FA 5 Progress Report WV-INBRE - Joan C. Edwards School of ...

Program Director/Principal Investigator (Last, First, Middle): Rankin, Gary O 48remains unclear, but as in striated muscle, it is reasonable to postulate that SM titin may contributeto stiffness. Unlike striated muscle, SM cells respond to stretch in a complex manner that involvesan extensive change in morphology and reorganization of their cytoskeleton. SM cells may thusrespond to changes in mechanical load with alterations in titin expression. In this study, titinexpression was determined (qRT-PCR) in A7r5 cells which were exposed to cyclic (dynamic) orstatic (step) stretch. There was an early decrease in expression of titin in response to 1hr (19%) and2hrs (77%) of 15% unidirectional cyclic stretch (UCS). It is during this time that the actincytoskeleton is relatively unloaded as it is breaking down and reforming. At 6hrs of UCS, when thecytoskeleton had remodeled to a lower stretch orientation, the decline in titin mRNA expression wasless than the 2hr value (38% vs 77%). In contrast to UCS, cells responded to 1hr of 15% staticstretch with an increase in titin expression (35%). In cells treated with cytochalasin D to blockpolymerization of actin stress fibers, there was a marked increase in titin expression in unstretchedcells (335%) which increased further after 6hrs of UCS (530%). Together, these data suggest thatSM cells may alter expression of titin in order to modulate internal stiffness. We believe that SM titinmay play a sensory role for the cell.Hypertrophy of VSMC leads to increased vascular stiffness and an increased risk ofcardiovascular disease. Although not well understood, recent data suggests that resveratrol (RV)may have beneficial effects for the prevention of cardiovascular disease. RV is a polyphenoliccompound found in high concentrations in grape skin and red wine and is known to haveanti-oxidant and anti-carcinogenic properties. Here we investigate the potential of RV to inhibit thegrowth of VSMC following stimulation with the PGF2α analog fluprostenol (Fp). It has beensuggested that PGF2α is a mediator of vascular cell growth through a process that may involve theproduction of intracellular reactive oxygen species (ROS). Increased ROS production has beenlinked to VSMC hypertrophy via a cell signaling pathway that is not well understood. One goal wasto investigate the potential of resveratrol in preventing hypertrophy of VSMC that occurs in responseto a physiologically relevant stimulus, namely activation of PGF2α receptors. VSMC hypertrophy isassociated with a number of vascular pathologies. It can have detrimental effects on vascularremodeling which can bring about changes in circulation and an increase in the metabolicdemands that are placed on the left ventricle. Ultimately, VSMC hypertrophy can lead to systolichypertension, an alteration in coronary perfusion, and eventually hypertrophy of the left ventricle. Inaddition, it often contributes to mortality that is associated with end-stage renal failure. Theseimportant clinical considerations highlight the need to expand our knowledge and develop a moredetailed understanding of the molecular mechanisms that underlie the onset of VSMC hypertrophyand to investigate ways in which responsible signaling mechanisms may be altered.Incubation of A7r5 VSMCs with 1µM Fp resulted in a 35% increase (P0.05) in cell size in 48hours as measured by flow cytometry. This Fp treatment was associated with markedly increasedintracellular ROS levels as determined by incubation of cells with hydroethidium. However, whencells were pre-treated with 1 or 20 µM RV, FP-induced hypertrophy was completely attenuated.Resveratrol treatment at both levels was associated with a decline in intracellular ROS. The resultsof the present study suggest that very low levels of resveratrol (1µM) can completely attenuatehypertrophy that is induced by exposure to fluprostenol through a mechanism that involvessuppression of the activation of ERK1/2 and ribosomal protein s6. On the other hand, resveratrol’sreduction in hypertrophy in this study was associated with a slight increase, rather than theexpected decrease, in Akt pathway activation. These results further our understanding of howresveratrol may exert its cardio-protective effects.PROTECTION AGAINST RESEARCH RISKSN 1. Will human subjects be involved next year?N 2. Will vertebrate animals be used next year?N 3. Will recombinant DNA experiment(s) be conducted next year?N 4. Are there potential hazards to laboratory workers (carcinogens, pathogens, ionizing radiation, etc.)involved in the proposed research for next year? If yes, identify:PHS 2590 (Rev. 06/09)Continuation Format Page