Statins, EPCs, and Capillary Density Figure 8. Pravastatin Increases Myocardial Capillary Density in Animals with Chronic Myocardial Ischemia In Both Hibernating Myocardium and the Non-ischemic Remote Myocardium. Panel A-Representative immunofluorescence images of myocardial tissue from untreated or Pravastatin treated pigs with hibernating myocardium. Scale bar = 500 microns. Panel B-Quantitative assessment of CD31+ cells per square millimeter in chronically ischemic hibernating myocardium in the territory of the left anterior descending artery, and the opposite non- ischemic posterolateral wall, n = 6 animals in pravastatin treated group, n = 5 non-statin treated animals, p,0.05. doi:10.1371/journal.pone.0024868.g008 established [19], the relationship between cardiovascular disease risk and LOEPC growth remain to be defined. Given the fact that LOEPCs proliferate in culture, this trait facilitates studies of the effects of aging of LOEPC growth and activity while EOEPCs cannot be studied in this manner. Furthermore, the clonogenic growth assay for LOEPCs may be applied as we have used it to study effects of statins to identify novel agents that promote their growth and survival in vitro, that may then be translated angiogenesis in vivo. The Origins of Late Outgrowth EPCs Early outgrowth EPCs are identified by expression of CD34, VEGFR2 and CD133. Recent evidence indicates that these cells also express CD45 (indicating a leukocyte cell type) and cell sorting experiments have illustrated that these cells are a hematopoietic progenitor and form monocytes in vitro [20]. Additionally, cell sorting experiments of mononuclear cells from umbilical cord and adult blood has shown that LOEPCs can be obtained from CD34+ VEGFR2+ CD45- and CD133- cell populations, while CD34+ CD45+ cells also express CD133, and generate monocytic cells that have properties consistent with early outgrowth EPCs [20]. Since late outgrowth EPCs lack specific identifying cell surface markers, genetic marking to facilitate fate mapping studies in vivo are currently not feasible. It is plausible that LOEPCs are derived from the bone marrow, or they arise directly from the vessel wall [21]. Endothelial cells with hemogenic potential have been described in the embryonic yolk sac, dorsal aorta, and fetal liver that originate from the vessel walls [22]. Yolk sac derived hemangioblasts, bi-potential cells producing both hematopoietic and endothelial cells have been described in vitro from embryonic stem cells [23]. In vivo fate mapping experiments indicate that multiple cellular sources contribute to yolk sac blood islands in development [24]. LOEPCs in adults may similarly be derived from a poly- clonal origin. Specific identifying features to track the fate of LOEPCs in vivo are required to determine their contribution to vessel homeostasis under both normal conditions and in atherosclerotic vascular disease, as well as neoplastic and ischemic angiogenesis. PLoS ONE | www.plosone.org 9 September 2011 | Volume 6 | Issue 9 | e24868