Statin Potency and Dose Dependence on Late Outgrowth EPC Growth, Relevance to Angiogenesis In Vivo Statins are recommended therapy for the primary and secondary prevention of coronary artery disease [25,26,27]. Most recent guidelines advocate treating to a target LDL cholesterol ,70 mg/dL, because of the effect on cardiovascular disease outcomes in large clinical trials [27,28]. However, several groups have reported an apparent biphasic or differential effect of statins, in that low concentrations of statins appear to promote endothelial cell proliferation, migration and differentiation, while higher doses impair these effects [3,17,29]. One mechanism may be that lower dose statin therapy may activate AKT signaling, but impair the function of isoprenylated proteins of the Ras family or lamins, which have essential functions in cell proliferation and division [30]. An alternative hypothesis would be that the effect of statins on angiogenesis is not tied to statin dose necessarily, but to differences in lipophillicity [31] and the intracellular concentration of statins. In our study, we found effects of pravastatin that promoted clonogenic growth of LOEPCs (10 mM), while the same dose of simvastatin had toxic effects on the cells. The effects of statins in this cell based assay cannot alter lipoprotein levels (i.e. low density lipoprotein), but may function through direct effects on cholesterol biosynthesis affecting lipid modifications of prenylated proteins. This suggests the difference in chemical properties of these two statins may underlie the differences in LOEPC growth and survival. Our findings show that statins increase both the derivation of LOEPCs from blood and growth under clonogenic conditions. While AKT activity plays a role in their clonogenic growth, antagonism of AKT does not completely inhibit the effect of statins to promote LOEPC growth. Additionally, in the chronically ischemic heart, we find that pravastatin therapy increases the myocardial capillary density in both chronically ischemic myocar- dium, and the less affected remote myocardial territory. These findings indicate that both in vitro and in vivo statins promote the growth of nascent endothelial cells. Additionally, recent evidence suggests that intracoronary injection of LOEPCs in pigs that have sustained a myocardial infarction may have a beneficial effect on References 1. Gheorghiade M, Bonow RO (1998) Chronic heart failure in the United States. Circulation 97: 282–289. 2. 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Statins, EPCs, and Capillary Density heart remodeling as compared with similar therapy with mesenchymal stem cells [32], suggesting a potential role for LOEPC therapy in animal models and patients with acute myocardial infarcts. LOEPCs may have an advantage over other adult stem cell therapies for acute heart disease including mesenchymal stem cells or cardiosphere derived cells in that they can readily obtained from venous blood. This application of LOEPCs adds significance to our findings that LOEPCs can be expanded in vitro by the addition of pravastatin. Study Limitations Our study shows that LOEPCs can be derived from young pigs and have markers, activity and growth properties that match LOEPCs derived from humans. The success of these studies may be due to the age of the animals used in this study. Remarkably, LOEPCs in humans have both low and high proliferative potential, and cells derived from adults predominantly have low proliferative potential. 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Yoder MC (2010) Is endothelium the origin of endothelial progenitor cells? Arterioscler Thromb Vasc Biol 30: 1094–1103. PLoS ONE | www.plosone.org 10 September 2011 | Volume 6 | Issue 9 | e24868