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60 Recent Advances in Angiogenesis and Antiangiogenesis, 2009 Bertolini et al. a b 0.7 mm c d Fig. (1). Transmission electron microscopy images of sorted DNA+,CD45-CD31+CD146+ CECs (from Bertolini et al, 2009, modified). a) Low-magnification overview of the sorted cell population, showing the presence of apoptotic/necrotic cells most likely derived from vessel wall turnover along with lymphocyte-like cells (arrows). b) Endothelial (precursor?) cell undergoing cellular division (a centriole is present). The arrow points to a Weibel-Palade body seen more detailed in the inset. c) A mature endothelial cell with a Weibel-Palade body (arrow). d) An overtly apoptotic cell. blood. The majority of sorted CECs, in fact, were found to be apoptotic or necrotic cellular fragments, most likely lost at count after the cell processing involved in IHC enumeration. Along with apoptotic CECs, however, TEM showed the presence of small, viable and lymphoid-like cells that are compatible with a progenitor cell morphology (Fig. 2). TEM will most likely be of help for the next crucial steps in CEC and CEP studies, namely, to dissect the functions of candidate CEC and CEP subpopulations. Both these cell families, in fact, encompass subpopulations with different roles. Multiparametric FC has shown that among DNA+,CD45- ,CD31+,CD146+ CECs there are some expressing other EC-related antigens such as CD143, CD144, VEGFR1, VEGFR2, VEGFR3, along with activation antigens such as CD105 (endoglin), among others [8]. The need for a detailed phenotypic profile is particularly urgent for CEPs, because CD34 and VEGFR2 antigens, used by many investigators for CEP enumeration by FC [5, 8, 27-28], are expressed 10 mm also by mature CECs, and the use of CD133 antigen for CEP identification [29-30] has led to the sorting of cells that not all laboratories were able to differentiate in vitro and in vivo along the endothelial lineage [31- 32]. Even more controversies exist for the enumeration of CEPs in mice, because the expression and function of CD34 and CD133 antigens are not well characterized in mice compared to humans. Thus a candidate phenotype for CEPs in mice is CD45-, VEGFR2(Flk)+, CD117+ [22-24], and antibodies reacting with a particular configuration of CD144 are also used [33-34]. 3. CEC NUMBER AND VIABILITY IN CANCER TREATMENT A variety of FC and IHC studies have indicated that in some types of cancer patient CEC numbers and viability are increased when compared to healthy controls [9-21]. Possible explanations for these findings involve the angiogenic switch associated with cancer growth and the robust production of angiogenic
Contribution of Circulating Endothelial Cells Recent Advances in Angiogenesis and Antiangiogenesis, 2009 61 Fig. (2). Overview of cellular events with an EC phenotype enumerated by flow cytometry (from Bertolini et al, 2009, modified). The majority of CD45-,CD31+CD146+ events are apoptotic or necrotic cellular fragments (1-10/L in the blood). The use of Syto16 allows the enumeration of DNA-containing CD45-CD31+CD146+ CECs (0.01-1/L). Along with CECs, TEM showed the presence of some small, viable and lymphoid-like cells that are compatible with a progenitor cell morphology, lack CD45 expression and may express CD133 and/or CD34 (Mancuso et al, in press). growth factors such as VEGF, bFGF, HGF and many others by cancer cells and/or various host cells [8]. The recent and unexpected finding of an autocrine loop in ECs [35] is of particular interest, because it might be that the increase of viable CECs in the blood of cancer patients mirrors an aberrant vascular turnover/remodeling associated with high local levels of VEGF produced by cancer cells. Following the preclinical evidence that CEC count can be used as a surrogate biomarker for angiogenesis and anti-angiogenic drug activity by means of determining the optimal biological dosage of anti-angiogenic drugs [24-26], their numbers and viability have also been measured in different clinical trials involving cancer patients treated with various anti-angiogenic therapies [10-21]. An increase in the number of apoptotic CECs after 60 days of therapy was associated with prolonged progression-free survival and overall survival in metastatic breast cancer patients treated with a doublet low-dose metronomic (antiangiogenic) chemotherapy regimen [16]. When the humanized anti-VEGF antibody, bevacizumab, was added to the metronomic chemotherapy for the treatment of metastatic breast cancer patients who showed a clinical response in a phase II clinical trial (as well as a larger population of patients who had a clinical benefit from the treatment) had significantly greater baseline levels of viable CECs than did patients who failed to respond; furthermore, the number of apoptotic CECs before therapy initiated was associated with prolonged progression-free survival [36]. In patients with renal cancer treated with the small molecule anti-angiogenic agent, sunitinib, changes in CECs differed between the patients with clinical benefit and those with progressive disease [18]. Taken together, these data suggest that the investigation of CEC number and viability by FC has potential for the stratification of cancer patients who are more likely to benefit from anti-angiogenic treatments [8, 25, 37]. This possibility awaits confirmation in prospective randomized clinical trials. 4. THE CATALYTIC CEP ROLE IN DEVELOPMENT OF MACROMETAS- TASES AND RESISTANCE TO CHE- MOTHERAPY, VDAS AND ANTI- ANGIOGENIC DRUGS Asahara et al first proposed the concept of CEPdependent new blood vessel formation in adult life in 1997 [27], a few months before a similar proposal from Shi et al in 1998 [28]. These two seminal studies identified endothelial progenitors as cells that are capable of generating mature ECs in vitro and in vivo. A study from Lyden et al [38] was the first to identify a role for CEPs in cancer growth using Id (Id1 +/- Id3 -/- ) deficient mice. These mice have a severe CEP defect leading to impaired angiogenesis and tumor growth. More recently it was found that CEP generation depends on the ability of Id to restrain the expression of its target gene p21 [39]. Genetic ablation of p21 rescued CEP generation in Id1 deficient mice, reestablishing normal tumor growth [39]. Understanding whether all types of tumor rely, at least in part, on
Page 2 and 3: Recent Advances in Angiogenesis and
Page 4 and 5: FOREWORD It has been known for a ve
Page 6 and 7: single proangiogenic protein. Howev
Page 8 and 9: v Vito Pistoia Head, Laboratory of
Page 10 and 11: 2 Recent Advances in Angiogenesis a
Page 12 and 13: 10 Recent Advances in Angiogenesis
Page 16 and 17: The Role of Mesenchymal Stem Cells
Page 22 and 23: Thymus and Angiogenesis Recent Adva
Page 32 and 33: Role of Thymidine Recent Advances i
Page 34 and 35: Role of Stromal Cells Recent Advanc
Page 40 and 41: Tumor Targeting with Transgenic End
Page 44 and 45: Tumor Vascular Disrupting Agents Re
Page 50 and 51: Tumour Vascular Disrupting Agents R
Page 52: Index Recent Advances in Angiogenes

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