteins is associated with an increased risk ofcoronary artery disease. Dr Karnik is seeking toidentify polymorphisms within an angiotensinreceptor gene and how these are linked to cardiovasculardisease. Dr. Bond uses gene chip analyses toidentify genes that are altered in human failing hearttissues, to determine if gene expression patterns canbe used to predict the course of heat failure, and toidentify molecular targets that may have a causativerole. Many analyses in the Department involve theuse of transgenic mice in which gene products areoverexpressed or inactivated to dissect how specificproteins known to contribute to cardiovasculardisease exert their functions.Structural BiologyThe above synopsis of ongoing researchactivities within the Department of MolecularCardiology indicates a heavy emphasis on peptideand protein functional analyses. To complementthese endeavors, Dr. Qin, a structural biologist,who studies basic aspects of protein structure andfunction and represent the fifth area of researchemphasis in the Department. Dr. Qin usesnuclear magnetic resonance spectroscopy toanalyze the three-dimensional structure of a widevariety of proteins. Together with facultymembers in various departments throughout theLerner Research Institute, Dr. Qin is a member ofa growing body of structural biologists withinCleveland’s structural biology community, whoseek to bring these state-of-the-art approaches toprovide high resolution understanding of howprotein molecules work. Knowledge of thethree-dimensional structure of proteins not onlyprovides insights into their modus operandi but alsois key to the future of drug design to target theseproblems.Dept. website: http://www.lerner.ccf.org/moleccard/119

THE BERKNERLABORATORYPOSTDOCTORAL FELLOWSWen Qian, Ph.D.Mark Rishavy, Ph.D.TECHNICAL ASSOCIATEKevin HallgrenKathleen L. Berkner, Ph.D.The Department of Molecular CardiologyInsight into Carboxylation of Vitamin K-DependentProteins Offers Hope for DesigningCoagulation Therapies, Hemophiliac HelpWe are interested in how vitamin K-dependent (VKD) proteins are carboxylated to render them active and in therole vitamin K plays in different biologicalsystems. Carboxylation involves the conversionof clusters of Glu residues to gamma-carboxylatedGlu residues in VKD proteins, using vitaminK as a cofactor. This modification generatescalcium-binding modules in VKDproteins, allowing them to exert theireffects in hemostasis, calcium homeostasis,bone development, cellular proliferation,vascular remodeling and signaltransduction. The enzyme that modifiesthe VKD proteins, gamma-carboxylase,resides in the endoplasmic reticulum,and carboxylation occurs during thesecretion of VKD proteins by amechanism that is poorly understood.Our recent work has revealed insightsinto the molecular mechanism forconversion of Glu residues to gammacarboxylatedGlu residues. In studiesthat represent a breakthrough formapping functional residues of thecarboxylase, we identified the active siteand implicated a mechanism ofsubstrate-regulated catalysis. Thesestudies are a critical first step towarddesigning superior anticoagulants thatdirectly target the carboxylase, ratherthan the indirect and nonspecific onescurrently in use. VKD proteins requiremultiple Glu to gamma-carboxylatedGlu conversions for their activity; forexample, 12 such conversions occur in thehemostatic VKD protein factor IX. We recentlyshowed that the carboxylase uses a processivemechanism to effect the multiple carboxylations,Pudota, B.N., Miyagi, M., Hallgren, K.W., West, K.A., Crabb, J.W., Misono, K.S., andK.L. Berkner (2000) Identification of the vitamin K-dependent carboxylase active site:Cys-99 and Cys-450 are required for both epoxidation and carboxylation. Proc. Natl.Acad. Sci. USA 97:13033-13038.Berkner, K.L. (2000) The vitamin K-dependent carboxylase. J. Nutr. 130:1877-1880.Stenina, O., Pudota, B.N., McNally, B.A., Hommema, E.L., and K.L. Berkner (2001)Tethered processivity of the vitamin K-dependent carboxylase: factor IX is efficientlymodified in a mechanism which distinguishes Gla’s from Glu’s and which accounts forcomprehensive carboxylation in vivo. Biochemistry 40:10301-10309.Pudota, B.N., Hommema, E.L., Hallgren, K.W., McNally, B.A., Lee, S., and K.L. Berkner(2001) Identification of sequences within the gamma-carboxylase that represent a novelcontact site with vitamin K-dependent proteins and that are required for activity. J. Biol.Chem. 276:46878-46886.Hallgren, K.W., Hommema, E.L., McNally, B.A., and K.L. Berkner (2002) Carboxylaseoverexpression effects full carboxylation but poor release and secretion of factor IX:implications for the release of vitamin K-dependent proteins. Biochemistry 41:15045-15055.i.e., all modifications occur as a consequence of asingle binding event. We are now determininghow processive carboxylation is accomplished,including whether a novel, second site of contactbetween VKD proteins and the carboxylase wediscovered is important for processivity.Our studies showing efficient processivityin vitro are of interest with regard to the poorefficiency of carboxylation that is sometimesobserved in vivo. Although normal physiologicalconditions result in full VKD protein carboxylation,there are instances when carboxylation issaturated, for example, in the expression ofrecombinant VKD proteins in mammalian cells.At low production levels, fully carboxylatedproteins are secreted, but at increased levels,undercarboxylated proteins are observed. Thislimitation has confounded the production ofVKD proteins, many of which have therapeuticpotential. The reason for such saturation is notunderstood, in part because the events involvedin VKD protein carboxylation and processing bythe secretory machinery are poorly defined. VKDprotein carboxylation places exceptional demandson the quality control mechanisms that facilitatesecretion: protein assembly is unusual because anenzyme-substrate complex is formed, and thecomplex must achieve full VKD proteincarboxylation. In addition, multiple VKDproteins (e.g., at least 10 in liver) engage onecarboxylase, creating a potentially competitivestate. To understand how this process is normallyaccomplished with such remarkable fidelity, wehave begun analyzing the intracellular events thatoccur during VKD protein carboxylation andsecretion. Our recent in vivo studies show, forexample, that saturation of carboxylation is notdue to limiting amounts of carboxylase. Theysuggest that the limitation in expressing fullycarboxylated proteins is due to vitamin Kbioavailability, and we are currently investigatingthis possibility. We are also determining howVKD protein carboxylation is regulated in vivo.We discovered that the carboxylase itself is aVKD protein that undergoes autocarboxylation.Preliminary studies indicate that carboxylasecarboxylation may function to downregulate theenzyme when VKD substrate levels are low.120

Role of Integrins in Thrombosis,Angiogenesis and Tumor BiologyThe integrin family of cell-surface moleculesmediates multiple, diverse cellularresponses, ranging from cell adhesion,migration and reorganization of extracellularmatrix to gene expression and apoptosis. Ourlaboratory’s overall goal is to understand the roleand the mechanisms of regulation of integrins inthe broad spectrum of physiological andpathophysiological responses, such as hemostasis/thrombosis, angiogenesis and tumor growth. Ourresearch focus is on the regulation of thefunctional activities of the integrin family ofadhesion receptors,specifically the beta-3integrins α vβ 3(expressedprimarily on vascular andtumor cells) and α IIbβ 3(platelet GPIIb-IIIacomplex).Our major project isto define the mechanismsof communication betweentwo receptor-ligandsystems, integrins/extracellular matrix andgrowth factors and theirreceptors. We recentlydescribed a new mechanismof vascular endothelialgrowth factor (VEGF)action in physiological andpathological responses(Byzova et al., MolecularCell, 2000). We demonstratedthat VEGF candirectly activate integrin α vβ 3, leading toenhanced adhesion and migration of endothelialcells (ECs) to a variety of ligands. We found thatVEGFR2 (flk-1) ligation, but not VEGFR1 (flt-1) ligation, is responsible for integrin activation.Using several in vivo models of angiogenesis, wedemonstrated that VEGFR-2-specific (andVEGFR-3-specific) VEGFs can induce completeangiogenic or lymphangiogenic response (Byzovaet al., Blood, 2002). Because VEGF exerts strikingeffects on ECs, we are interested in the signaltransduction pathways leading from occupiedVEGF receptors to downstream responses such asintegrin activation, cell proliferation andapoptosis. We have generated substantial dataindicating the direct involvement ofphosphatidylinositol 3'-kinase (PI3k)/proteinkinase B (Akt) pathway, which is negativelyregulated by the phosphatase and tensin homologuedeleted from chromosome 10 (PTEN)tumor suppressor protein, in integrin activationby VEGFs (Byzova et al., Molecular Cell, 2000).We are also investigating the role ofVEGFs and their receptors in pathology,specifically in prostate tumor growth and in thedevelopment of bone metastasis. We found thatThe Department of Molecular CardiologyTatiana V. Byzova, Ph.D.enhanced recognition of bone matrix proteins bythe activated integrins contributes to prostatecancer osteotrophism. The angiogenic growthfactors may be released from tumor cells andengage receptors, which are also expressed by thetumor cells, leading to an autocrine mechanism oftumor-cell stimulation. To extend these ourstudies into the clinical arena, we are analyzingthe expression levels of VEGF/VEGFR andintegrins on prostate cancer cells and in the tumorvasculature to determine whether the autocrinemechanism of tumor cells stimulation is operativein vivo. These studies areconducted in collaboration withDrs. W. Heston and J. Brainard.Some of our studies focuson the role of thrombospondin(TSP)/platelet interactions in theprocess of thrombus formation.A recent large-scale genetic studyconducted at the Cleveland Clinicrevealed that novel variants inthe TSP gene family areassociated with familial prematuremyocardial infarction.Patients with the mutationN700S in TSP-1 have a nine-foldhigher risk of early coronaryartery disease. This novelobservation prompted us toinitiate a study focused on thefunction of TSP-1 in plateletaggregation and in EC biology.Since we have access to manypatients who are homozygous forthe N700S mutation, we are uniquely positionedto investigate the role of this mutation in TSP-1biology. The overall goal of this study is toinvestigate the molecular mechanism responsiblefor differential activities of N700S and WT TSP-1 that can contribute to the development of earlycardiovascular disease.THE BYZOVALABORATORYPOSTDOCTORAL FELLOWSJuhua Chen, M.D., Ph.D.Sarmistha De, Ph.D.Natalia Narijneva, Ph.D.TECHNICAL ASSOCIATESVicky Byers-Ward, M.S.COLLABORATORSMarc Achen, Ph.D. 1Deepak L. Bhatt, M.D. 2Jennifer Brainard, M.D. 3Graham Casey, Ph.D. 4Nissim Hay, Ph.D. 5Warren (Skip) Heston, Ph.D. 4Ronald Midura, Ph.D. 6Maria Siemionow, M.D., Ph.D. 7Sanford J. Shattil, M.D. 8Steven Stacker, Ph.D. 1Eric J. Topol, M.D. 21Angiogenesis Laboratory, LudwigInst. for Cancer Res.,Melbourne, Australia2Dept. of CardiovascularMedicine, CCF3Dept. of Anatomic Pathology,CCF4Dept. of Cancer Biology, CCF5Dept of Molecular Genetics,Univ. of Illinois at Chicago6Dept. of Biomedical Engineering,CCF7Dept. of Plastic and ReconstructiveSurgery, CCF8Scripps Research Institute, LaJolla, CAByzova, T.V., and E.F. Plow (1998) Activation of α Vβ 3on vascular cells controlsrecognition of prothrombin. J. Cell Biol. 143:2081-2092.Topol, E.J., Byzova, T.V., and E.F. Plow (1999) Of platelets, integrin of α IIbβ 3and GPIIb-IIIa blockers: past, present and future perspectives. Lancet 143:227-231.Byzova, T.V., Kim, W., Midura, R.J., and E.F. Plow (2000) Activation of integrin α Vβ 3regulates cell adhesion and migration to bone sialoprotein. Exp. Cell Res. 254:299-308.Byzova, T.V., Goldman, C.K., Pampori, N., Thomas, K.A., Bett, A., Shattil S.J., and E.F.Plow (2000) A mechanism for modulation of cellular responses to VEGF: activation ofthe integrins. Mol. Cell 6:851-860.Byzova, T.V., Goldman, C.K., Jankau, J., Chen, J., Cabrera, G., Achen, M.G., Stacker,S.A., Carnevale, K.A., Siemionow, M., Deitcher, S.R., and P.E. DiCorleto (2002)Adenovirus encoding vascular endothelial growth factor-D induces tissue-specificvascular patterns in vivo. Blood 99:4434-4442.121

THE J. FOXLABORATORYPROJECT SCIENTISTSTimothy O'Toole, Ph.D.RESEARCH ASSOCIATEKatarzyna Bialkowska, Ph.D.POSTDOCTORAL FELLOWSSucheta Kulkarni, Ph.D.Shan Wu, M.D.TECHNOLOGISTSHuiqin Nie, B.S.Andriy PodnolikovPetro RyobokonJoan E.B. Fox, Ph.D.Afirst step following integrin-inducedadhesion is the transmission of signalsthat lead to the extension of filopodiaand lamellipodia and the resultant spreading ofcells. Spreading cells are able to assemble bundlesof actin and myosin filaments and exertcontractile forces that allow them to migrate. Inspreading cells, contact with the extracellularmatrix is through complexes of integrin andsignaling molecules that continuously form andbreak down as actin filament networks formingbeneath the membrane cause the membrane’slamellipodia to extend.In more fully spread cells, integrin complexes,known as focal adhesions, are larger andinteract with the bundles of actin and myosinfilaments known as stress fibers. Focal adhesionsalso form and break down as cells migrate. Ourwork has focused on identifying mechanismsinvolved in the formation and breakdown ofintegrin complexes. We have identified a previouslyunrcognized type of integrin complex that formsprior to the focal complexes and focal adhesions:integrin clusters. Moreover, we have identifiedthree potential mechanisms whereby formation ofthese integrin clusters is regulated, one involvingcalpain, another the cytoskeletal protein skelemin,and another involving the SH3 domain of spectrin.Calpain, a Ca 2 -dependent protease presentin most cells, has two forms: one requiresmicromolar concentrations of Ca 2+ to beactivated, the other millimolar concentrations.Until recently, it was not known whether calpainis involved in signal transduction mechanisms,because these concentrations of calcium are notthought to be normally attained within cells.However, we have shown that calpain isactivated as a consequence of signaling acrossintegrins. We have demonstrated that themicromolar form of calpain is activated; itcleaves proteins that are present within theintegrin clusters. Moreover, we have observedBialkowska, K., Kulkarni, S., Du, X., Goll, D.E., Saido, T.C., and J.E.B. Fox (2000) Evidence thatβ3 integrin-induced Rac activation involves the calpain-dependent formation of integrin clustersthat are distinct from focal complexes and focal adhesions that form as Rac and RhoA becomeactive. J. Cell Biol. 151:685-696.Reddy, K.B., Bialkowska, K., and J.E.B. Fox (2001) Dynamic modulation of cytoskeletalproteins linking integrins to signaling complexes in spreading cells: role of skelemin in initialintegrin-induced spreading. J. Biol. Chem. 276(30):28300-28308.Fox, J.E. (2001) Cytoskeletal proteins and platelet signaling. Thromb. Haemost. 86:198-213.Kulkarni, S., Goll, D.E., and J.E.B. Fox (2002) Calpain cleaves RhoA generating a dominantnegativeform that inhibits integrin-induced actin filament assembly and cell spreading. J. Biol.Chem. 277:24435-24441.Bialkowska, K., Zaffran, Y., Meyer, S.C., and J.E.B. Fox (2003) 14-3-3zeta mediates integrininducedactivation of cdc42 and Rac: platelet glycoprotein Ib-IX regulates integrin-inducedsignaling by sequestering 14-3-3zeta. J. Biol. Chem. 278:33342-33350.The Department of Molecular CardiologySpectrin SH3 Domain, Calpain RegulateFormation of Focal Complexes inIntegrin-Induced Cell Spreadingthat calpain is present in the integrin clusters butnot in the focal complexes or adhesions. Previously,Rac1, a member of the family of RasGTPases, has been shown to be involved in theformation of focal complexes. We have shownthat calpain is essential for integrin clusterformation and for integrin-induced cell spreadingand that it acts upstream of Rac1. Thus, wepropose that calpain is a signaling moleculeinvolved in integrin-induced spreading ofadherent cells and that it acts early, prior toactivation of Rac1.Our studies on proteins that may berequired in integrin-induced signaling have alsoinvolved identifying cytoskeletal proteins thatinteract with the cytoplasmic domain of ligandoccupiedintegrin. Previously, we used the twohybridsystem to show that skelemin interactswith the cytoplasmic domain of β1- and β3-containing integrins. We have shown that thisinteraction occurs within spreading cells and thatskelemin interacts with integrins in integrinclusters, but not in focal complexes or in focaladhesions. We have identified the domains ofskelemin and integrin involved in the interactionand used this information to ablate the interactionand to show that skelemin plays an essential rolein allowing the very first steps of integrin clusterformation and cell spreading.Finally, we have investigated the potentialrole of the SH3 domain in spectrin. Previously,we showed that spectrin was present in integrincytoskeletalcomplexes in platelets. Using thetwo-hybrid system, we identified a proteincontaining a proline-rich region that interactedwith spectrin. We have now shown that bothspectrin and this protein are present in integrinclusters, but not in focal complexes or focaladhesions of spreading cells. Overexpression ofthe SH3 domain of spectrin and the proline-richdomain of the protein that interacts with spectrindid not affect the formation of integrin clustersbut prevented the formation of focal complexesand the spreading of cells.Our studies have focused on the mechanismsinvolved in integrin-induced signaling inspreading cells. We have identified an early typeof integrin cluster and shown that proteinspresent in these clusters include the Ca 2+ -dependent protease calpain, the cytoskeletalproteins skelemin and spectrin, and a putativesignaling molecule that contains a proline-richmolecule that interacts with spectrin’s SH3domain. All of these proteins are present inintegrin clusters but absent from focal complexesand focal adhesions. Moreover, calpain, spectrin,and the spectrin-interacting protein all appear toplay an essential role in regulating focal complexformation and integrin-induced cell spreading.122

Role of the Plasminogen System in Stress:Induced Behaviors and Hormone ProcessingStroke and cerebral vascular disease areamong the leading causes of cardiovasculardiseases (CVD) and death. There is substantialevidence to infer a role of the plasminogen(Plg) system in brain function and neurologicaldevelopment. The goal of this project is todetermine the role of the Plg system in theextracellular matrix remodeling required forselective stress-induced behavior. Our resultsindicate that Plg-deficient mice exhibit amarkedly reduced acoustic startle reflex, a stressresponse behavior. Plg-deficient mice havedisturbances in the hypothalamic–pituitary axishormones, α−MSH and β-endorphin, thatmodulate behavior. The acoustic startle reflexresponse in Plg-/- mice can be restored by directinjection of Plg into the brain. We are currentlyexamining the role of extracellular matrixremodeling in the brain at sites involved in themodulation of acoustic startle reflex andhormone secretion.Molecular Basis for the Pathogenetic Riskof Lipoprotein(a)Elevated Lp(a), particularly with smallisoforms, is a risk factor for cardiovascular disease(‘CVD). Apo(a), the apoprotein unique to Lp(a),is structurally similar to plasminogen, and likeplasminogen can express lysine-binding-site (LBS)activity. The LBS function of plasminogenmediates its binding to substrates, cells, andextracellular matrices. Isolated Lp(a) has variableLBS activity, which could influence its pathoge-Hoover-Plow, J., Wang, N., and V. Ploplis(1999) Growth and behavioral development inplasminogen gene-targeted mice. GrowthDev. Aging 63:13-32.Simó, J.M., Joven, J., Vilella, E., Ribas, M.,Pujana, M.A., Sundaram, I.M., Hammel, J.P.,and J.L. Hoover-Plow (2001) Impact ofapolipoprotein(a) isoform size heterogeneityon the lysine binding function oflipoprotein(a) in early onset coronary arterydisease. Thromb. Haemost. 85:412-417.Hoover-Plow, J., Skomorovska-Prokvolit, O.and S. Welsh (2001) Selective behaviors alteredin plasminogen-deficient mice are reconstitutedwith intracerebroventricularinjection of plasminogen. Brain Res. 898:256-264.Hoover-Plow, J., and L. Yuen (2001) Plasminogenbinding is increased with adipocytedifferentiation. Biochem. Biophys. Res. Commun.284:389-394.Hoover-Plow, J., Ellis, J., and L. Yuen(2001) In vivo plasminogen deficiency reducesfat accumulation. Thromb. Haemost.87:1011-1019.The Department of Molecular Cardiologynetic activities and interfere with the functionsof plasminogen. The goals of this project are todetermine: whether apo(a) interacts with vascularsites via its LBS (sites which could be occupied byplasminogen but not low-density lipoprotein);whether apo(a) and plasminogen compete for thesame set of binding sites within the vessel wall;and whether apo(a) size alters apo(a) competitionwith Plg. Accordingly, we have developed micethat are Plg deficient and express apo(a). We willtest the role of LBS activity on the deposition ofapo(a) within blood vessels in thepresence or absence of plasminogen.In addition, we have developeda model of atherosclerosis, acopper-cuff carotid model, toevaluate the competition of apo(a)and Plg in the development ofatherosclerosis.THE HOOVER-PLOWLABORATORYPOSTDOCTORAL FELLOWSJingfeng Sha, M.D., M.S.Aleksy Shchurin, M.D.Olena Skomorovska-Prokvolit, Ph.D.TECHNICAL ASSOCIATESBryan McCullough, B.S.Jennifer Volle, B.A.Role of the PlasminogenSystem in Adipose TissueAccumulationObesity is a major riskfactor for the development andprogression of non-insulin diabetesmellitus and CVD. Obesity iscorrelated with hyperinsulinemia,reduced fibrinolysis, and elevatedcomponents of the Plg system, PAI-1 and t-PA. The overall objectiveof this project is to determine therole of the Plg system in theJane Hoover-Plow, Ph.D.extracellular matrix remodeling thatoccurs during development and maintenance ofadipose tissue. In Plg-deficient mice, adiposetissue accumulation is impaired. This reduced fataccumulation is not due to decreased food intakeor increased activity. Adipose tissue contains largecells with a single lipid droplet and a stromalfraction with endothelial cells, precursor fat cells,and immature fat cells. In Plg-/- mice, thenumber of cells in this stromal fraction isincreased, and when these cells are cultured,development of the mature cells is markedlydelayed compared with cells from wild-type mice.In the adipose tissue sections of the Plg-/- mice,the number of blood vessels is reduced comparedwith those of wild-type mice, suggesting impairedangiogenesis. Alterations of cell migration andangiogenesis during development are beinginvestigated.123

THE KARNIKLABORATORYRESEARCH ASSOCIATESupriya Patil, Ph.D.POSTDOCTORAL FELLOWSAditi Bandyopadhyay, Ph.D.Camelia Gogonea, Ph.D.Yasser Saad, Ph.D.Takanobu Takazako, M.D., Ph.D.TECHNICAL ASSISTANTAnita Shukla, M.S.COLLABORATORSChristine Schomisch Moravec, Ph.D. 1Walter G. Thomas, Ph.D. 21Dept. of Cardiology and Mol.Cardiology2Baker Medical Research Inst.,Melbourne, AustraliaOur research focuses on analyzing thestructure-function and signal transductionmechanisms of angiotensin II (Ang II)receptors. Ang II, an octapeptide hormone, mediatesblood-pressure regulation, salt-water balance, steroidgenesis, reactive oxygen production, proliferativeresponse, and matrix deposition. The AT 1and AT 2isoforms of Ang II receptors, members of the G-protein-coupled receptor (GPCR) superfamily,mediate these responses. In addition to G-proteincoupledreceptor responses, the AT 1receptor initiatesintracellular signal transduction pathways that areusually activated by cytokine and growth-factorreceptors. The AT 2receptor mediates differentiation,inhibition of growth and cell apoptosis.The AT 1receptor is the prototype for peptidehormone GPCRs. Its cytoplasmic domain binds andactivates the G-protein and interacts with differentkinases and adapter proteins that act as signalingplatforms. The extracellular domain, in combinationwith the seven transmembrane (7TM) helical bundle,binds the hormone and selective drugs that areclinically important. The ligand/G-protein interactionconforms to allosteric regulation across the membranebarrier. This general mechanism for transduction ofsignals by the 7TM structural motif is important forthe actions of therapeutic drugs and pathologic agentstargeting GPCRs. Therefore, elucidating theintramolecular communication between the twofunctional domains situated on opposite sides of themembrane is vital.Our current studies of the AT 1receptor areaimed at elucidating, at the molecular level, thefollowing: (i) the receptor’s interactions with Ang II,(ii) the mechanism of receptor inhibition by differentclasses of Ang II-receptor blockers, (iii) specificconformational changes that govern AT 1receptoractivation,and (iv) interactions of G-protein withthe activated AT 1receptor. To understand the in vivoconsequences of “constitutive activation” of the AT 1receptor, we have developed transgenic mousemodels expressing wild-type and constitutivelyThe Department of Molecular CardiologyAnalogs, Mutant Receptors Help DefineAng II Activation Mechanismsactivated mutant AT 1receptors. These models are usedfor: (i) evaluation of inverse agonists and antagonistsof the AT 1receptors, (ii) study of growth-factor andcytokine changes induced during AT 1receptormediatedhypertrophy, (iii) profiling of gene expressionchanges associated with hypertrophy, and (v)evaluation of dominant-negative AT 1receptormutants.The AT 2receptor is an important regulator ofphysiological ontogenesis in the developing fetus and inadult tissue-remodeling. Familial mutations in the AT 2receptor gene cause congenital abnormality of kidneyand urinary tract in humans. The AT 2receptor can beupregulated by physiological and pathological stimuli infailing and infarcted hearts, in neointima formationafter vascular injury, in atretic ovarian follicles, inuterine endometrium and in healing skin wounds. Mostoften, the high levels of AT 2receptor re-expression arelocalized to remodeling sites. We have shown that denovo overexpression of the AT 2receptor inducesapoptosis through activation of caspase and p38MAPK. The AT 2receptor cytoplasmic domaininteracts with novel adapter molecules to induceapoptosis. The goal of AT 2receptor research is toidentify the components and signal transductionmechanisms leading to apoptosis. We will use theprotein-interaction analysis by mass spectrometry toaccomplish this goal.Molecular variants of the AT 1and AT 2receptors and their role in the human cardiovasculardisease process are not fully defined. We haveundertaken a genetic analysis of the AT 1and AT 2receptor loci. Direct sequencing and genotyping of AT 1and AT 2receptor genes, in diseased and non-diseasedhuman subjects, was undertaken. The genomicsequence provided the genotypic and allelic frequencydata for the single-nucleotide polymorphisms that arenecessary for association studies. Functional studies todetermine the identity of the genetic variantsassociated with disease states and the molecularmechanisms by which genetic variants influencereceptor expression and function are in progress.Miura, S., and S.S. Karnik (2000) Ligand-independent signals from the angiotensin II type-2 receptor induce apoptosis.EMBO J. 19:4026-4035.Karnik, S.S. (2002) Analysis of structure-function from expression of G protein-coupled receptor fragments. Meth.Enzymol. 343:248-259.Sadashiva S. Karnik, Ph.D.Holloway, A.C., Qian, H., Pipolo, L., Ziogas, J., Miura, S., Southwell, B.R., Lew, M.J., Thomas, W.G., and S.S. Karnik(2002) Side-chain substitution within angiotensin II reveal different requirements for signaling, internalization andphosphorylation of type 1A angiotensin receptors. Mol. Pharmacol. 61:768-777.Miura, S., and S.S. Karnik (2002) Constitutive activation of angiotensin II type 1 receptor alters the orientation oftransmembrane Helix-2. J. Biol. Chem. 277:24299-24305.Saad, Y., Durkin, S., Hwang, J.Y., Boros, J., Moravec, C.S., and S.S. Karnik (2002) Haplotype variation at the humanangiotensin II receptor loci. In: Stillman, B., ed. Abstracts of papers presented at the LXVII Cold Spring HarborSymposium on Quantitative Biology: May 29-June 3, 2002. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory,p. 105.Miura, S., Zhang, J., Boros, J., and S.S. Karnik (2003) TM2-TM7 interaction in coupling movement of transmembranehelices to activation of the angiotensin II type-1 receptor. J. Biol. Chem. 278:3720-3725.124

Atrial natriuretic peptide (ANP) is a hormonesecreted by the heart in response toelevation in blood pressure and volume.ANP stimulates excretion of salt (natriuresis) andfluid (diuresis) and dilates arterial blood vessels,thereby exerting hypotensive effects. ANP alsosuppresses actions of neuronal and hormonalfactors that cause high bloodpressure. Thereby, ANP plays amajor role in regulating bloodpressure and body-fluid volumehomeostasis.ANP’s hormonal actionsare mediated by the cell surfacereceptor that carries intrinsicguanylyl cyclase (GCase)activity. Binding of ANPactivates GCase catalysis andelevates intracellular cGMPlevels. In turn, cGMP mediateshormonal actions of ANPthrough cGMP-regulated ionchannels,protein kinases, andphosphodiesterases. The ANPreceptor is a single-spantransmembrane receptor andoccurs as a dimer of atransmembrane polypeptidecontaining an extracellularANP-binding domain and anintracellular domain consistingof an ATP-binding regulatorydomain and a GCase catalyticdomain. ANP binding to the extracellular domainactivates GCase catalysis by an as yet unknownmechanism. The ANP receptor belongs to thefamily of membrane-bound, GCase-coupledreceptors having a similar molecular topology and,presumably, a similar signaling mechanism. TheseGCase-coupled receptors belong to the superfamilyof single-span transmembrane receptors, for whichthe mechanism of signal transduction has not beenwell defined.Our goal is to determine the ANPreceptor’s structure and to elucidate the mechanismsof hormone-binding and transmembranesignaling at atomic resolutions. We applystructural biology (X-ray crystallography), proteinbiochemistry, molecular biology, and biophysicsThe Department of Molecular CardiologyStructure and Transmembrane SignalingMechanism of the ANP Receptor:Development of Drugsand More Effective Treatmentstechniques to pursue our research goals.Our recent studies include identification of aconserved structural motif in the ANP receptor,termed GCase-signaling motif; expression andpurification of the hormone-binding domain(ANPR); determination of its complete covalentstructure by LC/MS; discovery of the chloridedependenceof ANPbinding; and determinationof ANP binding andreceptor dimerizationequilibria. We have alsodetermined the X-raystructures of the apo- andANP-bound ANPRcomplex and haveproposed a novel structuralmechanism for transmembranesignal transductionby the ANP receptor(submitted).Besides its majorrole in blood pressure andvolume regulation, ANP isinvolved in the pathogenesisof hypertension, heartfailure, and othercardiovascular diseases.Thus, the ANP receptorpresents an ideal target fordrugs used in treating suchdiseases. Elucidation ofthe ANP receptor’sstructure and mechanism of signaling will allowrational drug design. These studies will also give usbetter understanding of the pathophysiologicalroles of ANP in the cardiovascular system andpromote development of more effective diagnosisand treatment protocols.Kunio S. Misono, Ph.D.THE MISONOLABORATORYINVESTIGATORSP. Haruo Ogawa, Ph.D.Yue Sally Qiu, M.D.Xiaolun Zhang, M.S.COLLABORATORSEwa Folta-Stogniew, Ph.D. 1Earnest Freire, Ph.D. 2Craig Ogata, Ph.D. 3John Philo, Ph.D. 4Kenneth Williams, Ph.D. 11Dept. of Biochem. andBiophys., Yale Univ., NewHaven, CT2Dept. of Biology, JohnsHopkins Univ., Baltimore, MD3Argonne Natl. Laboratory,Chicago, IL4Alliance Protein Laboratories,Inc., Thousand Oaks, CAMisono, K.S., Sivasubramanian, N., Berkner, K., and X. Zhang, (1999) Expression andpurification of the extracellular ligand-binding domain of the atrial natriuretic peptide(ANP) receptor. Biochemistry, 38:516-523.Misono, K.S. (2000) Atrial natriuretic factor binding to its receptor is dependent onchloride concentration: A possible feedback-control mechanism in renal salt regulation.Circ. Res. 86:1135-1139.van den Akker, F., Zhang, X., Miyagi, M., Huo, X., Misono, K.S., and V.C. Yee (2000)Structure of the dimerized hormone-binding domain of a guanylyl-cyclase-coupledreceptor. Nature 406:101-104.Misono, K.S. (2002) Natriuretic peptide receptor: structure and signaling (Review). Mol.Cell. Biochem. 230:49-60.Ogawa, H., Zhang, X., Qiu, Y., Ogata, C., and K.S. Misono (2003). Structuralmechanism for transmembrane signaling by the atrial natriuretic peptide receptor(Submitted).125

THE MORAVECLABORATORYRESEARCH SCHOLARNancy A. DiIulio, Ph.D.LEAD TECHNOLOGISTWendy SweetPOSTDOCTORAL FELLOWSLouise Aquila Pastir, M.S.Karin Mauer, M.D.GRADUATE STUDENTCassandra Talerico-KaplinUNDERGRADUATE STUDENTSBrad MartinKim VargasMaria YaredCOLLABORATORSMeredith Bond, Ph.D. 1Robert Fairchild, Ph.D. 2Gary Francis, M.D. 3Larry R. Jones, Ph.D. 4Evangelia Kranias, Ph.D. 5Patrick McCarthy, M.D. 6Charles McTiernan, Ph.D. 7Muthu Periasamy, Ph.D. 8Peter Reiser, Ph.D. 8Nicholas Smedira, M.D. 6Mark Sussman, Ph.D. 5Vincent Tuohy, Ph.D. 2David Van Wagoner, Ph.D. 1Jean-Pierre Yared, M.D. 2James Young, M.D. 31Dept. of Molec. Cardiol., CCF2Dept. of Immunology, CCF3Dept. of Cardiovasc. Med., CCF4Krannert Inst. of Cardiol.,Indiana Univ. Sch. of Med.,Indianapolis, IN5Univ. of Cincinnati, Cincinnati,OH6Dept. of Thoracic andCardiovascular Surgery, CCF7Univ. of Pittsburgh, Pittsburgh,PA8Dept,. of Physiology, OhioState Univ., Columbus, OHOur laboratory focuses on excitationcontractioncoupling in human heartfailure, especially the phenotypicremodeling that accompanies end-stage heartfailure, and its functional ramifications. We haveshown that significant alterations occur in thecontractile properties of cardiac muscle removedfrom failing human hearts and that these changesare linked to decreased calciumstores.We hypothesize that thesealtered calcium stores contributeto impaired contractility andtherefore decreased cardiacfunction in heart failure. In thesame failing hearts, there isdecreased expression of theproteins that control calciumcycling within the cardiacmyocyte. Significant decreaseshave been shown in the steadystatemRNA for the sarcoplasmicreticulum (SR) Ca 2+ ATPase.These changes have been observedin the failing human heart, but notin hearts having left ventricularhypertrophy with preserved ventricular function,suggesting that the decreased levels may be anend-stage phenomenon.To more directly study the effects ofalterations in these calcium regulatory proteins,we have examined the hearts of transgenic micewith alterations in these proteins. In collaborationwith Dr. Evangelia Kranias of the Universityof Cincinnati, we have measured calciumwithin the SR of mice in which the regulatoryprotein phospholamban has been knocked out.We have directly demonstrated that eliminatingphospholamban significantly increases theamount of calcium that can be stored in the SR.In collaboration with Dr. Larry Jones of theKrannert Institute, we have also shown thatoverexpression of the protein calsequestrin, acalcium-binding protein found in the SR’s lumen,increases the amount of calcium stored in theThe Department of Molecular CardiologyDecreased Expression of Proteins RegulatingCalcium Cycling Compounds Heart FailureChristine SchomischMoravec, Ph.D.SR. In an ongoing study, we are examining theeffects of overexpression of the Ca 2+ ATPase,working with Dr. Muthu Periasamy of the OhioState University.In addition to these studies, we are testingthe broader hypothesis that some of thesephenotypic changes that accompany end-stageheart failure may actually be reversible. This is animportant question, because thepotential for therapy other thanheart transplantation is directlyrelated to whether any of thestructural/functional changesoccurring in heart failure can bereversed. Using a uniquepopulation of patients, weobtain failing human heart tissuefrom surgeries implanting a leftverntricular assist device withina patient as a “bridge totransplant.” Later, when thepatient receives the new heart,after a variable number ofweeks during which the devicetakes over the mechanical loadof the circulatory system, weobtain the explanted failing heart. We are thusable to compare structural, functional, andbiochemical properties of human heart musclethat has experienced end-stage failure and asignificant period of mechanical unloading in thesame patient. Our exciting preliminary datasuggest that unloading the heart actually leads torecovery of its contractile properties, the abilityof the muscle to respond to inotropic stimulationvia the beta-adrenergic nervous system, and someof the molecular markers of heart failure,including SR Ca 2+ ATPase. Current work in thelaboratory focuses on further investigation ofthese changes, as well as determination of themechanisms that may be responsible for thisreversibility. We hope to elucidate the phenotypicchanges that are reversible and determinetheir functional significance for the heart failurepatient.Ogletree-Hughes, M.L., Stull, L.B., Sweet, W.E., Smedira, N.G., McCarthy, P.M., and C.S. Moravec (2001) Mechanicalunloading restores beta-adrenergic responsiveness and reverses receptor downregulation in the failing human heart.Circulation 104:881-886.Tan, F.L., Moravec, C.S., Li, J., Apperson-Hansen, C., McCarthy, P.M., Young, J.B., and M. Bond (2002) The geneexpression fingerprint of human heart failure. Proc. Natl. Acad. Sci. USA 99:11387-11392.Melendez, J., Welch, S., Schaefer, E., Moravec, C.S., Avraham, S., Avraham, H., and M.A. Sussman (2002) Activation ofpyk2/related focal adhesion tyrosine kinase and focal adhesion kinase in cardiac remodeling. J. Biol. Chem. 277:45203-10.Aquila-Pastir LA, DiPaola NR, Matteo RG, Smedira NG, McCarthy PM, Moravec CS. uantitation and distribution of β-tubulinin human cardiac myocytes. J. Mol. Cell. Cardiol. 34:1513-1523.Oh, H., Wang, S.C., Prahash, A., Sano, M., Moravec, C.S., et al. (2003) Telomere attrition and Chk2 activation in humanheart failure. Proc. Natl. Acad. Sci. USA 100:5378-5383.126

Our laboratory investigates the structurefunctionand physiological action of α 1-adrenergic receptors (α 1-ARs). Theseseven-transmembrane-spanning proteins belong tothe G-protein-coupled receptor superfamily,through which over 80% of all hormones signaland carry out their physiological functions. ARsmodulate the sympathetic nervous system bybinding epinephrine and norepinephrine andcontrol cardiovascular functions such as bloodpressure homeostasis and cardiac contractility.Three subtypes of the α 1-AR family (α 1A, α 1B, andα 1D) derive from separate gene products and bindseveral synthetic agonists and antagonists withdifferent affinities. We study the amino acidsinvolved in this ligand selectivity between ARsubtypes, seeking to develop better therapeutics(Waugh et al., J. Biol. Chem., 2001). We do notyet know which α 1-AR subtypes control thevarious physiologies attributed to these receptors,whether the subtypes differ physiologically, orwhether abnormalities in their function lead todisease. To this end, we recently performedmicroarray studies in transfected cells expressingindividual α 1-AR subtypes and found that theycan couple to interleukin-6, JAK/STAT, and cellcycleregulation pathways (Gonzalez-Cabrera etal., Mol. Pharmacol., 2003). Ongoing experimentswith transgenic mice that systemically overexpressα 1-AR subtypes are addressing these questions.Overexpression of the α 1B-AR receptorcauses neurodegeneration consistent withmultiple system atrophy (MSA), a parkinsoniansyndrome (Zuscik et al., Nat. Med., 2000).Afflicted patients die within 9 years of diagnosis,and no effective treatment is available. As inhuman disease, our mice show major degenerationin the cerebellum, olive/pons, and spinal cord.The mice exhibit parkinsonian traits, since there istyrosine hydroxylase loss in the substantia nigra.They have autonomic failure, a characteristic ofMSA (i.e., low plasma levels of catecholamines,cortisol, ACTH and corticotropin-releasing factor,low blood pressure, bradycardia, reproductiveproblems, and weight loss) (Zuscik et al., J. Biol.Chem., 2001). Cytoplasmic inclusion bodies fromthese mice stain positive for α-synuclein andubiquitin in oligodendrocytes, a hallmark of MSA(Papay et al., J. Neurochem., 2002). In this model,α 1-AR blockers can partially rescue thedegradative phenotype (i.e., improve motorperformance). In work with CCF’s Departmentof Neurology, clinical trials are testing whetherα 1-AR blockers benefit MSA patients. We alsoplan studies to find the molecular basis for thisneurodegeneration, which may occur through anThe Department of Molecular CardiologyMouse Models of α 1-Adrenergic ReceptorOverexpression Offer Insights intoNeurodegeneration, Epilepsy,and Heart Disordersapoptotic mechanism (Yun et al., Brain, 2003) orthrough abnormal forms of α-synuclein (Papay etal., J. Neurochem., 2002).Interestingly, the α 1B-AR transgenic miceare epileptic. With our Neurology colleagues, wehave shown that these mice show abnormal waveforms by EEG (Kunieda et al., Epilepsia, 2002).Associated with the age onset of seizures, certainNMDA receptor subunits are upregulated andGABA receptors downregulated; both are criticalto maintaining the brain’s balance of excitatory/inhibitory signals. Understanding how seizures areinduced may provide clues to the onset ortreatment of human epilepsy.These transgenic mice show many cardiovascularabnormalities. Cardiac hypertrophy(enlargement of the heart) can sometimesprogress to heart failure. Understanding theinduction and progression of cardiac hypertrophyis crucial to preventing heart failure. Using genechip microarray analysis on transgenic hearts, wehave investigated the changes in gene expressionthat occur as hypertrophy caused by overexpressionof the α 1B-AR progresses (Yun et al.,Cardiovasc. Res., 2003). Notably, in young mice,we saw early events of apoptosis and changes intyrosine kinase signaling, whereas older micedisplayed genes associated with embryogenesisand inflammation, suggesting that an etiology ofapoptosis and Src-related signaling may be crucialto initiating hypertrophy. Induction of hypertrophy-associatedgenes, such as gp130, wasdownregulated in transgenic hearts. Loss ofgp130 has been associated with the transition toheart failure. We are exploring the α 1B-AR’s rolein modulating cardiac contractility and rhythm.THE D. PEREZLABORATORYPOSTDOCTORAL FELLOWSPedro Gonzalez-Cabrera, Ph.D.Dan McCune, Ph.D.Boyd Rorabaugh, Ph.D.June Yun, Ph.D.TECHNICAL ASSOCIATESRobert Gaivin, B.A.Robert Papay, B.S.COLLABORATORSWarren (Skip) Heston, Ph.D. 1Wendy Macklin, Ph.D. 2Imad Najm, M.D. 3Michael T. Piascik, Ph.D. 4Thyagarajan Subramanian, M.D. 3James Thomas, M.D. 51Dept. of Cancer Biology, CCF2Dept. of Neurosciences, CCF3Dept. of Neurology, CCF4Dept. of Pharmacol., Univ. ofKentucky, Lexington5Dept. of Cardiovasc. Med., CCFDianne M. Perez, Ph.D.Kunieda, T., Zuscik, M.J., Boongird, A., Perez, D.M., Lüders, H.O., and I.M. Najm (2002)Systemic overexpression of the α 1B-adrenergic receptor in mice: an animal model of epilepsy.Epilepsia 43:1324-1329.Papay, R., Zuscik, M.J., Ross, S.A., Yun, J., McCune, D.F., Gonzalez-Cabrera, P., Gaivin,R., Drazba, J., and D.M. Perez (2002) Mice expressing the α 1B-adrenergic receptor induces asynucleinopathy with excessive tyrosine nitration but decreased phosphorylation. J.Neurochem. 83:623-34.Yun, J., Zuscik, M.J., Gonzalez-Cabrera, P., McCune, D.F., Ross, S.A., Gaivin, R., Piascik,M.T., and D.M. Perez (2003) Gene expression profiling of α 1B-adrenergic receptor-inducedcardiac hypertrophy by oligonucleotide arrays. Cardiovasc. Res. 57:443-455.Gonzalez-Cabrera, P.J., Gaivin, R.J., Yun, J., Ross, S.A., Papay, R.S., McCune, D.F.,Rorabaugh, B.R., and D.M. Perez (2003) Genetic profiling of α 1-adrenergic receptor subtypesby oligonucleotide microarrays: coupling to interleukin-6 secretion but differences in STAT 3phosphorylation and gp-130. Mol. Pharmacol. 63:1104-1116.Yun, J., Gaivin, R.J., McCune, D.F., Atthaporn, B., Papay, R.S., Ying, Z., Gonzalez-Cabrera,P.J., Najm, I., and D.M. Perez (2003) Gene expression profiles of neurodegeneration inducedby the α 1B-adrenergic receptor: NMDA/ GABAA dysregulation and apoptosis. Brain Aug 22[epub aheadof print].127

THE PLOWLABORATORYCOLLABORATORSTatiana Byzova, Ph.D.Jun Qin, Ph.D.Olga Stenina, Ph.D.Tatiana Ugarova, Ph.D.RESEARCH ASSOCIATESElzbieta Pluskota, Ph.D.Valentin Yakubenko, Ph.D.POSTDOCTORAL FELLOWSBin Hu, Ph.D.Michelle Kish, Ph.D.Dmitry Solovjov, Ph.D.Carmen Swaisgood, Ph.D.Valentin Ustinov, Ph.D.PREDOCTORAL FELLOWNataly Podolnikova, M.S.TECHNICAL ASSOCIATESTimothy BurkeCarla DrummValeryi Lishko, Ph.D.Daniel TrepalShiying WangVISITING SCIENTISTSCzeslaw Cierniewski, Ph.D. 1Steve Busuttil, M.D. 21Univ. of Lodz, Poland2Case Western Reserve Univ.and Veterans Adm. Med. Ctr.,Cleveland, OHByzova, T.V., Goldman, C.K., Pampori, N., Thomas, K.A., Bett, A., Shattil, S.J., and E.F.Plow (2000) A mechanism for modulation of cellular responses to VEGF: activation of theintegrins. Mol. Cell 6:851-860.Plow, E.F., Haas, T.A., Zhang, L., Loftus, J., and J.W. Smith (2000) Ligand binding tointegrins. J. Biol. Chem. 275:21785-21788.Forsyth, C.B., Solovjov, D.A., Ugarova, T.P., and E.F. Plow (2001) Integrin α Mβ 2-mediatedcell migration to fibrinogen and its recognition peptides. J. Exp. Med. 193:1123-1134.Vinogradova, O., Velyvis, A., Velyviene, A., Hu, B., Haas, T., Plow, E., and J. Qin (2002) Astructural mechanism of integrin α IIbβ 3“inside-out” activation as regulated by its cytoplasmicface. Cell 110:587-597.Ustinov, V.A., and E.F. Plow (2002) Delineation of the key amino acids involved inneutrophil inhibitory factor binding to the I-domain supports a mosaic model for the capacityof integrin α Mβ 2to recognize multiple ligands. J. Biol. Chem. 277:18769-18776.Swaisgood, C.M., Schmitt, D., Eaton, D., and E.F. Plow (2002) In vivo regulation ofplasminogen function by plasma carboxypeptidase B. J. Clin. Invest. 110:1275-1282.128Edward F. Plow, Ph.D.Integrin and Plasminogen ReceptorsRegulate Cell Adhesion and MigrationCell adhesion and migration are essential for theformation, development and survival of allmulticellular organisms. Aberrations in theadhesive status of cells have major pathogeneticconsequences, including thrombosis, tumor growthand metastasis, and infection. Research within thislaboratory seeks to delineate molecular mechanismsthat regulate cell adhesion and migration. In particular,our efforts are directed toward analyzing thecontributions of two specific receptor systems to thesecellular responses: the integrin and plasminogenreceptor systems.The integrins are a large and broadly distributedfamily of adhesion receptors (>20 members, occurringon virtually every cell type). Each member is anoncovalent heterodimeric complex composed of anα and a β subunit. Key to many functions ofintegrins is their capacity to rapidly modulate theiraffinity for ligands, i.e., they can exist in ligandcompetentand in resting states, in which they do notbind ligands with high affinity. Also characteristic isthe capacity of each integrin to recognize multiple andstructurally unrelated ligands. Development of amolecular understanding of these two centralproperties of integrins is a general direction of thelaboratory.Integrin α IIbβ 3(GPIIb-IIIa) mediates plateletaggregation, an essential event in thrombus formation.We are attempting to understand the molecular basisfor ligand binding to this integrin. A combination ofmolecular biology, protein chemistry and biophysicalanalyses are being developed to identify the cation andligand contact sites within the polypeptide chains ofthe receptor. Our goal is to determine how cation andligand coordination sites cooperate to achieve receptorfunction. In parallel, we are seeking to understandhow the cytoplasmic tails of the α IIband β 3subunitstransmit signals to the extracellular domain to activatethe receptor’s ligand binding functions. Cell-permeablesynthetic peptides, corresponding to the cytoplasmictail of each subunit, have been shown to interact witheach other to form a functional cytoplasmic domain,and the structure of this cytoplasmic domain has beensolved by nuclear magnetic resonance spectroscopy.The Department of Molecular CardiologyThese studies are conducted collaboratively with thelaboratories of Dr. Jun Qin.Integrin α Mβ 2is expressed on leukocytes andplays a pivotal role in the transmigration of these cellsduring intravascular inflammatory responses, leading toatherosclerosis and restenosis, and extravascularinflammatory responses. The ligand repertoire of α Mβ 2is extremely broad. We are testing the hypothesis thatmany of the α Mβ 2ligands bind to overlapping but notidentical segments within a 200-amino-acid stretch, theI domain of the α Msubunit; and other regions of theα Mand β 2subunits influence subsequent responsessuch as adhesion and migration once ligand has beenengaged by the I domain. Since the crystal structure ofthe I domain is known, incisive mutagenesis strategiescan be implemented to test this hypothesis. Four α Mβ 2ligands of current interest are the fungal pathogenCandida albicans, neutrophil inhibitory factor (NIF),platelet membrane protein GPIb, and fibrinogen. Asoluble factor released from C. albicans, which supportsα Mβ 2-dependent cell adhesion and migration, is beingisolated and characterized. Studies of fibrinogenrecognition by α Mβ 2are conducted collaboratively withDr. Tatiana Ugarova, who is seeking the define the siteswithin fibrinogen recognized by the receptor as well asthe sites with α Mβ 2that recognize fibrinogen.In addition to its central role in fibrinolysis, theplasminogen system is also involved in cell migration.This latter function depends upon the interaction ofplasminogen with cell surface receptors. When bound,plasminogen is efficiently activated to plasmin, and thebound enzyme can cleave a variety of pericellularsubstrates to facilitate cell migration. Our currentefforts emphasize how plasminogen receptor expressionis modulated. We are investigating the capacity of celladhesion, proteolysis and apoptosis to modulateexpression of plasminogen receptors. Such modulationcan result in 5- to 10-fold changes in the expression ofnaturally occurring cells, such as neutrophils, to bindplasminogen. At the same time, we are also challengingthe hypothesis that plasminogen is crucial for cellmigration. These latter studies are being conducted inmice in which the gene for plasminogen has beeninactivated. These knockout mice are being used inmodels for the inflammatory response, atherosclerosisand restenosis, in which cell migration may contributeto pathogenesis. Recent studies have focused on therole of plasminogen in asthma and of a molecule,TAFI, which suppresses plasminogen binding to itscellular receptors.Recent large-scale genetic studies have identifiedparticular single nucleotide polymorphisms (SNPs)within members of the thrombospondin gene family asbeing associated with an increased risk of prematureatherosclerosis. The thrombospondins are large,extracellular matrix proteins that are reported to exert avariety of effects on blood and vascular cells. Incollaboration with Dr. Olga Stenina, we are determininghow SNPs in the thrombospondins alter structureand function as a potential means to define a mechanismunderlying atherosclerotic disease.

The Department of Molecular CardiologyACE: Biosynthesis and SecretionTHE I. SENLABORATORYAngiotensin-converting enzyme (ACE), adipeptidyl carboxypeptidase, is a keycomponent of the renin-angiotensinsystem that regulates blood pressure. Studieswith ACE knock-out mice have revealedadditional roles of ACE in renal physiology andmale fertility. Although ACE exists primarily as amembrane-bound cell-surfaceprotein, a soluble form ispresent under normalconditions in serum and otherbody fluids. Becauseinformation in the literaturesuggests that the specificphysiological function of cellboundACE may differ fromthose of ACE in circulation,production of soluble ACEfrom cell-bound ACE couldbe a significant point ofbiological regulation.We have been studyingthe process of ACE cleavagesecretionusing natural ACEproducingcells and cellstransfected with expressionvectors of ACE or itsmutants. Our studiesrevealed that the ectodomainof ACE is cleaved at aspecific site near the plasmamembrane by a membraneanchored metalloprotease, theACE-secretase. The cleavage specificity ismaintained not by amino acid sequence at oraround the cleavage site but by the presence ofthe distal ectodomain of the ACE protein thatactivates the ACE-secretase, suggesting that it isan atypical protease. The activity of themammalian secretase can be upregulated bytreatment of cells with phorbol esters,calmodulin inhibitors or protein tyrosinephosphatase inhibitors. Our research effort isdirected toward (1) identifying the sequences inectodomain of ACE thatactivates the secretase, (2)determining how thecleavage-secretion processis regulated, and (3)cloning and characterizationof ACE-secretase.These studies will providean understanding of howcell-bound ACE is releasedto the circulation in aregulated fashion.Our study is highlysignificant beyond theACE system as well.Although the cleavagesecretionprocess forproduction of solubleproteins from membraneboundforms is widelyused in biology, theresponsible secretases havenot been identified inmost systems. Thus,Indira Sen, Ph.D.identification andcharacterization of ACEsecretase will considerably advance our knowledgeof the secretases whose primary role is toselectively cleave and release many biologicallysignificant ectoproteins from the cell surface.POST DOCTORAL FELLOWSS. Sengupta, Ph.D.G. Karan, Ph.D.COLLABORATORSRoy A. Black, Ph.D. 1Janice G. Douglas, M.D. 2Ganes C.Sen, Ph.D. 31Immunex Corp., Seattle, WA2Dept. of Medicine, CaseWestern Reserve Univ.,Cleveland, OH3Dept. of Molecular Biology,CCFRamchandran, R., Kasturi, S., Douglas, J.G., and I. Sen (1996) Metalloprotease-mediated cleavage secretionof pulmonary ACE by vascular endothelial and kidney epithelial cells. Am. J. Physiol. 271:H744-H751.Sadhukhan, R., Sen, G.C., and I. Sen (1996) Synthesis and cleavage-secretion of enzymatically active angiotensin-convertingenzyme in Pichia pastoris. J. Biol. Chem. 271:18310-18313.Sadhukhan, R., Sen, G.C., Ramchandran, R., and I. Sen (1998) The distal ectodomain of angiotensin-convertingenzyme regulates its cleavage-secretion from the cell surface. Proc. Natl. Acad. Sci. USA 95:138-143.Sadhukhan, R., Santhamma, K.R., Reddy, P., Peschon, J.J. , Black, R.A., and I. Sen (1999) Unalteredcleavage and secretion of angiotensin-converting enzyme in tumor necrosis factor-α-converting enzyme-deficientmice. J. Biol. Chem. 274:10511-10516.Santhamma, K.R., and I. Sen (2000) Specific cellular proteins associate with angiotensin-converting enzymeand regulate its intracellular transport and cleavage-secretion. J. Biol. Chem. 275:23253-23258.129

THE S. SENLABORATORYPOSTDOCTORAL FELLOWSSudhiranjan Gupta, Ph.D.Pryan Prem, Ph.D.Sagartirtha Sarkar, Ph.D.TECHNICIANDavid YoungCOLLABORATORSOscar Bing, M.D., Ph.D. 1Joe Hollyfield, Ph.D. 2Anning Lin, Ph.D. 3Jun Qin, Ph.D. 4Mary Rayborn, M.S. 2Norman B. Ratliff, M.D. 5James D. Thomas, M.D. 6Qing Wang, Ph.D. 7M. Hilal Yamani, M.D. 61Dept. of Medicine, Tufts Univ.Sch. of Med., Boston, MA2Cole Eye Inst., CCF3Ben May Inst. for Cancer Res.,Univ. of Chicago, Chicago, IL4Dept. of Molecular Cardiology,CCF5Dept. of Anatomic Pathology,CCF6Dept. of Cardiovascular Medicine,CCF7Ctr. for Molecular Genetics, CCFYang, Y., Nanduri, S., Sen, S., and J. Qin (1998) The structural basis of ankyrin-like repeatfunction as revealed by the solution structure of myotrophin. Structure 6:619-626.Sen, S. (1999) Myocardial response to stress in cardiac hypertrophy and heart failure:effect of antihypertensive drugs. Ann. N.Y. Acad. Sci. 874:125-133.Mitra, S., Timur, A.A., Gupta, S., Wang, Q., and S. Sen (2001) Assignment of myotrophinto human chromosome band 7q33→q35 by in situ hybridization. Cytogenet. Cell Genet.93:151-152.Pathak, M., Sarkar, S., Vellaichamy, E., and S. Sen (2001) Role of myocytes in myocardialcollagen production. Hypertension 37:833-840.Gupta, S., Purcell, N.H., Lin, A., and S. Sen (2002) Activation of nuclear factor-κB isnecessary for myotrophin-induced cardiac hypertrophy. J. Cell Biol. 159:1019-1028.130Biochemical, Cellular, and Genetic StudiesReveal Molecular Aspects of MyocardialHypertrophyCardiac hypertrophy in hypertension, withsubsequent heart failure, is a major killerworldwide, whose causes remain unclear.Blood-pressure control mechanisms alone cannotexplain initiation/regression of such hypertrophy.We contend that it is initiated by (mechanicalor humoral) signals to the mycardium, whichin turn produce a factor that triggers proteinsynthesis.Since we first identified the 12-kDa proteinmyotrophin from hearts of spontaneously hypertensiverats (SHRs) and humans with myocardialhypertrophy, we have focused on why myotrophinlevels surge in hypertrophy. Webelieve myotrophin stimulatesmyocyte protein synthesis and maybe a common pathway for drugaction and workload stimulation.Recombinant myotrophinstimulates myocyte growth asactively as natural myotrophindoes. Myotrophin increasestranscript levels of protooncogenes(e.g., c-myc, c-fos, and c-jun) and known hypertrophymarkers (e.g., β-myosin heavychain, atrial natriuretic factor, andconnexin). Via radioimmunoassay,we found elevated myotro-phinlevels in hearts of SHRs andcardiomyopathic humans. Thesefindings demonstrate thatmyotrophin is a controlling factorfor myocyte growth.In 2000, we obtained exciting findings fromour newly established line of transgenic mice thatexpress myotrophin 10- to 100-fold normal,specifically in the heart, as measured by mRNA andproteins. Like humans, these mice exhibit leftventricular (LV) hypertrophy, cardiac myocytenecrosis, multiple focal fibrosis, and compromisedLV cardiac function with significantly reducedejection fraction. By 6 months, they develophypertrophy that worsens to heart failure, closelymimicking the human experience with cardiachypertrophy. This model provides a new investigationaltool to study molecular changes during thetransition of hypertrophy to failure.The Department of Molecular CardiologySubha Sen, Ph.D., D.Sc.Our laboratory combines molecular, genetic,and physiological approaches: (1) to define whatmolecular changes occur in these mice, thendetermine which changes result from myotrophinexcess; (2) to eludcidate myotrophin’s mechanism ofaction in vitro; and (3) by echocardiography, to definewhich cardiac functions correlate with observedmolecular changes.Our long-term goal is to understand howprotein synthesis is turned “on”/“off ” by selectivetherapy. We will then hold a key to therapeuticplanning for patients with hypertensive heartdisease, especially for developing appropriateantagonists to prevent/controlhuman cardiac hypertrophy.Collagen and Regression ofHypertrophyRegression of cardiovascularhypertrophy cannot beexplained by mechanical loadalone; it arises from the interplayof cardiac pressure load, thecardioadrenergic system, andvarious humoral factors.Each antihypertensive drughas a unique effect on thebiochemical composition ofcardiac collagen. Functionalconsequences vary according to thetype(s) of cardiac collagen ormyosin. Our focus is on cardiaccollagen production, determiningwhether regression of hypertrophyis beneficial or harmful.We hypothesize that functional/structuralremodeling of the heart’s interstitial matrix inhypertrophy and heart failure and the heart’s reremodelingafter regression reflect altered collagenproduction, which influences cardiac function. Westudy collagen phenotypes at the cellular andmolecular levels to evaluate their functionalconsequences.We recently identified fibroblast-derived factor(FDF), which stimulates angiotensin II (Ang II)-mediated myocyte growth. Ang II strongly stimulatesFDF secretion by fibroblasts, but its action canbe blocked by losartan, a specific Ang II receptorblocker. We were first to show that, via fibroblastmyocytecrosstalk, fibroblasts largely mediate AngII’s effect on myocyte growth. We are nowcharacterizing FDF and elucidating its mechanismsof action.These studies outline abnormalities duringdevelopment/regression of myocardial hypertrophyand explore effects on cardiac function. Oncederangements are found, we may suggest appropriatetreatments to correct changes in collagen formation/metabolism and evaluate whether directed alterationsin myocardial collagen formation can ameliorate thehypertrophied heart’s compromised function.

The overall objectives of our laboratory areto discover key genes and molecularmechanisms for human disease. Areas ofinterest include cardiacdisease, vascular malformations,eye disease, andneurological disorders.Cardiac arrhythmiascause >300,000 suddendeaths each year in the UnitedStates alone. We havepioneered the field of thegenetic studies of cardiacarrhythmias includingidiopathic ventricularfibrillation (IVF) and long QTsyndrome (LQT). We havecloned three genes for LQTand discovered the first genefor IVF. We continue to findnew arrhythmia genes, andperform genotype-phenotypecorrelation studies. Furthermore,we have created amouse model for LQT,ventricular tachycardia/ventricular fibrillation/sudden cardiac death by targeting a human LQTgene. Continueing characterization of this mousemodel as well as biochemical dissection of LQTgenes will reveal fundamental molecularmechanisms for pathogenesis of lethalarrhythmias.The second major project in our laboratoryis to clone a new gene for congenital heart diseaseThe Department of Molecular CardiologyGenetics and Molecular Biologyof Human DiseaseQing Wang, Ph.D.,M.B.A.Director, Center forCardiovascular Genetics(CHD), which is the most common birth defect.We are also using biochemical and cell biologicaltools to functionally characterize TBX5 andNKX2.5, two key genesinvolved on cardiacdevelopment and CHD.The third major areaof our research is on vascularbiology (vasculogenesis andangiogenesis). By studyingKlippel-Trenaunay syndrome,a congenital vascular disease,we have identified two genesinvolved in vascularmorphogenesis. Furtherstudies of these genes willlead to significant insightinto molecular mechanismsunderlying vasculogenesisand angiogenesis.The fourth project ison genetics of coronaryartery disease (CAD) andmyocardial infarction (MI),the No. 1 killer disease in theUnited States and otherdeveloped countries. By linkage analysis, we haveestablished six genetic loci for CAD and MI.Ongoing positional cloning will lead to theidentification or cloning of major genes for CADand MI.Finally, our laboratory is using linkageanalysis and positional cloning to identify genesfor retinitis pigmentosa, restless legs syndrome,and epilepsy.Chen, Q., Kirsch, G., Zhang, D., Brugada, R., Brugada, J., Brugada, P., Potenza, D., Moya, A., Borggrefe, M.,Breithardt, G., Oritz-Lopez, R., Wang, Z., Anzalevitch, C., O’Brien, R.E., Schultz-Bahr, E., Keating, M.T., Towbin,J.A., and Q. Wang (1998) Genetic basis and molecular mechanism for idiopathic ventricular fibrillation. Nature392:293-296.Wang, Q., Timur, A., Szafranski, P., Sadgephour, A., Jurecic, V., Cowell, J., Baldini, A., and D.J. Driscoll(2001) Identification and molecular characterization of de novo translocation t(8;14)(q22.3;q13) associated with avascular and tissue overgrowth syndrome. Cytogenet. Cellular Genet. 95:183-188.Chen, S., Chung, M. K., Martin, D., Rozich, R., Tchou, T.J., and Q. Wang (2002) SNP S1103Y in the cardiacsodium channel gene SCN5A is associated with cardiac arrhythmias and sudden death in a Caucasian family. J.Med. Genet. 39:913-915.Wu, L., Nishiyama, K., Hollyfield, J.G., and Q. Wang (2002) Localization of Na v1.5 sodium channel protein inthe mouse brain. Neuroreport 13:2547-2551.Fan, C., Duhagon, M. A., Oberti, C., Chen, S., Hiroi, Y., Komuro, I., Duhagon, P.I., Canessa, R., and Q. Wang(2002) Novel TBX5 mutations and molecular mechanism for Holt-Oram syndrome. J. Med. Genet. 40:E29.Wang, Q., Gu, Y., Ferguson, J.M., Chen, Q., Boatwright, S., Gardiner, J., Below, C., Espinosa, J., Nelson, D.L.,and L.G. Shaffer (2003) Cytogenetic analysis of obsessive-compulsive disorder (OCD): Identification of aFRAXE fragile site. Am. J. Med. Genet. 118A:25-28.THE Q. WANGLABORATORYPROJECT SCIENTISTShaoqi Rao, Ph.D.RESEARCH ASSOCIATESRajkumar Kadaba, Ph.D.Carlos Oberti, M.D.Gong-Qing Shen, M.D., Ph.D.RESEARCH FELLOWSShenghan Chen, M.D.Lejin Wang, M.D.POSTDOCTORAL FELLOWSChun Fan, M.D., Ph.D.Xiaoli Tian, Ph.D.Anyle Anil Timur, Ph.D.Sandro Yong, Ph.D.Sun-Ah You, Ph.D.Lin Li, Ph.D.NEUROLOGY FELLOWJocelyn Bautista, M.D.RESEARCH TECHNICIANZhaohui Tang, B.S.GRADUATE STUDENTSSteve Archaki, M.D.Wei Du, B.S.Mugen Liu, M.S.Ling Wu, M.S.STUDENTSGenise Owens, B.S. 1Josephine Williams 21Cuyahoga CommunityCollege, Cleveland, OH2John Hay High School-CCF/NIH/HHMI Training ProgramCOLLABORATORSMina K. Chung, Ph.D. 1David J. Driscoll, Ph.D. 2William G. Ondo, M.D. 3Patrick J. Tchou, Ph.D. 1Eric J. Topol, M.D. 11Dept. of CardiovascularMedicine, CCF2Dept. of Pediatrics, MayoMedical School, Rochester, MN3Baylor Coll. of Med., Houston,TX131

The Department of Molecular CardiologyMolecular Cardiology Project and Staff Scientistswith Independent FundingOlga Stenina, Ph.D.,I am interested in mechanisms of initiation of atherosclerotic lesions,and currently I am studying the role of thrombospondins in theseprocesses. Thrombospondins are regulating the interaction ofvascular cells with cellular matrix. These proteins have beenassociated with premature familial coronary artery disease at thegenetic level1. Our investigation of functional consequences ofproatherogenic single nucleotide polymorphisms in thrombospondingenes revealed that decreased adhesion and proliferation of endothelialcells in presence of mutant thrombospondin-4 may be a cause foraccelerated development of atherosclerosis. The expression ofthrombospondin-1, a potent anti-angiogenic and proatherogenic agent,increases in vascular wall in diabetes, a condition strongly predisposingto atherosclerosis. This protein may represent an important linkbetween diabetes, accelerated atherogenesis and increased restenosis.Tatiana Ugarova, Ph.D.Our laboratory studies the structure of integrins and their functionsin adhesion and migration of blood cells. The major object is integrinα Mβ 2(Mac-1, CD11b/CD18), a multi-ligand receptor which plays acentral role in the inflammatory response and host defense. Weexamine the mechanism which allows α Mβ 2to exhibit broad ligandspecificity and explore the biological value of its ligand promiscuity.We are testing the hypothesis that translocation of α Mβ 2to thesurface of neutrophils from internal pools and its capacity to bindnumerous extracellular matrix proteins control an overall process ofneutrophil migration. Another project examines the molecular basisfor binding of fibrinogen by platelet integrins in thrombus formationand remodeling.132

Neurosciences

DEPARTMENT OFNEUROSCIENCESCHAIRMANBruce D. Trapp, Ph.D.STAFFWendy Macklin, Ph.D.ASSOCIATE STAFFMark Perin, Ph.D.ASSISTANT STAFFKeiko Hirose, M.D.Hitoshi Komuro, Ph.D.Masaru Nakamoto, M.D., Ph.D.Susan Staugaitis, M.D., Ph.D.STAFF SCIENTISTClara Pelfrey, Ph.D.PROJECT SCIENTISTSTatyana Gudz, Ph.D.Yulong Han, Ph.D.Grahame Kidd, Ph.D.Sandhya Rani, Ph.D.Jerome Wujek, Ph.D.Xinghua Yin, M.D.Neuroscientists Examine BrainDevelopment, Neuronal Function,and Pathogenesis of Human DiseaseThe Department of Neurosciences, foundedin 1994, is chaired by Dr. Bruce Trapp andcomprises a core of internationallyrecognized scientists who investigate the cellularand molecular biology of brain development andneuronal and glial function. In collaboration withresearch programs in the Clinic’s Departments ofNeurology and Neurological Surgery, staffscientists also participate in clinically relevantresearch in the areas of myelin disease,neurodegeneration, neuro-oncology, epilepsy andcerebrovascular disease.Glial Development and Myelin FormationThe glial program includes Drs. Trapp,Wendy Macklin, Susan Staugaitis, RichardRansohoff and Richard Rudick. Drs. Trapp andMacklin investigate basic questions related to thecellular and molecular biology of glial developmentand myelin formation. Dr. Susan Staugaitisis a board-certified neuropathologist who hasjoint appointments in the Departments ofNeurosciences and of Pathology and LaboratoryThe Department of NeurosciencesMedicine. Dr. Staugaitis investigates glialprogenitor cells as the source of glial tumors inthe central nervous system (CNS). Dr. Trapp alsoinvestigates mechanisms responsible for destructionof myelin and axons in individuals withmultiple sclerosis (MS), and Dr. Macklininvestigates the glial response in animal models ofhypoxia. The glial research program has close tieswith physicians in the Cleveland Clinic’s MellenCenter for Multiple Sclerosis Treatment andResearch, which houses one of the largest clinicalMS programs in the world.Drs. Ransohoff and Rudick hold jointappointments in the Departments of Neurologyand Neurosciences. Dr. Ransohoff ’s researchfocuses on the regulation and function ofchemokines as they pertain to the pathogenesis ofdemyelinating diseases. Dr. Rudick directs clinicaltrials in MS therapeutics and is interested inoutcome measures and surrogate markers forContinued on Page 135RESEARCH ASSOCIATESAna Flores, Ph.D.DeRen Huang, M.D.JOINT APPOINTMENTSManjunatha Bhat, Ph.D.Nicholas Boulis, M.D.Mark Luciano, M.D.Marc Mayberg, M.D.Erwin Montgomery, M.D.Erik Pioro, M.D., Ph.D.Richard Ransohoff, M.D.Richard Rudick, M.D.Thyagarajan Subramanian, M.D.ADJUNCT STAFFTomasz Kordula, Ph.D. 1Robert H. Miller, Ph.D. 21Dept. of Biological, Geological,and EnvironmentalSciences, Cleveland StateUniv., Cleveland, OH2Dept. of Neurosciences, CaseWestern Reserve Univ.,Cleveland, OHBruce D. Trapp, Ph.D.Dept. website: http://www.lerner.ccf.org/neurosci/134

The Department of NeurosciencesContinued from Page 134disease progression in MS patients and inmechanisms of interferon action in MS therapeutics.The MS group is supported by an NIHProgram Project Grant.Neuronal Development and FunctionThe neuronal development and functionprogram includes Drs. Mark Perin, HitoshiKomuro, Masaru Nakamoto and Keiko Hirose.Dr. Perin’s research focuses on molecularmechanisms of neurotransmitter release andrecycling. Dr. Komuro investigates cellularmechanisms of neuronal cell migration in thedeveloping cerebellum. Dr. Nakamoto exploresthe role of ephrins and EPH receptors duringaxonal targeting in the developing cerebellum.Dr. Hirose studies auditory physiology andcellular mechanisms associated with hearing lossand deafness.Neurodegenerative DiseaseThe neurodegenerative disease programincludes Drs. Erwin Montgomery, Erik Pioro andThyagarajan Subramanian. Dr. Pioro is aphysician investigator who has joint appointmentsin the Departments of Neurology andNeurosciences. He investigates mechanisms ofneuronal degeneration in amyotrophic lateralsclerosis (ALS)(Lou Gehrig’s disease) and thepathogenesis of neurodegeneration in an animalmodel of ALS. Dr. Erwin Montgomery directsthe Parkinson’s Research Program at CCF. Hisbasic science research utilizes primate models tounderstand the complexity of neurosystems thatregulate motor function. His clinical researchfocuses on early diagnosis and surgical treatmentof Parkinson’s disease by deep brain stimulation.Dr. Subramanian is investigating the role ofsubstantia nigra reticulata (SNr) in the pathophysiologyof parkinsonism and the efficacy ofgene therapy to prevent the progression ofParkinson’s disease (PD). The MovementDisorders Program is testing novel medicationsand drug delivery systems that are designed tofacilitate neuroprotection and neuroregenerationin patients with PD, dystonia and spasticity.The strategic plan for the Department ofNeurosciences is to recruit additional staff whoseresearch focuses on neuronal development,neuronal function and the pathogenesis ofneurodegenerative diseases. The department is oneof the few neuroscience departments in theUnited States that has as its major mission thegoal of investigating and understanding thepathogenesis of human CNS diseases. Theinteractions between faculty in the Departmentsof Neurosciences, Neurology, NeurologicalSurgery and Radiology provide a unique environmentfor reaching this goal.Dept. website: http://www.lerner.ccf.org/neurosci/Specialized areas of muscle surface surround nerve terminalsand convert nerve firing into muscle contractions. From studies byXinghua Yin, M.D., and Bruce Trapp, Ph.D..135

THE BOULISLABORATORYPOSTDOCTORAL FELLOWSQinshan Teng, M.D., M.Sc.Diana Tanase, M.D., Ph.D.GRADUATE STUDENTJames Liu, B.S.TECHNICIANMary Garrity-Moses, B.S.RESEARCH COORDINATORRose AndersonThe Boulis Laboratory is focused on thedevelopment of viral gene transfer forneuroprotection and neuromodulation.The laboratory is organized by disciplines so thatconcepts in gene transfer can be efficientlytransferred from vector construction to in vitrotesting to vectoramplification andpurification forapplication in animalmodels. The laboratoryuses adenoviral, adenoassociatedviral vectors(AAV) and lentiviralvector systems.The laboratory’schief target forneuroprotection is motorneuron disease, includingboth amyotrophic lateralsclerosis and spinalmuscular trophy. In thisarea, we have constructedAAVs for thedelivery of growthfactors (IGF-I) and antiapoptoticfactors (BclxL).In addition, wehave constructedadenoviral vectors forthe delivery of thenewly characterized inhibitors of apoptosis (IAP)family. Further, through a collaboration with thebiotech firm Biomedica, we have constructed amotor neuron targeted vector for the delivery ofIGF-I. These vectors are being tested in an invitro glutamate excitotoxicity assay utilizingThe Department of NeurosciencesViral Vectors Point to Drug Delivery forMotor Neuron Disease, BiodefenseNicholas M. Boulis, M.DSHSY5Y neuroblastoma cells and E15 spinalmotor neurons. Finally, efforts are under way totest the spinal cord delivery of these vectors inrodents. We are beginning to merge these deliverystrategies with the neuroprotective vectors in theSOD1 mutant mouse model of motor neurondisease.The laboratory’s focus ingene-based neuromodulation isto develop viral vectors capableof synaptic control. We havecompleted experimentstargeting eating behaviorsthrough the expression ofglutamate decarboxylase in thelateral nucleus of the hypothalamus.In addition, we havedeveloped an adenoviral vectorfor the delivery of the tetanuslight chain fragment. In vitroand in vivo testing reveals thisvector to be capable of synapticinhibition through proteolyticdigestion of synaptobrevin, akey vesicle docking protein.To improve motorneuron vector targeting, thelaboratory has pursued astrategy of phage display toidentify small peptides capableof high-affinity and highspecificitymotor neuron terminal binding. Initialexperiments have revealed a 12-amino-acidsequence that appears to specifically bind theclostridial toxin receptor. This amino acid hasapplication to vector targeting as well asbiodefense.Boulis, N.M., Turner, D.E., Imperiale, M.J., and E.L. Feldman (2002) Neuronal survival following remoteadenoviral gene delivery. J. Neurosurg. 96(2 Suppl):212-219.Boulis, N.M., Willmarth, N.E., Song, D.K., Feldman, E.L., and M.J. Imperiale (2003) Intraneural colchicineinhibition of adenoviral and adeno-associated viral vector remote spinal cord gene delivery. Neurosurgery52:381-387.Rubin, A., Mobley, B., Hogikyan, N., Bell, K., Sullivan, K., Boulis, N., and E. Feldman (2003) Deliveryof an adenoviral vector to the crushed recurrent laryngeal nerve. Laryngoscope 113:985-989.Noordmans, A.J., Song, D.K., Noordmans, C.J., During, M.J., Fitzsimons, H., Imperiale, M.J., and N.M.Boulis (2003) Adeno-associated viral glutamate decarboxylase expression in the lateral nucleus of therat hypothalamus reduces feeding behavior. Gene Ther. (in press).Boulis, N.M., Noordmans, A.J., Song, D.K., Rubin, A., Leone, P., During, M., Feldman, E.L., and M.J.Imperiale (2003) Adeno-associated viral vector gene expression in the adult rat spinal cord following remotevector delivery. Neurobiol. Dis. (in press).136

The primary goal of our research is toelucidate the cellular mechanisms ofhearing loss by studying agents known tocause damage to the cochlea. The majority ofindividuals who suffer from hearing loss cannot behelped by any therapies because hair cells, thesensory cells of the cochlea, are not amenable tocurrent medical or surgical interventions. With abetter understanding of the steps that lead to haircell destruction and loss of hearing, we hope toimprove our ability to intervene in cases ofprogressive hearing loss.One area that has not been well investigatedis the role of the inflammatory response andthe immune system in the inner ear. Traditionalthinking holds that the inner ear is an immunologicallyprivileged site, much like the brain or the eye.However, the validity of this concept has notbeen clearly demonstrated. The aim of ourresearch is to determine what inflammatoryprocesses occur in the inner ear after tissuedestruction and to evaluate whether inflammatorycells facilitate repair or contribute to degenerationof the inner ear after noise trauma. Our first goalis to determine the effect of noise trauma on theactivation of macrophages in the inner ear. Cellsresembling macrophages have been reported in theinner ear following acoustic trauma. However,the cells that mediate inflammation in themammalian ear have not been systematicallystudied after acoustic injury. It is possible thatthese cells are residents of the inner ear itself orthat they migrate into the ear through thecirculation. Using noise levels that are known tocause significant tissue destruction in the inner ear,we are examining the influx and activation ofphagocytic cells, their proliferation, theirlocalization within the inner ear and how thesefactors correlate to tissue repair and return ofhearing function.Activation of inflammatory cells, such asmacrophages, typically results in the expression ofcytokines, which alter the local environmentthrough changes in vascular permeability, tissueThe Department of NeurosciencesInflammatory Mediators Play anIntegral Part in the Cochlea'sResponse to Noise Damageswelling, and recruitment of other immune cells.Cytokines can be both beneficial, inducing repair,and destructive, inducing cascades that result inrecruitment of other inflammatory mediators. Incollaboration with the laboratory of Dr. RichardRansohoff, we are exploring the role of inflammationin the inner ear by performing quantitativereverse transcription polymerase chain reaction(RT-PCR) analysis of cochlear tissue followingacoustic trauma. This technique will allow us todetermine if any of the traditional pathways fromcellular injury to repair are recapitulated in theinner ear, thus further challenging the concept thatthe inner ear is an immunologically privileged site.Our collaboration with the laboratory ofDr. Vincent Tuohy focuses on developing ananimal model of autoimmune sensorineural hearingloss. Although the clinical picture of autoimmunedeafness has been well described in the clinicalliterature, our scientific understanding of themechanism of this disease is very poor. The Tuohylaboratory has generated two peptides that bindthe T-cell receptor and induce sensorineural hearingloss in mice by sensitizing T cells to two knownligands in the inner ear, alpha-tectorin and Cochprotein. Our preliminary physiologic datademonstrate that these mice develop elevations intheir hearing thresholds at 5 weeks after antigenicexposure; the condition of such mice over longerterms is being studied. Further pursuit of theseexperiments will include a detailed histopathologicstudy of the cochleas of these animals todetermine which area of the inner ear is affected.Also, we are performing immunohistochemicalstudies to determine whether T cells actually enterthe inner ear and whether or not antibody isgenerated against these two proteins.Ultimately, we hope not only to betterunderstand the immune system and its interactionwith the neurosensory portion of hearing, but alsoto provide a better fundamental knowledge ofhow the inner ear degenerates and how it repairsitself to preserve the function that remains afterdamage to the cochlea.THE HIROSELABORATORYPOSTDOCTORAL FELLOWJames Chan, M.D.Christopher M. Discolo, M.D.TECHNICIANJodi R. KeaslerCOLLABORATORSRichard M. Ransohoff, M.D. 1Ganesh C. Sen, Ph.D. 2Bruce D. Trapp, Ph.D. 1Vincent K. Tuohy, Ph.D. 31Dept. of Neurosciences, CCF2Dept. of Molec. Biology, CCF3Dept. of Immunology, CCFKeiko Hirose, M.D.Hirose, K., and P.H. Chan (1993) Blockade of glutamate excitotoxicity and its clinical applications.Neurochem. Res. 18:479-483.Hirose, K., Hockenbery, D.M., and E.W. Rubel (1997) Reactive oxygen species in chick hair cells aftergentamicin exposure in vitro. Hear. Res. 104:1-14.Hirose, K., Wener, M.H., and L.G. Duckert (1999) Utility of laboratory testing in autoimmune inner eardisease. Laryngoscope 109:1749-1754.Hirose, K., Westrum, L.E., Stone, J.S., Zirpel, L., and E.W. Rubel (1999) Dynamic studies of ototoxicityof mature avian auditory epithelium. Ann. N.Y. Acad. Sci. 884:389-409.Wang Y, Hirose K, and M.C. Liberman (2002) Dynamics of noise-induced cellular injury and repair in themouse cochlea. J. Assoc. Res. Otolaryngol. 3:248-268.137

THE KOMUROLABORATORYCOLLABORATORSArmando A. Genazzani, Ph.D. 1Pasko Rakic, M.D., Ph.D. 21Dept. of Pharmacol., Univ. ofCambridge, UK2Sect. of Neurobiology,Yale Univ. Sch. of Med.,New Haven, CTThe long-term goal of our laboratory is toelucidate the cellular and molecularmechanisms underlying neuronalmigration in the developing brain.(1) At present, we are determiningwhether somatostatin, a neuropeptide, controlsthe transition of cerebellar granule cell migrationduring the translocation from the cells’ birthplaceto their final destination. We are determiningcharacteristic features of the migratingbehavior of granule cells in their natural cellularmilieu with the use of confocal microscopy andacute slice preparationsobtained from the earlypostnatal mouse cerebellum.We will determine whetherand how activation and/orinhibition of somatostatinreceptors affect the granulecell’s behavior and itsmorphology in each cerebellarcortical layer. Furthermore,we will determine howintracellular messengers,including Ca 2+ and cAMP,which are modified byactivation of somatostatinreceptors, differentiallyregulate the behavior of thecell body and the process ofmigrating granule cells.Transient expression ofsomatostatin and its receptorsis a characteristic feature ofdeveloping neurons duringperiods of neurogenesis and brain differentiation.The function of somatostatin in neuronalmigration, however, is not known. Answers tothese questions will elucidate the undiscoveredrole of somatostatin and its receptors in theformation of brain and provide new insights forunderstanding the effects of various genetic andenvironmental factors in the pathogenesis ofbrain malformations that are implicated invariety of functional disorders, such as epilepsy.(2) Maternal alcohol consumption duringpregnancy can cause serious birth defects, ofThe Department of NeurosciencesCellular Mechanisms UnderlyingCerebellar Granule Cell Migrationwhich fetal alcohol syndrome (FAS) is the mostdevastating. Recognized by characteristiccraniofacial abnormalities and growth deficiency,this condition includes severe alcohol-induceddamage to the developing brain. FAS childrenexperience deficits in intellectual functioning anddifficulties in learning, memory, problem-solving,and attention; problems with mental health andsocial interactions also affect these children.In our laboratory, to elucidate the cellularand molecular mechanisms underlying alcoholinducedmalformation of brain, we are determiningthe effects of alcohol oncerebellar granule cell migration.First, we will determine when,where and how alcohol altersthe migration of cerebellargranule cells in real time withthe use of acute cerebellar slicepreparations and microexplantcultures. In particular, we willexamine the relationshipbetween the amounts anddurations of alcohol administrationand the inhibition of cellmovement. Second, we willdetermine whether changes inintracellular Ca 2+ fluctuationsand the membrane potential ofmigrating granule cells areinvolved in alcohol-inducedalteration of neuronal migration.Third, we will determineHitoshi Komuro, Ph.D. whether manipulations ofintracellular Ca 2+ fluctuationsand membrane potentials by activating NMDAreceptor or inhibiting K + channel activity canovercome the alcohol-induced changes in cellmigration.The fundamental mechanisms wherebyethanol administration leads to disturbances inbrain development have not been delineateddefinitively. Answers to the questions raised in thisproject will provide new insights for postnataltreatments and understanding how prenatal andearly postnatal exposure to alcohol causesmalformation of brain.Kumuro, H., Yacubova, E., and P. Rakic (2001) Mode and tempo of tangential cell migration in the cerebellar externalgranular layer. J. Neurosci. 21:527-540.Yacubova, E., and H. Komuro (2002) Stage-specific control of neuronal migration by somatostatin. Nature 415:77-81.Yacubova, E., and H. Komuro (2002) Intrinsic program for migration of cerebellar granule cells in vitro. J. Neurosci.22:5966-5981.Yacubova, E., and H. Komuro (2003) Cellular and molecular mechanisms of cerebellar granule cell migration. Cell.Biochem. Biophys. 37:213-234.Komuro, H., and E. Yacubova (2003) Recent advances in cerebellar granule cell migration. Cell. Mol. LifeSci.60:1084-1098.138

The Department of NeurosciencesCerebral Blood Flow, Shunting Examinedin Chronic Hydrocephalus ResearchCombined efforts in clinical medicine andbasic research are being used to investigatethe pathophysiology, diagnosis, andtreatment of chronic hydrocephalus. Compellingevidence from our laboratory, as well as others,suggests that brain hypoxia, through vesselcompression, may play an important role in thispreventable neurological injury. Our directobjective is to understand cerebrovascularresponse to ventricular enlargement, optimizecurrent treatment such as cerebrospinal fluid(CSF) shunting, and develop new techniques and/or methods to alleviate symptoms related tochronic hydrocephalus.Recently Dr. Luciano has been awarded afour-year; NIH-R01 grant entitled “CerebralBlood Flow Response to Chronic Hydrocephalus.”Using a canine model developed in ourlaboratory, we are able to study the pathophysiol-ogy of chronic hydrocephalus by measuringventricular size and pressure, and their relationshipto histological changes, cerebral blood flow(CBF) and oxygen delivery to the brain. Regionalchanges in CBF will be studied with microsphereinjection technique and patterns of oxygendelivery by measuring brain and CSF oxygensaturation. The mechanisms of CBF changes andtheir effect on CNS tissue are studied throughquantitative histological examination of thecerebrovascular morphology and neural parenchyma.This study focuses on the naturalprogression of chronic hydrocephalus and theeffect of surgical treatment (CSF shunting) onthe vascular response of early and late shunting tonon-hydrocephalic animals and untreated animals.We hope to expand our understanding of chronichydrocephalus through basic research and applythis knowledge in clinical medicine.THE LUCIANOLABORATORYRESEARCH ASSOCIATEStephen Dombrowski, Ph.D,Director of PCNS ExperimentalStudiesPostdoctoral FellowsSamer Elbabaa, M.D.Zhicheng Li, M.D.Mark Luciano, M.D.,Ph.D.Director,PCNS and Cleveland Clinic Hydrocephalus ProjectJohnson, M.J., Ayzman, I., Wood, A.S., Tkach, J.A., Klauschie, J., Skarupa, D.J., McAllister, J.P., andM.G. Luciano (1999) Development and characterization of an adult model of obstructive hydrocephalus. J.Neurosci. Methods 91:55-65.Luciano, M.G., Skarupa, D.J., Booth, A.M., Wood, A.S., Brant, C.L., and M.J. Gdowski (2001) Cerebrovascularadaptation in chronic hydrocephalus. J. Cereb. Blood Flow Metab. 21:285-294.Fukuhara, T., Luciano, M.G., Brant, C.L., and J. Klauscie (2001) Effects of ventriculoperitoneal shunt removalon cerebral oxygenation and brain compliance in chronic obstructive hydrocephalus. J. Neurosurg.94:573-581.Fukuhara, T., Luciano, M.G., and R.J. Kowalski (2002) Clinical features of third ventriculostomy failuresclassified by fenestration patency. Surg. Neurol. 58:102-110.139

THE MACKLINLABORATORYPROJECT STAFFTatyana Gudz, Ph.D.Mika Yoshida, M.D.RESEARCH ASSOCIATEAna Flores, Ph.D.POSTDOCTORAL FELLOWYuko Fujita, Ph.D.TECHNICAL ASSISTANTSChristine AgaibiEid DarwishCindy KangasKapila NavaratneElizabeth ShickCOLLABORATORSMartha J. Miller, M.D., Ph.D. 1Robert H. Miller, Ph.D. 2Stephen A. Stohlman, Ph.D. 3Bruce L. Trapp, Ph.D. 41Dept. of Pediatrics, CaseWestern Reserve Univ.,Cleveland, OH2Dept. of Neurosciences, CaseWestern Reserve Univ.,Cleveland, OH3Dept. of Molec. Biol./Immunol., Univ. of SouthernCalif., Los Angeles, CA4Dept. of Neurosciences, CCFThe Department of NeurosciencesGenes, Molecular Signaling Regulationof Normal Brain DevelopmentThe long-range goal of my research programis understanding the molecular signals thatregulate normal brain development. Themain research effort is directed to oligodendrocytedifferentiation and myelin biogenesis. We focus ona gene that is primarily expressed in myelinatingoligodendrocytes, the myelin proteolipid protein(PLP), and the closely related DM20 protein,which are the most abundant proteins of the CNSmyelin membrane. Point mutations in this gene arelethal, and the affected animals die by 3-4 weeksof age. Mutations in other oligodendrocyte genesthat generate important proteins for the myelinmembrane do not kill the animals at young ages.We are particularly interested in why mutations inthe PLP protein should be so devastating.We have used the PLP gene promoter togenerate transgenic animals overexpressingenhanced GFP (EGFP) in oligodendrocytes andhave used these transgenic animals to track PLPgene expression in developing oligodendrocytesfrom normal and mutant animals. The EGFPtransgenic mice are being used because PLPfluoresces in live cells, so we can identify oligodendrocytesin live tissue. In the PLP-EGFP mice, allcells in the lineage from oligodendrocyte progenitorto mature myelinating oligodendrocytes aredetected. In addition, we detect strong expressionin the sciatic nerve and the developing embryo.The embryo expression is in nonmyelinating cellsof both the central and peripheral nervous systems.Thus, we are able to track these cells to investigatenormal development of glial cells in both the PNSand CNS, and to study their differentiation inmutant animals or other pathological environments.We study their migration and differentiationby live confocal imaging of optic nerve.We are investigating why the PLP gene isexpressed in nonmyelinating, migrating cells earlyin development. We have demonstrated that thePLP protein interacts with integrins, an interactioncontrolled by neurotransmitter receptors on thesurface of oligodendrocytes. This inside-outsignaling of integrins in response to neurotransmittersinduces a complex of PLP withα v-integrin and reduces binding of fibronectin tooligodendrocytes. This altered binding to anextracellular matrix protein also alters themigration of oligodendrocyte progenitor cells.In the PLP mutant mice, the oligodendrocytesdie by apoptosis. Other problems in additionto the PLP mutations can induce oligodendrocyteapoptosis, e.g., growth factor deprivation. Survivalfactors can protect cells from apoptosis by blockingcell death signals. Thus, the balance of death andsurvival signals determines cell fate. We havestudied the neuregulin/erbB receptor system,which is an ideal candidate to provide survivalsignals from neurons to oligodendrocytes in vivo. Wehave established that the survival function ofneuregulins acts through the PI3 kinase/Aktpathway. We are currently investigating Aktsubstrates in oligodendrocytes and haveoverexpressed Akt in transgenic mice, which arejust now being analyzed.In other studies on oligodendrocytedifferentiation, we are studying oligodendrocyte/neuron interactions during development, using anewly identified mouse mutant with defectivePurkinje cell development. Purkinje cells are amajor set of neurons in the cerebellum, and theonly neurons that are myelinated. These cells beginto differentiate, but even at 6 days of age, somedifferences are noted between these cells and thePurkinje cells in wild-type animals. Our particularinterest in these animals is in the interactionbetween the oligodendrocytes and their targets, thePurkinje cells. What happens to oligodendrocyteswhen their target is altered in development? Wehave demonstrated a delay in oligodendrocytedifferentiation and an overall downregulation inmyelin gene expression in oligodendrocytes thatwould normally have myelinated the Purkinje cellaxons.In overview, these studies all focus ondifferent aspects of the ability of cells in thedeveloping nervous system to differentiatecorrectly and to find the appropriate site and targetfor their functions.Wendy B. Macklin, Ph.D.Mallon, B.S., Shick, H.E., Kidd, G.J., and W.B. Macklin (2002) Proteolipid promoter activity distinguishestwo populations of NG2-positive cells throughout neonatal cortical development. J. Neurosci. 22:876-887.Gudz, T.I., Schneider, T.E., Haas, T.A., and W.B. Macklin (2002) Myelin proteolipid protein participates in integrinreceptor signaling in oligodendrocytes. J. Neurosci. 22:7398-7407.Kahle, P.J., Neumann, M., Ozmen, L., Mueller, V., Jacobsen, H., Spooren, W., Fuss, B., Mallon, B., Macklin,W.B. Fujiwara, H., Hasegawa, M., Iwatsubo, T., Kretzschmar, H.A. and C. Haass (2002) Hyperphosphorylationand insolubility of a-synuclein transgenic mouse oligodendrocytes, EMBO Rep. 3:583-588.Mallon, B.S., and W.B. Macklin (2002) Overexpression of the 3’-untranslated region of myelin proteolipid proteinmRNA leads to reduced levels of endogenous proteolipid transcripts. Neurochem. Res. 27:1349-1360.Baracskay, K.L., Duchala, C.S., Miller, R.H., Macklin, W.B., and B.D. Trapp (2002) Oligodendrogenesis isdifferentially regulated in gray and white matter of jimpy mice. J. Neurosci. Res. 70:645-654.140

Our broad research interest is to understandthe molecular and cellular mechanismsthat establish the spatial pattern of thevertebrate nervous system. During developmentof the nervous system, neuronal differentiation,migration, axon guidance, and specificsynaptogenesis take place in a well-organizedmanner.Considering the complexity of the nervoussystem, it is clear that many of the importantmolecules that control neuronal behavior duringdevelopment have not yet been identified. Onefocus of our research is, therefore, to identifynew molecules that are involved in developmentof the nervous system. The approaches include(1) identification of ligands for orphan receptorsand cell adhesion molecules and of receptors forpeptide growth factors, and (2) subtractioncloning and differential library screening.In addition to identifying new molecules,we are interested in understanding how thesemolecules function during development of thenervous system. In particular, we are exploringthe formation of neuronal networks. Function ofthe nervous system is critically dependent uponthe establishment of appropriate connectionsbetween neurons and their target cells. The initialdevelopment of these connections typicallyoccurs before neurons become functionally active,and it is believed to be established by molecularguidance cues. First, axons must follow theircorrect pathway and reach the target region.Second, within the target region, each axon mustfind and recognize the correct target cells andform specific connections. In the vertebratenervous system, this step typically involvestopographic mapping, whereby axons ofneighboring neurons project to neighboring areasin the target region so that the spatial order ofthe projecting neurons is maintained in the target.About half a century ago, Sperry proposed thattopographic mapping could be guided bycomplementary positional labels in gradientsacross pre- and post-synaptic fields. AlthoughSperry’s idea has been widely accepted, molecularThe Department of NeurosciencesMolecular Mechanisms ofVertebrate Neural Developmentidentification of the positional labels has longremained an elusive goal.The Eph receptor family is by far thelargest known family of receptor tyrosine kinasesand contains 14 members in vertebrates. Recently,a family of ligands for the Eph receptors, namedephrins, has been identified, with eight membersthus far cloned in vertebrates. Most of the Ephreceptors and ephrins are predominantlyexpressed in the nervous system, with distinct andoverlapping patterns. Ourprevious work has demonstratedthat ephrin-A2 andits high-affinity receptorEphA3 can act as molecularlabels during the developmentof a topographicprojection. More recentwork by other groups hasrevealed the functions ofthe Eph receptors andephrins in rhombomereformation and neural crestcell migration. Consideringthe large number anddiversity of expressionpatterns, the Eph receptorsand ephrins are likely to playcrucial roles in many aspectsof neural development. Weare further investigating thefunctions of the Ephreceptors and ephrins inspatial patterning ofconnections and cellpositions in the nervoussystem.THE NAKAMOTOLABORATORYRESEARCH FELLOWSMarc Jones, M.S.Hiroshi Matsuoka, M.D., Ph.D.RESEARCH TECHNOLOGISTZhufeng Ouyang, M.D., M.S.RESEARCH TECHNICIANYukari Izutani, B.A.Many congenitaldiseases affect networkMasaru Nakamoto, M.D., Ph.D.formation and cell migrationduring neural development.In addition, the mechanisms of neuronalregeneration after injury is of great clinicalinterest. The study in this field, therefore, willalso produce many important insights in clinicalmedicine.Cheng, H-J., Nakamoto, M., Bergemann, A.D., and Flanagan, J.G. (1995) Complementary gradient in expressionand binding ELF-1 and Mek4 in development of the topographic retinotectal projection map. Cell82:371-381.Nakamoto, M., Cheng, H-J., Friedman, G.C., McLaughlin, T., Hansen, M., O’Leary, D.D.M., and Flanagan,J.G. (1996) Topographically specific effects of ELF-1 on retinal axon guidance in vitro and retinal axonmapping in vivo. Cell 86:755-766.Nakamoto, M. (2000) Eph receptors and ephrins. Int. J. Biochem. Cell Biol. 32:7-12.Nakamoto, M., and A.D. Bergemann (2002) Diverse roles for the Eph family of receptor tyrosine kinasesin carcinogenesis. Microsc. Res. Tech. 59:58-67.Nishida, K., Flanagan, J.G., and M. Nakamoto (2002) Domain-specific olivocerebellar projection regulatedby the EphA-ephrin-A interaction. Development 129:5647-5658.141

THE NAJMLABORATORYCOLLABORATORSWilliam Bingaman, M.D. 1Damir Janigro, M.D. 2Dianne Perez, Ph.D. 31Dept. of Neurological Surgery,CCF2Center for CerebrovascularResearch, CCF3Dept. of Mol. Cardiology, CCFWithin the Epilepsy section of theDepartment of Neurology, the AdultEpilepsy Center comprises pediatricand adult neurologists; neurosurgeons;neuroradiologists; nuclear medicine physicians;nurse specialists; pharmacologists; physical,occupational, and speech therapists; dietitians;neuropsychologists and psychiatrists; educationalcounselors and social workers; and an array ofscientists and technologists. For both childrenand adults, this team provides state-of-the-artdiagnosis and treatment in a caring environment.Their goal remains constant – to help patientsmanage their disease so that they, in turn, canenjoy fuller, more productive lives. Thisdedicated team of experts diagnoses and treatsmore than 2,000 patients with epilepsy eachyear, has established comprehensive researchprograms for testing anticonvulsant drugs andmonitoring blood-drug levels, and has developedextensive facilities for evaluating patients andfor performing epilepsy surgery.A key focus of the Najm laboratory is theThe Department of NeurosciencesGamma Knife Radiosurgery Focuses onMedically Refractory Epilepsyclinical trial “Gamma Knife Radiosurgery forMedically Refractory Mesial Temporal LobeEpilepsy,” with co-Principal Investigators ImadNajm, M.D., and Nancy Foldvary, D.O., a jointeffort of the Departments of Neurology andNeurological Surgery. The trial’s populationcomprises patients >16 years old with medicallyrefractory focal epilepsy due to documentedmesial temporal sclerosis. Its aim is to determinethe effectiveness of gamma knife surgery inpatients with temporal lobe epilepsy due tomesial temporal sclerosis that is incompletelycontrolled with antiepileptic medications.Aspects to be evaluated include (1) preoperativetesting, including Video-EEG monitoring, MRI,FDG-PET, MRS, intracarotid amytal test(WADA), neuropsychological testing, and formalvisual fields; (2) Gamma Knife treatment in anoutpatient setting; and (3) serial neurologicassessments in the outpatient clinic, MRI,neuropsychological battery, MRS, PET, and visualfield testing 2 years from the time of GammaKnife treatment.Imad Najm, M.D.Najm, I., Ying, Z., and D. Janigro (2001) Mechanisms of epileptogenesis. Neurol. Clin. 19:237-250.Ruggieri, P.M., and I.M. Najm (2001) MR imaging in epilepsy. Neurol. Clin. 19:477-489.Ying, Z., and I.M. Najm (2002) Mechanisms of epileptogenicity in focal malformations caused by abnormalcortical development. Neurosurg. Clin. N. Am. 13:27-33, vii.Najm. I.M., Bingaman, W.E., and H.O. Luders (2002) The use of subdural grids in the management of focalmalformations due to abnormal cortical development. Neurosurg. Clin. N. Am. 13:87-92, viii-ix.Kunieda, T., Zuscik, M.J., Boongird, A., Perez, D.M., Luders, H.O., and I,M. Najm (2002) Systemicoverexpression of the alpha 1B-adrenergic receptor in mice: an animal model of epilepsy. Epilepsia43:1324-1329.Luders, H., Najm, I., and E. Wyllie (2003) Reply to “Of Cabbages and Kings: Some Considerations onClassifications, Diagnostic Schemes, Semiology, and Concepts”. Epilepsia 44:6-7.142

We are focusing on three main projectsrelating to multiple sclerosis:Longitudinal Autoreactivity to Central NervousSystem AntigensVery little is known about the relationship betweenimmune cytokine responses (particularly towardsbrain components, such as myelin proteins) and clinicalparameters that are used to assess disease progression inmultiple sclerosis. We performed a longitudinal study thatinvolved mapping of IFNγ and IL-10 cytokine responsesto myelin peptides in MS patients/controls. Cytokine expressionpatterns were correlated with clinical findings,demographic factors and diagnostic measures includingquantitative MRI. Our results show that following MS patientsover time by analysis of their cytokine patterns tellsus that these important immune chemicals can be linkedto MS patients’ disability but do not appear linked to anyparameter in healthy controls. Certain immune tissuetypes are strongly associated with MS and now appear tobe strongly associated with specific immune cytokineresponses that we can observe at multiple points in time.Immune responses to specific regions of myelin proteinswere highly dynamic over one year of observation andappeared to show bursts of activity coordinated withgadolinium enhanced MRI lesions. We have identified 3patterns of longitudinal reactivity to myelin proteolipidprotein (PLP): I = no reactivity at any timepoint; II =isolated peptide reactivity; and finally, III = generalized“bursts” of reactivity across the entire PLP molecule.Interestingly, no MS patients fell into category I, whereas85% of MS patients showed pattern III compared to only59% of controls. These findings suggest that our methodsare useful for tracking immune events that are linkedto disease disability and may provide beneficial markersfor clinical trials in MS. In addition, these data suggest thatinflammatory cytokines may play a role in progression ofMS and should be considered as targets for therapy.Gender Differences in Immune Response in MSWomen develop MS almost three timesmore often than men. To better understand this“gender gap in autoimmunity,” we are examiningsex differences in multiple immunologicalparameters that may show sexually dimorphicresponses. One of these parameters includesmeasuring inflammatory and regulatory cytokines.Our results show that IFNγ secretion to myelinpeptides and proteins, although heterogeneous,tends to support our previous findings: thatThe Department of NeurosciencesInflammatory, Immunomodulatory CytokinesStudied in Triad of MS Clinical Projectsfemales secrete more IFNγ than males. Anotheraim was to determine if the addition ofexogenous sex hormones can alter the expressionof cell-surface molecules that promote cellularinteractions–known as co-stimulatory moleculeslike CD40L, CD86 and CD80. We observe thatexpression of some co-stimulatory molecules insome individuals respond to in vitro exposure tosex hormones, and specifically to hormones thatare elevated in pregnancy. Finally, measuringexpression of chemokine receptors on lymphocyteswill help us to learn if these importantchemical messengers play a role in male/femaledifferences in MS. A better understanding of thegender bias in MS may allow development of newtherapies that capitalize on the differentimmunological responses in women versus men.Immunomodulatory Effects of MS TreatmentPatients with the most rapidly debilitatingform of multiple sclerosis (MS), primaryprogressive MS, have virtually no therapiesavailable to them. A newly approved therapy forthese MS patients is mitoxantrone; however, itsunderlying mechanisms of action are poorlyunderstood. Mitoxantrone has been found toregulate the generation of T-helper and T-suppressor cells via effects on macrophages.Several soluble mediators elaborated by macrophagescould be responsible for this effect. We aremeasuring communicating cytokines thatmacrophages secrete to upregulate/downregulateT-cell functions. We also examine the effect ofmitoxantrone treatment on soluble levels ofinflammatory mediators in the serum of primaryprogressive MS patients. Chemokine receptorsare frequently correlated with inflammatoryprocesses and have been linked to diseasepathogenesis in MS. Our recent studies focus onthe effect of mitoxantrone treatment on thesurface expression of chemokine receptors onlymphocytes. Understanding the mechanisms ofaction of mitoxantrone will help in improvingtherapy for this highly debilitating form of MS.THE PELFREYLABORATORYADVANCED POSTDOCTORALFELLOWIoana Moldovan, M.D., Ph.D.SENIOR RESEARCHTECHNOLOGISTAnne Cotleur, M.S.TECHNOLOGISTNatacha Zamor, B.S.COLLABORATORSRobert Fox, M.D. 1Matt Karafa, Ph..D. 2Jar-Chi Lee, M.S. 2Paul Lehmann, M.D., Ph.D. 3Richard A. Rudick, M.D. 1Lael Stone, M.D. 1Vincent Tuohy, Ph.D. 4Brian Weinshenker, M.D. 51Mellen Ctr. for Multiple Sclerosis,CCF2Dept. of Biostatistics andEpidemiol., CCF3Case Western Reserve Univ.,Dept. of Pathol., Cleveland,OH4Dept. of Immunology, CCF5Mayo Clinic, Dept. of Neurol.,Rochester, MNClara M. Pelfrey,Ph.D.Pelfrey, C.M., Rudick, R.A. , Cotleur, A.C., Lee, J.-C., Tary-Lehmann, M., and P.V. Lehmann(2000) Quantification of self-recognition in multiple sclerosis by single-cell analysis of cytokineproduction. J. Immunol. 165:1641-1651.Liu, Z., Pelfrey, C.M., Cotleur, A., Lee, J.-C., and R.A. Rudick (2001) Immunomodulatory effectsof interferon β-1a in multiple sclerosis. J. Neuroimmunol. 112:153-162.Pelfrey, C.M. (2001) Sexual dimorphism in autoimmunity: a focus on Th1/Th2 cyto-kines andmultiple sclerosis. Clin. Appl. Immunol. Rev. 1:331-345.Pelfrey, C.M., Cotleur, A.C., Lee, J-C., and R.A. Rudick (2002) Sex differences in cytokineresponses to myelin peptides in multiple sclerosis. J. Neuroimmunol. 130:211-223.Moldovan R.R., Rudick, R.A., Cotleur, A.C., Born, S.E., Lee, J.C., Karafa, M.T., and C. M.Pelfrey (2003) Interferon-γ responses to myelin peptides in multiple sclerosis correlate with anew clinical measure of disease progression. J. Neuroimmunol. (in press).143

THE PERINLABORATORYPOSTDOCTORAL FELLOWXiaoqin Liu, M.D., Ph.D.TECHNICAL ASSOCIATESStudies Focus on Neural Synaptic ProteinInteractions Regulating NeurotransmitterRelease, Membrane Trafficking EventsTheMichael Dentlerresearch interests of my laboratory focusJennifer Preziosoon nerve terminal membrane traffic. Thisspans the biochemistry and molecularbiology of neurotransmitter release, the recyclingof synaptic vesicle membrane and the uptake ofmaterial that occurs during synaptic remodeling.The first area is broadly a question of signaltransduction. The other areas concern events thatare likely to be important in thedevelopment of synapses and of thenervous system.My interests center on thesynaptic proteins involved with thedocking/fusion complex thatmediates neurotransmitter release.My laboratory has predominantlyfocused on the synaptic vesicleprotein synaptotagmin and thepresynaptic proteins with which itmay interact. Synaptotagminmediates the calcium activation ofneurotransmitter release. We haveshown that synaptotagmin containsthree evolutionarily conserveddomains that likely mediate itspotential function in docking,activation and fusion of synapticvesicles.My laboratory is investigatingthe function of the third conserveddomain, which consists of thecarboxyl-terminal 35 amino acids ofMark S. Perin, Ph.D.synaptotagmin 1. We have shownthat this domain can interact with afamily of presynaptic proteins, theneurexins, and with calmodulin. This domain isshared with other synaptotagmins (1-9) andsynaptotagmin-like proteins such as rabphillin 3A.This finding opens up new avenues for studies onPerin, M.S., Fried, V.A., Mignery, G.A., Jahn, R., and T.C. Südhof (1990) Phospholipidbinding by a synaptic vesicle protein homologous to the regulatory domain of proteinkinase C. Nature 345:260-263.Perin, M.S. (1996) Mirror image motifs mediate the interaction of the COOH terminusof multiple synaptotagmins with the neurexins and calmodulin. Biochemistry 43:13808-13816.Schlimgen, A.K., Helms, J.A., Vogel, H., and M.S. Perin (1995) Neuronalpentraxin, a secreted protein with homology to acute phase proteins of the immunesystem. Neuron 14:519-526.Dodds, D., Omeis, I., Cushman, S.J., Helms, J.A., and M.S. Perin (1997) Neuronalpentraxin receptor, a novel integral membrane pentraxin that interacts with neuronalpentraxin 1 and 2 and taipoxin-associated calcium binding protein 49. J. Biol. Chem.272:21488-21494.Kirkpatrick, L.L., Matzuk, M.M., Dodds, D.C., and M.S. Perin (2000) Biochemical interactionsof the neuronal pentraxins: Neuronal pentraxin (NP) receptor binds to taipoxinand taipoxin-associated calcium-binding protein 49 via NP1 and NP2. J. Biol. Chem.275:17786-17792.The Department of Neurosciencesthe regulation of synaptotagmin and on additionalcalcium-dependent steps in neurotransmitterrelease.The other major area of interest of mylaboratory is the nerve terminal membranetrafficking events of endocytosis/recycling ofsynaptic vesicle membrane and how theseprocesses might also mediate uptake of synapticmaterial. We have become interested in this via agroup of snake venom toxins that blockneurotransmitter release by inhibiting thereuptake of synaptic vesicle membrane. We arestudying the most lethal of these toxins, taipoxin,as a potential tool to characterize the reuptake ofsynaptic vesicle membrane and to identifymolecular components that may mediate thisuptake. Using affinity chromatography, we haveidentified and cloned four interacting proteinsthat appear to mediate the uptake and action oftaipoxin. These include three neuronal proteins(Neuronal Pentraxin 1, Neuronal Pentraxin 2 andNeuronal Pentraxin Receptor) that havehomology to each other and to the pentraxinfamily of proteins.The pentraxin family includes the acutephase protein C-reactive protein, which isthought to mediate a general recognition ofbacteria and cell debris. We suggest that thesethree neuronal proteins mediate uptake of thetoxin. The fourth protein (Taipoxin-associatedCalcium Binding Protein-49) is a luminal calciumbindingprotein with six EF hand motifs that mayactivate the toxin. We hypothesize that theseproteins outline a novel pathway for synapticuptake and that these proteins mediate removalof degraded synaptic material during synapseformation, remodeling or elimination. Given thelack of information on how synapses areremodeled, we believe that the identification ofthese proteins may be an important step towardscharacterizing how synapse remodeling is initiatedand, as such, could have important implications inneuronal development. We are using stem celltechnology to investigate the in vivo function ofour identified taipoxin binding proteins. We arealso pursuing biochemical studies of theinteractions of these proteins to understand howtaipoxin binding results in blockade of synapticvesicle membrane endocytosis/recycling and howthis action may illuminate mechanisms ofrecycling and uptake of synaptic material.144

Amyotrophic lateral sclerosis (ALS, LouGehrig’s disease) is probably the mostdreaded of neurodegenerative diseases.There is no cure for this disorder, which causesprogressive paralysis, muscle atrophy, and death,usually from respiratory complications. In theUnited States, it occurs as frequently as multiplesclerosis, with over 5,000 new cases occurring peryear. However, only about 30,000patients are living, becausesurvival averages three to fiveyears after symptom onset.The cause of ALS isunknown, and clinical diagnosis isusually difficult, especially in earlystages. My studies focus on twoseparate but complementary linesof research, which address theseissues.• Molecular mechanismsof motor neuron degeneration arestudied in the spontaneous mutantwobbler mouse and transgenicmouse that overexpresses amutant human Cu,Zn-superoxidedismutase (mH-SOD1) gene.Experimental therapies aimed atblocking these mechanisms aretested for rescuing their phenotypes.• Neuroimaging techniquesof magnetic resonance imaging (MRI) andproton magnetic resonance spectroscopy ( 1 H-MRS) are used to identify motor pathwayabnormalities in the brains of patients with ALSand the mouse models; postmortem studies revealthe histopathologic correlates.The wobbler mouse and mH-SOD1transgenic mouse provide excellent substrates toinvestigate the pathogenic mechanisms of motorneuron degeneration in ALS, including oxidativestress, excitotoxicity, mitochondrial dysfunction,and aggregation of ubiquitinated proteins. Workin the laboratory focuses on examining thesignificance of ubiquitinated aggregates,intermediate filament accumulation, andmitochondrial dysfunction in wobbler motorneurons. Based on these findings, we testtherapies that prevent these abnormalities andpotentially rescue the phenotype, includingpharmacologic treatments and cross-breedingwobbler mice with mice transgenic for specificgenes.We are examining whether abnormalities inthe ATP-dependent ubiquitin-proteasome systemof wobbler cervical spinal cord motor neuronsThe Department of NeurosciencesImaging, Molecular Mechanism StudiesAim to Identify Causes of ALScause inadequate proteolysis and intraneuronalaccumulation of proteins. This condition wouldbe detrimental to cellular functioning and wouldlikely contribute to neurodegeneration. Ourstudies compare the mH-SOD1 mouse and humanALS spinal cord tissue to determine if relatedabnormalities occur (e.g., in mitochondria) and ifa ubiquitinated protein can be characterized inALS tissue. We are alsocharacterizing mitochondrialabnormalities in the brain andcervical spinal cord ofwobbler mice, which probablyresult in deficient ATPproduction.In vivo MR imaging ofbrain and spinal cord inpatients with ALS revealsevidence of motor neuronand motor pathway (corticospinaltract) degeneration.We have demonstrated thiswith 1 H-MRS and moretraditional MRI. There isneurochemical evidence from1H-MRS of glutamateglutamineexcess in brainstemregions, which correlates withbulbar (speech and swallowing)dysfunction, supportingErik P. Pioro, M.D., Ph.D.an excitotoxic pathogenicmechanism. Similar 1 H-MRSchanges in the wobbler mouse brain in vivo,confirmed by immunohistochemistry, revealfurther similarities with ALS. Correlative MRIand histopathologic studies of brain from ALSpatients and mouse models are providingsignificant insights into the evolution andmechanisms of motor neuron degeneration.THE PIOROLABORATORYINVESTIGATORSVladymyr Kostenko, M.D.Jialin Zhang, M.D.COLLABORATORSMichael Coleman, Ph.D. 1Douglas A. Gray, Ph.D. 2Andrei Gudkov, Ph.D. 3Michael T. Kinter, Ph.D. 4Michael Phillips, M.D. 5Dennis J. Stuehr, Ph.D. 6Scott M. Wilson, Ph.D. 71Cologne, Germany2Ottawa Health Research Inst.,Ottawa, Ont., Canada3Dept. of Molecular Biology,CCF4Dept. of Cell Biology, CCF5Dept. of Neuroradiology, CCF6Dept. of Immunology, CCF7Dept. of Neurobiology, Univ.of Alabama at BirminghamPioro, E.P., Wang, Y., Moore, J.K., Ng, T.C., Trapp, B.D., Klinkosz, B., and H. Mitsumoto(1998) Neuronal pathology in the wobbler mouse brain revealed by in vivo protonmagnetic resonance spectroscopy and immunocytochemistry. NeuroReport9:3041-3046.Pioro, E.P., Majors, A.W., Mitsumoto, H., Nelson, D.R., and T.C. Ng (1999) 1 H-MRSevidence of neurodegeneration and excess glutamate+glutamine in ALS medulla. Neurology53:71-79.Tsuzaka, K., Ishiyama, T., Pioro, E.P., and H. Mitsumoto (2001) Role of brain-derivedneurotrophic factor in wobbler mouse motor neuron disease. Muscle Nerve 24:474-480.Mitsumoto, H., Klinkosz, B., Pioro, E.P., Tsuzaka, K., Ishiyama, T., O’Leary, R.M.,and D. Pennica (2001) Effects of cardiotrophin-1 (CT-1) in a mouse motor neuron disease.Muscle Nerve 24:769-777.Dal Bello-Haas, V., Andrews-Hinders, D., Richer, C.B., Blakely-Adams, C., Hanson,J., Hammel, J., Kelly, D., Kloos, A., Pioro, E.P., Powazki, R.D., Wheeler, T., and H.Mitsumoto (2001) Development, analysis, refinement, and utility of an interdisciplinaryamyotrophic lateral sclerosis database. Amyotroph. Lateral Scler. Other MotorNeuron Disord. 2:39-46.145

THE RANSOHOFFLABORATORYThe Department of NeurosciencesASSISTANT STAFFJavier Provencio, M.D.PROJECT STAFFSandhya Rani, Ph.D.RESEARCH ASSOCIATEDeRen Huang, M.D., Ph.D.POSTDOCTORAL FELLOWSMelissa Callahan, Ph.D.Astrid Cardona, Ph.D.Pia Kivisakk, M.D., Ph.D.Don Mahad, M.D.Richard M. Ransohoff, M.D.TECHNICAL ASSOCIATESTao He, M.S.Zaachary LeibsonMeggan SasseBarbara Tucky, B.S.Tao Wei, M.D.COLLABORATORSElizabeth Fisher, Ph.D. 1Andrzej R. Glabinski, Ph.D. 2Hans Lassmann, M.D. 3Claudia F. Lucchinetti, M.D. 4Robert H. Miller, Ph.D. 5Richard A. Rudick, M.D. 6George R. Stark, Ph.D. 7Bruce D. Trapp, Ph.D. 71Dept. of Biomed. Engineering,CCF2Medical Univ. of Lodz, Poland3Univ. of Vienna, Austria4Mayo Clinic, Rochester, MN5Case Western ReserveUniv.Sch. of Med., Cleveland, OH6Div. of Clin. Res.; Mellen Ctr.for Multiple Sclerosis, CCF7Dept. of Molecular Biology,CCF8Dept. of Neurosciences, CCFChemokines, Chemokine Receptors andPhysiology of the Nervous SystemChemokines are small peptides that governleukocyte trafficking and activation. Thereis a substantial and growing literatureconcerning their biological function in development,inflammation and degeneration of thenervous system.The core hypothesis of our research is thatchemokines and their receptors are significantlyinvolved in leukocyte invasion, differentiation,activation, tissue destruction and repair in theCNS. Furthermore, resident neural cells respondto locally produced chemokines both duringdevelopment and disease.To address this hypothesis and identifymolecular targets for therapy, we examinechemokine production and function. Thesestudies comprise tissue culture systems, diseasemodels and material from patients with neurologicaldisease. We make extensive use of transgenicand knockout mice to clarify how chemokinesexert remarkably specific effects in vivo, in the faceof apparent functional redundancy in vitro.Tsai, H.H., Frost, E., To, V., Robinson, S., Ffrench-Constant, C., Geertman, R., Ransohoff, R.M., and R.H. Miller(2002) The chemokine receptor CXCR2 controls positioning of oligodendrocyte precursors in developing spinal cordby arresting their migration. Cell 110:373-383.Kieseier, B.C., Tani, M., Mahad, D., Oka, N., Ho, T., Woodroofe, N., Griffin, J.W., Toyka, K.V., Ransohoff, R.M.,and H.P. Hartung (2002) Chemokines and chemokine receptors in inflammatory demyelinating neuropathies: a centralrole for IP-10. Brain 125:823-834.Ransohoff, R.M., Wei, T., Pavelko, K.D., Lee, J.C., Murray, P.D., and M. Rodriguez (2002) Chemokine expressionin the central nervous system of mice with a viral disease resembling multiple sclerosis: roles of CD4+ and CD8+ Tcells and viral persistence. J. Virol. 76:2217-2224.Rani, M.R., Hibbert, L., Sizemore, N., Stark, G.R., and R.M. Ransohoff (2002) Requirement of phosphoinositide 3-kinase and Akt for interferon-beta -mediated induction of beta -R1 (SCYB11) gene. J. Biol. Chem. 277:38456-38461.Trebst, C., Staugaitis, S.M., Kivisakk, P., Mahad, D., Cathcart, M.K., Tucky, B., Wei, T., Rani, M.R., Horuk, R., Aldape,K.D., Pardo, C.A., Lucchinetti, C.F., Lassmann, H., and R.M. Ransohoff (2003) CC chemokine receptor 8 inthe central nervous system is associated with phagocytic macrophages. Am. J. Pathol. 162:427-438.Huang, D., Tani, M., Wang, J., Han, Y., He, T.T., Weaver, J., Charo, I.F., Tuohy, V.K., Rollins, B.J., and R.M. Ransohoff(2002) Pertussis toxin induced reversible encephalopathy dependent on monocyte chemoattractant protein-1over-expression in mice. J. Neurosci. 22:10633-10642.Kivisäkk, P., Mahad, D.J., Callahan, M.K., Trebst, C., Tucky, B., Wei, T., Wu, L., Baekkevold, E.S., Lassmann, H.,Staugaitis, S.M., Campbell, J.J., and R.M. Ransohoff (2003) Human cerebrospinal fluid central memory CD4+ T-cells:Evidence for trafficking through choroid plexus and meninges via P-selectin. Proc. Natl. Acad. Sci. USA. 2003 June26 [Epub ahead of print].Ransohoff RM, Kivisäkk P, and G. Kidd (2003) Three (or more) ways for leukocytes into the CNS. Nat. Rev. Immunol.3:569-581.146

Our primary focus is on four main projectsrelating to multiple sclerosis:MRI Monitoring TechniquesIn collaboration with Dr. Elizabeth Fisher, wecontinue to develop MRI parameters as a moremeaningful measure of MS disease activity and severity.We focus on the use of a normalized measure of wholebrain atrophy–the brain parenchymal fraction. We areconducting longitudinal studies in patients with firstattack, relapsing-remitting MS, and both primary andsecondary progressive MS.Longitudinal Autoreactivity to Central NervousSystem AntigensThe relationship between immune cytokineresponses towards brain proteins and clinical measuresof disease progression in MS is poorly understood. In alongitudinal study mapping cytokine responses to myelinpeptides in MS patients/controls, we find that cytokineexpression patterns correlated with clinical findings,demographic factors and quantitative MRI. Our resultsshow that following MS patients over time by analysisof their cytokine patterns suggests that these importantimmune chemicals can be linked to MS patients'disability. Immune responses to specific regions ofmyelin proteins were highly dynamic over time andshowed bursts of activity coordinated with gadoliniumenhancedMRI lesions. These findings suggest that ourmethods are useful for tracking immune events and mayprovide beneficial markers for clinical trials in MS.Immunomodulatory Effects of MS TreatmentFew therapies are available for patients with themost rapidly debilitating form of multiplesclerosis, primary progressive MS (PPMS).Mitoxantrone is a newly approved therapy forMS, however, its mechanisms of action arepoorly understood. We measure macrophagesecretedcytokines that upregulate/downregulateT-cell functions as well asThe Department of NeurosciencesClinical Approach Tests Immune Monitoring,Advanced Imaging to Understand andTreat Multiple Sclerosiseffects of mitoxantrone treatment on solublelevels of inflammatory mediators in the serum ofPPMS patients. Our recent studies focus on theeffect of mitoxantrone treatment on thelymphocyte surface expression of chemokinereceptors, molecules implicated in diseasepathogenesis in MS. Understandingmitoxantrone's mechanisms of action will help toimprove therapy for PPMS.Oxidative injury during CNS inflammationmight be linked to the rate of MS progression and brainatrophy. Thus, we are collaborating with Dr. StanHazen to examine products of oxidative tissue injury inMS. We quantify molecular markers specific foroxidative damage (e.g. nitrotyrosine) by HPLC with onlineelectrospray ionization tandem mass spectrometryusing methods developed in Dr. Hazen's lab. If our datashow consistently elevated CSF levels of nitrotyrosine,then it would suggest that the CNS in MS subjects is aunique site for oxidative damage. Inhibiting pathwayscontributing to nitrative stress could provide noveltherapeutic strategies for treatment of CNS oxidativeinjury in MS.Gender Differences in Immune Response in MSWe examine sex differences in multipleimmunological parameters (e.g., inflammatory andregulatory cytokines) to understand why more womenget MS than men. Our results show that IFNγ secretionto myelin peptides and proteins is elevated in females.We have examined whether exogenous sex hormonesalter the expression of cell-surface molecules thatpromote cellular inter-actions, known as co-stimulatorymolecules. We observe that expression of some costimulatorymolecules respond to in vitro exposure tosex hor-mones, and specifically to hormones that areeleva-ted in pregnancy. Finally, we are measuringexpres-sion of chemokine receptors on lymphocytes tolearn if these important chemical messengers play a rolein male/female differences in MS. Overall, learningmore about the gender differences in immune responseswill help improve MS therapy.THE RUDICKLABORATORYSTAFF SCIENTISTClara Pelfrey, Ph.D.ADVANCED POSTDOCTORAL FELLOWIoana Moldovan, M.D., Ph.D.SENIOR RESEARCH TECHNOLOGISTAnne Cotleur, M.S.TECHNOLOGISTNatacha Zamor, B..A.COLLABORATORSJeff Cohen, M.D. 2Elizabeth Fisher, Ph.D. 1Robert Fox, M.D. 2Jar-Chi Lee, M.S. 5Clara Pelfrey, Ph.D. 3Richard M. Ransohoff, M.D. 3Jack Simon, M.D. Ph.D. 4Lael Stone, M.D. 2Bruce D. Trapp, Ph.D. 3Stanley Hazen, Ph.D. 61Dept. of Biomed. Eng., CCF2Dept. of Neurology, MellenCenter, CCF3Dept. of Neurosciences, CCF4Dept. of Radiology-MRI, Univ. ofColo. Health Sci. Ctr., Denver5Dept. OF Biostat./Epidem., CCF6Dept. of Cell Biology, CCFRudick, R.A., Fisher, E., Lee, J.C., Duda, J.T., and J. Simon (2000) Brain atrophy in relapsing multiple sclerosis:relationship to relapses, EDSS, and treatment with interferon β-1a. Mult. Scler. 6:365-372.Richard A. Rudick, M.D.Pelfrey, C.A., Cotleur, A.C., Lee, J.-C., and R.A. Rudick (2002) Sex differences in cytokine responses to myelinpeptides in multiple sclerosis. J. Neuroimmunol. 130:211-223.Kivisakk, P., Trebst, C., Liu, Z., Tucky, B.H., Sorensen, T.L., Rudick, R.A., Mack, M., and R.M. Ransohoff (2002)T-cells in the cerebrospinal fluid express a similar repertoire of inflammatory chemokine receptors in the absenceor presence of CNS inflammation: implications for CNS trafficking. Clin. Exp. Immunol. 129:510-518.Chang, A., Tourtellotte, W.W., Rudick, R., and B.D. Trapp (2002) Premyelinating oligodendrocytes in chronic lesionsof multiple sclerosis. N. Engl. J. Med. 346:165-173.Rudick, R.A., Cutter, G., and S. Reingold (2002) The multiple sclerosis functional composite: a new clinical outcomemeasure for multiple sclerosis trials. Mult. Scler. 8:359-365.Fisher, E., Rudick, R.A., Simon, J.H., et al. (2002) Eight-year follow-up study of brain atrophy in patients withMS. Neurology 59:1412-1420.147

THE STAUGAITISLABORATORYCOLLABORATORSGene Barnett, M.D. 1Richard Ransohoff, M.D. 2Olga Tchernova, Ph.D. 1Bruce D. Trapp, Ph.D. 2Raymond Tubbs, D.O. 31Brain Tumor Institute, CCF2Dept. of Neurolosciences, CCF3Dept. of Clinical Pathology,CCFMy research focuses on the molecularcharacterization of human neurologicaldisease. This approach includescharacterization of specific candidate targetmolecules identified through basic research onanimal models, screening of molecules identifiedthrough large-scale analysis gene expression, anddevelopment of clinical laboratory tests.Through my joint appointments in the Departmentsof Neurosciences and Anatomic Pathology,I facilitate the close lines of communicationsThe Department of NeurosciencesHuman Neurological SpecimenResearch Leads to Clinical TestsUsed in Treatment Planningamong clinical, laboratory, and basic sciencedepartments that are essential to achieve correctdiagnosis and optimal utilization of excess tissuespecimens for research. My specific activitiesinclude genotypic and phenotypic analysis ofgliomas for use as clinical tests in treatmentplanning. In addition, I work closely with theMultiple Sclerosis research group in the Departmentof Neurosciences to facilitate acquisitionand analysis of human surgical and postmortemspecimens.Susan M. Staugaitis, M.D., Ph.D.Pedraza, L., Fidler, L, Staugaitis, S.M., and D.R. Colman (1997) The active transport of myelin basic proteininto the nucleus suggests a regulatory role in myelination. Neuron 18:579-589.Shoshan, Y., Nishiyama, A., Mork, S., Barnett, G.H., Cowell, J.K., Trapp, B.D., and S.M. Staugaitis (1999)Expression of oligodendrocyte progenitor cell antigens by gliomas: Implications for the histogenesis ofbrain tumors. Proc. Natl. Acad. Sci. USA 96:10361-10366.Staugaitis, S.M., Zerlin, M., Hawkes, R., Levine, J.M., and J.E. Goldman (2001) Aldolase C/zebrin II expressionin the neonatal rat forebrain reveals cellular heterogeneity within the subventricular zone and earlyastrocyte differentiation. J. Neurosci. 21:6195-6205.Trebst, C., Staugaitis, S.M., Kivisakk, P., Mahad, D., Cathcart, M.K., Tucky, B., Wei, T., Rani, M.R., Horuk,R., Aldape, K.D., Pardo, C.A., Lucchinetti, C.F., Lassmann, H., and R.M. Ransohoff (2003) CCchemokine receptor 8 in the central nervous system is associated with phagocytic macrophages. Am. J.Pathol. 162:427-438.Chahlavi, A., Kanner, A., Peereboom, D., Staugaitis, S.M., Elson, P., and G. Barnett (2003) Impact ofchromosome 1p status in response of oligodendroglioma to temozolomide: preliminary results.Neuro-Oncology 61:267-73.148

Experimental Therapeuticsin Parkinson’s Disease andRelated DisordersOur research focus is on neuroprotectionand recovery of function in the nervoussystem. Several neurological disordersare characterized by degeneration of neurons ortheir processes, resulting in loss of function anddisability. Such neurological disorders can bebroadly classified as primary neurodegenerativedisorders, e.g., Parkinson’s disease (PD), andsecondary neurodegenerative disorders, e.g.,neurodegeneration following central nervoussystem ischemia. Our laboratory is interested inthe pathophysiology of both primary andsecondary neurodegeneration and in experimentaltherapies that promote recovery of function orprovide neuroprotection.Cell Transplantation and Gene TherapyUsing specific targeted transplants of cellssecreting different neurotransmitters, directinfusion of neurotransmitters or neuroprotectivesubstances, we examine the behavioral, electrophysiological,neurochemical and moleculareffects in animal models of neuronal injury. Wehave shown that transplantation of retinalpigment epithelial (RPE) cells into the striatumin animal models of PD resulted in significantbehavioral recovery of function compared withanimals that received placebo transplants. Basedon these robust results, several PD patients havebeen transplanted with RPE cells into thestriatum in preliminary clinical trials, resulting insustained clinical benefits. In ongoing cell-culturestudies, we are evaluating the biochemical andmolecular properties of RPE cells to bettercharacterize these cells and perhaps improve theirtherapeutic efficacy. Our preliminary studiesindicate that RPE cells may secrete smallamounts of a dopamine-like substance and avariety of trophic substances that may prevent ordelay neurodegeneration. In parallel experiments,we are investigating the use of recombinantadenoassociated and lentiviral vectors totransduce glial derived neurotrophic factor(GDNF) expression in the brain and to examinethe neuroprotective effects of GDNF in PD.Intranigral TransplantationIt is probable that dopamine replacementin the striatum alone is not sufficient toameliorate all parkinsonian signs and symptoms.Replacement of dopamine or other neurotransmittersat sites outside the striatum in addition torestoration of dopamine inputs into the striatummay be a viable alternative to improve clinicaloutcome. One of the promising extrastriataltargets for dopamine replacement is thesubstantia nigra reticulata (SNr). Our electrophysiologicalstudies indicate that the primateSNr is involved in parkinsonian pathophysiology.The Department of NeurosciencesWe recently examined the effects of intranigralallogenic dopaminergic tissue transplantation inparkinsonian monkeys. The results from this pilotstudy suggest that the SN in primates may be auseful target for continuous dopamine replacement.We are also evaluating the role of SNr andthe subthalamic nucleus (STN) in the pathophysiologyof PD and the therapeutic efficacy of dualtransplants into the striatum and the SN toameliorate parkinsonism. These studies involvebehavioral training of animals, MRI- andmicroelectrode-guided mapping of the brain,single-cell neuronal recordings, cell transplantation,microdialysis and histological assessment ofthe brain after transplantation.Translational ResearchWe are translating several preclinicalresearch studies through active clinical trials inpatients with primary and secondaryneurodegeneration. For example, in patients withmultisystem atrophy (MSA), we are assessing theeffects of oral administration of terazosin, anagent which has been shown to improvesymptoms in a mouse model of MSA by our coinvestigatorDr. Dianne Perez. In PD patients, weare testing the effects of rasagiline, a newlycharacterized monoaminooxidase inhibitor, toameliorate disability. In patients with dystonia,we are investigating the effects of botulinumtoxin injections to ameliorate symptoms and toassess the risk of developing neutralizingantibodies. In patients with post-stroke spasticity,we are assessing the effects of intrathecalbaclofen to improve function. This research studyuses a novel pump technology that we previouslytested in laboratory-based animal studies.THE SUBRAMANIANLABORATORYRESEARCH ASSOCIATEKala Venkiteswaran, Ph.D.POSTDOCTORAL FELLOWJai Perumal, M.D.RESEARCH SPECIALISTSErin Gilbert, B.S.Patrick Redman, B.S.RESEARCH STUDY COORDINATORRuthie KolbCOLLABORATORSMandar Jog, M.D. 1Bala S. Manyam 2Dianne Perez, Ph.D. 3Thomas Wichmann, M.D. 41Univ. of Western Ontario,London, Ont., Canada2Texas A&M Univ., Temple, TX3Dept. of Molec. Cardiol., CCF4Emory Univ., Atlanta, GAThyagarajan Subramanian,M.D.Wichmann, T., Bergman, H., Starr, P.A., Subramanian, T., et al. (1999) Comparison of MPTPinducedchanges in spontaneous neuronal discharge in the internal pallidal segment and in thesubstantia nigra pars reticulata in primates. Exp. Brain Res. 125:397-409.Starr, P., Subramanian, T., Bakay, R.A., and T. Wichmann (2000) Electrophysiological localizationof the substantia nigra in the parkinsonian primate. J. Neurosurg. 93:704-710.Subramanian, T. (2001) Cell transplantation for the treatment of Parkinson’s disease: an update.Semin. Neurol. 21:103-115.Fowler, K.A., Huerkemp, M.J., Pulliam, J.K., and T. Subramanian (2001) Anesthetic protocol:Propofol use in Rhesus macaques (Macaca mulatta) during magnetic resonance imaging withstereotactic head frame application. Brain Res. Brain Res. Protocols 7:87-93.Subramanian, T., Marchionini, D., Potter, E.M., et al. (2002) Striatal xenotransplantation of humanretinal pigment epithelial cells attached to microcarriers in hemiparkinsonian rats amelioratesbehavioral deficits without provoking a host immune response.Cell Transplant.11:207-214.149

THE TRAPPLABORATORYINVESTIGATORSAnsi Chang, M.D.Ranjan Dutta, Ph.D.Susan FoellYasuhisa Fujii, Ph.D.Grahame Kidd, Ph.D.Jennifer McDonough, Ph.D.Micke MooneyKim Moran-Jones, Ph.D.Jackie Morris, Ph.D.Therese SvarovskySam TaylorJerome Wujek, Ph.D.Vijay Yadav, Ph.D.Xinghua Yin, M.D.GRADUATE STUDENTSKaren BaracskayJohn PetersonCOLLABORATORSLars Bö, M.D., Ph.D. 1Wendy Macklin, Ph.D. 2Albee Messing, V.M.D., Ph.D. 3Sverre Mörk, M.D. 4Richard Ransohoff, M.D. 5John Roder, Ph.D. 6Richard Rudick, M.D. 7Elizabeth Fisher, Ph.D. 81Dept. of Neurology, Univ. ofBergen, Bergen, Norway2Dept. of Neurosciences, CCF3Dept. of PathobiologicalSciences, U. of WisconsinSch. of Veterinary Med., WI4Dept. of Neuropathology,Univ. of Bergen, Bergen,Norway5Depts. of Neurology andNeurosciences, CCF6Mt. Sinai Hospital, ResearchInst., Toronto, Ont., Canada7Div. of Clin. Research; Dept.of Neurosciences and MellenCtr., Dept. of Neurology, CCF8Dept. of Biomed. Eng., CCFTrapp, B.D., Peterson, J., Ransohoff, R.M., Rudick, R., Mörk, S., and L. Bö (1998) Axonaltransection in multiple sclerosis lesions. N. Engl. J. Med. 338:278-285.Yin, X., Kidd, G.J., Wrabetz, L., Feltri, M.L., Messing, A., and B.D. Trapp (2000) Schwann cell myelinationrequires timely and precise targeting of P 0protein. J. Cell Biol. 148:1009-1020.Peterson, J.W., Bö, L., Mörk, S., Chang, A., and B.D. Trapp (2001) Transected neurites, apoptoticneurons, and reduced inflammation in cortical multiple sclerosis lesions. Ann. Neurol. 50:389-400.Wujek, J.R., Bjartmar, C., Richer, E., Ransohoff, R.M., Yu, M., Tuohy, V.K., and B.D. Trapp(2002) Axon loss in the spinal cord determines permanent neurological disability in an animal modelof multiple sclerosis. J. Neuropath. Exp. Neurol. 61:23-32.Chang, A., Tourtellotte, W.W., Rudick, R., and B.D. Trapp (2002) Premyelinating oligodendrocytesin chronic lesions of multiple sclerosis. New Engl. J. Med. 346:165-173.Bjartmar, C., Wujek, J.R., and B.D. Trapp (2003) Axonal loss in the pathology of MS: Consequencesfor understanding the progressive phase of the disease. J. Neurol. Sci. 206:165-171.150Genetic Manipulation of Glial Developmentand Myelin Formation Helps ElucidatePathogenesis of Myelin DiseasesThe objective of our research effort is twofold.The first is to obtain a betterunderstanding of cellular and molecularevents involved in glial cell development andmyelin formation in the central and peripheralnervous systems. The second is to understandhow myelin, myelin-forming cells, and axons aredestroyed in autoimmune andinherited diseases of myelin.A common theme of theseresearch programs is thatnovel information about thenormal function of myelinformingcells and myelinaxoninteractions will help usunderstand the mechanismsinvolved in the pathogenesisof permanent neurologicaldisability in human disease.Cellular and MolecularBiology of MyelinationA major objective ofthese studies is to obtain abetter understanding ofcellular and molecular eventsthat regulate oligodendrocyteproduction and differentiationand CNS myelination.Current studies use a varietyof transgenic mice to studythe role of axons on the initial production andsurvival of oligodendrocyte progenitors andpremyelinating oligodendrocytes. We are alsoinvestigating whether remyelination in the adultCNS involves the production of new oligodendrocytesin a manner that resembles that indevelopment. We use zebrafish to study themolecular interactions, which regulate differentiationof myelin-forming cells and the maturationand survival of axons. Other studiesconcentrate on the developmental appearanceand location of glial and myelin proteins innormal development and set the stage for furtherelucidation of their function in gene knockout ortransgenic animals. We are currently investigatingphenotypes in mice that overexpress P 0protein inthe PNS or in the CNS and in mice thatoverexpress PDGFα.Pathogenesis of Neurological Deficits inMultiple SclerosisThe overall aim ofthese studies is todetermine the cause ofMS and to therapeuticallyprevent irreversibleneurological deficits in MSpatients. MS is aninflammatory demyelinatingdisease of the CNS.Historically, it has beenassumed that there wasrelative sparing of axonsfrom the pathologicalconsequences of inflammatorydemyelination.We have describedabundant axonal transectionin MS lesions. Moreimportantly, our dataindicate that axonaltransection begins atdisease onset and is lateraccompanied by degeneration of chronicallydemyelinated axons. We propose that irreversibleaxonal loss represents the underlying pathogenicprocess responsible for permanent neurologicaldeficits in MS patients and for the conversion ofrelapsing-remitting MS to secondary progressiveMS. The therapeutic correlate to this hypothesis isthe likelihood that anti-inflammatory andneuroprotective strategies should be applied earlyin the disease course and continued during periodsof apparent disease quiescence. Current studiesare investigating cellular and molecular mechanismsof myelin and oligodendrocyte destruction,the potential of oligodendrocyteprogenitor cells to repopulate MSlesions with oligodendrocytes,mechanisms responsible for axonalBruce D. Trapp, Ph.D.degeneration in MS, pathologyresulting from demyelination of thecerebral cortex, molecules andmolecular interactions that mediateentry of immune cells into MS lesions,and animal models of inflammatorydemyelination that include axonaltransection and neuronal pathology.Continuation of these studies shouldprovide direction for therapeuticintervention that may delay or stopprogression of MS.

CentersofResearch

CENTER FORANESTHESIOLOGYRESEARCHDIRECTORPaul A. Murray, Ph.D.The Carl E. Wasmuth ChairASSISTANT STAFFManjunatha Bhat, Ph.D.Derek S. Damron, Ph.D.Balakrishnan Gopakumaran,Ph.D.CLINICAL INVESTIGATORSCARDIOTHORACIC ANESTHESIANorman Starr, M.D., ChairmanC. Allen Bashour, M.D.Paula Bokesch, M.D.Randall Correia, M.D.Pierre DeVilliers, M.D.Andra Duncan, M.D.Steven Insler, D.O.Colleen Koch, M.D.Erik Kraenzler, M.D.Emad Mossad, M.D.Julie Tome, M.D.Lee Wallace, M.D.Jean-Pierre Yared, M.D.GENERAL ANESTHESIOLOGYArmin Schubert, M.D., ChairmanJames Dolak, M.D., Ph.D.D. John Doyle, M.D., Ph.D.Zeyd Ebrahim, M.D.Shahpour Esfandiari, M.D.Alexandru Gottlieb, M.D.Sam Irefin, M.D.Ali Jahan, M.D.Julie Niezgoda, M.D.Jerome O’Hara, Jr., M.D.Brian Parker, M.D.Marc Popovich, M.D.Karen Steckner, M.D.John Tetzlaff, M.D.Jonathan Waters, M.D.PAIN MANAGEMENTNagy Mekhail, M.D., Ph.D.,ChairmanAyman Basali, M.D.Teresa Dews, M.D.Salim Hayek, M.D., Ph.D.Leonardo Kapural, M.D., Ph.D.Osama Malak, M.D.Michael Stanton-Hicks, M.D.Center forAnesthesiology ResearchThe Center for Anesthesiology Researchcoordinates and administers all basic andclinical research activities within theDivision of Anesthesiology and Critical CareMedicine. The Center provides a structured,interactive environment to perform research thatis both clinically relevant and fundamentallyimportant. The clinical staff, fellows and residentshave the opportunity to participate in an ongoing,productive, thematic research enterprise. TheCenter fosters meaningful collaborations betweenbasic scientists and clinical anesthesiologists andintensivists and provides a forum to establishcollaborative relationships with other clinicaldivisions and Lerner Research Institute departments.The Center currently emphasizes two areasof basic research: cardiovascular regulation andpain. In addition, there are plans to develop basicresearch in critical care medicine, neuroanesthesia,and clinical engineering. Dr. Paul Murrayinvestigates neural, humoral and local mechanismsthat are involved in the fundamental regulationof the normal pulmonary circulation. This workserves as a foundation to investigate the effectsof anesthetic agents on mechanisms of pulmonaryvasoregulation, as well as to characterize chronicchanges in the pulmonary circulation that areassociated with lung transplantation. A major goalis to identify the cellular mechanisms that mediatechanges in the pulmonary circulation in responseto physiological and pharmacological activation,anesthetic agents, lung transplantation andhemodilution.Dr. Derek Damron’s laboratory usesisolated cardiac and vascular smooth muscle cellsto investigate the extent to which generalanesthetics alter signal transduction pathwaysassociated with the regulation of myocardialcontractility and vascular smooth muscle tone.These studies primarily involve the simultaneousmeasurement of myocyte contractility andintracellular Ca 2+ cycling or intracellular pH infreshly isolated ventricular myocytes and vascularsmooth muscle cells. Isolated sarcoplasmicreticulum vesicles are used to examine changes inCa 2+ uptake, and isolated myofibrils are used toexamine phosphorylation of contractile proteinsand activity of the myofibrillar actomyosinATPase. The major goal is to identify the cellularsites of action and cellular mechanisms by whichanesthetic agents alter the signal transductionpathways that regulate intracellular free Ca 2+availability and myofilament Ca 2+ sensitivity.Dr. Manju Bhat’s research has focused onthe structure-function relationship of theryanodine receptor, which plays an important rolein maintaining intracellular Ca 2+ homeostasis. Dr.Bhat has specifically investigated the role of theryanodine receptor and apoptotic regulatoryproteins in programmed cell death. He usessimilar molecular and cellular techniques toelucidate the signal transduction mechanismsinvolved in neuropathic pain, diabetic neuropathy,cancer and inflammatory pain. The long-termgoal is to identify potential therapeutic targets fornovel pain medications.In addition to basic research, the Centercoordinates a variety of clinical researchprotocols. These studies include the assessment ofinvestigational cardiovascular drugs, the safetyand efficacy of blood substitutes in theperioperative setting, studies with new anestheticsand muscle relaxants, pain therapy, outcomestudies and antibiotic therapy. These studies areperformed in the operating room, the cardiac andsurgical intensive care units, the outpatientcenter, and the pain management center. Clinicalresearch nurses support the Staff in the performanceof these clinical protocols. The Center alsohas an active training program at the pre- andpostdoctoral levels.http://www.lerner.ccf.org/research/anesthesiology.htmlhttp://www.clevelandclinic.org/anesthesia/research/152

Mechanisms of Pulmonary Vasoregulation:In Vivo and In Vitro StudiesThe primary focus of our research is toelucidate fundamental mechanisms ofpulmonary vascular regulation. Thesestudies provide a foundation for investigating theacute effects of inhalational and intravenousanesthetics on pulmonary vasoregulation, as wellas chronic changes in the pulmonary circulationthat occur following lung transplantation.The pulmonary circulation is unique inthat it receives 100% of the right ventricularoutput. Thus, it represents the total “afterload”against which the right ventricle must ejectblood. Any increase in pulmonary vascularresistance will increase the work of the rightventricle, which under normal circumstances hasrelatively little inotropic reserve. An acute orchronic increase in pulmonary vascular resistancecan be particularly deleterious in the setting ofright ventricular hypertrophy and heart failure.Compared with cellular mechanisms thatregulate the systemic circulation, those thatregulate pulmonary vasomotor tone are much lesswell understood. An increase in pulmonaryvasomotor tone can be achieved by increasingintracellular calcium concentration and/orincreasing myofilament calcium sensitivity. Wehave compelling evidence that endogenousvasoconstrictor stimuli (alpha adrenoreceptoractivation, angiotensin II, endothelin) that workthrough the same signal transduction pathwaycan have markedly different effects on these twocellular mechanisms that regulate pulmonaryvasomotor tone.We use a variety of in vitro techniques todelineate the cellular mechanisms that mediatethe effects of endogenous vasoconstrictorstimuli, as well as to elucidate the cellularmechanisms by which anesthetic agents and lungtransplantation modify these responses. Thesetechniques include:• Isolated sarcoplasmic reticulum (SR)vesicles to directly measure pulmonary arterysmooth muscle (PASM) SR calcium uptake,release, and content;• Purified myofibrils to directly measurePASM actomyosin ATPase activity;• Individual PASM cells to measureintracellular calcium concentration, membranepotential, ion currents, inositol phosphateproduction, and phosphorylation of contractileproteins;• Western blot analysis to measureprotein tyrosine phosphorylation;• Immunofluorescence techniques tomeasure translocation of protein kinase Cisoforms; and• PASM strips to simultaneously measurechanges in the calcium-tension relationship andintracellular pH.Our results indicate that anesthetic agentsand lung transplantation can alter multiplecellular mechanisms of pulmonary vasoconstriction.We have also developed a technique tomeasure the effects of vasoconstrictor stimuli,alone and in combination with anesthetic agents,on the intact pulmonary microcirculation. Themajor focus of these studies is to assess the roleof endogenous endothelial dilator mechanisms inmodulating microvascular responses to vasoconstrictorstimuli and to determine whetheranesthetics exert their effects on these endothelialmechanisms or directly on pulmonary vascularsmooth muscle. These studies represent the firstsystematic investigation of the effects ofanesthetics on the pulmonary microcirculation.Finally, we perform in vivo studies inchronically instrumented dogs to assess the effectsof anesthetics on the pulmonary vasoconstrictorresponses to alveolar hypoxia, circulatoryhypotension, and normovolemic hemodilution.We believe it is imperative to characterize theseresponses in the whole animal, with all mechanismsthat either mediate or modulate thepulmonary vascular responses to these stimuliintact. It is important to note that hypoxia,hypotension, and hemodilution are frequentoccurrences during anesthesia for cardiac surgery.We believe that our experimental approachis unique in that it integrates the in vitro, microcirculatory,and in vivo methodologies. The resultsfrom each type of study are complementary andare used to delineate the effects and mechanismsof action of anesthetics on the pulmonarycirculation. These studies yield fundamentalinformation about cellular mechanisms ofpulmonary vascular regulation, which providesinsight about mechanisms of pulmonary vasculardisease. Our results also elucidate cellularmechanisms of anesthetic action, which providesinsight about the optimal choice of anestheticagent to minimize increases in right ventricularafterload in patients with right ventriculardysfunction or failure.THE MURRAYLABORATORYASSISTANT STAFFBalakrishnan Gopakumaran, Ph.D.RESEARCH FELLOWSXueqin Ding, M.D., Ph.D.Hiroshi Ito, M.D.Woon-Seok Roh, M.D.Sachiko Shimizu, M.D.TECHNOLOGISTDawn Farrar, B.S.CLINICAL RESEARCH NURSESCarrie Beven, R.N.Michael Beven, B.AChristine Cribbs, R.N.Deborah Manke R.N.Stephanie Ziegman, R.N.Paul A. Murray, Ph.D.Ogawa, K., Tanaka, S., and P.A. Murray PA (2001) Propofol potentiates phenylephrine-inducedcontraction via cyclooxygenase inhibition in pulmonary artery smoothmuscle. Anesthesiology 94:833-839.Tanaka, S., Kanaya, N., Homma, Y., Damron, D.S., and P.A. Murray (2002) Propofolincreases pulmonary artery smooth muscle myofilament calcium sensitivityvia the protein kinase C signaling pathway. Anesthesiology 97:1557-1566.Damron, D.S,, Kanaya, N., Homma, Y., Kim, S.O., and P.A. Murray (2002) Roleof PKC, tyrosine kinase and rho-kinase in a-adrenoreceptor-mediated pulmonary arterysmooth muscle contraction. Am. J. Physiol. 283:L1051-L1064.Sato, K., Seki, S., and P.A. Murray (2002) Effect of halothane and enflurane onsympathetic b adrenoreceptor-mediated pulmonary vasodilation in chronically-instrumenteddogs. Anesthesiology 97:478-487.Sohn, J.-T., and P.A. Murray (2003) Inhibitory effects of etomidate and ketamineon ATP-sensitive K + channel relaxation in canine pulmonary artery. Anesthesiology98:104-113.153

Role of Receptors and Ion Channels inNeuropathic PainManju Bhat, Ph.D.THE BHATLABORATORYRESEARCH FELLOWSSeok Kon Kim, M.D.Hongyu Zhang, M.D.RESEARCH TECHNOLOGISTKristen Yankura, B.S.COLLABORATORSLiliana Berti-Mattera, Ph.D. 1Derek S. Damron, Ph.D. 2Salim M. Hayek, M.D., Ph.D. 3Anoopa Kumar, Ph.D. 1Minh Lam, Ph.D. 1Ram Nagaraj, Ph.D. 1Anna-Liisa Nieminen, Ph.D. 1Andrea Romani, M.D., Ph.D. 1Basil D. Roufogalis, Ph.D. 4Hiroshi Takeshima, Ph.D. 51Case Western Reserve Univ.,Cleveland, OH2Ctr. for Anesthesiology Res.,CCF3Dept. of Pain Management,CCF4Tohoku Univ., Sendai, Japan5Univ. of Sydney, AustraliaAccording to the World Health Organization,90% of all illnesses are associatedwith pain, and patients with chronic painuse health services up to five times as frequentlyas the rest of the population. Treating symptomaticpain is challenging because of its subjectivenature, yet adverse side effects of existing painmedications demand further research to developnew protocols for treating different types of pain.Neuropathic conditions, including peripheralneuropathy, are the most commonly encounteredgroup of chronic complications associated withdiabetes. The actual prevalence of diabeticneuropathy is not known, but 60-70% of diabeticpatients develop neuropathy at some stage. It isestimated that by 2010, over 220 million peopleworldwide will suffer from these diseases. It isimportant to understand the underlying pathophysiologyto identify novel therapeutics forneuropathic and other related pain. Significantprogress has recently been made in understandingthe neurobiology of pain, with the identificationof new receptors, ion channels and signalingmolecules that may serve as potential therapeutictargets. However, the molecular mechanism(s)of the function and regulation of these cellulartargets are not clearly understood.Our research focus is to understand thefunction and regulation of cell surface receptorsinvolved in nociception, such as opioid andopioid- like receptors (m, d, k and nociceptin/orphanin FQ) and capsaicin receptors and toelucidate the pathophysiologic role of calcium(Ca 2+ ) as an intracellular second messenger in thetransduction and modulation of nociceptivesignals. Considerable evidence exists for theinvolvement of Ca 2+ in the transmission ofnociceptive signals within the peripheral andcentral nervous systems. For example, agents thatincrease the concentration of intracellular Ca 2+decrease the analgesic potency of opioid drugs,whereas calcium chelators and calcium channelantagonists potentiate opioid-induced antinociception.Furthermore, altered intracellularCa 2+ homeostasis is thought to be one of thereasons for decreased anti-nociceptive response toopioid receptor agonists in diabetic neuropathy.We are investigating sensory neuronal Ca 2+signaling mechanism(s) and the role of Ca 2+ in thepathophysiology of diabetic neuropathy. Insensory neurons from dorsal root ganglia (DRG),activation of Ca 2+ release from the endoplasmicreticulum via ryanodine receptors (RyR) triggersCa 2+ influx through the plasma membrane via amechanism called capacitative Ca 2+ entry (CCE).Current research efforts are aimed at characterizingthe molecular properties and regulation ofRyR and proteins involved in CCE pathway.Preliminary results indicate that this functionalinteraction between different Ca 2+ transportpathways is altered in diabetes. The role of Ca 2+and Ca 2+ channels in the function and regulationof nociceptive receptors is also being investigated.Molecular and cellular techniques are beingused to investigate the signal transductionmechanism(s) involved in such disease models asneuropathic pain, including diabetic neuropathy.The function and regulation of receptors and ionchannels involved in nociception are beingstudied using both freshly isolated DRG neuronsand immortalized sensory neurons in culture.Ion-selective fluorescent dyes and confocalmicroscopy are used to examine the changes inintracellular ionic fluxes. Electrophysiologicaltechniques (patch-clamp and planar lipid bilayerreconstitution) will be used to examine singleion-channelfunction and its regulation byreceptors and other modulators. Our long-termgoal is to pursue the molecular findings in animalmodels and eventually patients, with the hope ofidentifying potential therapeutic targets that mayhelp in developing “mechanism based” treatmentparadigms.Chipuk, J.E., Bhat, M., Hsing, A.Y., Ma, J., and D. Danielpour (2001) Bcl-xL blocks transforming growthfactor-beta 1-induced apoptosis by inhibiting cytochrome c release and not by directly antagonizingApaf-1-dependent caspase activation in prostate epithelial cells. J. Biol. Chem. 276:26614-26621.Bhat, M.B., and J. Ma (2002) The transmembrane segment of ryanodine receptor contains an intracellularmembrane retention signal for Ca 2+ release channel. J. Biol. Chem. 277:8597-8601.Paul-Pletzer, K., Yamamoto, T., Bhat, M.B., Ma, J., Ikemoto, N., Jimenez, L.S., Morimoto, H., Williams,P.G., and J. Parness (2002) Identification of a dantrolene-binding sequence on the skeletal muscle ryanodinereceptor. J. Biol. Chem. 277:34918-34923.Shin, D.W., Pan, Z., Bandyopadhyay, A., Bhat, M.B., Kim, D.H., and J. Ma (2002) Ca 2+ -dependent interactionbetween FKBP12 and calcineurin regulates activity of the Ca 2+ release channel in skeletal muscle.Biophys. J. 83:2539-49.Shin, D.W., Pan, Z., Kim, E.K., Lee, J.M., Bhat, M.B., Parness, J., Kim do H, and J. Ma (2003) A retrogradesignal from calsequestrin for the regulation of store-operated Ca 2+ entry in skeletal muscle. J.Biol. Chem. 278:3286-3292.154

Anesthesia and Excitation-Contraction Couplingin the Cardiovascular System: Signal Transductionand Cellular Mechanisms of ActionInduction of anesthesia is known to exert aprofound negative influence on cardiovasculardynamics, typically resulting in cardiacdepression and hypotension. In many instances,anesthesia-induced cardiovascular depression is alimiting factor for surgical maneuvers in patientswith limited or compromised cardiovascularreserve and could result in mortality.The mechanisms by which anestheticagents alter cardiovascular dynamics have notbeen clearly delineated, but appear to bemultifactorial, involving alterations in neural,humoral and local mechanisms of cardiovascularregulation. In addition, anesthetic agents mayalso alter cardiovascular dynamics via directactions on the heart and/or vasculature. Becauseof concomitant changes in preload, systemicresistance, baroreflex activity and central nervoussystem activity following induction of anesthesia,the direct actions of general anesthetics onintrinsic myocardial contractility or vascularreactivity are difficult to assess in vivo. Therefore,the laboratory uses a variety of in vitro approachesat the cellular level to examine theextent to which anesthetic agents alter signaltransduction pathways associated with theregulation and/or modulation of myocardialcontractility and vasomotor tone.Recent data from our laboratory hasdemonstrated that a variety of anesthetic agentshave direct actions on cardiac and vascularsmooth muscle consistent with cardiovasculardepression. The cellular mechanisms by whichthese anesthetic agents exert their direct effectson the heart and vasculature are not known andare the focus of investigations ongoing withinthe laboratory.Because intracellular free Ca 2+ concentrationand myofilament Ca 2+ sensitivity are keydeterminants of myocardial contractility andvasomotor tone, the overall working hypothesisof the laboratory is that anesthetic agents alterthe signal transduction pathways which regulatethe availability of intracellular free Ca 2+ and/ormyofilament Ca 2+ sensitivity in cardiac andvascular smooth muscle cells. Through the use ofintracellular fluorescent probes, the laboratorytests the hypothesis that general anesthetics alterCa 2+ signaling in cardiac and vascular smoothmuscle cells in response to inotropic and vasoconstrictorstimuli. Electrophysiological techniquesare used to test the hypothesis that generalanesthetics alter sarcolemmal K + and/or Ca 2+channels.We also use a variety of subcellularpreparations to directly assess mechanisms ofanesthetic action. Isolated cardiac and smoothmuscle myofibrils are used to test the hypothesisthat general anesthetics alter myofibrillar proteinphosphorylation and Ca 2+ -activated actomyosinATPase activity, leading to changes in myofilamentCa 2+ sensitivity. Isolated sarcoplasmicreticulum (SR) vesicles are used to test thehypothesis that general anesthetics directly alterCa 2+ uptake by the SR Ca 2+ pump or release ofCa 2+ from the SR via interactions with theryanodine receptor.The results from these studies will provideimportant fundamental information regarding thecellular mechanisms of anesthetic action incardiac and vascular smooth muscle cells. Thesedata will be useful for the future design anddevelopment of novel anesthetic agents that maybe of greater benefit to patients with limitedcardiovascular reserve.THE DAMRONLABORATORYPOSTDOCTORAL FELLOWSToshiya Shiga, M.D.Hiroyuki Tanaka, M.D.TECHNICAL ASSISTANTSDawn Ferrar, B.A.Chirag Patel, B.S.COLLABORATORSManju Bhat, Ph.D. 1Meredith Bond, Ph.D. 2Linda Graham, M.D. 3Christine Schomisch Moravec,Ph.D. 4Paul Murray, Ph.D. 1Charles Pilati, Ph.D. 5Yan Xu, Ph.D. 61Ctr. for Anesthesia Res., CCF2Dept. of Molecular Cardiology,CCF3Depts. of Vascular Surgery andBiomed. Eng., CCF4Dept. of CardiovascularMedicine, CCF5Northeastern Ohio UniversitiesColl. of Med., Rootstown, OH6Dept. of Cancer Biology, CCFKurokawa, H., Murray, P.A., and D.S. Damron (2001) Propofol attenuates β-adrenoreceptor-mediatedsignal transduction via a protein kinase C-dependent pathway in cardiomyocytes. Anesthesiology96:688-698.Derek S. Damron, Ph.D.Kang, S.K., Kim, D.K., Damron, D.S., Baek, K.J., and M.J. Im (2002) Modulation of intracellular Ca 2+via α 1B-adrenoreceptor signaling molecules, Gα h(transglutaminase II) and phospholipase C-d1. Biochem.Biophys. Res. Commun. 293:383-390.Damron, D.S., Kanaya, N., Homma, Y., Kim, S.-O., and P.A. (2002) Role of PKC, tyrosine kinases,and Rho kinase in α-adrenoreceptor mediated PASM contraction. Am. J. Physiol. 283:L10451-L1064.Kanaya, N., Murray, P.A., and D.S. Damron (2002) The differential effects of midazolam and diazepamon intracellular Ca 2+ transients and contraction in adult rat ventricular myocytes. Anesth. Analg.95:1637-1644.Tanaka, S., Kanaya, N., Homma, Y., Damron, D.S., and P.A. Murray (2002) Propofol increases pulmonaryartery smooth muscle myofilament calcium sensitivity: role of protein kinase C. Anesthesiology97:1557-1566.Chaudhuri, P., Colles, S.M., Damron, D.S., and L.M. Graham (2003) Lysophosphatidylcholine inhibitsendothelial cell migration by increasing intracellular calcium and activating calpain. Arterioscler. Thromb.Vasc. Biol. 23:218-223.155

CENTER FORCEREBROVASCULASRESEARCHCenter forCerebrovascular ResearchDIRECTORDamir Janigro, Ph.D.INVESTIGATORSBenedict Albensi, Ph.D. 1Gene Barnett, M.D. 1Nicholas Boulis, M.D. 1Lily Krizanac-Bengez, M.D., Ph.D. 1Luca Cucullo, Ph.D. 1Shailesh Desai, Ph.D. 1Miranda Kapural, M.D. 1Matteo Marroni, Ph.D. 1Marc Mayberg, M.D. 1Imad Najm, M.D. 1Shobu Namura, M.D., Ph.D. 1Fiona Parkinson, Ph.D. 2Peter Rasmussen, M.D. 1Mike Vogelbaum, M.D., Ph.D. 31Dept. of Neurological Surgery,CCF2Univ. of Manitoba, Winnipeg,MB, Canada3Dept. of Cancer Biology, CCFFELLOWSEmil Zeynalov, M.D.Tamer A. Mohammed Attia, M.D.TECHNICIANJoseph Waterman, B.S.COLLABORATORSJoan Abbott, Ph.D. 1Marco DeCurtis, M.D. 2Paul DiCorleto, Ph.D. 3Howard Fine, M.D. 4Gerry Grant, M.D., Ph.D. 5Claudia Martini, Ph.D. 6Jay Nelson, Ph.D. 7P.A. Schwartzkroin, Ph.D. 8Dana Stanimirovic, M.D., Ph.D. 91King’s College, CambridgeUniv., London, UK2Univ. of Milano, Italy3Dept. of Cell Biology, CCF4National Cancer Institute, NIH,Bethesda, MD5Univ. of Washington, Seattle6Univ. of Pisa, Italy7Oregon Health Sciences Univ.,Portland8Univ. of California/Davis, CA9National Research Council,Ottawa, Ont., Canada156The Center for Cerebrovascular Researchwas created in 1998 as part of the bridgeprogram between the Departments ofNeurosurgery and Neurology and the LernerResearch Institute. The main goal of this effortwas to promote cerebrovascular research, anoften neglected but clinically relevant field of theneurosciences. The Center provides state-of-theartfacilities to interactively perform a broadvariety of experiments, ranging from animalmodels of acute neurological disorders tomolecular and electrophysiological investigationof the mechanisms of human disease. The Centerenjoys a vast array of local and extramuralcollaborations, and members of the Center havebeen actively involved in efforts aimed atpromoting awareness of the importance ofcerebrovascular research to treat and preventneurological diseases. The pre-clinical (“translational”)relevance of the Center for CerebrovascularResearch is emphasized by the almost equaldistribution of M.D. and Ph.D. staff among itsinvestigators.Although the understanding of hownonneuronal mechanisms participate in theoverall complexity of neuronal function andneuropathogenesis constitutes the main focus ofthe Center, individual investigators are approachingthis task from different perspectives. Themajor ongoing efforts include the following.The Effect of Shear Stress on the Blood-Brain BarrierCurrent research investigation focusesprimarily on glia-endothelial interactions at theblood-brain barrier during ischemia, using adynamic in vitro model of blood-brain barrier(DIV-BBB). The goal is to investigate therespective roles of ischemic components - shearstress with/without hypoxia and/or hypoglycemiain the modulation of the inflammatory responseof the vessel wall, and subsequent alterations inthe blood-brain barrier. There is substantialevidence linking BBB disruption to inflammatoryprocesses involving cytokines, chemokines andleukocyte-endothelial cell interactions. Thefollowing aims have been studied:• NO-modulated cytokine production byastrocytes or WBC-dependent cytokinerelease by WBC, endothelium and astrocytesas critical precedents to subsequentinflammation.• Expression of cell adhesion molecules duringflow cessation/reperfusion correlated toleukocyte adhesion and subsequent BBBdisruption.• The nature of WBC-endothelial celladhesion examined in terms of cytokinestimulatedprostaglandin synthesis andrelease of reactive oxygen species.• The role of matrix metalloproteinases(MMP-2, -3 and -9) in inflammationmediatedBBB disruption.This research project should provide abetter understanding of the relationship betweenmicrovascular blood flow reductions and BBB,and may lead to effective therapies to preventBBB disruption after stroke.Synaptic Organization and PhysiologyRsearch LabFocus Area: The exploration of cellularand molecular mechanisms associated with normalvs. pathologic synaptic transmission and plasticityin the hippocampus and cortex. The hippocampalformation is a critical brain region for memoryconsolidation that is highly sensitive to acuteinjury. Of particular interest is how acute formsof brain injury, such as ischemia, disrupt processesbelieved to be associated with synaptic plasticityand cellular memory encoding and also, how acuteinjury may lead to long-term changes, such aswith Alzheimer’s dementia and/or epilepticseizures. Past and present investigations include:• Temporal evolution of hypoxia-ischemia andtrauma in the neonatal vs. adult hippocampus.• Mitochondrial dysfunction in stroke/trauma,and hippocampal synaptic plasticity.• Tumor necrosis factor and hippocampalsynaptic plasticity.• Natural products, NMDA/AMPA-receptormediated transmission, and hippocampalsynaptic plasticity.• Suppression of epileptiform activity byprolonged electrical stimulation in thehippocampus.• Gene-transfer based neuromodulation ofGABA-mediated inhibition in the hippocampusIn vitro electrophysiological techniques(extracellular, intracellular, multielectrode array,and patch clamp recordings) have been performedin human and animal cells, brain slices, andorganotypic cultures for studies involving braininjury and long-term potentiation/depression(LTP/LTD), a molecular model of learning/memory. Other research has employed functionaland conventional in vivo MRI techniques, whichallows an important comparison with the abovein vitro techniques. Furthermore, investigationshave attempted to identify therapeutic targets inCNS diseases and pathology that may havepotential for drug development.This research bridges clinical expertise withlaboratory investigation for studying basicpathophysiological mechanisms underlying stroke,trauma, epilepsy, and dementia.Dynamic in vitro Model of BBBFocus Area: Studies performed on viable invitro models are set to accelerate the design ofdrugs that selectively and aggressively can targetthe CNS. Several systems in vitro attempt toContinued on Page 161

Continued from Page 160reproduce the physical and biochemical behaviorof intact BBB, but most fail to reproduce thethree-dimensional nature of the in vivo barrierand do not allow concomitant exposure ofendothelial cells to abluminal (glia) and luminal(flow) influences. For this purpose, we havedeveloped a new dynamic in vitro blood-brainbarrier model (NDIV-BBB) designed to allow forextensive pharmacological, morphological andphysiological studies. This new dynamic modelof the BBB allows for longitudinal studies of theeffects of flow and co-culture in a controlledand fully recyclable environment that alsopermits visual inspection of the abluminalcompartment and manipulation of individualcapillaries. Further the system closely mimics thehemodynamic conditions present in pre- andpost-capillaries in vivo. Past and presentinvestigations include:• Development of new dynamic in vitro BBBmodels.• Molecular and cellular mechanisms ofblood-brain barrier induction and maintenance.• Molecular mechanism and site of action ofglucocorticoids.• Inflammatory processes at the BBB level,role of matrix metalloproteinase andproteinase inhibitors.• Isolation of human endothelial cells (EC)and human astrocytes.The Neurovascular Research LabThe Neurovascular Research Lab is heavilyinvolved in numerous research activities, such asbasic laboratory and applied animal research. Weoffer an outstanding environment for researchwith an infrastructure of research laboratoriesand animal facilities. Outstanding support isavailable for basic research and neurosiences withone of the best Cerebrovascular Centers in thecountry under the leadership of NeurologicalSurgery and Interventional Radiology departments.The laboratory specializes in research andanalysis of cerebral, abdominal and peripheralangiograms as well as MRI and CAT scan imagingon animals of all sizes. An excellent equipmentand variety of anesthetics allowing us to providea general anesthesia during a surgery/procedure aswell as monitoring and control of live functionsare available. The staff of the Laboratory andAnimal Husbandry Facility is LAC and GLPcertified. The Animal Critical Care Unit isavailable 24 hours a day, 7 days a week.Ongoing Current Projects:• Implantation of Encapsulated Islet Cells intoLiver/Gastric Wall (Diabetes).• Implantation of Encapsulated Tumor Cellsinto Brain.• Intrarterial Drug Delivery.• Blood Brain Barrier Damage in Stroke andIntracranial Hemorrhage.• Cerebral Aneurysms.• S-100 as a Serum Marker for distribution ofthe Blood Brain Barrier.• The Blood Brain Barrier and its Significancein Neuroimaging.• Markers of Blood Brain Barrier Formation.• Mechanisms of Osmotic Opening of theBlood Brain Barrier.Available Imaging Equipment:• MRI- SIEMENS (1.5 and 3T).• CT, CTA, CTP- SIEMENS 16.• Angiography- SIEMENS Angiostar (AxiomArtis BA and 3-D Reconstruction).157

THE JANIGROLABORATORYPROJECT SCIENTISTSBenedict Albensi, Ph.D.Lily Krizanac-Bengez, M.D., Ph.D.Thomas Masaryk, M.D.Shobu Namura, M.D., Ph.D.Fiona Parkinson, Ph.D.Peter Rasmussen, M.D.RESEARCH ASSOCIATELuca Cucullo, Ph.D.RESEARCH FELLOWSTamer A. Mohammed Attia, M.D.Gabrielle Dini, Ph.D.Andrew Kanner, M.D.Nicola Marchi, Ph.D.Gabriel Moddel, M.D., Ph.D.Emil Zeynalov, M.D.TechnologistsVincent Fazio, M.S.Kerri Hallene, B.S.Mohammed Hossain, M.S.Kelly Kight, B.S.Joseph Waterman, B.S.CLINICAL FELLOWEdwin Cunningham, M.D.COLLABORATORSN. Joan Abbott, Ph.D. 1Gene Barnett, M.D. 2Giorgio Battaglia, M.D. 3William Bingaman, M.D. 2Marco De Curtis, M.D. 3Anthony Furlan, M.D. 4Gerry Grant, M.D., Ph.D. 5Imad Najm, M.D., Ph.D. 4Erik Pioro, M.D., Ph.D. 4Peter Rasmussen, M.D. 2P.A. Schwartzkroin, Ph.D. 5Annamaria Vezzani, Ph.D. 3Michael Vogelbaum, M.D.,Ph.D. 21King’s College, London, UK2Dept. of Neurol. Surg., CCF3Univ. of Milan, Italy4Dept. of Neurology, CCF5Univ. of Washington, SeattleBlood-Brain Barrier in Health and Disease:Clinical ImplicationsWork in Dr. Janigro’s laboratory focusesprimarily on the investigation of glialendothelialinteractions in the ontogenesisand failure of the blood-brain barrier. To thisend, he developed a three-dimensional model ofthe blood-brain barrier in vitro that has become afundamental tool used to study how endothelialcells grown under physiological conditions reactto intra- and extraluminal signals. The research hasexpanded from the initial pharmacological andphysiological examination of the development ofthe blood-brain barrier to molecular analysis ofgene expression changes that characterizeendothelial-glial differentiationduring vasculogenesis. Thepossibility that some forms ofneurological disorders arise fromdevelopmental failure of thesemechanisms is also being investigatedusing transgenic animals withevident neuronal migrationdisorders. A novel non-invasiveperipheral blood test has beendeveloped to assess integrity of theblood-brain barrier. This test isbased on the extravasation of acerebrospinal fluid protein inperipheral blood when the bloodbrainbarrier becomes leaky. Thisdiagnostic procedure is now beingevaluated as a predictor of brain tumor recurrence,brain metastasis, and propensity to chronicneurological disease, as well as for intraoperativeassessment of neurologic deterioration.Peripheral markers of brain damageS100β is a protein that is constitutivelyexpressed by brain astrocytes and physiologicallydoes not appear in minimal concentration in thesystemic circulation. Recent evidence from thislaboratory demonstrates that S100β is a sensitivemarker of blood-brain barrier failure that doesDamir Janigro, Ph.D.not necessarily indicate brain damage.With this in mind, Dr. Janigro and hiscollaborators, Nicola Marchi, Ph.D., and AndrewKaner, M.D., prospectively studied serum S100βlevels in patients undergoing hypersomatic bloodbrainbarrier (BBB) disruption for intra-arterialchemotherapy for primary central nervous systemlymphoma. These studies indicated that S100?directly correlated with the degree of clinical andradiological signs of BBB disruption in patientswho were enrolled in the hyperosmotic study.Furthermore, in patients with neoplastic brainlesions, Gadolinium enhancement on MRIcorrelated with elevated S100βlevels versus non-enhancing scans.Primary brain tumors orCNS metastases presented withsignificantly elevated serum S100β.The same researchers havealso identified a protein markerthat could detect disease anddetermine when the body may bereceptive to medication. Thisprotein marker, dubbedtranstyretin monomer (TTR),could deliver potentially lifesavingmedications when thebody’s natural defense mechanismsare temporarily breached.The defense mechanisms to whichDr. Janigro refers protect the brain from foreignsubstances by creating barriers between the bloodand the brain, as well as between the blood andcerebrospinal fluid (CSF). This finding may havesignificant implications in the development ofsimple diagnostic tools for conditions affectingthe central nervous system. Additionally,discovery of this marker may help us manage ordiagnose disorders such as stroke, brain tumors,inflammations of the nervous system and othercerebrovascular disorders.Marroni, M., Agaral, M., Kight, K., Hallene, K.L., Hossain, M., Cucullo, L., Signorelli, K., Namura, S,and D. Janigro (2003) Relationship between expression of multiple drug resistance proteins and p53 tumorsuppressor gene proteins in human brain astrocytes. Neuroscience 2003 in press.Kanner, A.A., Marchi, N., Fazio, V., Mayberg, M.R., Koltz, M.T., Siomin, V., Stevens, G.H., Masaryk, T.,Ayumar, B., Vogelbaum, M.A., Barnett, G.H., and D. Janigro (2003) Serum S100beta: a noninvasivemarker of blood-brain barrier function and brain lesions. Cancer 97:2806-13.Cucullo, L., Marchi, N., Marroni, M., Fazio, V., Namura, S., and D. Janigro (2003) Blood-brain barrierdamage induces release of alpha 2-macroglobulin. Mol Cell Proteomics 2003.Marroni, M., Marchi, N., Cucullo, L., Abbott, N.J., Signorelli, K., and D. Janigro (2003) Vascular and parenchymalmechanisms in multiple drug resistance: a lesson from human epilepsy. Curr Drug Targets4:297-304.Marchi, N., Fazio, V., Cucullo, L., Kight, K., Masaryk, T., Barnett, G., Vogelbaum, M., Kinter, M., Rasmussen,P., Mayberg, M.R., and D. Janigro (2003) Serum transthyretin monomer as a possible markerof blood-to-CSF barrier disruption. J Neurosci 23:1949-55.158

Molecular, Genetic Function During CerebralArtery Response to Ischemia, Low Flow andRadiation Link Clinical and Basic ResearchDr. Marc Mayberg leads a group ofclinician-scientists who are investigatingbasic pathophysiologic mechanismsunderlying stroke. The clinician-scientists in thisgroup all maintain clinical practices in thetreatment of patients with cerebrovasculardiseases and bring clinical expertise into laboratoryinvestigation and vice versa. Dr. Mayberg’sresearch program concernsthe response of cerebralarteries to various injuries,including ischemia, lowflow and radiation.Ljiljana Bengez, M.D.,Ph.D., and EdwinCunningham, M.D., use anin vitro model of theblood-brain barrier toinvestigate the respectiveroles of hypoxia,hypoglycemia, flow andsteroids in the modulationof the inflammatoryresponse by elements ofthe vessel wall andsubsequent alterations inthe blood-brain barrier.Young-Soo Kim, M.D.,uses the same model toinvestigate the role ofmatrix metalloproteaseactivation and thebreakdown of bloodbrainbarrier in the same settings. Both of theseresearch efforts are essential in understanding thepathophysiology of important clinical conditionssuch as hemorrhagic stroke, reperfusion injury,and ischemic pre-conditioning in the brain. Dr.Mayberg is directing a research program toinvestigate the response of cerebral arteries toMarc Mayberg, M.D.radiation. Using in vitro and in vivo models, hisresearch program will characterize gene activation,blood-brain barrier dynamics, and cellturnover in the blood vessel wall after exposureto ionizing radiation. John Perl, M.D., isinvestigating the application of new endovasculartechnologies to treatment of cerebrovasculardisorders. His research, in association with thatof Younghua Dong, M.D.,involves the developmentof mechanical thrombolysisand magnetically guidedcatheters to treat strokeand aneurysms in previouslyinaccessible parts ofthe cerebral circulation.Peter Rasmussen, M.D., isinvestigating disruption ofthe blood-brain barrierafter intracerebralhemorrhage in an animalmodel and the potentialutility of rapid minimallyinvasive hematomaevacuation using mechanicalthrombolysis. MichaelDeGeorgia, M.D., andDerk Krieger, M..D., areinvestigating the cellularmechanisms of hypothermicprotection againstcerebral ischemia and newapplications of intraarterialcooling devices in animal models ofintracerebral hemorrhage and stroke. Observationsfrom these investigations will be directlyapplicable to concurrent Intensive Care Unitprotocols using hypothermia in Neuro-ICUpatients.THE MAYBERGLABORATORYSTROKEINTERVENTION/HYPOTHERMIARESEARCHCLINICIAN INVESTIGATORSMichael DeGeorgia, M.D. 1Derk Krieger, M.D. 1John Perl, M.D. 2Peter Rasmussen, M.D. 21Dept. of Neurological Surgery,CCF2Dept. of Neurology, CCFPROJECT SCIENTISTYounghua Dong, M.D.RESEARCH FELLOWSLjiljana Bengez, M.D., Ph.D.Edwin Cunningham, M.D.Young-Soo Kim, M.D.Alberts, M.J., Hademenos, G., Latchaw, R.E., Jagoda, A., Marler, J.R., Mayberg, M.R., Starke, R.D., etal. (2000) Recommendations for the establishment of primary stroke centers. Brain Attack Coalition.JAMA 283:3102-3109.Moon, C.T., Gajdusek, C., London, S., and M.R. Mayberg (2001) Expression of endothelial nitric oxidesynthase after exposure to perivascular blood. Neurosurgery 48:1328-1332.McAllister, M.S., Krizanac-Bengez, L., Macchia, F., Naftalin, R.J., Pedley, K.C., Mayberg, M.R., Marroni,M., Leaman, S., Stanness, K.A., and D. Janigro (2001) Mechanisms of glucose transport at the bloodbrainbarrier: an in vitro study. Brain Res. 904:20-30.Gajdusek, C., Onoda, K., London, S., Johnson, M., Morrison, R., and M. Mayberg (2001) Early molecularchanges in irradiated aortic endothelium. J. Cell Physiol. 188:8-23.Krieger, D.W., De Georgia, M.A., Abou-Chebl, A., Andrefsky, J.C., Sila, C.A., Katzan, I.L., Mayberg,M.R., and A.J. Furlan (2001) Cooling for acute ischemic brain damage (COOL AID): an open pilotstudy of induced hypothermia in acute ischemic stroke. Stroke 32:1847-1854.159

THE HOLLYFIELDLABORATORYPROJECT SCIENTISTSVera Bonilha, Ph.D.Quiyun Chen, Ph.D.Mary E. Rayborn, M.S.Preenie Senanayake, Ph.D.OPHTHALMOLOGY FELLOW/RESIDENTKo NakataLEAD RESEARCH TECHNOLOGISTKaren Shadrach, M.S.COLLABORATORSDean Bok, Ph.D. 1Anthony Calabro, Ph.D. 2John W. Crabb, Ph.D. 3Vincent Hascall, Ph.D. 2Motohiro Kamei, M.D. 4Ronald Midura, Ph.D. 2Daniel Organisciak, Ph.D. 5Neal Peachey, Ph.D. 3Elisabeth Rungger, Ph.D. 6Hiro Sakaguchi, M.D. 4Robert G. Salomon, Ph.D. 71Jules Stein Eye Inst., Univ.of California, Los Angeles2Dept of Biomedical Eng.,CCF3Dept of Ophthalmic Res.,Cole EyeInst., CCF4Osaka Univ. Med. Sch.,Osaka, Japan5Dept. of Biochem./Mol. Biol.,WrightState Univ., Dayton, OH6Univ.of Geneva, Dept. ofOphthalmology,Geneva, Switzerland7Dept. of Chemistry, CaseWestern ReserveUniv., Cleveland, OHCenter forOphthalmic ResearchIPM-Specific Glycoprotein andProteoglycan Genes as Candidates forRetinal Disease and Drusen Studies inAge-Related Macular DegenerationThe interphotoreceptor matrix (IPM) is thepart of the eye that ophthalmologists callthe subretinal space, the compartmentlocated between the outer retinal surface and theapical border of the retinal pigment epithelium(RPE). Our laboratory focuseson the IPM and its role inhealth and disease. This highlyspecialized extracellular matrixlinks the retina and the RPE.Structure-function activities,such as transport and communicationbetween these twotissues, photoreceptor protection,retinal attachment, andsynthesis and secretion ofvarious molecules, occur at thisinterface. The molecularinteractions responsible forthese activities are not known,but our laboratory recentlyidentified two unique proteinsin the IPM: SPACR(SialoProtein Associated withCones and Rods) was identifiedin the human and monkey(primates) IPM as a glycoproteinand in nonprimates (bovine, mouse and rat)as a proteoglycan; and a novel protein we namedSPACRCAN was identified as a chondroitinsulfate proteoglycan in both primates andnonprimates. The function of these highlyconserved IPM moleculesremains to be determined.We are defining therole of hyaluronan (HA), aglycosaminoglycan that isproposed as the basicorganizer of the IPM, byestablishing the distributionand regulation of thespecific hyaluronansynthases (HASs) thatcontribute HA to thiscompartment. With thisapproach, we are alsodefining the role of twoplasma membranereceptors for HA presenton cells bordering theIPM: RHAMM, localizedto the RPE’s apicalmembranes; and CD44,localized to theJoe G. Hollyfield, Ph.D.Continued on Page 161Marmorstein, A.D., Marmorstein, L.Y., Rayborn, M., Wang, X., Hollyfield, J.G., and K. Petrukhin (2000)Bestrophin, the product of Best vitelliform macular dystrophy gene (VMD2), localizes to the basolateralmembrane of the retinal pigment epithelium. Proc. Nat. Acad. Sci. USA 97:12758-12763.Sakaguchi, H., Miyagi, M., Shadrach, K.G., Rayborn, M.E., Crabb, J.W., and J.G. Hollyfield (2002)Clusterin is present in drusen in age-related macular degeneration. Exp. Eye Res. 74:547-549.Marmorstein, A.D., Marmorstein, L.Y., Sakaguchi, H., and J.G. Hollyfield (2002) Spectral profiling ofautofluorescence associated with lipofuscin, Bruch’s Membrane, and sub-RPE deposits in normal andAMD eyes. Invest. Ophthalmol. Vis. Sci. 43:2435-2441.Nishiyama, K., Sakaguchi, H., Hu, J.G., Bok, D., and J.G. Hollyfield (2002) Claudin localization in ciliaof the retinal pigment epithelium. Anat. Rec. 267:196-203.Crabb, J.W., Miyagi, M., Gu, X., Shadrach, K., West, K.A., Sakaguchi, H., Kamei, M., Hasan, A., Yan,L., Rayborn, M.E., Salomon, R.G., and J.G. Hollyfield (2002) Drusen proteome analysis: an approach tothe etiology of age-related macular degeneration. Proc. Natl. Acad. Sci. USA 99:14682-14687.Chen, Q., Lee, J.W., Nishiyama, K., Shadrach, K.G., Rayborn, M.E., and J.G. Hollyfield (2003) SPACR-CAN in the interphotoreceptor matrix of the mouse retina: molecular, developmental and promoter analysis.Exp. Eye Res. 76:1-14.160

Continued from Page 160microvillae of retinal Müller cells. We are testingthe hypothesis that HA in the IPM is the productof the combined synthetic activities of HAS-1,-2, and -3. We are also interested in establishingwhether HA recovery from the IPM fordegradation is mediated by CD44 and/orRHAMM; for this, we will analyze HA recoveryin CD44- and RHAMM-knockout mice.An ongoing focus is to define in molecularterms the linkage between the accumulation ofsoft drusen below the RPE in the macula and theincreased risk of developing age-related maculardegeneration (AMD). The presence of softdrusen in the macula is the hallmark risk factorfor developing AMD. Surprisingly little is knownof the origin or composition of drusen. To thisend, a novel method for drusen isolation has beendeveloped that allows the collection of microgramquantities of drusen from donor eye tissue.At the time of isolation, different drusen subtypescan be identified and separated for use in studiesthat will characterize their molecular composition.Because of the relationship of drusen andAMD, understanding the composition of differentdrusen subtypes will provide important informationon possible pathways that are causallyinvolved in drusen development. Novel proteinsor common modifications of proteins present indrusen should provide insight as to potential drugtargets of therapeutic agents to treat AMD.161

THE ANAND-APTELABORATORYPOSTDOCTORAL FELLOWSQuteba Ebrahem, M.D.Philip Klenotic, Ph.D.Jian Hua Qi, Ph.D.Anand-Apte B., and B. Zetter (2000) Biological principles of angiogenesis. In:D’Amore, P., and E. Voest, eds. Tumor Angiogenesis and Microcirculation. New York:Marcel Dekker, pp. 59-72.Anand-Apte, B. and P. Fox (2001) Melanoma: Translating biologicals into effectivetherapies: In: Borden, E., ed. Tumor Angiogenesis. New York: Humana Press.Karasarides M, Anand-Apte B, Wolfman A. (2001) A direct interaction between oncogenicHa-Ras and phosphatidylinositol 3-kinase is not required for Ha-Ras-dependenttransformation of epithelial cells. J. Biol. Chem. 276:39755-39764.Qi, J.H., Ebrahem, Q., Yeow, K., Edwards, D.R., Fox, P.L., and B. Anand-Apte (2002)Expression of Sorsby’s fundus dystrophy mutations in human retinal pigment epithelialcells reduces matrix metalloproteinase inhibition and may promote angiogenesis. J.Biol. Chem. 277:13394-13400.Qi, J.H., Ebrahem, Q., Moore, N., Murphy, G., Claesson-Welsh, L., Bond, M., Baker,A., and B. Anand-Apte (2003) A novel function for tissue inhibitor of metalloproteinases-3(TIMP3): inhibition of angiogenesis by blockage of VEGF binding to VEGF receptor-2.Nat. Med. 9:407-415.162Bela Anand-Apte, Ph.D.Choroidal Neovascularization Regulatedby Extracellular MatrixThe clinical significance of ocular angiogenesis is enormous, because in the West,retinal neovascularization resulting fromdiabetic retinopathy is the most common cause ofnew blindness in young patients, and choroidalneovascularization (CNV) resulting from agerelatedmacular degeneration (AMD) is the chiefcause of severe, irreversible loss of vision inelderly patients. Recently, much progresshas been made in angiogenesis research,fueled by the hypothesis that inhibition ofangiogenesis would be a useful strategy totreat cancers.Pathologic angiogenesis plays a rolein a number of other diseases. Retinalneovascularization involves the developmentof sprouts from retinal vessels,which usually penetrate the inner limitingmembrane and grow into the vitreous.Retinal neovascularization is observed inischemic retinopathies such as diabeticretinopathy, retinopathy of prematurity,central vein occlusion and branch retinalvein occlusion. CNV refers to theformation of new vessels in the subretinalor sub-retinal pigment epithelial (RPE)space, which arises from thechoriocapillaris. CNV is seen in oculardiseases such as AMD, presumed ocularhistoplasmosis, high myopia and angioidstreaks.Angiogenesis (formation of newblood vessels) is a multi-step processrequiring degradation of the basementmembrane of the parent vessel, endothelial cellmigration, capillary tube formation and endothelialcell proliferation. A precise spatial andtemporal regulation of extracellular proteolyticactivity appears to be important inneovascularization. The matrix metalloproteinases(MMPs), a major group of degradative enzymes,can break down most components of theextracellular matrix. Under physiologicalconditions, the matrix’s integrity is maintained byan orchestrated balance between MMPs and theirendogenous inhibitors, tissue inhibitors ofmetalloproteinases (TIMPs).TIMP-3 is a novel TIMP expressed by RPEcells and is unique in being a component of theextracellular matrix. It has been established thatSorsby’s fundus dystrophy (SFD), a dominantlyinherited form of blindness, is caused by Timp-3gene mutations. SFD is characterized bydevelopment of CNV, subretinal hemorrhages andchanges consistent with disciform degeneration.Although rare, SFD is of considerable interest, asit is the only genetic disease in which degenerationoccurs in most affected patients.We have shown that TIMP-3 is a potentinhibitor of angiogenesis. Since CNV is aprominent feature of SFD, we propose thatmutations in SFD may affect the angiogenesisinhibitory function of the wild-type protein andinduce the formation of new blood vessels. Weare investigating whether endothelial cells arenormally maintained in a quiescent state becauseof a regulated balance between angiogenesisinducers and inhibitors. Wild-type TIMP-3protein is secreted by RPE cells and deposited inBruch’s membrane, where it acts as an efficientangiogenesis inhibitor. During pathological retinalneovascularization, as seen in SFD, mutantTIMP-3 is either unable to inhibit angiogenesis byacting directly on the endothelial cells or is aninefficient inhibitor of MMPs, resulting in anincreased breakdown of matrix, release ofsequestrated angiogenic factors such as vascularendothelial growth factor, and increasedneovascularization. To this end, we havegenerated adenovirus-expressing wild-type TIMP-3 and mutated versions of the protein (TIMP-3156, and TIMP-3 181) to test in vivo in a mousemodel and in vitro in cell cultures. We are alsopurifying recombinant protein from Baculovirus totest in similar models.The goal of the laboratory is to gain anunderstanding of the mechanism(s) by whichalterations in matrix integrity may regulate retinalneovascularization. We have identified a novelADAM-TS (A Disintegrin-like AndMetalloprotease domain with ThromboSpondintype I modules)-like molecule that is expressed inthe retina and may play a role in angiogenesis. Weare attempting to identify other novel endogenousinducers and inhibitors of angiogenesis tounderstand the basic biology ofneovascularization and to design therapeuticapproaches to combat this process in diseasestates. Our ultimate goal is to prevent and/orreverse this process in an effort to control thedevastating consequences of CNV.

Oxidative Protein Modifications Situatedat Focal Point of Proteomic Studies onAge Related Macular DegenerationResearch in the Crabb laboratory seeks abetter understanding of the biochemistryof vision in two broad areas: proteomechanges associated with retinal degenerativediseases and the mammalian visual cycle.ProteomicsOur objective is to identify proteins andprotein chemical modifications associated withthe pathogenesis of age-related macular degeneration(AMD). AMD, the most common cause ofblindness in the U.S. forthose over age 55, is acomplex disease withboth genetic andenvironmental contributions.Significant riskfactors for AMDdevelopment areextracellular deposits ordebris termed drusen,which form beneath theretina on Bruch’smembrane. We arepursuing proteomestudies to test thehypothesis that proteinoxidative modificationscontribute to drusenformation and Bruch’smembrane thickening inAMD. Our proteomicsresearch involves avariety of methods.Microdissection methodsare used to isolate drusenand Bruch’s membrane.Proteins in tissue samples are fractionated by 1Dand 2D chromatographic and electrophoreticmethods. Mass spectrometry (MS) tools,including matrix-assisted laser desorption/ionization time-of-flight (MALDI-Tof) MS, triplequadrupole electrospray MS, capillary liquidchromatography quadrupole time-of-flight (QTof)MS and liquid chromatography MS/MS, are usedfor protein identification. Oxidative proteinmodifications are identified using bioinformatictools and a battery of antibodies. Immunocytochemistryprovides confirmation of drusen andBruch’s membrane localization. We anticipatethat this protein chemical approach will provideinsights into the etiology of AMD and newopportunities for finding cures for the disease.Visual Cycle StudiesThe rod visual cycle is the process bywhich all-trans-retinal, released from rhodopsinJohn W. Crabb, Ph.D.during bleaching, is enzymatically isomerized to11-cis-retinal in the retinal pigment epithelium(RPE), then shuttled back to the rod photoreceptorcells for rhodopsin regeneration. Themechanism for regeneration of bleached visualpigments in cone photoreceptors appears to bedifferent than in rods and may involve retinalMüller cells. Our research focuses on the cellularretinaldehyde-binding protein (CRALBP), whichendogenously carries 11-cis-retinol and/or 11-cisretinaland is expressed inboth the RPE and Müllercells. CRALBP functionsin the rod visual cycle asan 11-cis-retinol acceptorin the isomerization stepand as a substrate carrierfor 11-cis-retinoldehydrogenase. We areprobing CRALBPstructure-functionrelationships using acombination of proteinchemical, nuclearmagnetic resonance andmolecular biologicalapproaches. In additionto characterizing thestructure of CRALBPfunctional domains,CRALBP proteininteractions are sought.Using immunoaffinitypurification and MSmethods, we haveidentified an RPE visualcycle protein complex. Current efforts focusupon identifying additional CRALBP interactionsand the mechanisms controlling CRALBPexpression.THE CRABBLABORATORYPROJECT SCIENTISTBruce LevisonRESEARCH ASSOCIATESanjoy Bhattacharya, Ph.D.POSTDOCTORAL FELLOWSXiaorong GuJian SunLEAD RESEARCH TECHNOLOGISTKaren A. West, M.S.GRADUATE STUDENTSZhaoyan Jin 1Regganathan Kutralanathan 2Zhiping Wu 11Cleveland State Univ.,Cleveland, OH2Case Western Reserve Univ.,Cleveland, OHUNDERGRADUATE STUDENTJohn S. CrabbCase Western Reserve Univ.,Cleveland, OHCOLLABORATORSSuneel S.Apte, M.B.B.S., D.Phil. 1Charles L. Bevins, M.D., Ph.D. 2Joe G. Hollyfield, Ph.D. 3John C. Saari, Ph.D. 4Robert J. Salomon, Ph.D. 5Dennis J. Stuehr, Ph.D. 21Dept. of Biomed. Eng., CCF2Dept. of Immunology, CCF3Ctr. for Ophthalmic Res., CCF4Univ. of Washington, Seattle5Case Western Reserve Univ.,Cleveland, OHCrabb, J.W., O’Neil, J., Miyagi, M., West, K., and H.F. Hoff (2002) Hydroxynonenalinactivates cathepsin B by forming Michael adducts with active site residues.Protein Sci. 11:831-840.Miyagi. M., Sakaguchi, H., Darrow, R.M., Yan, L., West, K.A., Aulak, K.S., Stuehr,D.J., Hollyfield, J.G., Organisciak, D.T., and J.W. Crabb (2002) Evidence that lightmodulates protein nitration in rat retina. Mol. Cell. Proteomics 1:293-303.Sakaguchi, H., Miyagi, M., Darrow, R.M., Crabb, J.S., Hollyfield, J.G., Organisciak,D.T., and J.W. Crabb (2002) Intense light exposure changes the crystallin content inretina. Exp. Eye Res. 76:131-133.Crabb, J.W., Miyagi, M., Gu, X., Shadrach, K., West, K.A., Sakaguchi, H., Kamei,M., Hasan A., Yan, L., Rayborn, M.E., Salomon, R.G., and J.G. Hollyfield (2002)Drusen proteome analysis: an approach to the etiology of age-related macular degeneration.Proc. Natl. Acad. Sci. USA 99:14682-14687.Kennedy, B.N., Li, C., Ortego, J., Coca-Prados, M., Sarthy, V.P., and J.W. Crabb(2003) CRALBP transcriptional regulation in ciliary epithelial, retinal Müller and retinalpigment epithelial cells. Exp. Eye Res. 76:257-260.163

THE HAGSTROMLABORATORYSENIOR TECHNOLOGISTGayle T. Pauer, B.S.POSTDOCTORAL FELLOWTULP1 Under Scrutiny forRole in Early OnsetProgressive Retinal DegenerationRetinalQuansheng Xi, Ph.D.degeneration encompasses a myriadof genetically and phenotypically heterogeneous diseases. The majority of these blindingSTUDENTdisorders are inherited. The retina’s light-sensitiveAndrea Crabbphotoreceptor cells degenerate due to defects inmany different genes required for the normalphysiology of these cells. The broad long-term goalsof my laboratory are to identify novel genes causingretinal degeneration in patients and to define theunderlying pathogenic mechanisms responsible forphotoreceptor degeneration.In collaboration with ophthalmologists in ourdepartment, a program has been designed to recruitlarge numbers of patients with a variety of types ofretinal degeneration. DNA samples from hundredsof patients are deposited into a centralized database.My laboratory has implemented highthroughputmutation screeningtechniques, allowing us to efficientlyanalyze many genes for mutations inthe DNA of these patients. Our goalis to identify genetic alterations andcorrelate these results with theclinical phenotype of the patient.Recently, we identifiedmutations in a novel gene, TULP1, inpatients with an autosomal recessiveform of retinitis pigmentosa, a groupof progressive retinal degenerationsleading to blindness. TULP1 is amember of a conserved family offour proteins of unknown functionnamed tubby-like proteins (TULPs).We have begun to explore theStephanie A. Hagstrom, Ph.D.physiologic properties of TULP1 inthe retina by analyzing the tissuedistribution of the protein in normalHagstrom, S.A., North, M.A., Nishina, P.A., Berson, E.L., and T.P. Dryja (1998) Recessive mutations inthe gene encoding the tubby-like protein TULP1 in patients with retinitis pigmentosa. Nat. Genet. 18:174-176.Hagstrom, S.A., and T.P. Dryja (1999) Mitotic recombination map of 13cen-13q14 derived from an investigationof loss of heterozygosity in retinoblastomas. Proc. Natl. Acad. Sci. USA 96:2952-2957.Hagstrom, S.A., Duyao, M., North, M.A., and T. Li (1999) Retinal degeneration in tulp1-/- mice: accumulationof extracellular vesicles in the interphotoreceptor space. Invest. Ophthalmol. Vis. Sci. 40:2795-2802.Hagstrom, S.A., Neitz, M., and J. Neitz (2000) Cone pigment gene expression in individual photoreceptorsand the chromatic topography of the retina. J. Optical Soc. Am. A 17:527-537.Hagstrom, S.A., Adamian, M., Scimeca, M., Pawlyk, B.S., Yue, G., and T. Li (2001) A role for the tubby-likeprotein 1 in rhodopsin transport. Invest. Ophthalmol. Vis. Sci. 42:1955-1962.Xi, Q., Pauer, G.J.T., West, K.A., Crabb, J.W., and S.A. Hagstrom (2003) Retinal degenerations causedby mutations in TULP1. In Anderson, R.E., Lavail, M.M., and J. Hollyfield, eds. New Insights into RetinalDegenerative Deseases. Kluwer Academic/Plenum.mice and the photoreceptor disease phenotype intulp1-knockout mice. We generated antibodiesagainst Tulp1 and determined that it is aphotoreceptor-specific protein. It is predominantlylocalized in two specialized compartmentsof photoreceptors, named the inner segment andthe connecting cilium. In addition, we generatedtulp1-knockout mice and determined that theydevelop an early-onset, progressive photoreceptordegeneration that parallels that seen in patientswith TULP1 mutations. Based on the localizationof Tulp1 and the features of the retinalphenotype in knockout mice, we hypothesize thatTULP1 is involved in the transport of proteinssynthesized in the inner segment compartment toits final location in the outer segment compartmentof photoreceptor cells.The steps and proteins involved in thetransport of proteins to the outer segments ofphotoreceptors is an essential but not wellunderstoodaspect of photoreceptor cell biology.The outer segment is connected to the innersegment via a narrow compartment, the connectingcilium. Outer segment-bound proteins aresynthesized in the inner segment and must beefficiently transported to their target location.The molecular basis of this transport systemremains unclear. Our localization of Tulp1 andanalysis of knockout mice suggest that Tulp1 maybe involved in this transport process.We are currently testing this hypothesis viaseveral approaches. We are generating an in vitrosystem in which to study the subcellular localizationand properties of wild-type and mutantforms of Tulp1. We are also searching forinteracting proteins by using yeast two-hybrid andimmunoprecipitation analysis. We are continuingto characterize the physiologicproperties of Tulp1 in vivo bystudying the knockout mice.We anticipate that results fromthese experiments will provideinsights into the pathogenicmechanism causing retinitispigmentosa and help us assignfunction(s) to a novelphotoreceptor-specific protein.As we continue tosearch and catalogue novelgenes causing retinal degeneration,the ultimate goal of ourresearch is to provide thefoundation for future studiesaimed at evaluating therapeuticmodalities that might slow,stop, or reverse the course ofretinal degeneration.164

Functional Consequences of HereditaryRetinal DiseaseOur laboratory is interested in hereditarydisorders that affect the outer retinallayers. These layers contain the rod andcone photoreceptors and the main second-orderretinal neuron, the bipolar cell. By usingappropriate stimulus conditions, the activity ofeach of these cell types can be monitored usingthe electroretinogram(ERG). In the mammalianretina, this method isparticularly useful fordiscerning the cones andbipolar cells, which arefar less numerous thanare rod photoreceptors,yet are key to manyblinding disorders.An importantfocus of our research ison congenital stationarynight blindness (CSNB),a class of retinal disorderin which communicationbetween photoreceptorsand bipolar cells issomehow disrupted. Wehave identified twomouse models of CSNB.One model, which wenamed nob (no b-wave),involves a naturallyoccurring X-linked trait.We have recently identified the nob gene product(nyctalopin), confirming prior results that the nobmouse might provide a model for CSNB Type 1.A closely related form of CSNB involvesnull mutations in the α 1Fsubunit of the L-typecalcium channel. Mice lacking the β 2subunit ofthis calcium channel in the central nervoussystem closely model the functional abnormalitiesfound in CSNB Type 2. In addition to indicatingthat the α 1Fsubunit depends critically upon theβ 2subunit to form a functional channel, thismutant mouse provides a model in which todefine the pathophysiological mechanismsNeal S. Peachey, Ph.D.underlying this rare retinal disorder. Besidescharacterizing these models, we have establishedan ERG-based screen with which to identifyadditional mutant lines generated by large-scalemutagenesis and to use these animals to identifynovel candidate genes for human CSNB.A second avenue of research involves thepossibility that visualfunction can be restored to aretina blinded by photoreceptordegeneration.Although this situation isencountered in retinitispigmentosa and age-relatedmacular degeneration, thereare few treatment options tooffer affected patients. Incollaboration withOptobionics Corporation, weare carrying out studies toevaluate prototype implantdevices that are designed toelectrically stimulate theouter retina from thesubretinal space. Implantedanimals are evaluated postoperativelyusing a variety oftechniques to establishimplant durability andbiocompatibility. Theseresults are used to identifyimplant designs suitable forapplication to animal models of photoreceptordegeneration, where the potential of this retinalprosthetic may be better evaluated.A general focus of the laboratory continuesto be development and evaluation of assays withwhich to better understand the functional effectsof gene manipulation on the mouse retina. Wehave now established protocols for evaluatingfunction of every cell type in the neural retina,and for the retinal pigment epithelium. As aresult, we can now thoroughly examine theimpact of experimental manipulation in themouse retina.THE PEACHEYLABORATORYPROJECT SCIENTISTMarc Schiavone, Ph.D.RESEARCH ASSOCIATESSherry Ball, Ph.D.Jiang WuTECHNICAL ASSOCIATESMelissa BlumBrett HanzlicekBoth at VA Medical Ctr.,Cleveland, OHCOLLABORATORSKenneth Alexander, Ph.D. 1Alan Chow, M.D. 2Steven Fliesler, Ph.D. 3Ronald Gregg, Ph.D. 4Vance Lemmon, Ph.D. 5Maureen McCall, Ph.D. 4Machelle Pardue, Ph.D. 61UIC Eye Center, Univ. ofIllinois, Chicago, IL2Optobionics Corporation,Wheaton, IL3Eye Inst., St. Louis University,St. Louis, MO4Univ. of Louisville, Louisville,KY5Case Western Reserve Univ.,Cleveland, OH6Veterans Affairs Medical Ctr.and Emory Univ., Atlanta, GABall, S.L., Powers, P.A., Shin, H.-S., Morgans, C.W., Peachey, N.S., and R.G. Gregg (2002) Role of the β 2subunit of voltage dependent calciumchannels in the retinal outer plexiform layer. Invest. Ophthalmol. Vis. Sci. 43:1595-1603.McCall, M.A., Lukasiewicz, P.D., Gregg, R.G., and N.S. Peachey (2002) Elimination of the ρ1 subunit abolishes GABA Creceptor expressionand alters visual processing in the mouse retina. J. Neurosci. 22:4163-4174.Krishna, V.R., Alexander, K.R., and N.S. Peachey (2002) Temporal properties of the mouse cone electroretinogram. J. Neurophysiol. 87:42-48.Peachey, N.S., Stanton, J.B., and A.D. Marmorstein (2002) Noninvasive recording and response characteristics of the rat DC electroretinogram.Vis. Neurosci. 19:693-701.Gregg, R.G., Mukhopadhyay, S., Candille, S.I., Ball, S.L., Pardue, M.T., McCall, M.A., and N.S. Peachey (2003) Identification of the gene andthe mutation responsible for the mouse nob phenotype. Invest. Ophthalmol. Vis. Sci. 44:378-384.165

THE V. PEREZLABORATORYTECHNOLOGISTAndrew Hsia, B.S.COLLABORATORSRobert Fairchild, Ph.D. 1Peter Heeger, M.D. 1Bruce Ksander, Ph.D. 2Ann Marshak-Rothstein, Ph.D. 3Eric Pearlman, Ph.D. 4Luk Van Parijs, Ph.D. 51Dept. of Immunology, CCF2Schepens Eye Res. Inst.,Harvard Med. School,Boston, MA3Boston Univ. Sch. of Med.,Boston, MA4Dept. of World HealthMedicine, Case WesternReserve Univ., Cleveland, OH5Ctr. for Cancer Res., Mass.Inst. of Technol., Cambridge,MAVictor L. Perez, M.D., Ph.D.Immunological Regulation ofInflammatory Responses in the CorneaThe cornea is the structure of the eyeresponsible for allowing the entrance ofexternal images into the eye. Cornealclarity and transparency are the unique qualitiesof this tissue critical for visual function.Moreover, the cornea also serves as a mechanicalbarrier against infections and trauma, and isconsidered part of the bio-defense system of theeye. For these reasons, the immune-regulatorymechanisms used by this immune privilege tissue,are key in maintaining a balance between thesetwo important functions of the cornea. Failureof these mechanisms to control inflammation willresult in uncontrolled corneal inflammation,vascularization, fibrosis and scarring, that lead tosevere functional compromise and even blindness.Common and clinically relevant examples ofwhere the dysregulation of immuno-inflammatoryresponses of the cornea are critical for a goodvisual outcome include; microbial keratitis,herpetic keratitis, contact lensrelated ulcers, autoimmuneulcerative keratitis and cornealtransplant allograft rejection.One major interest in mylaboratory is the understandingof immune-regulatory mechanismsused by the eye during thedevelopment of an immuneresponse in the cornea. To studythis, we have developed an invivo model where animal corneascan be transfected with adenovirusvector containing any gene ofinterested and achieve proteinexpression in the corneal tissue.This technique has allowed us totest the role of differentimmunological molecules in theregulation of corneal inflammation.We have focused ourattention in Fas ligand (FasL), animportant molecule known toregulate immune responses inimmune privilege sites, whichalso can differentially regulate immune responsesin the cornea. Like other members of the TNFfamily, matrix metalloproteinase enzymes cancleave FasL from the cell surface to produce a 26-kD soluble protein. Therefore, FasL can be existsas a membrane-only (non-cleaved) or soluble(cleaved) form. We have used vectors containingthese different forms of FasL to demonstrate thatmembrane and soluble FasL have opposing effectson neutrophils-mediated inflammation in thecornea. Our studies show that the two forms ofFasL have opposite regulatory roles in the cornea:(i) membrane-only FasL initiates innate immunityand causes keratitis, and (ii) soluble FasLterminates innate immunity, protecting the corneafrom an uncontrolled inflammatory response thatwould jeopardize the function and integrity ofthe cornea. We believe that these two moleculeswill allow us to understand better the mechanismsof immune-regulation used by the corneaand could also represent a new modality oftreatment for inflammatory diseases of the eye.The other area of interest studied in mylaboratory the use of the eye to visualize in vivoimmune responses. The eye provides a uniquewindow into the body. Its translucent natureallows the visualization of events, such as cellgrowth, cell death, migration, and transformationas they occur in vivo, in a non-invasive manner.We are establishing new experimental systems tomonitor gene expression and cellular migration inresponse to inflammation and other injuries in theeye. Using a retrovirus-based technique togenerate mice that express GFP in the majorityof hematopoietic cells, we can detect GFPpositive cells infiltrate the cornea of live micewithin 60 minutes after the induction ofinflammation. Using in vivo fluorescent biomicroscopywith digital imaging system, real timeimages of cellular interaction in the cornea willbe capture for analysis. This technique will allowus to characterize in vivo the migration patternand interaction of inflammatory cells with othercells in the cornea.Lu, M., Perez, V.L., Ma, N., Miyamoto, K., Peng, H.B., Liao, J.K., and A.P. Adamis (1999) VEGF increasesretinal vascular ICAM-1 expression in vivo. Invest. Ophthalmol. Vis. Sci. 40:1808-1812.Perez, V.L., Biuckians, A.J., and J.W. Streilein (2000) In-vivo impaired T helper 1 cell development insubmandibular lymph nodes due to IL-12 deficiency following antigen injection into the anterior chamberof the eye. Ocul. Immunol. Inflamm. 8:9-24.Perez, V.L., and C.S. Foster (2001) Uveitis with neurological manifestations. Int. Ophthalmol. Clin.41:41-59.Segall, A.I., Vitale, M.F., Perez, V.L., and M.T. Pizzorno (2003) HPLC analysis of 5H-benzo[a]carbazolewith antifungal activity. J. Pharm. Biomed. Anal. 31:1021-1026.Thakker, M.M., Perez, V.L., Moulin, A., Cremers, S.L., and C.S. Foster (2003) Multifocal nodular episcleritisand scleritis with undiagnosed Hodgkin’s lymphoma. Ophthalmology 110:1057-1060.166

Center forSurgery ResearchCENTER FORSURGERY RESEARCHDIRECTORSuyu Shu, Ph.D.The Center for Surgery Research wasestablished in 1995 under the administrationof the Division of Surgery at theCleveland Clinic Foundation (CCF). Since then, acontinued emphasis of our activity and developmenthas been translational research in the areaof tumor immunotherapy. Consistent with thisphilosophy, the Center’s scientific pursuit hasfocused on the analysis of the fundamentalprinciples of host immune responses to a varietyof malignant tumors. It is hoped that throughthis understanding, strategies to enhance tumorantigen recognition by the cells of the immunesystem can be improved. Therefore, the overallgoal and mission of the Center is to provideexcellence in research and education throughbench investigation, preclinical animal experimentationand clinical trials.Our currently have full Staff members(Suyu Shu, Ph.D., Director, Peter A. Cohen,M.D., Julian A. Kim, M.D., and Julian A. Kim,M.D.) and one Associate Staff (David E. Weng,M.D., Ph.D.). In addition, two former researchfellows (Jorgen Kjaergaard, Ph.D., and LiaominPeng, M.D.) have recently been promoted toProject Scientists. Dr. Cohen is a board-certifiedmedical oncologist. His research has beencentered on the mechanisms and functions ofantigen-presenting cells (APCs) present in tumormass. He is also interested in delineating theantitumor effects and mechanisms mediated bytumor-sensitized CD8 T lymphocytes. His workis supported by an R01 investigation grant fromthe National Cancer Institute.Dr. Julian Kim is a surgical oncologist whogained significant experience in clinical T-cellimmunotherapy of colon cancer while at OhioState University in Columbus. Now, Dr. Kimprovides surgical expertise to our group and isinvolved in a number of clinical trials. He hasalso developed his own research projects closelyrelated to immunotherapy of cancer. During thepast two years, Dr. Kim’s laboratory research hasdefined the effects of anti-CD40 monoclonalantibodies on the sensitization of immune T cellsagainst the murine breast cancer 4T1. Anotherline of his investigation has been the inhibitionof vascular endothelial growth factor (VEGF) topromote immune responses to the 4T1 breastcancer. This tumor model has the potential tospontaneously metastasize to various visceralorgans and to the brain, thus providing anexcellent opportunity to investigate the effects ofimmunotherapy against naturally occurring tumormetastases.Dr. David E. Weng has had a primaryappointment at the Center since January 2001;previously, his primary position was in theDepartment of Hematology/Oncology, where hespent 50% of his time in clinical patient care.Transfer to the Center has allowed him to focuson developing scientific research programs andprocedures for experimental clinical trials. Dr.Weng received his undergraduate education fromHarvard University and completed his M.D. andPh.D. degrees at Johns Hopkins University. Hehas already participated in several of our ongoingclinical immunotherapy protocols. In thelaboratory, Dr. Weng has begun work onanalyzing the expression of chemokines and theirreceptors at the site of tumor regression mediatedby transferred T cells. Much progress has beenmade towards delineating the expression of IP-10(interferon [IFN]-inducible protein-10), Mig(monokine induced by IFN-gamma) andRANTES (regulated on activation normal Tlymphocyte expressed and secreted), as well aschemokine receptors such as CCR2, CCR5 andCXCR3. It is an important area of researchbecause the role that chemokines play in thetrafficking and interactions of T cells with tumorhas not been defined.Dr. Jorgen Kjaergaard is a graduate ofUniversity of Aarhus, Denmark, where heobtained his Ph.D. degree in Microbiology andImmunology in 1994. During graduate study, hebecame interested in tumor immunology, workingintensively on the biology and functions oflymphokine-activated killer (LAK) cells. He wasa postdoctoral fellow in Dr. Shu’s laboratory atCCF from 1997-2000. He has since contributedto work on the pattern of systemically administeredT lymphocytes.Dr. Liaomin Peng graduated from the FirstMilitary Medical University, PRC, with an M.D.degree in 1986. He came to CCF in 1996 as apostdoctoral fellow working with Dr. JohnKrauss for about 3 years before working with Dr.Cohen. Prior to his coming, Dr. Peng hadconsiderable experience in molecular biology andtumor immunology. In the past 6 years, he hasworked on various projects analyzing T-cellreactivities against malignancies in preclinicalanimal models. Dr. Peng has defined thesuppression role of a particular T-cell subpopulationin the tumor-draining lymph nodes. He isalso studying the effects of ligation of thecostimulatory molecule OX-40R to enhance T-cell-mediated antitumor immune responses.Staff members in the Center have jointappointments in other CCF departments, such asthe Departments of Immunology, Cell Biology,Cancer Biology, Otolaryngology, Hematology/Oncology, and General Surgery and in the TaussigCancer Center. The Center for Surgery ResearchContinued on Page 168INVESTIGATORSWei Chen, M.D., Ph.D.Peter Cohen, M.D.Julian Kim, M.D.Gregory E. Plautz, M.D.ASSOCIATE STAFFDavid E. Weng, M.D., Ph.D.PROJECT STAFFJorgen Kjaergaard, Ph.D.Liaomin Peng, M.D.167

Continued from Page 167active training program for postgraduatelevel fellows. Currently, we have fourpostdoctoral fellows and one surgical residentfrom Otolaryngology. The training programfocuses on areas of tumor immunology, molecularbiology and clinical immunotherapy ofcancer.Research programs within the Centerconcentrate around the central theme of T-lymphocyte responses to autochthonous tumorcells. By applying immunological and molecularbiological techniques, we wish to define the roleof the host immune system in preventing,controlling and eradicating malignant T cells.Such an approach requires both extensive basicresearch utilizing animal tumor models andhuman experimentation through designed clinicalprotocols. In animal studies, the transfer oftumor-immune T lymphocytes has been demonstratedto be the most effective means oferadicating established primary as well asmetastatic tumors. This form of immunotherapyof cancer, referred to as “adoptive immunotherapy,”requires the generation of tumorsensitizedT cells in numbers sufficient fortreatment in clinical settings.A few years ago, results from animalstudies and laboratory investigations weresufficient to allow extrapolation for a plan of asystemic immunotherapy of humans withmalignancies. The basic procedures involvevaccination of patients with their own tumorcells, followed by retrieval of the draining lymphnodes to provide a source of tumor-reactiveimmune T lymphocytes. After further stimulationand propagation of these lymph node T cellsin vitro, they are reinfused into the patient forthe treatment of tumor. Since the approval bythe Food and Drug Administration in July 1995of an Investigational New Drug application, wehave extended this protocol to treat a number ofdiverse cancers including malignant gliomas,advanced renal cell carcinoma, squamous cellcarcinoma of the head and neck, and metastaticmalignant melanoma. This effort has been acollaborative interaction between the Center andseveral other departments, including NeurologicalSurgery, Otolaryngology, Urology, PlasticSurgery, General Surgery and Hematology/Oncology. Because of the collegial atmosphereand strong support provided by CCF, we havemade significant progress toward standardizationof the procedures and analysis of toxic sideeffects of the treatment.The first disease we proposed to treat withautologous T lymphocytes was glioblastomamultiforme (GBM). The procedure for generatingT cells has been modified several times, basedon laboratory research findings. Ten patients,most of them with recurrent GBM, have beentreated with modified procedures. Three of thosepatients showed MRI-documented tumorregression. Analysis of survival also suggests thatthere might be a prolongation of survival in thisgroup of patients as compared to the results of asingle-drug (carmustin polymer) trial in patients atsimilar stages of disease. Data generated fromthis trial provided preliminary results for asuccessful R01 grant application. In the meantime,we started to treat patients with metastaticrenal cell cancer and cancer of the head and neck.Since July 1997, 22 patients with renal cellcarcinoma have enrolled and completed thetreatment procedure. It may be too early to drawa clear conclusion on the outcome of thetherapeutic efficacy, but clearly, treatment withautologous T cells poses little or no toxicity. Ourprotocol differs from many others in a veryimportant way in that it is readily modifiedaccording to concurrent research results. One ofthe potential refinements of this protocol is theisolation of L-selectin-negative T cells from thedraining lymph nodes for generation of mosteffective tumor-sensitized lymphocytes. In animalstudies, L-selectin-negative T cells representapproximately 20% of the total lymph node TContinued on Page 169Suyu Shu, Ph.D.168

Continued from Page 168cells. However, the purified L-selectinnegativeT cells demonstrated a 30-fold increasein their therapeutic effects. Evidence alsosuggests that the L-selectin-negative T cells willsecrete a variety of cytokines in vitro whenstimulated with tumor cells. It is thereforepossible that the isolation of L-selectin-negativecells will improve therapeutic efficacy andprovide an in vitro assay to predict the anti-tumoreffects of T cells before infusion into patients.Because of lack of significant (> grade II)toxicity, we have began to conduct phase II trialsfor newly diagnosed malignant gliomas andmetastatic renal cell carcinoma. For head andneck cancer, adoptive immunotherapy will focuson patients with stage III or IV disease. Aftersurgical resection of their primary tumors, 70%of them are predicted to have a recurrence ofcancer, and most of the recurrences (>80%)occur within one year. Therefore, the benefits oftreatment can be rapidly analyzed, based onprolongation of disease-free intervals in a phase IItrial.More recently, we have tested the effectsof in vivo ligation of OX-40R on T cells topromote anti-tumor immune responses. OX-40R(CD134) is a lymphocyte-specific member of agrowing family of receptors for membrane-boundand soluble cytokines that has been termed thetumor necrosis factor receptor (TNFR) superfamily.A common function of the TNFR familyseems to be regulation of activation and/orproliferation or apoptosis of lymphocytes. Inanimal studies, although the use of OX-40Rmonoclonal antibody (mAb) showed antitumoreffects, those effects were only seen with limitedtumor burden. We have tested the ability of theOX-40R mAb to enhance T-cell immunotherapy.In intracranial tumor models, treatment inconjunction with the OX-40R mAb has demonstratedenhancement of T-cell function. For thetreatment of day-10 established brain tumor, theuse of OX-40R mAb and as few as 5 x 10 6immune T cells resulted in cure of all treatedmice. In collaboration with Cantab Pharmaceuticals,Cambridge, United Kingdom, we arecurrently including the OX-40R mAb in thedesign of our next clinical trial of T-cellimmunotherapy for glioma.From reviewing our experimental andclinical results, we found that the adoptiveimmunotherapy approach might not rendersufficient stimulation of draining lymph nodes invivo for T-cell priming. Although we routinelyuse intact tumor cells as immunogens, it is likelythat antigen presentation relies on the host APCs.Among various APCs, dendritic cells (DCs) seemto have all the essential properties required foreliciting T-cell responses. In many laboratories,DCs are pulsed with peptides, proteins, tumorlysate or RNA derived from neoplastic cells tostimulate antitumor immunity. Althougheffective for stimulating a primary immuneresponse, the therapeutic effects have beenlimited to small tumor burdens and requirerepeated vaccine administrations.At the Center for Surgery Research, wehave recently been successful in fusing DCs andlive tumor cells to form hybrid cells by exposingthem to electric fields. Fusion hybrid cells shouldhave the ability to elicit both major histocompatibilitycomplex (MHC) class I and II restrictedresponses by processing and presenting bothknown and undefined tumor-associated antigens.Thus the fusion hybrids may represent the mostpotent immunogenic forms of tumor vaccine. Inthe past year, we studied the functions of thesecells in animal tumor models. In in vitro systemsin which immune T cells were stimulated withantigens to secrete IFN-gamma, we demonstratedthat DC-tumor chimeric fusion hybrids representedthe strongest stimulus to activatedsensitized T cells as compared with all othermeans of antigen presentation. Most significantly,mice with established tumors of 5 x 7 mmcould be cured after a single vaccination withDC-tumor fusion cells.Because of this remarkable observation,we have obtained Institutional Review Board(IRB) approval for active immunotherapy ofstage IV melanoma patients. This clinical trialwill be carried out in collaboration with Dr.Donald L. Morton of the John Wayne CancerInstitute, Santa Monica, California. We also haveIRB approval for using DC-tumor fusion hybridsto stimulate draining lymph nodes for adoptive T-cell immunotherapy of brain tumor patients,including those with GBM.The laboratory research and clinicalactivities have served well as an instrument foreducating young professionals. Currently, theCenter has five postgraduate fellows with variousmedical and biology backgrounds.The research and clinical trials of theCenter for Surgery Research receive support fromthe CCF’s Board of Governors. The T-celladoptive immunotherapy clinical trials are alsosupported by an R01 research grant from theNational Institutes of Health (NIH). We haveapplied for NIH support for carrying out clinicaltrials with electrofused DC-tumor hybrids. Basicresearch has been supported by three major NIHgrants (4 R01s). The active research pursuits andthe ability to extrapolate laboratory findings toclinical trials have made the Center for SurgeryResearch one of the most unique units in theUnited States with a focused effort on thedevelopment of immunotherapy of malignancies.169

J.J. JACOBS CENTERFOR THROMBOSIS ANDVASCULAR BIOLOGYJoseph J. Jacobs Center forThrombosis and Vascular BiologyDIRECTOREric J. Topol, M.D.HEAD OF RESEARCHEdward F. Plow, Ph.D.STAFFPaul E. DiCorleto, Ph.D.Joan E.B. Fox, Ph.D.Kandice Kottke-Marchant, Ph.D.ASSOCIATE STAFFKathleen L. Berkner, Ph.D.A. Michael Lincoff, M.D.ASSISTANT STAFFTatiana Byzova, Ph.D.Jane Hoover-Plow, Ph.D.Marc Penn, M.D.STAFF SCIENTISTTatiana Ugarova, Ph.D.PROJECT SCIENTISTSOlga Stenina, Ph.D.Timothy O’Toole, Ph.D.RESEARCH ASSOCIATESKasia Bialkowska, Ph.D.Elzbieta Pluskota, Ph.D.Valentin Yakubenko, Ph.D.POSTDOCTORAL FELLOWSJuhua Chen, Ph.D.Sarmistha De, Ph.D.Bin Hu, Ph.D.Sucheta Kulkarni, Ph.D.Natalia Narizhneva, Ph.D.Prakash Paragi, M.D.Wen Qian, Ph.D.Mark Rishavy, Ph.D.Jingfeng Sha, Ph.D.Aleksey Shchurin, Ph.D.Dmitry Solovjov, Ph.D.Carmen Swaisgood, Ph.D.Valentin Ustinov, Ph.D.Shan Wu, Ph.D.PREDOCTORAL FELLOWNataly Podolnikova, M.S.170The Center for Thrombosis and VascularBiology was founded in 1993 anddedicated in March 1995. Dr. Eric Topol isDirector and Dr. Edward F. Plow is Head ofResearch.The Center comprises 15 faculty membersand has a total staff of 39. Faculty members alsohold appointments in other CCF departments,including the Departments of Cell Biology andMolecular Cardiology in the Lerner ResearchInstitute and the Department of CardiovascularMedicine. The Center is committed to implementinga fully integrated, interdisciplinary approach tothe study of vascular biology and cardiovascularmedicine. Targets of research within the Centerinclude studies of basic molecular and cellularmechanisms, the occurrence and manipulation ofthese mechanisms in animal models, analyses ofthe genes and gene products that play a causativerole in cardiovascular disease and the basis fortheir action, and clinical research in patients, bywhich we seek to better diagnose and improvetherapy for cardiovascular disease.Research programs include studies of theparticipation of platelets in thrombosis andhemostasis. These studies seek to define themolecular basis for the adhesion of platelets to thesubendothelial matrix, to other platelets and toendothelial cells. Critical to platelet adhesion andaggregation are two receptors: integrin α IIbβ 3(GPIIb-IIIa) and GPIb-IX-V. The structure andfunction of these adhesion receptors are beingelucidated using molecular biology, proteinstructure, microscopic imaging, antibody mappingand synthetic peptide approaches. The mechanisms,which regulate activation of the integrinand the signaling events that are induced as aconsequence of ligand binding to both of theseadhesion receptors, are a particular emphasis ofresearch. The ligands for these receptors are beinganalyzed to understand how changes in theirconformation induced by biological events (such asproteolysis, deposition in the extracellular matrix orbinding to other cellular receptors) regulate theirrecognition by their platelet receptors. These basicstudies of platelets are being extended into patientsto determine whether inhibition of these receptorsor other platelet targets can prevent or be used totreat such cardiac diseases as myocardial infarctionsand unstable angina.A similar progression of studies, rangingfrom basic to clinical investigations, occurs in thefibrinolysis/thrombolysis area within the Center.Basic studies of the plasminogen system are beingconducted to identify receptors that regulate theconversion of plasminogen to plasmin and todefine mechanisms that influence the capacity ofplasmin to degrade fibrin. Transgenic mice havebeen developed in which various components ofthe plasminogen system have been inactivated.These transgenic knockout animals are being usednot only to critically evaluate the role of theplasminogen system in fibrinolysis but also to testother postulated functions of plasminogen,including its role in mediating various aspects ofthe inflammatory response and in brain function.At the same time, clinical studies in humanscontinue to compare the efficacy of variousthrombolytic agents in conjunction with othertherapeutic agents to optimize therapy.Our research efforts include analyses ofcardiovascular risk factors, such as fibrinogen andlipoprotein(a), seeking to define the molecular basisfor their pathogenic activities. Such insights maybecome the basis for new diagnostic and therapeuticapproaches. Recent studies of patients haveemphasized the importance of inflammation in thedevelopment of overt cardiovascular disease. Earlyevents in atherosclerosis include the recruitment ofinflammatory cells across the endothelium. Theadhesion receptors that mediate such leukocytetrafficking are the subject of investigation withinthe Center. How receptor activation by physiologicallyrelevant agonists on leukocytes and vascularcells influences the adhesion and migration of thesecells is under intensive investigation.An additional area of emphasis in the Centeris restenosis following angioplasty. Investigators areseeking to identify new molecular targets toprevent neointima formation. Not only new drugsbut also new methods for their delivery are beingtested in a number of different animal models toidentify therapeutic approaches that may beapplicable to limit restenosis in humans. How otherdiseases, such as diabetes, influence the need andoutcome of angioplasty is being evaluated as well.Three areas of emphasis have recently beenadded to the repertoire of research programs in theCenter: (1) A large-scale genetic study conductedin the Center led to the identification of singlenucleotidepolymorphisms (SNPs) within membersof the thrombospondin gene family as risk factorsfor premature atherosclerosis. Studies are beingconducted to determine the effects of thesesubstitutions on the structure and function of thethrombospondins. (2) A promising approach tosalvaging damaged heart tissue is to repopulate itwith progenitor cells that can develop untofunctional cardiomyocytes. Such stem-cell researchhas been initiated to understand, optimize andapply these approaches to patients. (3) Angiogenesis,the formation of new blood vessels, providesa promising approach to restore blood flow todamaged heart tissue. The mechanisms ofangiogenesis and ways to encourage this process arebeing analyzed in vitro and in vivo.Overall, the Center seeks to implement acomprehensive approach to define the basicmolecular mechanisms of cardiovascular diseaseand to extend these insights into new therapies.Web Site: http://www.lerner.ccf.org/tvb/

Center forCancer Drug Discovery and DevelopmentCENTER FOR CANCERDRUG DISCOVERY ANDDEVELOPMENTThe Center for Cancer Drug Discovery andDevelopment, founded in 1998, aims todevelop novel and effective therapeuticoptions for cancer patients. Our intent is todiscover and develop therapeutics with particularfocus on biological agents targeted at genes orgene products that determine the course ofcancer development. We strive to designinnovative approaches to stem the growth andmetastasis of tumors, and our efforts are gearedto translate our findings into rigorous clinicaltrials that test the efficacy of molecular medicalcancer treatments.Lerner Research Institute investigators areadept at discovery, development and evaluationof cancer therapeutics through the targeting ofspecific genes and gene products critical inneoplasia. We aim to translate findings frommolecular biological research into molecularmedical strategies through close collaborationswith investigators at the Cleveland Clinic’s TaussigCancer Center. This molecularapproach to medicine will focuson altering the course of generegulation or deregulation in thebiology of cancer. These novelstrategies offer physicians newapproaches to combat cancer.The Center provides a focusfor investigation of new moleculesin preclinical screeningsystems and to translate theinformation about novel cancerrelatedmolecules to clinical trials.By using the principles ofpharmacology and the knowledgeof cancer biology, we design anddevelop new compounds orcombinations of new compoundsthat reduce morbidity from thecomplications of cancer. Forexample, preclinical and clinicalresearch on new molecules isunder way in collaboration withThe Institute for PathologicalProducts (Shanghai, P.R. China),Igeneon Inc (Vienna, Austria),Immunicon (Philadelphia, PA),Schering-Plough, Inc.(Kenilworth, NJ), and RibozymePharmaceuticals, Inc. (Boulder,CO).We use innovativebiostatistical methodologies toexpedite evaluation of drugtherapies. Our efforts include the development ofonline networks to acquire clinical data for rapid,comprehensive analysis.Our objectives are to develop innovativedrug screening technologies while continuing ourresearch in the design of small molecules targetedat cellular signal transduction. We aim to usenormal programmed cell death (apoptosis) as ameans by which to curtail tumor developmentthrough induction of identified genes. Over thenext few years, new investigators will berecruited for this center to add to existingexpertise.The research community has developed awealth of findings on important cellular processescritical in the regulation of normal proliferationand the initiation, progression and metastasis ofcancer cells. The Center for Cancer DrugDiscovery and Development aims to integrateinformation from these data and use it to developnew approaches to the treatment of cancer.DIRECTORErnest C. Borden, M.D.ASSISTANT STAFFJulian Kim, M.D.Daniel J. Lindner, M.D., Ph.D.Jaroslaw P. Maciejewski,M.D., Ph.D.PROJECT SCIENTISTJoseph A. Bauer, Ph.D.ADMINISTRATORLynn Borzi, M.S.COLLABORATORSG. Thomas Budd, M.D. 1Ronald M. Bukowski, M.D. 1Maurie Markman, M.D. 1Ganes C. Sen, Ph.D. 2Robert H. Silverman, Ph.D. 3George R. Stark, Ph.D. 2Bryan R.G. Williams, Ph.D. 3Yan Xu, Ph.D. 3Taolin Yi, M.D., Ph.D. 3Maciej Zborowski, Ph.D. 41Taussig Cancer Center, CCF2Dept. of Molecular Biology,CCF3Dept. of Cancer Biology,CCF4Dept. of BiomedicalEngineering, CCFErnest C. Borden, M.D.Web Site: http://www.lerner.ccf.org/cancerdrug/171

THE BORDENLABORATORYRESEARCH ASSOCIATESMamta Chawla-Sarkar, Ph.D.Kevin Taylor, Ph.D .POSTDOCTORAL FELLOWSSoo-In Bae, Ph.D.Vadim Budagian, Ph.D.Yan-Fang Liu, Ph.D.Navneet Majail, M.D.Paul Masci, D.O.Fred Reu, M.D.Snehal Thakar, M.D.TECHNICAL ASSOCIATESRon Grane, B.S.Barbara Jacobs, M.S.COLLABORATORSDaniel Lindner, M.D., Ph.D. 1Ganes C. Sen, Ph.D. 2Robert H. Silverman, Ph.D. 3Bryan R.G. Williams, Ph.D. 3Taolin Yi, Ph.D. 31Taussig Cancer Center, CCF2Dept. of Molecular Biology,CCF3Dept. of Cancer Biology, CCF172Investigations of Interferon Add Insight forUse in Combined Modality Cancer TreatmentsInterferons (IFNs) have proven an importantparadigm for establishing the role ofbiologicals as effective therapy in humanmalignancies. Despite this substantial progress,we still do not understand the underlyingmechanisms of tumor sensitivity and resistance.How to overcome resistance to IFNs innonresponding patients has been little explored.It is our overall hypothesis that clinical resistanceresults from deficiencies in components of theJAK/STAT signal transduction pathway and/orexpression of specific interferon-stimulatedgenes (ISGs); conversely, tumor response will beaugmented by increases in signal transductionand/or expression of specific ISGs.IFNs have proven active as single agents insome tumors (melanoma, myeloma, renal cellcarcinoma, chronic leukemias, T- and B-celllymphomas, carcinoids, and Kaposi’s sarcoma)and are now being increasingly integrated intocombined modality therapy with clinicallybeneficial effects. IFNs are possibly the mostpotent modulators of gene expression used inclinical medicine. Since the clinical effects almostcertainly result from ISGs, to probe underlyingmechanisms of antitumor action, we havefocused on ISGs and their control. We havedemonstrated modulation of such genes and theirproducts in treated patients in vivo. These studieshave defined correlations between doses,schedule of administration, and cytokine typewith gene induction and cellular response.One goal is thus to define defects inexpression or activation of signal transductioncomponents and extend our studies of interventionsto correct abnormalities. We have identifiedantiestrogens, demethylating agents, phosphataseChawla-Sarkar, M., Leaman, D.W., and E.C. Borden (2001) Preferential induction ofapoptosis by interferon (IFN)-beta compared with IFN-alpha2: correlation withTRAIL/Apo2L induction in melanoma cell lines. Clin. Cancer Res. 7:1821-1831.Leaman, D.W., Chawla-Sarkar, M., Vyas, K., Reheman, M., Tamai, K., Toji, S., andE.C. Borden (2002) Identification of X-linked inhibitor of apoptosis-associated factor-1 as an interferon-stimulated gene that augments TRAIL Apo2L-induced apoptosis.J. Biol. Chem. 277:28504-28511.Chawla-Sarkar, M., Leaman, D.W., Jacobs, B.S., and E.C. Borden (2002) IFN-betapretreatment sensitizes human melanoma cells to TRAIL/Apo2 ligand-induced apoptosis.J. Immunol. 169:847-855.Padovan, E., Terracciano, L., Certa, U., Jacobs, B., Reschner, A., Bolli, M., Spagnoli,G.C., Borden, E.C., and M. Heberer (2002) Interferon stimulated gene 15 constitutivelyproduced by melanoma cells induces E-cadherin expression on humandendritic cells. Cancer Res. 62:3453-3458.Chawla-Sarkar, M., Leaman, D.W., Jacobs, B.S., Tuthill, R.J., Chatterjee-Kishore,M., Stark, G.R., and E.C. Borden (2002) Resistance to interferons in melanomacells does not correlate with the expression or activation of signal transducer andactivator of transcription 1 (Stat1). J. Interferon Cytokine Res. 22:603-613.Pathak, M.K., Dhawan, D., Lindner, D.J., Borden, E.C., Farver, C., and T. Yi(2002) Pentamidine is an inhibitor of PRL phosphatases with anticancer activity.Mol. Cancer Ther. 1:1255-64.inhibitors and cytokines as molecules thataugment IFN signal transduction and antitumoreffects in preclinical models. Such defects mayinfluence not only response of tumors to IFNsbut also to other cytokines that work via IFNassociatedsignal transduction pathways. Since,however, not all tumors will have signal transductionabnormalities, it seems likely that selectivelack of expression of specific ISGs will accountfor resistance in other tumors.A second goal is to identify specific ISGsthat are induced in human tumors in response toIFN-α2 and IFN-β. Through gene chip technologyin vitro, new genes have been identified thatmay be particularly critical in mediating theantitumor effects. Toward this end, we aredissecting the influence on cell function of thesegene products and are assessing newly identifiedISGs in patients receiving IFNs and otherbiological response modifiers. In collaborationwith Drs. Bryan Williams and Robert Silverman,our focus is particularly on newly identified ISGsthat influence cell proliferation and/or apoptosis.The pleiotropic effects of IFNs on cellfunction have led us to assess not onlyantiproliferative and apoptotic effects on tumorcells but also components of host response.Influences of IFNs on immune effector cellfunction that have been defined include effects onboth natural killer/lymphokine-activated killer(NK/LAK) cell activity and monocytes. We haveidentified a new lymphokine, ISG15 (itself anISG protein product), that influences NK/LAKcell function. Another important component ofhost-tumor interaction is endothelial cellproliferation. In collaboration with Dr. DanielLindner, we are continuing to assess the augmentedanti-angiogenic effects of IFNs incombination with other molecules in causingtumor inhibition. Finally, although apoptosis hasnot been considered a prominent part of themechanism of action of IFNs, our recent studiesdefine a caspase-mediated event induction in celllines of several histologies.The clinical and gene modulatory effects ofmost members of the family of IFN proteins andof IFN inducers have hardly begun to be assessed.Thus we are beginning to more extensivelyevaluate other members of the IFN multigenefamily in more depth. We are thus assisting, withDr. Silverman and Dr. Ganes Sen, in probing theantitumor effects of other molecules that harnessthe IFN system for potentially greater clinicaleffectiveness. We are also evaluating otherbiologicals or low-molecular-weight moleculesthat modify host response, such as other cytokines(interleukin [IL]-2, -6, -10, and -12), monoclonalantibodies, and tumor vaccines that havecomplementary mechanisms of cellular andantitumor effects of IFNs. It is our goal in all ofthese studies to translate important leads withIFNs and other biological modifiers into moreeffective therapies.

Development of Clinical Anti-TumorAgents Based on Antisense Knockout ofRID Genes, Interferon Role in CytotoxicityAbetter understanding of how interferons(IFNs) induce apoptosis may allow theirimproved clinical utilization as antitumoragents. Though they have traditionally beenconsidered cytostatic agents, IFNs inducecytotoxicity in several tumor cell lines in cultureand in vivo. The mechanism by which this occursrequires the function of IFN-induced geneproducts. To identifyfunctionally relevant deathassociatedgene products,our laboratory has employedan antisense technicalknockout strategy. In thisapproach, specific deathinducinggenes, termedRegulators of InterferoninducedDeath (RIDs), areinactivated by antisensegene products, thusproviding a growthadvantage to transfectedcells in the presence ofIFNs. Because an IFNstimulatedpromoter drivesthe expression of theantisense inserts, a functionalJAK-STAT pathwayis required. Therefore,antisense RNAs directedagainst the genes encodingIFN receptor chains, or JAKs, or STATs cannotaccount for cell survival in the presence of IFNs.Consistent with the possibility that multiple geneproducts participate in IFN-induced cell death,several different cDNAs have been isolated usingthis approach, many of them novel. One RIDgene is identical to inositol hexakisphosphatekinase 2, (IP6K2) a kinase that creates pyrophosphatelinkages on inositol phosphates.Overexpression of IP6K2 sensitizes cells to avariety of cytotoxic stimuli. Dr. Bei Morrisonperforms functional analysis of two RID genes toelucidate their enzymatic activity. In collaborationwith our clinical colleagues, we are performing aPhase II trial of IFN-beta in ovarian carcinoma.RID gene expression will be evaluated in bloodand tumor samples from these patients.To potentiate their clinical efficacy, IFNsare increasingly being used in combinationtherapy. In collaboration with Drs. ErnestBorden and Dhan Kalvakolanu, we have shownthat combining IFNs with antiestrogens (e.g.,tamoxifen) or retinoids (e.g., all-trans retinoicacid) results in enhanced antitumor effects, bothin cell culture and in athymic nude mice xenograftmodels. Part of the increased antitumor activityis due to direct anti-cellular effects mediated byDaniel J. Lindner, M.D., Ph.D.the drug combination, and a portion of theantitumor effect is secondary to enhanced hosteffects. One of the most important anti-tumoreffects mediated by IFNs is the inhibition oftumor-induced angiogenesis. On a molar basis,IFNs are the most powerful anti-angiogenicagents currently known. Current angio-genesisstudies are probing the antiangio-genic effects ofsecond generation IFNs such asPEG-IFN, a polyethylene glycolmodification of the IFNmolecule that results in aprolonged half life and hencemay provide enhancedbiological activity. We are alsotesting IFNs in combinationwith Angiozyme, an antisensemolecule directed againstVEGFR1/flt-1, a criticalreceptor that mediates bloodvessel growth.Our laboratory is alsoactive in the area of newcancer drug development. Dr.Joseph Bauer has synthesized anovel chemotherapeuticcompound, nitrosylcobalamin(NO-Cbl), that consists ofnitric oxide bound to vitaminB12. Inactive until taken up bycells, once inside lysosomes, thedrug releases nitric oxide and induces apoptosis.Importantly, NO-Cbl causes very little toxicity tonormal tissues. We believe this “Trojan Horse”approach may target tumors through their highrequirement for vitamin B12.THE LINDNERLABORATORYPRINCIPAL INVESTIGATORDaniel J. Lindner, M.D., Ph.D.RESEARCH ASSOCIATESJoseph A. Bauer, Ph.D.Bei Hu Morrison, M.D.COLLABORATORSAlexandru Almasan, Ph.D.Ernest C. Borden, M.D.Dhananjaya Kalvakolanu, Ph.D. 1Bellur Seetharam, Ph.D. 2Ganes C. Sen, Ph.D.Deborah J. Vestal, Ph.D.1Dept. of Microbiology, Univ. ofMaryland, Baltimore, MD2Dept. of Biochemistry, Med.College of Wisconsin,Milwaukee, WICLINICAL COLLABORATORSJerome Belinson, M.D.Maurie Markman, M.D.Kenneth Webster, M.D.Lindner, D.J. (2002) Interferons as antiangiogenic agents. Curr. Oncol. Rep. 4:510-514.Yi, T., Pathak, M.K., Lindner, D.J., Ketterer, M.E., Farver, C., and E.C. Borden(2002) Anticancer activity of sodium stibogluconate in synergy with IFNs. J. Immunol.169:5978-5985.Pathak, M.K., Dhawan, D., Lindner, D.J., Borden, E.C., Farver, C., and T. Yi(2002) Pentamidine is an inhibitor of PRL phosphatases with anticancer activity.Mol. Cancer Ther. 1:1255-1264.Bauer, J.A., Morrison, B.H., Grane, R.W., Jacobs, B.S., Borden, E.C., and D.J.Lindner (2003) IFN-alpha2b and thalidomide synergistically inhibit tumor-inducedangiogenesis. J. Interferon Cytokine Res. 23:3-10.Chawla-Sarkar, M., Lindner, D.J., Liu, Y.F., Williams, B.R., Sen, G.C., Silverman,R.H., and E.C. Borden (2003) Apoptosis and interferons: Role of interferon-stimulatedgenes as mediators of apoptosis. Apoptosis 8:237-249.173

CLEVELAND CENTERFOR STRUCTURALBIOLOGYCleveland Center forStructural BiologySTAFFKwaku Dayie, Ph.D.Jun Qin, Ph.D.RESEARCH ASSOCIATESOlga Vinogradova, Ph.D.Yanwu Yang, Ph.D.POSTDOCTORAL FELLOWSRune Hartmann, Ph.D.Sujay Ithychanda, Ph.D.Pius S. Padayatti, Ph.D.PREDOCTORAL FELLOWSPamela HallKaren KnausXiangming (Sean) Kong, M.S.Hua Li, M.S.Algirdas Velyvis, B.S.Xiaoxia (Susan) Wang, M.S.The Cleveland Center for StructuralBiology (CCSB) seeks to apply state-ofthe-arttechnologies to characterize andsolve the three-dimensional structures ofproteins and other biological molecules. Thisinformation is then used to provide insights intothe basic functions of molecules and theirinteractions and to help identify approaches toregulating these functions and interactions; i.e.,rational drug design. Originating as a jointventure between the Cleveland Clinic Foundationand neighboring Case Western ReserveUniversity, the program is aimed at acquiring themost modern and sophisticated equipment toanalyze macromolecular structures and forrecruiting key personnel to develop and applythese approaches. The endeavor to develop anduse structural biology has expanded to encompassthe Cleveland research community, includingCleveland State University, MetroHealth MedicalCenter, and University Hospitals.The structural biology initiative gainedfunding from two primary sources: the ClevelandFoundation and the State of Ohio Board ofRegents. With initial seed funding, CCSBinstalled an array of high-technology instrumentationto analyze structures of biomolecules.Nuclear magnetic resonance (NMR) equipmentacquired by the Center includes three machines (a600-MHz, a 500-MHz, and a solid-state 300-MHz instrument). Some of this NMR equipmentis located in a central facility donated by CCFand located between the Lerner ResearchInstitute and Case Western Reserve University.The Center also has x-ray crystallographyequipment, located in part in the Lerner ResearchInstitute. Mass spectroscopy, Raman spectroscopyand Biacore technologies are also available to theresearch community through the CCSB initiative.Recently, CCSB has made substantialadditions to its armamentarium of sophisticatedinstrumentation. CCSB has joined a synchrotronconsortium with 10 other academic institutions toaccess a new beam line. The Center’s participationwill allow for access to the highest resolutionx-ray crystallographic facility. In addition, CCSBhas completed an agreement to acquire new NMRequipment, including the highest field instrumentavailable, a 900-mHz NMR. Funds for theequipment were obtained from the ClevelandFoundation, the State of Ohio NMR Consortium,and the National Institutes of Health, as well asby substantial gifts from the two foundinginstitutions. Our access to these instrumentsplaces CCSB at a world-class position in structuralbiology.The success of the scientific enterpriserequires not only instrumentation but also capablescientists to deploy it. The Lerner ResearchInstitute’s structural biology effort includes Drs.Jun Qin and Kwaku Dayie, both of whom areNMR spectroscopists. The research programs ofthese investigators are described in the pages thatfollow.By defining the structure of molecules inthree dimensions, the most fundamental mechanismsof function and interaction can bedelineated in exquisite detail. As specificexamples, clarification of a specific proteinstructure enables a structural biologist todetermine how the active site on an enzymeaccommodates substrate, how an antibodyrecognizes its specific epitope, or how thedomains of one protein interact with those ofanother protein or with a nucleic acid to control aparticular function. Analysis of molecularstructure also enables researchers to modifybiomolecules in a rational way to alter theirfunction. Understanding of molecular structurealso facilitates the design of small molecules,which can inhibit or regulate molecular functionwithin living systems, thereby allowing fordevelopment of therapeutics to combat disease.The structural biologists at CCF have applied theirexpertise to a broad range of biomolecules inheart and vascular disorders, cancer and infectiousdiseases.Center Web Site: http://www.lerner.ccf.org/structbio/174

Structure, Dynamics, and Function ofRNAs and RibonucleoproteinsWe take a “ribocentric” view of the worldin my laboratory. How do RNAmolecules recognize their specific targetsto affect a myriad of biological processes in thecell, such as protein synthesis, enzyme catalysis,gene regulation, and viral infections? The currentgoal of my research program is to understand, atthe structural molecular level, howRNA molecules recognize theirtargets (such as other RNAs,proteins, and ions) with highaffinity and specificity. Knowledgeof the three-dimensional architectureof biological molecules isfundamental to parsing out thedeterminants of molecularrecognition and possibly toforming the basis for rationaldesign of new drugs. Experimentally,we apply a wide range ofbiochemical and biophysicalmethods but use state-of-the-artnuclear magnetic resonance(NMR) spectroscopy as theprimary structural tool tocharacterize specific modelsystems. Our research interest istherefore focused on two aspects: structureelucidation of biologically important moleculesand methodological developments to study largebiomolecules by NMR spectroscopy.We are presently investigating thestructures of RNAs derived from the catalyticcore of group II introns. These structural studies,coupled with probing the role of metal ions inRNA structure, may shed light on understandingthe catalytic mechanism of self-splicing and thepotential relationship to the mechanisms ofmRNA splicing in vivo.At present, detailed NMR spectroscopicinformation can only be obtained for relativelysmall biomolecules, up to a molecular mass of 20kDa, whereas protein-nucleic acid complexesoften are much larger. One main research effortof the group is therefore to develop newexperimental techniques to alleviate this problem.In the area of methodology development, wefocus on implementing and designing multidimensionalNMR experiments to obtain data onKwaku T. Dayie, Ph.D.large biomolecules. These data will be transformedinto realistic three-dimensional molecularstructures using computer calculations. Inaddition, because many biological processes areundergirded by conformational flexibility, we aredeveloping techniques to probe this phenomenon.These results will provide us with a window onthe impact of dynamics on the structure andfunction of biological molecules.THE DAYIELABORATORYGRADUATE STUDENTHua Li, M.S.TECHNOLOGISTXinxing Wang, M.S.COLLABORATORSRichard A. Padgett, Ph.D. 11Dept. of Molecular Biology,CCFDayie, K.T., and G. Wagner (1997) Carbonyl carbon probe of local mobility in 13 C, 15 N-enriched proteins using high resolution NMR. J. Am. Chem. Soc. 119:7797-7806.Walters, K.J., Dayie, K.T., Reece, R.J., Ptashne, M., and G. Wagner (1997) Structureand mobility of the PUT3 dimer: a DNA pincer. Nat. Struct. Biol. 4:744-750.Zhang, P., Dayie, K.T., and G. Wagner (1997) Unusual lack of internal mobility and fastoverall tumbling in oxidized flavodoxin from Anacystis nidulans. J. Mol. Biol. 272:443-455.Dayie, K.T., Tolbert, T.J., and J.R. Williamson (1998) 3D C(CC)H-TOCSY experimentfor assigning protons and carbons in uniformly 13 C and selectively 2 H-labeled RNA. J.Magn. Reson. 130:97-101.Dr. Dayie is a Staff member in the LRI Departmentof Molecular Biology.Dayie, K.T., Brodsky, A.S., and J.R. Williamson (2002) Base flexibility in HIV-2 TARRNA mapped by solution 15 N, 13 C NMR relaxation. J. Mol. Biol. 317:263-278.Hall, J.D., Dayie, K.E., and D. Fushman (2003) Direct measurement of the 15 N CSA/dipolarrelaxation interference from coupled HSQC spectra. J. Biomol. NMR 26:181-186.175

THE QIN LABORATORYPOSTDOCTORAL FELLOWSSujay Subbayya, Ph.D.Olga Vinogradova, Ph.D.Yanwu Yang, Ph.D.TECHNICAL ASSOCIATEAsta Velyviene, B.S.GRADUATE STUDENTSXiangming (Sean) Kong, M.S.Julia Vaynberg, B.S.Algirdas Velyvis, B.S.Xiaoxia (Susan) Wang, M.S.Dr. Qin is a Staff member inthe LRI Department ofMolecular Cardiology.NMR Depiction of Protein Complexes Leadto Design of Pharmaceutical EffectorsThe primary research aim of our laboratoryis to probe and understand the molecularmechanisms of key biological eventsinvolving biomolecular interactions, notablyprotein-protein and protein-nucleic acidinteractions. The major step toward this goal isthe study of three-dimensional structures anddynamics of proteins and protein complexes atatomic resolution. Our laboratory is engaged inthis study by using state-of-the-art nuclearmagnetic resonance spectroscopy(NMR) as a primary tool,combined with other modernbiology techniques. Severalprojects ongoing in thelaboratory include: (a)structural elucidation ofintegrin signaling involved incell adhesion and cell migration;(b) mechanistic investigationof protein kinase R(PKR) involved in interferonand cellular signaling; and (c)molecular mechanism of redox(oxidation/reduction)regulation. The current majorfocus is to explore themolecular basis of integrinsignaling involved in regulatingcell adhesion and cell migration.Integrins are known to be heterodimeric(α/β) cell surface receptors that mediateadhesion of the cells to one another and to theirsurroundings. Such adhesion is crucial for manybiological processes, such as embryogenesis,hemostasis, the immune response and themaintenance of tissue integrity; and its dysfunctionleads to numerous human disorders, such asthrombasthenia and chronic inflammatorydiseases. Therefore, understanding the mechanismof integrin adhesion is of both physiologicaland pathological importance. The integrins’ability to bind their extracellular ligands foradhesion is tightly regulated through a processtermed inside-out signaling, i.e., a cellular signalstimulates a conformational change in thecytoplasmic domain of an integrin, whichpropagates through its transmembrane region tothe extracellular domain, transforming it from alow-affinity to a high-affinity ligand-binding state(integrin activation). On the other hand, ligandoccupancy of the extracellular domain also elicitssignals back to the cytoplasmic face,which activate cascades ofintracellular responses, ultimatelyconnecting to cytoskeleton(outside-in signaling). In thismanner, the inside and outside ofthe cells are physically linked,resulting in a cooperative regulationof cell functions including celladhesion, migration, cell growthand differentiation. Although thecytoplasmic domains of integrinsare small and devoid of enzymaticactivities, they are the center of bidirectionalsignaling machinery andhence crucial for the control of theintegrin function. Our goal in thisstudy is to elucidate the structuralJun Qin, Ph.D.signalingbasis of the integrin bi-directionalas mediated by the cytoplasmicdomains. A working hypothesis is that thecytoplasmic face adopts distinct conformationsthat direct the inside-out and outside-in signalingprocesses. We are pursuing the structure of thea IIbb 3cytoplasmic complex and its interactionswith their target signaling network. We are alsoworking on determining the structure of theintegrin cytoplasmic domain coupled with thetransmembrane domain. These structures willprovide insights at the atomic level into thecomplex mechanism of integrin activation andsignaling.Nanduri, S., Carpick, B.W., Yang, Y., Williams, B.R., and J. Qin (1998) Structure of the double-strandedRNA-binding domain of the protein kinase (PKR) reveals the molecular basis of its dsRNA-mediated activation.EMBO J. 17:5458-5465.Vinogradova, O., Haas, T., Plow, E.F., and J. Qin (2000) A structural basis for integrin activation by thecytoplasmic tail of the α IIbsubunit. Proc. Natl. Acad. Sci. USA 97:1450-1455.Nanduri, S., Rahman, F., Williams, B.R., and J. Qin (2000) A dynamically tuned double-stranded RNAbinding mechanism for the activation of antiviral protein kinase PKR. EMBO J. 19:5567-5574.Velyvis, A., Yang, Y., Wu, C., and J. Qin (2001) Solution structure of the focal adhesion adaptorPINCH LIM1 domain and characterization of its interaction with the integrin-linked kinase ankyrin repeatdomain. J. Biol. Chem. 276:4932-4939.Vinogradova, O., Velyvis, A., Velyviene, A., Hu, B., Haas, T., Plow, E., and J. Qin (2002) A structuralmechanism of integrin α IIbβ 3‘inside-out’ activation as regulated by its cytoplasmic face. Cell 110:587-597.Shi, J., Krishnamoorthy, G., Yang, Y., Hu, L., Chaturvedi, N., Harilal, D., Qin, J., and J. Cui (2002)Mechanism of magnesium activation of calcium-activated potassium channels. Nature 418:876-880.176

Center forUrological ResearchThe Center for Urological Research comprisesbasic and clinical research laboratories withinthe Lerner Research Institute and theGlickman Urological Institute. Each of theselaboratories is directed by a full-time scientistfaculty member jointly appointed in a basic sciencedepartment of the Lerner Research Institute and theGlickman Urological Institute. Basic research studieswithin the Center for Urological Research arepredominantly focused in the areas of transplantationbiology, renal epithelial cell biology, urologiconcology and bladder physiology. Renal cellcarcinoma and prostate cancer are the two urologicmalignancies that are being extensively studied. Theemphasis within the Center for Urological Researchis on translational research studies that can enhanceour understanding of the pathogenesis, presentationand management of urologic diseases. All urologyresidents spend a full year training in one of theseresearch laboratories; the period of research trainingfor postgraduate urology fellows is two or threeyears. Members of the Center meet regularly duringmonthly research presentations. In addition, theresidents and fellows present their work during theannual Urology Research Day which includes twodayvisits by prominent Visiting Professors fromother institutions.Andrology Laboratory and Sperm Bank, andCenter for Advanced Research in HumanReproduction, Infertility, and SexualFunctionResearch work in the Reproductive ResearchCenter is mainly focused on elucidating themolecular mechanism associated with male andfemale infertility, and on studies involving themanagement of sexual dysfunction in male andfemale patients following urologic surgeries.Bladder Pathophysiology LaboratoryThis lab focuses on projects related tobladder and female pelvic floor disorders. Anumber of translational projects currentlyinvestigate the bladder remodeling under diabeticconditions in both animal models and humanbladder samples. These projects serve as model forstudies of other pathologies involving the bladdersuch as changes occurring with geriatric bladder.Recently an animal model for stress urinaryincontinence and sling procedure has been created.Validation and testing of this model for a numberof research questions related to treatment of stressurinary incontinence is under way.Renal Carcinoma Immunology LaboratoryThe overall goal of this laboratory researcheffort is to understand how renal tumors can inhibitthe development of an effective antitumor immuneresponse. Findings suggest that tumor derivedproducts can either sensitize T lymphocytes toactivation induced cell death or can directly induceapoptosis. The affected cells appear to includeantigen-specific T cells. The major projects includedefining the role tumor derived gangliosides andoxidized lipid products play in altering thesensitivity of T cells to apoptosis including theirmechanism of action. Additional studies areattempting to determine if the transgene expressionof various anti-apoptotic genes can protect T cellsfrom tumor-induced apoptosis.Transplant Immunology LaboratoryThe focus of this program is inflammatoryfactors directing T cells and other leukocytes intorenal allografts. Current projects are investigating: 1)the expression of inflammatory genes duringischemia and reperfusion of kidneys during clinicaltransplantation and in mouse models: 2) theexpression of inflammatory genes and proteins inurine as markers indicating the presence ofrejection in renal allografts; and, 3) the role ofadhesion molecules and chemokines in directingleukocyte infiltration into organ allografts.Transplant Immunology LaboratoryThis laboratory continues to focus attentionon transplantation immunobiology in mousemodels and translational studies of humanimmunology in transplant recipients. The animalstudies provided new and important information onthe role of memory T cells as barriers to transplanttolerance. They additionally identified a neweffector pathway for alloreactive T cells that wasrecently published in Nature Immunology. Humanstudies are progressing nicely as well. The grouprecently showed that posttransplant immunemonitoring of human peripheral blood is feasibleand that this approach may provide a reliablesurrogate marker for poor outcome lateposttransplant.Prostate Cancer LaboratoryThe laboratory’s research focus is onunderstanding the biology of abnormal prostategrowth. The laboratory cloned and has characterizeda novel protein that we named prostate specificmembrane antigen, PSMA. PSMA is overexpressedin prostate and increases in its expression in theaggressive forms of prostate cancer. We areidentifying the biological reasons for its expressionand are developing new methods to further targetPSMA for imaging prostate cancer and for targetingtherapy to prostate tumors. They discovered thatPSMA is strongly expressed in the new bloodvessels of all solid tumors. Thus PSMA is a targetnot only for prostate tumor cells, but a targettherapy development for all solid tumors.Epithelial Cell Biology LaboratoryUrology-related research in Dr. Weimbs’laboratory focuses on three themes. First, theinvestigation of the function of polycystin-1, theprotein affected in autosomal-dominant polycystickidney disease. Second, the identification of urinarymarkers for the detection of early-stage renal cellcarcinoma. Third, the investigation of basicmechanisms underlying the ability of renalepithelial cells perform “polarized membranetrafficking” which is essential to their function.CENTER FORUROLOGICAL RESEARCHINVESTIGATORSAshok Agarwal, Ph.D. 1,5Firouz Daneshgari, M.D. 1,6Robert L. Fairchild, Ph.D. 2,7James H. Finke, Ph.D. 2,8Peter Heeger, M.D. 2,7Warren Heston, Ph.D. 3,19Thomas Weimbs, Ph.D. 4,101Glickman Urological Institute2Department of Immunology3Department of Cancer Biology4Department of Cell Biology5Director, Andrology Laboratoryand Sperm Bank, andCenter for AdvancedResearch in HumanReproduction, Infertility, andSexual Function6Director, Bladder PathophysiologyLaboratory7Director, Transplant ImmunologyLaboratory7Director, Transplant ImmunologyLaboratory8Director, Renal CarcinomaImmunology Laboratory9Director, Prostate CancerLaboratory10Director, Epithelial CellBiology Laboratory177

The view from the skyway between the wingsof the Lerner Building.178

ScientificSupportServices

OFFICE OFSPONSORED RESEARCHEXECUTIVE DIRECTOR OFFINANCE, ACADEMIC AFFAIRSF. John Case, Ed.D.PROGRAM COORDINATORGinger VaughanFEDERAL AND FOUNDATIONGRANTS COORDINATORMartina SteeleCONTRACTS MANAGERPeggy Beat, R.N., J.D.OFFICE OF SPONSORED RESEARCHThe Office of Sponsored Research (OSR) is one of the administrative arms of the LernerResearch Institute and the Division of Clinical Research. OSR provides oversight of processes andprocedures used to administer all sponsored research grants and contracts funded by corporations,Federal, state, and local governments, private foundations, or CCF internal sources. The OSR, utilizedby basic science and clinical researchers throughout CCF, oversees sponsored research administrationthrough stages of project planning and development, pre-award negotiations, post-award management,interactions with sponsors, and project closeout and analysis. The OSR promotes effective interfaceswith key CCF committees and boards responsible for oversight of human patient and animal-basedstudies through the Institutional Review Board (IRB), the Institutional Animal Care and Use Committee(IACUC), and the Institutional Biosafety Committee (IBC). Inherent in the responsibilities of the OSRis developing and maintaining interfaces with CCF’s General Counsel, CCF Innovations, the FinanceDivision, Institutional Relations and Development, Internal Audit, and the Office of CorporateCompliance. OSR also maintains communications with division and departmental administrators,principal investigators and other research staff.OSR is physically located within the Lerner Research Institute. Last year the Office worked withbasic and clinical researchers who successfully competed for over $100 million in research funding fromexternal sources. These research funds are awarded from many federal, non-profit, private, and publicorganizations. The principal sponsors at CCF are the National Institutes of Health and private industry.OSR builds relationships with the investigators and sponsors to efficiently manage the funds whilemaintaining compliance with their respective regulations and guidelines.OSR oversees sponsored project administration with principal investigators from the earliest stagesof development by assisting researchers in planning, budgeting, assessing risk, interpreting applicableinstructions, negotiating and contracting, complying with policies and regulations, reviewing proposals,and submitting proposals or contracts to the sponsor. The OSR remains in close contact with thedepartments, divisions, and support groups during project inception and development. Working withthe Research Accounting Office, OSR maintains administrative oversight of research projects, onceawarded, through account management andprogress reporting. The Office ensures thatcompliance with regulations is maintained andtracks any changes in the terms of the awardor contract. At the completion of researchprojects, the OSR assists in project closeoutresponsibilities, including final projectreporting and invention disclosures, whenrequired. Project reporting includes compilationand maintenance of the OSR’s sponsoredresearch database. OSR coordinates investigator,departmental, and division reportspertinent to the research project as well asreports to the sponsor. Throughout thisprocess, OSR works closely with the FinanceDivision and Institutional Relations andDevelopment.The OSR and other administrativeoffices involved in the research mission ofCCF continue to review current issues inresearch administration. More efficient andeffective processes are reviewed to explorenew directions and offer expanded services toits people it serves: the researchers, departmentaladministrative personnel, and sponsors.Plans for increased service levels are critical tothe Office’s success and will help researchersand CCF continue in its growth in the researchmission of the organization.F. John Case, Ed.D.Executive Director of Finance, Academic Affairs180

RESEARCH CORE SERVICESThe Research Core Services provide investigators at the Lerner Research Institute (LRI) withaccess to technology and instrumentation that is too costly, complex, or otherwise inaccessible toindividual laboratories. The cores rely on feedback and direction from the research staff and arecommitted to enhancing the research performed by all LRI scientists and clinician-scientists of theCleveland Clinic as a whole.Demand for and usage of the Core Services continues to increase, especially for the GeneExpression Core, the Hybridoma Core, the Flow Cytometry Core, and the Central Cell Services Core,and requisite expansions are under way to meet the rising demand.A significant change in the Gene Expression Core involves the beginning of a new interinstitutionalcollaboration: The Affymetrix section of the Gene Expression Core has teamed up withthe Affymetrix Core of Case Western Reserve University (CWRU) to provided expanded capacity andservices to all Cleveland investigators. Drs. Martina Veigl and Patrick Leahy of the CWRU AffymetrixFacility have presented talks for investigators at CCF and, together with Dr. Colmenares, are planning onoffering a course on analysis of microarray data at CCF.Expanded software options for analysis of microarray data are provided both at CCF and throughthe CWRU Affymetrix service. These include GeneSpring, Spotfire, and Microarray Suite. TheAffymetrix Micro DB and Data Mining Tool are also available on computers at both CWRU and CCF.Expansion of the Biological ResourcesUnit (BRU) Core continues under the abledirection of Dr. Stan Dannemiller. The BRUwill move into its new vivarium in the fall of2003. This facility will supplement existinganimal housing and allow for expansion ofboth the animal census and of servicesprovided by the BRU for CCF investigator.As a consequence of the newvivarium’s construction, the Transgenic andKnockout Mouse Core operations have beentemporarily halted. The Core expects toreopen in its new quarters within the newvivarium building. In the interim, in anothernew interaction between CCF and CWRU,transgenic and knockout mouse services areavailable through core services at CWRU.The Imaging Core has recently added aLeica microdissection microscope.All the cores continue to explore newdirections and offer expanded services, whichare described in more detail on the followingpages.RESEARCH CORESERVICESDIRECTORClemencia Colmenares, Ph.D.COORDINATORJeanne InemanMission Statement of theResearch Core Services:To provide high qualityservices, expertise,education and resourcesin support of research atthe Cleveland ClinicFoundationClemencia Colmenares, Ph.D.Director, Research Core ServicesWebsite: http://info.lerner.ccf.org/services/181

BIOLOGICAL RESOURCES UNITThe Cleveland Clinic Foundation’s Biological Resources Unit (BRU; formerly Animal CoreFacilities) is an integral unit of the Lerner Research Institute. The BRU’s function is to give support inmatters related to animal experimentation to all the scientists of the Foundation who use animals intheir research projects. This function is carried out with full consideration of the animals’ welfare. Thus,all efforts are made to minimize the number of animals used and any physical and psychological distressexperienced.The facilities are located in multiple areas of the CCF, but centrally managed. A variety of animalspecies, from rodents to ruminants, are housed in those areas. State-of-the-art surgical facilities andequipment are provided in each of the sites with cineangiography units (with digitizing and freeze-framecapabilities) and general instrumentation support. Approximately 30 animal technicians staff the BRU,many of them certified by the American Association for Laboratory Animal Science (AALAS).The services provided by the BRU encompass all aspects related to animal husbandry andexperimental manipulation, ordering and procurement of animals, quarantine procedures, perioperativeanimal care, health surveillance and consultation on animals models. Implementation of, and compliancewith, the Animal Welfare Act and Public Health Services policies are conducted in conjunction with theInstitutional Animal Care and Use Committee (IACUC). In 2001, the CCF animal care and use programwas evaluated by the Association for the Assessment and Accreditation of Laboratory Animal Care,International (AAALAC) and received full accreditation as part of an ongoing program. The BRU’sfacilities have been AAALAC accredited since 1976. During the past year, the BRU has implemented anew animal users’ training and occupational health program to meet NIH initiatives. Implementation ofincreased security measures is under way.The BRU will move into its new vivarium in the fall of 2003. This facility will supplementexisting animal housing and allow for expansion of both the animal census and for services provided bythe BRU for all CCG investigators. It will also consolidate satellite housing areas that have providedlimited temporary expansion space.Website: http://info.lerner.ccf.org/services/bru/BIOLOGICAL RESOURCES UNITDIRECTORStanley D. Dannemiller, D.V.M., M.S., Dipl. A.C.L.A.M.ASSISTANT DIRECTORJori Leszczynski, D.V.M.182MANAGERSLinda McCort, A.S., L.A.T.G.Lonnie Thomas, B.A., L.A.T.G.RESEARCH COMPLIANCECOORDINATORNatalie L. Mays, B.A., L.A.T.G.CHIEF TECHNICIANHoward Roper, L.A.T., I.L.A.M.ANIMAL SURGERY SUPERVISORRobert LewisANIMAL SURGERY TECHNICIANSJames Howard, L.A.T.Terry Keller, L.A.T.Ronald Porter, L.A.T.MICHELLE WALSHCHRONIC CARE UNITTerry Fye (Research Monitor)VETERINARY TECHNICIANMelanie Hoffner, R.V.T.ANIMAL TECHNICIAN GROUP LEADERSJohn Bolden, A.L.A.T.China Morrison-KeetonScott Zeigler, L.A.T.ADMINISTRATIVE SUPPORTPeggy Buckner (Dept. Coordinator)Deborah Underwood (ClericalAsst.)ANIMAL TECHNICIANSPatrina BarlowDavid BredaDawn CadeJanie CallowayMary ColemanLaToya DavisPaul DevineStephanie EncarnacionDeborah FlorioJanice FranklinCharles FullerElizabeth HarmonDonnie HayesYvonne JamesYegeniy Kononov, L.A.T.Larry Kramer, Jr.Robert McCutcheonDrazen Mikulec, L.A.T.Mary MitchellRebecca ShrefflerJose SolanoDeAndre TerrellHenry Wyant

MOLECULAR BIOTECHNOLOGY COREThe Molecular Biotechnology Core laboratory provides consultation and technical support servicesin DNA sequencing protein analysis, peptide chemistry, amino acid analysis, and bioconjugate chemistry.These services are made available to all investigators of the Lerner Research Institute and the ClevelandClinic Foundation.High-throughput DNA sequencing systems based on capillary electrophoresis have revolutionizedthe genome sequencing projects dramatically in the last several years. The blueprints of human,Arabidopsis, Caenorhabditis elegans, Drosophila, yeast, Escherichia coli, Haemophilus influenzae, and severalother microbial genomes have now been sequenced. Similarly, the huge data-processing power ofcomputers and increasing availability of powerful laboratory data management software in recent yearshas made annotation of genome sequences possible in databases. This has stimulated mining of databases,using powerful technologies such as high-throughput mapping of single-nucleotide polymorphisms(SNPs) through the entire genome. SNPs are expected to provide clues to predisposition to disease andresponse to drugs. As a consequence, life scientists are using DNA sequencing as a routine researchpractice in their laboratories to understand fundamental biological processes at molecular level. Therefore,DNA sequencing will continue to be a major task in the foreseeable future as gene sequences aresurveyed more often. In the post genome era, the Molecular Biotechnology Core laboratory at the LernerResearch Institute will continue to play an important role by providing crucial research support in theareas of customized DNA sequencing and SNP identification. The DNA sequencing samples submittedby CCF investigators to the Core has increased steadily. To meet this demand, a MegaBACE 1000workstation, which uses capillary array electrophoresis, became operational in January 2001.The core laboratory also offers N-terminal amino acid sequence analysis by Edman degradationchemistry and amino acid composition analysis by the orthophthalaldehyde (OPA) method. Facilities forin-gel digestion and peptide separation (mapping) of N-terminal blocked protein for internal peptidesequence analysis are available, although mass spectrometry is lately becoming a method of choice inprotein identification. In the area of peptide chemistry, the laboratory performs peptide synthesis by thesolid-phase method using Fmoc chemistry. To exploit the full potential of synthetic peptides in biomedicalresearch, the Core laboratory has an ABI 431A synthesizer (Applied Biosystems) and an Omega 396multiple peptide synthesizer (Advanced ChemTech). With the installation of the new 396 Omegamultiple peptide synthesizer, CCF investigators have the flexibility of synthesis at the 50-, 100- or 250-mmole scale. Every peptide synthesized is rigorously evaluated routinely by mass spectrometry, HPLCanalysis and by N-terminal sequencing if necessary with the ultimate goal of providing the correctintended peptide sequence to the investigator.MOLECULARBIOTECHNOLOGYCOREDIRECTORSatya Yadav, Ph.D.SCIENTIFIC ADVISOREDWARD PLOW, PH.D.SUPPORT PERSONNELTalat Haqqi, M.S.Adriana Panciu, B.S.Wei-Zhen Shen, B.S.Xiaolan Zhao, M.D.Web site: http://www.lerner.ccf.org/services/molecbiotech/VIRUS COREThe Virus Core Facility provides services and expertise, including scientific consultations, relatedto infectious and noninfectious viruses. The Core provides such essential services as making virus stocks,inducing large-scale expression of cloned proteins in baculovirus, constructing retroviral and adenoviralvectors expressing cDNAs, and making respective recombinant virus stocks.Currently, the core maintains the following virus stocks: Human immunodeficiency virus (HIV-1), respiratory syncytial virus (RSV), parainfluenza virus (PIV), recombinant baculovirus, adenovirus,retrovirus and other viruses as requested. For protein expression in baculovirus, primary services includeconstructing recombinant baculovirus expressing cloned cDNA (with or without His-tag), makingrecombinant virus and performing small-scale infections to characterize gene expression, determining theoptimum conditions of harvest time and infection ratio that gives maximum protein yield, and carryingout large-scale infection to produce large quantities of protein. The Core also offers a complete range ofservices for the construction of recombinant adenoviruses, including cloning, plaque purification,screening, amplification, double cesium chloride gradient purification, and viral particle titration.The Virus Core Facility is involved in new collaborative projects that will generate valuableresearch materials for the research institute. The Virus Core has trained numerous scientists in how togenerate recombinant baculoviruses, adenoviruses and retroviruses and has designed experiments fortheir research work. We are also in the process of making new adenoviral vectors that will regulate geneexpression in a tissue-specific manner.VIRUS COREDIRECTORRatan K. Maitra, Ph.D.SCIENTIFIC CONSULTANTSAmiya K. Banerjee, Ph.D.Joseph DiDonato, Ph.D.SUPPORT PERSONNELDonald R. RempinskiWeb site: http://www.lerner.ccf.org/services/virus/183

IMAGING COREIMAGING COREDIRECTORJudith A. Drazba, Ph.D.HISTOLOGY MANAGERLinda VargoELECTRON MICROSCOPYMANAGERMei YinDIGITAL IMAGING TECHNOLOGISTSDmitry LeontievJoydeepSarkarPostdoctoral FellowAmit Vasanji, Ph.D.The Imaging Core Facility consists of three divisions: Digital Imaging, Histology, and ElectronMicroscopy. The facility provides a wide range of advanced imaging and scientific consultation servicesfor all investigators in the Lerner Research Institute.The primary mission of the facility is to assist investigators in producing high-resolution images ofcells and tissues using light and electron microscopy. For those requiring basic light microscopy the corewill help determine appropriate staining protocols and assist with the proper use of one of two newupright Leica DMR microscopes equipped for transmitted light and fluorescence microscopy. Thesemicroscopes are used for observing specimens mounted on slides. Digital images can be captured with ahigh-resolution Princeton Instruments MicroMax cooled CCD camera using ImagePro Plus Capture andAnalysis software. The microscopes are equipped with a full range of Chroma filters for fluorescentimaging. Living specimens in dishes or flasks can be observed on the new inverted Leica DMIRBfluorescence microscope. This microscope is also equipped with a PI MicroMax cooled CCD camera,ImagePro Plus Capture and Analysis software, and a full range of filters for standard fluorochromes andfluorescent protein tags. Another inverted Leica DMIRB fluorescence microscope is equipped for livetime-lapse imaging with a high-resolution Photometrics CoolSnap cooled CCD camera, a Sutter filterwheel, Uniblitz shutter, Prior Z-focus motor, stage-mounted heat/CO2 incubator and MetaMorphsoftware.Those requiring optical sectioning capability to better resolve the fluorescence in their samples canuse one of two new state-of-the-art Leica TCS-SP spectrophotometric laser scanning confocal microscopes.These instruments provide three-dimensional information from the sample and are eachequipped with four lasers for excitation at 351, 364, 457, 488, 514, 568 and 633 nm. Emitted light canbe detected interactively from 350-800 nm in 5-nm increments. We are now routinely imaging quadruple-labeledfluorescence specimens.The facility provides a full range of histology services including processing and embedding inparaffin, sectioning, H & E staining, a broad range of special stains, and frozen sectioning. Conventional(non-fluorescent) immunostaining of cells and tissues is also available.The EM services comprise conventional transmission electron microscopy, as well as antigenlocalization by immunogold labeling techniques. Conventional TEM services include routine processingof samples, (glutaraldehyde-osmium fixation, dehydration and plastic embedding), thick or ultra-thinsectioning, and photomicrography of thin sections. Possible samples include fresh tissue specimens,cultured cells growing on a substrate, cells in suspension, or subcellular fractions (for purity determinations).Sub-cellular localization of antigens within cells by immunogold localization can also be performed.In addition to the microscopy services investigators also have access to the Image Processing lab.This includes several NT workstations, a flatbed color scanner, a photographic quality Fujix Pictrographyprinter, and a high-resolution Polaroid Sprintscan 4000 slide scanner that can scan conventional 35 mmslides or specimens on microscope slides that are too large for conventional compound microscopy. Thisequipment provides investigators with sophisticated tools and assistance for preparing their images forpresentations and publication.This year, the facility has acquired a Leica MicroDissection microscope that will allow investigatorsto precisely excise cell groups, single cells or even parts of cells out of tissue sections or cultureswithout touching them or contaminating them. Such explants could then be used for RNA, DNA orprotein analysis.Web site: http://www.lerner.ccf.org/services/imaging/184

MASS SPECTROMETRY CORE I: PROTEIN SEQUENCINGThe first Mass Spectrometry Core laboratory was created in June 1999 with the goal of makingadvanced methods and instrumentation for protein sequencing available to the investigators in theCleveland Clinic. The laboratory is equipped with a Finnigan LCQdeca ion trap mass spectrometrysystem, with a capillary column liquid chromatography inlet and a microspray ionization source, and aMicromass TofSpec 2E matrix-assisted laser desorption/ionization time-of-flight mass spectrometrysystem. These systems are able to acquire a variety of sequence-specific information that is used tosearch the growing protein and genome sequence databases to identify a protein.The key aspect of the protein sequencing and identification experiment is the simple fact that,because of the sensitivity of the experiment, any protein band that can be visualized in a Coomassie bluestained gel can be sequenced and identified. The analysis begins by cutting the protein band of interestout of the gel and digesting it directly in that piece of polyacrylamide. This digest produces a relativelylarge number of peptides derived from the protein that are sequenced in each analysis. As a result, theexperiment is direct, rapid, sensitive, and identifies the protein based on the characterization ofapproximately one half of its sequence. The sensitivity of the experiment also allows one to considersequencing proteins in silver-stained gel bands, but extra care must be taken to use compatible stainingtechniques and to avoid contamination of the gel with background proteins.The laboratory also has a variety of electrophoresis systems that may be of interest to investigators.These systems include 2D electrophoresis systems that use immobilized pH gradient strips for theisoelectric focusing and high resolution, pre-cast gels for the second dimension. These systems areavailable for the development of 2D methods for complex protein separation problems.MASS SPECTROMETRYCORE I:PROTEIN SEQUENCINGDIRECTORMichael Kinter, Ph.D.SUPPORT PERSONNELBelinda B. Willard, Ph.D.Andrew Keightley, Ph.D.Lemin ZhengWebsite: http://www.lerner.ccf.org/services/mass_spec/MASS SPECTROMETRY CORE II: MOLECULES/SMALL COMPOUNDSIn 2001, the Lerner Research Institute established a second Mass Spectrometry Core, functioningas both an investigative core and a service core research facility. The main focus of this core is quantificationof molecules in complex matrices and structural characterization of small compounds. The coreis designed to meet the growing needs of investigators to develop analytical methods for detection andquantification of biomarkers in plasma, tissue and other biological materials.The core is equipped with a Micromass Quattro Ultima triple quadruple mass spectrometry systemand a Beckman HPLC equipped with autosampler, photodiode array and fluorescence detectors. Themass spectrometer has two ionization sources available: electrospray ionization (ESI) and atmosphericpressure chemical ionization (APCI). The effective mass range is 4180 Da for singly charged species andhigher for multiply charged species. Reverse phase HPLC/MS or online HPLC tandem mass spectrometry(LC/MS/MS) analysis is available with this instrument.The MS II Core, under the direction of Dr. Stanley Hazen, was made possible by the award of aninstrument grant from the NIH. The core manager, Dr. Renliang Zhang, has both biochemistry andanalytical chemistry background and extensive experience with HPLC and mass spectrometry.MASS SPECTROMETRYCORE II:MOLECULES / SMALLCOMPOUNDSDIRECTORStanley Hazen, M.D., Ph.D.MANAGERRenliang Zhang, M.D., Ph.D.http://www.lerner.ccf.org/services/ms2/185

HYBRIDOMA COREHYBRIDOMA COREDIRECTOREarl PopticThe Hybridoma Core was established to provide investigators at the Lerner Research Institutewith a resource for monoclonal antibody production. The facility also provides consultation services toassist the investigator in all aspects of monoclonal antibody production. Services are performed understrict quality-control guidelines appropriate for antibodies that may have commercial potential.Monoclonal antibodies are extremely valuable tools in both the research and clinical settings.There are two phases of monoclonal antibody production: development of the hybridoma and productionof the monoclonal antibody from the established hybridoma cell line. In brief, hybridoma developmentinvolves immunizing mice with the antigen of interest and fusing splenocytes of B-cell lineage witha mouse myeloma cell. Theoretically, fusion of these two cell types should result in a hybrid cell(hybridoma) that is immortalized and that secretes antibody. Single-cell cloning ensures that thehybridoma cell line is derived from a single clone. Analysis of the supernatant from the cultures by theinvestigators should determine if the resultant antibody has the desired specificity.The second phase of monoclonal antibody production is the actual production of the antibodyfrom the established hybridoma cell line. Several in vitro and in vivo methods are available. TheHybridoma Core offers three methods for monoclonal antibody production from hybridoma cell lines,two in vitro (tissue culture and the Integra system) and one in vivo (ascites production). In the last fewyears, there has been a move in the scientific community to seek alternative methods to avoid orminimize the use of animals in research. The Core will only use the ascites method if the in vitro methodsfail and if the investigators receive approval by the CCF’s Institutional Animal Care and Use Committee.The majority of hybridoma cell lines will grow in vitro; the choice of method depends on the amount,concentration, and purity of antibody required.The Hybridoma Core offers both stationary (T flasks) and suspension (roller bottle/spinner flasks)cultures. The Core is now able to offer both the Integra flask system and the hollow fiber bioreactormethod. In this system, the cells are maintained in a compartment separated from the media reservoir bya selective membrane, and the antibody is secreted in a relatively small volume of the extracapillaryspace. The advantage of these systems is that the antibody is much more concentrated and of highpurity.The Core also offers polyclonal antibody production, on a limited basis. Once the new BiologicalResources Unit facilities are completed in 2003, the Core plans to expand this service, increasing thenumber of species or offering both monoclonal and polyclonal antibody production.Website: http://www.lerner.ccf.org/services/hybridoma/FLOW CYTOMETRY COREFLOW CYTOMETRYCOREDIRECTORRobert L. Fairchild, Ph.D.MANAGERCathy StankoSUPPORT PERSONNELDolly KlingmanCONSULTANTAnne CotleurThe overall goal of the Flow Cytometry Core Facility is to provide investigators in the LernerResearch Institute, the Taussig Cancer Center, and clinical investigators throughout the Cleveland ClinicFoundation with a resource for analytical and preparative studies of cells using flow cytometry. The Coreutilizes two FACScans for analytical studies of cells and a FACSVantage for cell sorting and applicationsrequiring ultraviolet wavelengths of light.The Core offers many services for the analysis and/or isolation of cells following staining ofcellular proteins or DNA with fluorochrome-labeled reagents. These services include:• Single-, two-, and three-color analysis of cells. List mode acquisition and storage of dataallows for analysis and presentation in many different formats, according to the needs and preference ofthe investigator;• Sterile isolation of selected cell populations on the basis of specific protein expression, cellsize, or DNA content;• Cell cycle/DNA content analysis of cells;• Analysis of cells undergoing programmed cell death or apoptosis using the TUNEL assay;• Studies to detect intracellular Ca 2+ flux following cell surface receptor engagement with ligand.• In addition, the FACSVantage is equipped with an Autoclone device that allows sorting ofspecific cells and delivery of a single sorted cell to each well of a 96-well tissue culture plate.• In October 2001, the Core unveiled service using a 3-laser, 6-color analytical machine. TheBecton Dickinson LSR will allow investigators to stain and analyze up to 6 parameters on a single sampleof cells. This resource ill expand the analysis capability using rare samples of cells, such as those derivedfrom patientsAll acquired data is stored in files linked to the computer network to enable data retrieval fromlaboratory or office computers.Website: http://www.lerner.ccf.org/services/flow/186

CENTRAL CELL SERVICES COREThe Central Cell Services Core was established in 1999 to provide cell services, media and solutionpreparation, glassware washing and sterilization for all investigators at the Lerner Research Institute andthe Cleveland Clinic Foundation. The services offered are efficient and convenient.The primary goal of the Cell Services laboratory is to provide researchers with large and smallquantities of healthy viable cells and a variety of specialized cell culture techniques. In 2000, thelaboratory purchased an incubator that maintains a constant temperature of 27° to provide bulkquantities of insect cell cultures. The Cell Core successfully transformed white blood cells with Epstein-Barr virus (EBV). The Cell Services Core has carried out work on several projects, such as insect cellculture, EBV transformations, roller-bottle cultures, mycoplasma testing and embryonic stem (ES) cellculture.As part of a quality control program, the cell services core is equipped with a Mycoplasma andQuarantine facility to ensure that the cells provided are of the highest quality. Cells are originally fromthe American Type Culture Collection (ATCC), grown in media prepared by the media laboratory, frozenat an early passage and are routinely tested for mycoplasma and iso-enzyme analysis to ensure cells arecontaminant free. The Core tests for and eradicates mycoplasma from cell cultures. To date, the cellservices core has carried out mycoplasma testing for all departments of the Lerner Research Institute.The Media preparation facility is a convenient and economical service offered to investigators atthe LRI and the Cleveland Clinic Foundation. This facility continues to steadily increase its volumeoutput. Prior to release, each batch of prepared media undergoes quality control testing for a two-weekperiod to ensure quality and sterility. In addition to a variety of quality-controlled tissue culture andbacteriological media, the laboratory provides balanced salt solutions, buffers and tissue culture reagents.The media laboratory has also accommodated special order requests such as custom media, packaging andshipment of media/solutions to other institutions.The glassware facility offers glass washing and sterilization of all types of glassware and pipettesfrom the laboratories and maintains stocks of all types of sterile glassware in a common storage area.The glasswash core also offers an autoclaving service. Biological indicators are used in the sterilizationprocess to ensure proper sterilization has been reached.CENTRAL CELLSERVICES COREMANAGERCarmel BurnsSUPPORT PERSONNELLaquitta AndersonDorothy BanksMilagros BranchPamela ClarkArlene DuncanGloria Hunter-RodgersNatasa PopovicDora SimmsWeizhen WangLinda WashingtonCarolyn YoungGENE EXPRESSION COREWebsite: http://www.lerner.ccf.org/services/cell/The Gene Expression Core was established in 1998. The primary goal was to allow researchersaccess to state-of-the-art microarray technology for large-scale gene expression analysis. The Affymetrixsection of the Gene Expression Core has teamed up with the Affymetrix group at Case Western ReserveUniversity to provide expanded capacity and services to all Cleveland investigators. Drs. Martina Veigland Patrick Leahy, who oversee operations of the CWRU facility, have presented talks for investigatorsat CCF and, together with Dr. Colmenares, plan to offer a course on analysis of microarray data at CCF.Expanded software options for analysis of microarray data are provided both at CCF and theCWRU Affymetrix service; these include GeneSpring, Spotfire, and Microarray Suite. The AffymetrixMicro DB and Data Mining Tool are also available on computers at both institutions.The CCF Core also provides an ABI 7700 Sequence Detection System, used for real-timequantitative PCR. The instrument is widely used by numerous investigators at CCF.GENE EXPRESSIONCOREDIRECTORClemencia Colmenares, Ph.D.Website: http://info.lerner.ccf.org/services/genechip/PHOTOGRAPHIC SERVICES COREThe Photographic Services Core provides traditional and digital photography services with anemphasis on the rapid turnaround for research materials required for grants, publications, and presentationsby members of the Lerner Research Institute. The comprehensive services offered include copyingof gels, slides, and other hard copy, processing of microscopy films, and production of black/white orcolor slides and publication-quality prints.The Core uses digital photographic processing to supplement its traditional “wet-chemistry”services. The principal function of the facility is the production of publication-quality color or black/white prints and slides from files generated by either Macintosh- or PC-compatible computers. Digitalscanning of 35-mm slides, gels, and other hard copy for printing or transformation to a computerreadablefile is also available. Consultation on typographic and other technical issues is offered to allinvestigators.PHOTOGRAPHICSERVICES CORESUPPORT PERSONNELJames Lang187

LERNER RESEARCHINSTITUTECOMPUTING SERVICESDIRECTOREldon M. Walker, Ph.D.DESKTOP SUPPORT MANAGERJeff HowellINTEGRATION / NETWORKCOMPUTING MANAGERMichael BusheyUNIX ADMINISTRATION MANAGERStefan GanobcikWEB DEVELOPERSytze van der LaanSYSTEMS ANALYSTSJames FeldkircherOmar NepomucenoW. Charles SimciakTECHNICIANSPeter BalintStacey FearsLinda McConnellMark PedersenSantino RizzoNETWORKING SPECIALISTJames SettyCOMPUTING SERVICESThe Lerner Research Institute Computing Services group provides support for the desktop, server,and network computing infrastructure of the Institute. We also provide desktop and server support forthe Division of Education. The combined supported user base is greater than one thousand. Platformssupported include Windows 9x/NT/2000, Macintosh, Unix/Linux, and Netware. We provide supportfor shared network disk space and backup, web development, various Internet communication andcollaboration protocols, remote access, and application and print servers. We maintain domain nameservices for the /19 CIDR IP allocation (32 subnets) assigned to the Lerner Research Institute andadminister the lerner.ad.cchs.net subdomain of the Cleveland Clinic Health System Active Directoryforest.The mission of Lerner Research Institute Computing Services is to support computing in aheterogeneous Biomedical Science and Biomedical Engineering environment. Our Desktop groupprovides custom support of the scientific computing workstation across the Macintosh and Windowsplatforms. Integration/Project services specialize in supporting the interface between computers andscientific equipment and, in cooperation with Unix and Desktop support, works to provide a collaborativecross-platform computing environment. Unix workstations and servers currently participate in aunified computing structure supported by the Unix administration group. Unix variants supportedinclude Sun, SGI, and Linux.The infrastructure that provides connectivity for Lerner Research Institute computing is a highspeed,highly redundant meshed fiber and copper network running on an OC48 (2.4 Gbs) single-modefiber backbone. Service to the desktop is 155 Mbs ATM (copper or fiber), 100/10 Mbs ethernet(copper), or 622 Mbs ATM (fiber). LAN services (TCP/IP, IPX, Appletalk) are provided via ATM LANemulation (LANE).Online disk storage exceeds 2 terabytes, which is fully replicated in hardware. Backup is providedby a dual-head 30-cartridge LTO tape autoloader and two dual-head 20-cartridge DLT-8000 autoloaders.Shared advanced computing resources include a quad-processor SGI 3400 with 4 gigabytes of memory, aquad-processor SGI Origin 200 with 4 gigabytes of memory, and a dual processor SGI Origin 200 with 2gigabytes. Other resources for computing and calculation include a quad-processor Intel Xeon-basedWindows NT server with 4 gigabytes of memory, and a 14 node Linux cluster. Other Windows NT /2000, Linux, SGI, and Solaris server resources are available for special hardware or software needs.It is clear that scientific resources and applications will become more highly network-oriented anddistributed in nature, and that the need for raw computing power will continue to increase. It is the goalof Lerner Research Computer Services to move with the shifting paradigms. To that end, we encouragethe investigators we support to maintain a dialog with us. Contact information, support request forms,breaking news, and information of general interest concerning computing in the Lerner ResearchInstitute can be found on our website:http://computing.lerner.ccf.orgEldon M. Walker, Ph.D.188

ELECTRONICS CORE AND MECHANICAL PROTOTYPE LABORATORYThe Electronics Core and the Mechanical Prototype Laboratory are among the Lerner ResearchInstitute’s core services providing support for physicians, surgeons, and investigators throughout TheCleveland Clinic Foundation. These areas also form two of four subgroups within the Design &Technology Group (DTG) in the Department of Biomedical Engineering. The other functional groups inthe DTG include the Polymer Laboratory and the Engineering Design & Analysis Group. The mission ofthis integrated cross-functional team is twofold: (1) provide technical support for CCF research andclinical activities and (2) promote the development of innovative medical devices that can advancepatient care. Working closely with the CCF’s technology transfer office (CCF Innovations), the DTGdesigns and prototypes new medical device concepts with the goal of bringing these new inventions tothe medical marketplace.MECHANICAL PROTOTYPE LABORATORYThe Mechanical Prototype Laboratory (MPL) provides a wide range of design, fabrication,customization and repair services for mechanical devices and equipment in support of CCF research andclinical activities. MPL personnel are available to meet with researchers and clinicians to discuss theirideas and to develop ways to implement them. Our staff is experienced in precision machining ofmetals and polymers, welding of structural and exotic metals and mechanical repair of existing equipment.The MPL is located in ND1-27, on the first floor, West Wing of the Lerner Research Institute,near the corner of Carnegie and East 96th Street.The MPL features state-of-the-art Computer Numerical Control (CNC) equipment for thefabrication of highly complex devices or when identical multiple components are required. The CNCequipment list includes: a 5-axis vertical machining center, a 4-axis lathe, and a 4-axis Wire ElectrodeDischarge Machine (W-EDM). A Coordinate Measurement Machine (CMM), which enables precisioninspection of devices fabricated, is also located in the laboratory. Additional machining equipmentincludes: 2 ½-axis CNC milling centers, a 2-axis CNC lathe, 3-axis manual milling machines, manuallathes, universal tool grinder, and surface and sectioning grinders. Sheet-metal work equipment, weldingcapabilities, and heat treating of metals are also available.MPL personnel have years of practical design experience and can provide the mechanicalexpertise needed by clinicians and researchers. The laboratory personnel can work from conceptsketches to design and fabricate new devices, test fixtures, and equipment. Documentation of thenewly created part or device is also available through the generation of two- or three-dimensionalcomputer-aided design (CAD) drawings. These CAD drawings can then be converted into computeraidedmanufacturing (CAM) programs that are directly fed into the CNC machines to fabricate thedesired parts.A cornerstone of the laboratory is the growing capability for the customization, refurbishing,sharpening, and repair of a variety of surgical devices and instruments. This service can reduce the needto replace costly instruments and provide “personalization” of instruments to meet the needs of thesurgeon. The MPL has built replicas of discontinued devices. The MPL can help staff membersdevelop a custom device that may enable them to complete projects or procedures.The equipment at our disposal allows us to create high-precision devices for a wide range ofclinical or research applications. Examples of recent projects include: (1) design and fabrication of adevice to simulate slipping injuries for humans; (2) extensive design and development of test fixtures formicro material testing of cartilage, tendon and ligaments; (3) design and fabrication of components andphantoms for a micro-CT system as well as a 10' w x 14' l x 7' h lead-lined room to enclose the CTsystem; and (4) design, development, and preliminary testing of new medical devices for laparoscopicorthopedic and urologic surgeries.MECHANICALPROTOTYPELABORATORYMANAGERAnthony ShawanSUPPORT PERSONNELEdward CloesmeijerHelmuth KotschiJames ProudfitBrian SauerWalter ZimmerWeb site: http://www.lerner.ccf.org/bme/dtg/prototype/189

ELECTRONICS COREELECTRONICS COREMANAGER AND SENIORPRINCIPAL RESEARCH ENGINEERBarry KubanSENIOR RESEARCH ENGINEERFarhad BahrehmandENGINEERING RESEARCHTECHNICIANSRaymond DessoffyKevin WatersThe Electronics Core provides electronic design, fabrication, repair, and surgical support servicesfor all departments within The Cleveland Clinic Foundation. The Electronics Core can design and buildcustom electronic devices that are unavailable from any other source and, in many cases, can produceelectronics that are better suited to an investigator’s needs at a lower cost than similar commerciallyavailable items. Timely on-site support for the life of the product, including repairs, changes andupgrades, is a significant benefit of this service.Services:• Complete electronic circuit design and fabrication, from concept to finished product• Documentation of projects, including computer-generated schematics and circuit boardlayouts, which greatly reduces the cost of replication and future development• Experienced microprocessor-based design capabilities for simplifying complex functions• Resident computerized printed-circuit prototyping equipment provides accurate and reproduciblecircuit boardsData Acquisition System Design:• Consultation on transducer selection and custom transducer/sensor design• Design and assembly of computer-operated or stand-alone data acquisition systems• Design and setup of data capture and storage systemsLaboratory and Office Equipment Repair:• Repair of most electronic and electromechanical equipment at rates that are significantlylower than those of outside vendorsAnimal Surgical Support Services:• Blood (or other in vivo) pressures• EKG• EEG• CMAP (compound muscle action potential)• Arterial and venous blood flow• Cardiac output• Other signals from experimental devices and instrumentation, including area and volumesignals from ultrasonic imaging devices• X-ray services• Consultation for experiment design• Custom cabling/wiring for interconnection of instrumentationWeb site: http://www.lerner.ccf.org/bme/dtg/electronics/190

RESEARCH EDUCATION OFFICEThe 2002-03 development of ResearchEducation Office (REO) represents amajor commitment of the LernerResearch Institute to training and researcheducation programs of nearly 250 postdoctoralresearch fellows and over 100 graduate students.The mission of this office is to recruit qualifiedindividuals who wish to further their scientificcareers by participating in and contributing toleading-edge biomedical research and toencourage career development and facilitate thetransition into a permanent position.The creation and maintenance of afunctional and dynamic website is among themany responsibilities of the REO. With theexpertise of Research Computing Services, theDirector, Dr. Marcia Jarrett, has developed theREO working website (http://www.lerner.ccf.org/education/) to advertisepostdoctoral and graduate student researchtraining opportunities in LRI laboratories and toinform visitors of the newly created support andcareer development programs of the REO. Inaddition, a current electronic PostdoctoralFellow CV directory was created and is maintainedfor use by LRI principal investigators.Recruiting materials, campus tours andinformational interviews for prospectivecandidates are now available to LRI principalinvestigators recruiting postdoctoral fellows andgraduate students for their laboratories throughthe REO. In addition, the REO sponsors andorganizes graduate student symposia showcasingthe research efforts of graduate students fromregional universities as well as faculty researchsymposia designed to introduce new studentsenrolled in affiliate graduate programs to thedepth and variety of research programs availablethroughout the LRI.The REO assists the departments of theLRI with administrative support for all LRIgraduate students and advisors. Principalinvestigators of the LRI support the research ofgraduate students in LRI laboratories throughjoint faculty appointments at three areauniversities: Cleveland State University (AppliedBiomedical Engineering, Biology, and Chemistry),Case Western Reserve University (BiomedicalEngineering, Biomedical Scientist TrainingProgram, Physiology and Biophysics), and KentState University (Biochemistry and Pathobiology).Each new postdoctoral fellow and graduatestudent meets with Dr. Jarrett when he or shearrives for a research orientation into ourscientific community and is provided withprocessing-in instructions, maps, contact lists andother materials. In addition, each postdoctoralfellow and graduate student schedules an REOexit interview upon completion of their researchtraining. As a result, the new LRI traineeMarcia Takacs Jarrett, Ph.D.database maintained by the REO containscurrent, complete and relevant data on allpostdoctoral fellows and graduate students inaddition to historical and forwarding information.The database is an invaluable resource for salaryreviews, institutional grant applications, careerdevelopment programs and other supportinitiatives for our trainees.The REO also functions on behalf of theresearch trainee programs as a central communicationsource between the LRI and CCF’s Divisionsof Education and Marketing and the Office ofProfessional Staff Affairs.RESEARCH EDUCATIONOFFICEDIRECTORMarcia Takacs Jarrett, Ph.D.ADMINISTRATIVE ASSISTANTRobin WebsterWEB MASTERSytze van der Laanhttp://www.lerner.ccf.org/education/191

MEDICAL SCIENTIFIC COMMUNICATIONSMEDICAL SCIENTIFICCOMMUNICATIONSMEDICAL SCIENTIFIC WRITERRussell J. Vanderboom, Ph.D.The Lerner Research Institute addresses the need to provide information to several receptiveaudiences through our Medical Scientific Communications. The bimonthly newsletter Notations providescurrent news regarding staff, programs, grants and activities within the Lerner Research Institute.Notations is directed toward scientific staff and clinical physicians within the Cleveland Clinic Foundation.The Lerner Research Institute Scientific Report is a comprehensive compilation of researchprograms conducted within the laboratories of more than 100 LRI principal investigators in our departments,centers and bridge programs. Support services are also summarized in the annual report. This report is intendedto inform scientific peers, industrial associates, and interested scientific professionals regarding details ofthe Institute’s research projects and the individuals who direct those studies.The Lerner Research Institute Web Site offers many facets of in-depth information about theInstitute. The site continues to be reorganized on a streamlined organizational tree, allowing easy touchlink access to program descriptions, employment opportunities, staff information, administration,contacts, news and general information (accessed at: http://www.lerner.ccf.org/notations).Medical Scientific Communications facilitates dissemination of news information to the nonscientificaudience through news releases to print, radio, television and general news organizations. Newsabout the Lerner Research Institute, our scientists, our findings, and our accomplishments are targeted atlocal, regional and national readers and viewers.This office functions in close collaboration with the Cleveland Clinic Foundation’s Department ofPublic and Media Relations to most effectively broadcast information about our scientists.Since September 1998, Russell J. Vanderboom, Ph.D., has been the Lerner Research Institute’sMedical Scientific Writer. Dr. Vanderboom previously conducted breast cancer research as a Fellow at theMayo Clinic Scottsdale. He is a graduate of the University of Wisconsin-Madison, where he completedhis doctorate in Endocrinology/Reproductive Physiology. Dr. Vanderboom was a journalist prior to hisgraduate education and worked in public relations and as a newspaper and magazine editor. He can becontacted at:Lerner Research Institute /NB219500 Euclid AvenueCleveland, Ohio 44195Office: 216-444-5830Fax: 216-444-3279E-mail: 192

LERNER RESEARCH INSTITUTEDIVISION AND DEPARTMENTAL ADMINISTRATIONDIVISION OFFICE (NB2-52, MAIL CODE NB21)Paul E. DiCorleto, Ph.D. - Division ChairmanJenienne Geist – Executive SecretaryJames Ellis – Financial Manager, Research DivisionClemencia Colmenares, Ph.D. – Director, Research Core ServicesJeanne Ineman – Coordinator, Core ServicesMartina Steele – Research CoordinatorDiane Bruosta – Secretary, Research Programs CouncilJody Kiss – Secretary, Institutional Animal Care and Use CommitteeRussell J. Vanderboom, Ph.D. – Medical Scientific WriterDEPARTMENT OF BIOMEDICAL ENGINEERING (ND2-06, MAIL CODE ND20)Peter Cavanagh, Ph.D. – Department ChairmanGeorge Sciortino – AdministratorLisa Maher – SecretaryIsabelita Delgado-Dembie – Department CoordinatorCatherine (Katie) Root – Education CoordinatorAdelaide Jaffe – Editorial AssistantChristine Kassuba - Editorial AssistantJudith Sedmak – SecretaryEleanora Voelkel – Coordinator/Orthopaedic Research CenterKathleen Vukovich – Department CoordinatorDEPARTMENT OF CANCER BIOLOGY (NB4-57, MAIL CODE NB40)Bryan R.G. Williams, Ph.D. – ChairmanMary Vavpetic – AdministratorMaxine Cox – SecretaryAudrey Brickenden – SecretaryGail Daniels – SecretaryDEPARTMENT OF CELL BIOLOGY (NC1-128, MAIL CODE NC10)Guy Chisolm, III, Ph.D. – Interim ChairmanJudy Schiciano – AdministratorTheresa Schanz – SecretaryCharlene Mitchell – SecretaryDEPARTMENT OF IMMUNOLOGY – (NB3-37, MAIL CODE NB30)Thomas A. Hamilton, Ph.D. – ChairmanGail Lannum – AdministratorJan Kodish – SecretaryDEPARTMENT OF MOLECULAR BIOLOGY (NC2-124, MAIL CODE NC20)Andrei Gudkov, Ph.D. – ChairmanLinda Webster – AdministratorMary Bartos – SecretaryRosemary Olson – SecretaryDEPARTMENT OF MOLECULAR CARDIOLOGY (NB5-57, MAIL CODE NB50)Edward F. Plow, Ph.D. – ChairmanEric Topol, M.D. – Vice ChairmanRebecca Zuti – AdministratorJoAnne Holl – SecretaryRobin Lewis – SecretaryCindy Davidson – SecretaryJeannette Poruban – SecretaryDEPARTMENT OF NEUROSCIENCES (NC3-137, MAIL CODE NC30)Bruce D. Trapp, Ph.D. – ChairmanCarol Haney, Ph.D. – AdministratorVictoria Pickett – CoordinatorMichelle Barnard – Secretary193

Keyword IndexACESen, I. 129Acoustic traumaHirose 137Adaptive immunityLi 95Adenovirus E1AHarter 106Adrenergic receptorPerez, D. 127Age-related maculardegenerationCrabb 163Hollyfield 160AGEsDaneshgari 52AggregatesPioro 145AgingRunge109AIDSQuiñones-Mateu 115AKTMacklin 140Sizemore 60Allograft rejectionHeeger 93AllograftSiemionow 96AlzheimerSmith, J. 80Amyotrophic lateralsclerosis (ALS)Pioro 145Anesthetic agentsDamron 155Murray 153AngiogenesisAnand-Apte 162Byzova 121AngiotensinKarnik 124Angiotensin-convertingenzymeSen, G. 110Sen, I. 129ANP-r structureMisono 125AntiangiogenesisLindner 173Antimicrobial peptideBevins 89Antiretroviral therapyQuiñones-Mateu 115AntisenseLindner 173AntiviralSilverman 59AortaGreenberg 26Aortic valveVesely 27Apolipoprotein ESmith, J. 80194Apolipoprotein-BDriscoll 72ApoptosisAlmasan 48Chisolm 70Howe 75Larner 94Maytin 33Vogelbaum 61Wolfman 83ArthritisApte 34AsthmaAronica 88ErzurumHazen 74AstronautDavis 19AtherosclerosisCathcart 69Chisolm 70DiCorleto 71Hazen 74Smith, J. 80Atrial natriuretic peptidereceptorMisono 125AutoimmuneHirose 137AutoimmunityTuohy 100Axon guidanceNakamoto, M. 141Basal gangliaSubramanian 149BiomechanicsCavanagh 18Davis 19van den Bogert 20Biomedical DevicesSmith, W. 25BioMEMSFleischman 21Roy 21BladderDaneshgari 52Blood pumpSmith, W. 25BoneKnothe Tate 37Powell 29Bone formationMidura 40Bone graftingMuschler 41Bone lossDavis 19Bone repairMuschler 41Brain developmentKomuro 138Brain imagingFisher 28Brain parenchymal factorFisher 28Rudick 147Brain tumorsVogelbaum 61Breast cancerCasey 50Bruch’s membraneHollyfield 160Ca 2+ availabilityDamron 155Ca 2+ sensitivityDamron 155Calcium homeostasisMurray 153Calcium regulatory proteinsMoravec 126Calcium storesMoravec 126CalciumBhat 154CalmodulinStuehr 97CancerApte 34Banerjee, S. 49Borden 172Chumakov 105Sizemore 60Yi 64Cancer related genesGudkov 104CarboxylationBerkner 120Cardiac diseaseWang.131Cardiac hypertrophySen, S.130Cardiac muscleDamron 155Cardiac myocyteMoravec 126Cardiovascular diseaseHoover-Plow 123Cell adhesionByzova 121Plow 128Cell CycleStacey 111Cell differentiationLefebvre 38Cell divisionWeimbs 82Cell migrationFox, P. 73Cell polarityWeimbs 82Cell sortingZborowski 22Cell spreadingAdams 68Cell therapyZborowski 22Cell-cycle controlAlmasan 48Cerebellar granule cellKomuro 138Cerebral blood flowLuciano 139Cerebrospinal fluidLuciano 139CeruloplasminFox, P. 73Chemokine expressionHamilton 92Chemokine receptorsRansohoff 146ChemokineFairchild 90Ransohoff 146Shu 168CholesterolSmith, J. 80Cholesterol homeostasisMorton 78ChondrocytesBallock 34ChromatinLuse 107ChromosomeRunge 109Clinical laboratory testsStaugaitis 148CNSRansohoff 146Yue 32CoagulationBerkner 120CochleaHirose 137CollagenGraham 25ColonStrong 98Colon CancerCasey 50Composite tissue allograftSiemionow 96Congenital heart diseaseWang 131ContractilityMoravec 126CorAide tmGolding 23CorneaPerez, V. 166Coronary artery diseaseWang. 131Coronary artery morphologyVince 30Cyclin D1Stacey 111CytokineHaque 55Pelfrey 143Thomassen 81Tuohy 100Weimbs 82Yi 64CytoskeletonFox, J. 122

Keyword IndexCytotoxicityLindner 173Dab2Howe 75DefensinBevins 89Dendritic cellsShu 168DevelopmentApte 34Colmenares 51DiabeticDaneshgari 52Davis 19Diabetic footCavanagh 18DifferentiationColmenares 51DNA polβBanerjee, S. 49DNA ReplicationPellett 114Dominant-negative mutantBanerjee, S.49Drug developmentYi 64Drug discoveryBorden 172Gudkov 104EAE/MSTuohy 100Early detection of cancersXu, Y. 63ElectroretinogramPeachey 165Endothelial cellGraham 25Endothelial dysfunctionJacobsen 76EndotheliumDiCorleto 71Endovascular graftingGreenberg 26EntericDiDonato 53EosinophilHazen 74Epithelial cellsWeimbs 82ExonsPadgett 108Extracellular MatrixAdams 68Apte 34Hascall 36Midura 40Fas ligandPerez, V. 166FascinAdams 68FibrosisSen, S. 130FolateJacobsen 76Folate hydrolaseHeston 56FootDavis 19Forward GeneticsStark 112Free radicalsHazen 74G Protein coupled receptorsXu, Y. 63G1, G2 phase of cell cycleStacey 111Gaitvan den Bogert 20Gender differencesPelfrey 143Gene expressionDiCorleto 71Padgett 108Gene therapySubramanian 149GeneticsCasey 50Smith, J. 80Vogelbaum 61Wang, Q. 131Genotoxic stressAlmasan 48GenotypeStaugaitis 148GlioblastomaHaque 55GliomaStaugaitis 148Vogelbaum 61Glutamate carboxypeptidaseHeston 56Glutathione peroxidase-4Chisolm 70GlycosylationDaneshgari 52GM-CSFThomassen 81GPCrKarnik 124Growth PlateBallock 34Harmonic imagingVince 30Hearing lossHirose 137Heart failureGolding 23Heart failureMoravec 126Heart valvesVesely 27Hemodynamic forcesGreenbergHerpesvirusesPellett 114High-density lipoprotein(HDL)Morton 78High-frequency ultrasoundVince 30HIVQuiñones-Mateu 115HomingPenn 79HomocysteineJacobsen 76Host-virus interactionBanerjee, A. 113Human parinfluenza virusBanerjee, A. 113Human tissueStaugaitis 148HyaluronanStrong 98Hascall 36HydrocephalusLuciano 139HypercholesterolemiaGraham 25HypertensionKarnik 124Misono 125HypothermiaMayberg 159IκB KinaseSizemore 60IFN-αQuiñones-Mateu 115IL-1R/TLRs superfamilyLi 95IL-4Haque 55Image analysisFisher 28Image and signal processingVince 30ImagingPowell 29Immune toleranceHeeger 93ImmunityWilliams 62InfarctionPenn 79InflammationAronica 88Cathcart 69DiDonato 53Fairchild 90Fox, P. 73Haque 55Hazen 74Hirose 137Perez, V. 166Ransohoff 146Rudick 147Strong 98Thomassen 81Inflammatory bowel disease(IBD)Bevins 89Strong 98Innate immunityLi 95Inotropic agentsDamron 155InstrumentationFleischman 21Roy 21IntegrinFox, J. 122Macklin 140Plow 128Qin, J. 176Integrin receptorsPlow 128InterferonBorden 172Sen, G. 110Larner 94Lindner 173Silverman 59Stark 112Williams 62Interferon stimulated genesBorden 172InterleukinFinke 91Intravascular ultrasoundVince 30Intravital neurodegenerationPerez, V. 166IntronsPadgett 108Ionizing radiationAlmasan 48Iron metabolismFox, P. 73IschemiaPenn 79Kidney cancerFinke 91Weimbs 82KneeMcDevitt 39Leukocyte adhesionStrong 98LeukocyteRansohoff 146Lipid transferMorton 78LipidCathcart 69Lipoprotein oxidationChisolm 70Lipoprotein remodelingMorton 78Lipoprotein(a)Hoover-Plow 123Low-density lipoprotein (LDL)Morton 78Lysophosphatidic acidXu, Y. 63LysophospholipidsXu, Y. 63195

Keyword IndexMacrophagesHamilton 92Magnetic nanoparticleZborowski 22Magnetic resonance imagingFisher 28Magnetic separationZborowski 22MagnetophoresisZborowski 22MalignancyBorden 172Vogelbaum 61Mass spectrometryCrabb 163Kinter 77Mast cellsHaque 55Membrane fusionWeimbs82Membrane traffickingPerin 144MEMSFleischman 21Roy 21MeniscusMcDevitt 39MetalloproteasesApte 34MetastasisCasey 50MicroarraysPerez, D. 127Micro-CTPowell 29MicroelectromechanicalsystemsFleischman 21Roy 21MicrogravityCavanagh 18MitochondriaPioro 145MitoxantronePelfrey 143MonocyteCathcart 69Motor-function recoveryYue 32Mouse developmentLefebvre 38Movement disordersSubramanian 149mRNA EditingDriscoll 72mRNA StabilityHamilton 92Multiple sclerosisFisher 28Perez, V. 166Trapp 150Pelfrey 143Ransohoff 146Rudick 147Musculoskeletal functionvan den Bogert 20MutagenesisStark 112Myelin proteinsPelfrey 143MyelinationPerez, V. 166Trapp 150MyeloperoxidaseHazen 74Myocyte growthSen, S. 130MyoDHarter 106MyofilamentDamron 155Myofilament calciumsensitivityMurray 153MyotrophinSen, S. 130Myxomatous mitral valvediseaseVesely 27NanotechnologyFleischman 21Roy 21Nerve regenerationSiemionow, M. 96Neural developmentNakamoto, M. 141Neural Synaptic ProteinPerin 144Neural transplantationSubramanian 149Neural controlYue 32NeurodegenerationFisher 28Trapp 150Neurodegenerative diseaseRudick 147NeuroimagingPioro 145Neuromuscular controlvan den Bogert 20Neuromuscular rehabilitationYue 32NeuronMacklin 140Bhat 154Neuronal migrationKomuro 138NeuroprotectionPioro 145NeurotransmitterPerin 144NFkBLi 95Stark 112DiDonato 53Night blindnessPeachey 165Nitric OxideErzurum 54Hazen 74Stuehr 97Thomassen 81Nitric Oxide SynthaseStuehr 97NMRQin, J. 176nobPeachey 165Non-pulsatile pumpsSmith, W. 25Novel gene discoveryGudkov 104Nuclear Hormone ReceptorsBallock 34ObesityHoover-Plow 123Ocular neovascularizationAnand-Apte 162OligodendrocyteMacklin 140OncogeneWolfman 83Organ transplantationPellett 114Orthopaedic bioengineeringKnothe Tate 37OsteoblastKnote Tate 37Midura 40Wolfman 83OsteoporosisMidura 40Muschler 41Powell 29Ovarian cancerXu, Y. 63OxidationHazen 74Cathcart 69Oxidized LDLGraham25Oxidative stessKinter 77p53Chumakov 105Gudkov 104Stark 112PainBhat 154Paneth cellsBevins 89Parathyroid hormoneMidura 40Parkinson’s diseaseSubramanian 149PathogenDiDonato 53PathophysiologyKnothe Tate 37PentraxinPerin 144Peroxisome Proliferatoractivatedreceptors (PPARs)Ballock 34PhosphatasesLarner 94PhotoreceptorHagstrom 164Hollyfield 160PKRWilliams 62PlasminogenHoover-Plow 123Plasminogen ReceptorsPlow 128PlateletsFox, J. 122PoliovirusBanerjee, A. 113Polycystic kidney diseaseWeimbs 82PolycystinWeimbs 82Promoter clearanceLuse 107Prostate cancerByzova 121Casey 50Heston 56Protein identificationKinter 77Protein modificationKinter 77Protein sequencingKinter 77Protein structureQin, J. 176Protein synthesisSen, S. 130ProteoglycansHascall 36ProteolipidMacklin 140ProteomicsCrabb 163proto-oncogeneColmenares 51PTENSizemore 60PTPaseYi 64Pulmonary circulationMurray 153pulmonaryAronica 88Thomassen 81Pulsed electromagnetic fieldsWolfman 83Quantitative microscopyPowell 29RasStacey 111Ras isoformsWolfman 83196

Keyword IndexReceptorBhat 154Receptor tyrosine kinasesNakamoto, M. 141Remodeling mechanismsKnothe Tate 37Renal cell carcinomaFinke 91Weimbs 82ReplicationBanerjee, A. 113RetinaHollyfield 160Hagstrom 164Peachey 165Retinal degenerationHagstrom 164Retinal pigment epitheliumHollyfield 160Peachey 165Reverse geneticsBanerjee, A. 113RibozymesDayie 175RNA functionDayie 175RNA in drug discoveryDayie 175RNA Polymerase IILuse 107RNA ProcessingPadgett 108RNA-Protein interactionsDayie 175RNAse LSilverman 59RNomicsDayie 175RyanodineBhat 154SecretaseSen, I. 129SeleniumDriscoll 72SelenocysteineDriscoll 72SensorsFleischman 21Roy 21Signal transductionKarnik 124Li 95Qin, J. 176Yi 64SignalingFox, J. 122Signalling PathwayHowe 75Larner 94DiDonato 53SilencingRunge 109Skeletal muscle differentiationHarter 106SkeletogenesisLefebvre 38skiColmenares 51SkinMaytin 33Small molecule p53 inhibitorGudkov 104SNARE proteinsWeimbs 82snoColmenares 51Sodium stibogluconateYi 64Quiñones-Mateu 115Sox genesLefebvre 38SplicingPadgett 108Sports injuryvan den Bogert 20STATsStark 112Stem cellsMuschler 41Penn 79Perez, V. 166Trapp 150Stent graftsGreenberg 26StrokeMayberg 159StuctureStuehr 97Structure-functionKarnik 124SurfactantThomassen 81SynaptotagminPerin 144T lymphocytesHeeger 93Aronica 88T CellAronica 88Finke 91Fairchild 90Rudick 147Siemionow, M. 96Shu 168Tuohy 100TelomereRunge 109TGF-βHowe 75ThrombosisByzova 121Plow 128Thrombospondin-1Adams 68Thyroid hormone receptorBallock 34Tissue characterizationVince 30Tissue engineeringKnothe Tate 37Muschler 41Vesely 27Tissue factorChisolm 70Tissue inhibitor of metalloproteinases-3(TIMP-3)Anand-Apte 162TLR signalingSen, G. 110T-lymphocyteFairchild 90TNFr superfamilyLi 95Topoisomerase IIStacey 111Total artificial heartSmith, W. 25Transcript elongationLuse 107Transcript initiationLuse 107TranscriptionDiCorleto 7Transcription factorMaytin 33Sizemore 60Lefebvre 38TranscytosisJacobsen 76Transgenic miceBanerjee, S. 49TransgenicsPerez, D. 127Translational controlFox, P. 73Translational regulationSen, G. 110Translational researchPellett 114TransplantFairchild 90Transplant toleranceSiemionow, M. 96TransplantationHeeger 93Perez, V. 166trypsinBevins 89TULP1Hagstrom 164Tumor immunotherapyShu 168Tumor InfiltratingLymphocytes (TILs)Finke 91Tumor SuppressorSilverman 59Williams 62tumorLindner 173Type VI collagenMcDevitt 39Tyrosine KinaseLarner 94UrologyDaneshgari 52UV lightMaytin 33Vascular biologyFox, J. 122Wang 131Vascular endothelial growthfactor (VEGF)Anand-Apte 162Byzova 121Vascular graftsGraham 25Vascular regulationMurray 153Ventricular assist deviceGolding 23Smith, W. 25Ventricular remodelingPenn 79Vesicular stomatitis virusBanerjee, A. 113Viral mechanismsHarter 106Viral stressSen, G. 110VirologyPellett 114VisionCrabb 163Visual cycleCrabb 163Vitamin B 12Jacobsen 76Vitamin KBerkner 120Vitamin K-dependentproteinsBerkner 120Voluntary motor actionYue 32Wilms tumorWilliams 62Wound healingMaytin 33McDevitt 39YeastRunge 1092-5ASilverman 59197

Illustrations LegendsBIOMEDICAL ENGINEERING—PAGE 11Top Left Triad: Stem Cell technology, the laboratory of George Muschler, M.D. Left Image: In Situ hybridizationexpression of Collagen Type 1 on day 6 Human CTPs in vitro. Control hybridized with sense to bothCbfa 1 and BMP6. Center Image: Proliferation of Human CTPs and Expression of Alkaline Phosphatase onLoaded Coralline HA disks, day 9 culture. Graphic Image on right: Schematic diagram of the osteoblasticstem cell system. This conceptual drawing illustrates the primary candidate populations of stem cells and transitcells thought to be associated with bone formation and remodeling: Vascular pericytes (green), Westen-Baintoncells (orange), type I or pre-osteoblasts (pink), secretory osteoblasts (maroon), osteocytes (brown), liningcells (purple), and adipocytes (yellow). Vascular pericytes may give rise to the Westen-Bainton cells. Pericytesand Westen-Bainton cells may contribute to the formation of pre-osteoblasts and also adipocytes. New osteoblastare added in the region immediately behind the advancing front of osteoclastic resorption. Secretory osteoblastsproduce new bone matrix until they become quiescent on the surface of bone as a lining cells (purple)or become embedded in the matrix as osteocytes (brown), or die via apoptosis. Osteoclast formation is also illustrated.A fraction of the monocytes population in systemic circulation (blue) will become resident in thebone marrow space. Osteoclasts are formed by fusion of monocytes resident in bone marrow to form multinucleatedfunctional units. The nuclei in active osteoclasts continue to be turned over as a result of nuclear lossand ongoing fusion events with new marrow derived monocytes. The black arrow indicates the direction of boneresorption by the osteoclastic front, followed by bone formation.Double panel, top right: Normal human femur contrasted with Human femur degenerated by osteoporosis(Top right twin panels). Images generated with high-resolution micro-CT, a 3D x-ray imaging technology, to evaluatebone microarchitecture in early bone loss and bone formation in the laboratory of Kimerly Powell, Ph.D.Central image: An array of microneedles (center image) 30 mm wide by 300 mm high in development in theBiological Micro Electrical Mechanisms Systems laboratory of Shuvo Roy, Ph.D. and Aaron Fleischman, Ph.D.Lower panel: “Biomechanics of a walk.” Graphic (lower panel) provided by Ton van den Bogert, Ph.D., BiomechanicsLaboratory.CANCER BIOLOGY—PAGE 44Top figure: Diagram illustrating the progression of prostate cancer. Human prostate cancer involves stagesthat correlate with loss of tumor suppressor genes. From Robert Silverman, Ph.D.Middle figure:Model hereditary prostate cancer family. From Graham Casey, Ph.D.Bottom figure: Immunohistochemical analysis of the expression of RNase L protein in a prostate tumor specimenfrom a mutation carrier. The cytoplasm of normal prostate epithelium stains positively (arrow on right),whereas the tumor cells are negative (arrow on left). From Robert Silverman, Ph.D.CELL BIOLOGY—PAGE 65Top image: Distribution of EGFP-fascin in a syndecan-1-activated cell, (top image). Activation of syndecan-1,by cell attachment to surfaces coated with either syndecan-1 antibody or thrombospondin-1, results in lamellipodialcell spreading and recruitment of fascin into the core F-actin bundles of microspikes and filopodia. Illustrationfrom the laboratory of Jo Adams.Lower Panels: Confocal micrographs showing surface binding (lower left) and internalization (lower right) ofHDL 2(red) and HDL 3(green) mediated by the scavenger receptor Bl in cultured adrenal cells. Areas of HDL 2and HDL 3colocalization are yellow. Images by Diane Green, B.S0., from the laboratory of Rick Morton, Ph.D.,with Judy Drazba, Ph.D., Imaging Core.IMMUNOLOGY — PAGE 85Top image: Normal T cells (nuclei DAPI stained, small blue) become trapped in the Hyaluronic Acid (HA) (FITCstained, green) cables formed on a renal cell carcinoma cell line (nuclei DAPI stained, large blue), SK-RC-45.The HA ligand, CD44 (Alexa 568 stained, red), can be seen on both the RCC line and the T cells.Mark Thornton, from Dr. Jim Finke’s lab.Lower image: Confocal micrograph of poly I:C-treated mouse colon parenchymal cells. Hyaluronan (green),TNF-stimulated gene 6 (TSG-6) (red) and nuclei (blue) are fluorescently labeled in this image. From de laMotte, C., Drazba, J., Hascall, V., Day, A., and S. Strong.198

MOLECULAR BIOLOGY — PAGE 101Confocal micrograph of mouse colon tissue from an animal treated with dextran sulfate to induce experimentalcolitis. Sections were fluorescently labeled for hyaluronan (green), inter-alpha inhibitor (red) and nuclei (blue).Image produced in the laboratory of Ganesh Sen, Ph.D. From Kessler, S., de la Motte, C., Drazba, J., Sen,G., and S. Strong.MOLECULAR CARDIOLOGY — PAGE 118Top image: Molecular surface of PINCH LIM domain involved in mediating cell adhesion. The regions coloredin blue are involved in protein-protein recognition in focal adhesion assembly. From the laboratory of Jun Qin,Ph.D.Middle image on right: Agonist binding pocket of the α1-Adrenergic Receptor. A molecular model of theα1A-AR as shown from the extracellular surface. Alpha-carbon coordinates were taken from the bacteriorhodopsinmodel and adjusted based upon the results of several mutagenesis studies from the laboratory of DiannePerez, Ph.D. Residues that been identified to be involved in agonist binding are shown in space-filled representationand are listed under its respective transmembrane domain (TM) in order from the extracellular surface.Amino acid residues are numbered according to the rat α1A-AR sequence.Lower image: Vitamin K-dependent (VKD) proteins are modified by the VKD- or gamma-carboxylase, an integralmembrane enzyme that resides in the endoplasmic reticulum. Carboxylation occurs during the secretion ofVKD proteins in a process that is poorly understood. The VKD proteins have a sequence (the pink rectangle)that the carboxylase binds with high affinity, which selectively targets VKD proteins for carboxylation. Thecarboxylase uses the oxygenation of vitamin K hydroquinone (KH 2, illustrated by the orange napthoquinone) tovitamin K epoxide (KO) to convert glutamic acid residues in VKD proteins to carboxylated glutamic acids (indicatedby white Y’s). Clusters of glutamic acids are modified (3 in this example) to render the VKD proteinsactive in functions that include hemostasis, growth control, bone metabolism and signal transduction. Normally,fully carboxylated VKD proteins are generated; however, conditions that limit the supply of KH 2block carboxylationand result in the secretion of uncarboxylated- and partially-carboxylated forms of inactive VKD proteins.Warfarin limits KH 2by inhibiting the reductase that regenerates KH 2from KO and is a commonly-used anticoagulant.From the laboratory of Kathy Berkner, Ph.D.NEUROSCIENCES — PAGE 133Lower left and right, uppermost central panel: Cultured hippocampal neurons immunofluorescently stainedfor syntaxin (red) and neurofilament (green). Images by Xiaoquin Liu, from the laboratory of Mark Perin, Ph.D.Central middle and lower panel: In situ hybridization for ephrin mRNAs and ligands in embryonic chick cerebellum,detected using alkaline phosphatase substrate. Reproduced from Nishida et al., 2002, Development129:5647-58 with permission.Background and remaining panels: Oligodendrocytes and oligodendrocyte progenitors expressing enhancedgreen fluorescent protein and immunolabeled for NG2 proteoglycan (red). Reproduced from Mallon et al., 2002,Journal of Neuroscience 22: 876-85 with permission.Page design by Graham Kidd, Ph.D., Department of Neuroscience.CENTERS OF RESEARCH — PAGE 153Top image: Molecular surface of PINCH LIM domain involved in mediating cell adhesion. The regions coloredin blue are involved in protein-protein recognition in focal adhesion assembly. From the laboratory of Jun Qin,Ph.D.Middle image: Three dimensional backbone trace of PINCH LIM domain involved in mediating cell adhesion.Regions involved in protein recognition are highlighted by amino side chains (blue). From the laboratory of JunQin, Ph.D.Lower image: A biological “Trojan Horse” utilizing receptor-mediated Cbl uptake as a means of targeting NO-Cbl to neoplasms. Nitrosylcobalamin (NO-Cbl) is delivered to cells bound to plasma transcobalamin II (TC II).TC II, a non-glycosylated plasma protein (43-kD), binds to specific cell surface receptors (TC II-R) that recognizethe TC II-Cbl complex (holo-TC II) preferentially to apoTC II (TC II alone). The TC II-R:TC II:NO-Cbl complexis internalized via endocytosis and TC II-NO-Cbl is delivered to lysosomes where NO is released fromCbl, and TC II is subsequently degraded. The chemotherapeutic effectiveness of NO-Cbl is based on the cytotoxicproperties of nitric oxide (NO). NO is oxidized from NO-Cbl in lysosomes at acidic pH. The NO free radicalinduces cytotoxicity through increased oxidative stress, inhibition of cellular metabolism, and direct DNAdamage, leading to apoptosis and/or necrosis. By Joseph Bauer, Ph.D., in the laboratory of Dan Lindner,M.D., Ph.D.Continued on Page 200199

Continued from Page 199SCIENTIFIC SUPPORT SERVICES— PAGE 181LRI Scientific Support: People-empowered Cores provide infrastructure support for CCF researchers.Clockwise from top left, Cathy Stanko, Manager Flow Cytometry Core; CarmelBurns, Director, Central Cell Services Core; Earl Poptik, Director, the Hybridoma Core; JeffHowell, smiling, and Eldon Walker, Ph.D., Director, Computer Services, also smiling; JamesProudfit, Mechanical Prototype Laboratory; and Pam Clark, Central Cell Services Core.BACK COVERTop Image: Full color 3-dimensional tomographic assessment of the human coronary arteryanatomy in vivo in real time. By Geoffrey Vince, Ph.D., the Whitaker Imaging Laboratory.Second image from top, on right: Human cystic fibrosis (CF) airway epithelial cells infectedwith human parainfluenza virus (HPIV3) 24 hr earlier. Immunofluorescence staining for ribonucleoproteinof HPIV3 identifies virus within single infected cells and in large multinucleated syncytia,which form through cell-cell fusion. There is increased viral replication in CF cells, providinga mechanism for understanding the severe respiratory symptoms frequently requiring hospitalizationof CF children. Increased virus is specifically due to lack of antiviral host defense inCF airway epithelial cells. Cover image from Immunity, (2003) 18:619-630. From an article byZheng, S. et al. Image produced in the laboratory of S.C. Erzurum, M.D. Used with permission.Third image from top, on left: Normal T cells (nuclei DAPI stained, small blue) becometrapped in the Hyaluronic Acid (HA) (FITC stained, green) cables formed on a renal cell carcinomacell line (nuclei DAPI stained, large blue), SK-RC-45. The HA ligand, CD44 (Alexa 568stained, red), can be seen on both the RCC line and the T cells.Mark Thornton, from Dr. Jim Finke’s lab.Fourth image from top: Normal human femur. Image generated with high-resolution micro-CT,a 3D x-ray imaging technology, to evaluate bone microarchitecture in early bone loss and boneformation in the laboratory of Kimerly Powell, Ph.D.Fifth image from top, on left: Confocal micrograph of human colon tissue from a patient withactive ulcerative colitis. Hyaluronan (green), CD-44 (red) and nuclei (blue) are fluorescently labeledin this section. By Carol de la Motte, with Judy Drazba, from the Laboratory of ScottStrong M.D.Sixth image from top: Molecular surface of PINCH LIM domain involved in mediating cell adhesion.The regions colored in blue are involved in protein-protein recognition in focal adhesionassembly. From the laboratory of Jun Qin, Ph.D.Seventh image from top: Confocal micrograph of poly I:C-treated mouse colon parenchymalcells. Hyaluronan (green), TNF-stimulated gene 6 (TSG-6) (red) and nuclei (blue) are fluorescentlylabeled in this image. By Carol de la Motte, with Judy Drazba, from the Laboratory ofScott Strong M.D.Lerner Research InstituteScientific Report 2003-2004RUSSELL J.VANDERBOOM, PH.D., EDITORCHRISTINE KASSUBA, EDITORIAL ASSISTANTJIM LANG, PHOTOGRAPHYDAVID SCHUMICK, MEDICAL ILLUSTRATOR200