Statins, EPCs, and Capillary Density Figure 6. Clonogenic Growth of LOEPCs is Sensitive to AKT inhibition, while the Effect of Pravastatin and Simvastatin on LOEPC Clonogenic Growth is Not. Panel A-Western blot analysis showing both pravastatin and simvastatin activate AKT phosphorylation, and AKT inhibitor IV blocks the phosphorylation of AKT in LOEPCs. Panel B-LOEPCs were grown under clonogenic conditions with or without an AKT inhibitor (100 nM), in the presence or absence of pravastatin or simvastatin (100 nM each) to determine if the effect of statins on LOEPC clonogenic growth as sensitive to AKT inhibition. n = 6 independent experiments. * p,0.05 vs. No Akt inhibitor IV. doi:10.1371/journal.pone.0024868.g006 untreated cells. The sum of vessel segments in untreated cells, or after treatment with Pravastatin, Simvastatin, and Atorvastatin was the same in all conditions, consistent with a lack of proliferation of endothelial cells when plated onto Matrigel as previously shown [16]. The findings are consistent with higher potency statins being more effective than Pravastatin in promotion of in vitro angiogenic branch formation in LOEPCs. Moreover, our findings indicate divergent effects of statins on LOEPC clonal growth versus in vitro angiogenesis. Pravastatin Therapy Increases Myocardial Capillary Density in Hibernating Myocardium To assess the in vivo significance of statin effects on porcine LOEPCs and cardiac angiogenesis, we examined the effect of statin treatment on porcine myocardium. Conflicting studies have suggested that statin therapy may augment or impair angiogenesis in chronically ischemic myocardium [3,17]. To determine the effect of pravastatin on angiogenesis in the chronically ischemic heart, we measured the capillary density in myocardial tissue obtained from the chronically ischemic anterior wall and non- ischemic posterolateral wall segments from both untreated animals and those treated with pravastatin for 30 days. We found that pravastatin treatment increased the number of CD31+ capillaries per square mm of myocardial tissue by 46% (1769.16116.7 vs. 1210.66151.6 capillaries/mm2, p#0.05) in the hibernating LAD territory, and 37% in the remote, non-ischemic territory (1495.2693.4 vs. 1095.16123.0, p#0.05, Figures 8A and B). We have performed similar analysis of CD31+ vessels in the heart using immunoperoxidase labeled secondary antibodies. We found that immunoperoxidase staining did not enhance the visualization of CD31+ cells in the heart. The CD31+ vessels appear small because they are cut in 90u cross section (transversely). The images shown are truly representative of the effect of pravastatin versus no treatment on capillary density in the chronically ischemic heart. The image shown in Figure 8A is representative of one of .400 microscopic fields that were quantified per animal to determine the capillary density. The quantification of the images in panel B reflects the statistically significant difference noted between untreated and pravastatin treated animals. In previously published work, we found that therapy with pravastatin increased myocyte nuclear density in the chronically ischemic heart with proliferation of small cardiac myocytes [2]. These findings are consistent with an effect of pravastatin that increases the myocardial capillary density to match the microcir- culation with formation of new and smaller cardiac myocytes. Discussion From these studies we identified the following novel conclusions: 1) LOEPCs are effectively mobilized into the blood after short-term therapy with pravastatin, 2) both pravastatin and simvastatin facilitate the derivation of LOEPCs from peripheral blood mononuclear cells, 3) low concentrations of both pravastatin and simvastatin enhance clonogenic growth of LOEPCs in vitro, while higher concentrations of simvastatin have toxic effects, 4) antago- nism of AKT signaling impairs clonogenic growth of LOEPCs, but does not impair the positive effect of statins on LOEPC growth, 5) in vivo pravastatin increases capillary density in chronically ischemic myocardium in parallel with increased myocyte nuclear density. PLoS ONE | www.plosone.org 7 September 2011 | Volume 6 | Issue 9 | e24868