Statins, EPCs, and Capillary Density Figure 1. Characterization of porcine late outgrowth endothelial progenitor cells (LOEPCs). Panel A: Phase contrast image of porcine LOEPC colony (upper image 2X magnification, lower image 10 x magnification, scale bar 500 microns). Panel B: Representative microscopic fields of LOEPCs are shown illustrating antigens characteristic of LOEPCs and endothelial cells; PECAM (CD31), VE-Cadherin (CD144), VEGFR2 (FLK1/KDR), c-Kit (CD117), endothelial specific nitric oxide synthase (eNOS), and Tie 2. Scale bars = 500 um. Results shown are representative of at least three independent experiments from separate animals. doi:10.1371/journal.pone.0024868.g001 In conclusion, statin therapy significantly improved the derivation of LOEPCs from MNCs, facilitating their isolation and expansion in culture. Statins Augment Clonal Growth of LOEPCs Predominantly by AKT Independent Effects A key difference between LOEPCs and EOEPCs is their ability to grow under limiting dilution or clonal conditions [5,6]. Hematopoietic cell progenitors are defined by their ability to form colonies based on differences in proliferative or differentia- tion potential, and the same principal of clonal growth was used to define LOEPCs from humans [7]. Clonogenic growth is a defining assay to distinguish EOEPCs from LOEPCs. The capacity for replating and growth under clonal conditions is a feature specific for the LOEPC and provides additional evidence to support our characterization data [12]. We hypothesized that one effect of pravastatin on LOEPCs was to promote their growth and survival in culture. Additionally, some reports suggested that different statins may have stimulatory vs. inhibitory effects on angiogenesis, but it remains unclear if this effect is true of all statins, or if more subtle chemical differences between compounds may underlie potential differential effects [13,14]. We compared the effects of the hydrophilic statin pravastatin vs. the hydrophobic statin simvastatin on LOEPC colony growth (see Figure 5). Both pravastatin and simvastatin augmented the growth of LOEPCs with the greatest effect noted at 100 nM. Pravastatin (100 nM) increased the number of LOEPC colonies 60% (95.8614 colonies formed/500 cells plated vs. 59.764.5 for untreated cells, p#0.05), while simvastatin (100 nM) increased the number of LOEPCs colonies obtained by 38% (82.167.3 colonies/500 cells plated). Interestingly, high concentrations of simvastatin ($10 mM) but not pravastatin resulted in cytotoxicity indicating a differential effect of statin drugs on LOEPCs colony growth. As statins have been shown to mediate their effects on early outgrowth EPCs via the PI-3 kinase/AKT pathway, we asked whether statin treatment of porcine LOEPCs induces AKT phosphorylation [12]. We found that statin treatment in vitro (100 nM) increased AKT phosphorylation 20% when compared with untreated cells (0.82360.04 densitometric units vs. 1.0560.08 for pravastatin treated cells, vs. 1.0260.1 for simvastatin treated cells). We verified the specificity of our western blot detection method using an inhibitor of AKT activity (Figure 6A). To determine the functional significance of AKT activity on clonal growth of LOEPCs, we treated control and statin exposed cells with an AKT-specific antagonist (AKT inhibitor IV). We used 100 nM AKT inhibitor IV for these experiments because we found higher concentrations resulted in cell death in the majority of the cell cultures. We found that inhibition of AKT activity in LOEPCs reduced the number of colonies formed by 70% (596 3.9 vs. 17.563.5 colonies/500 cells plated with AKT inhibitor (100 nM), p#0.05) in non-statin treated cells, 65% in simvastatin treated cells (8260.7 vs. 29.263.4, p#0.05), and 63% in pravastatin treated cells (95.860.5 vs. 37.363.7, p#0.05, Figure 6B). These findings indicate the majority of the growth and survival properties of LOEPCs are sensitive to AKT inhibition. In contrast to early outgrowth EPCs, statin treatment of LOEPCs in PLoS ONE | www.plosone.org 4 September 2011 | Volume 6 | Issue 9 | e24868