Statins, EPCs, and Capillary Density Figure 2. Flow cytometry analysis of porcine late outgrowth endothelial progenitor cells (LOEPCs). Representative flow cytometry plots of cultured late outgrowth endothelial progenitor cells expanded from initial clonal outgrowths from blood mononuclear cells. LOEPCs were analyzed with mouse monoclonal antibodies to the following antigens: Mouse IgG1, Mouse anti-pig CD31, CD105, CD133, and CD45. Primary antibodies were detected with Alexa Fluor 488 conjugated donkey anti-mouse antibodies. Results are representative of at least three independent experiments from separate animals. doi:10.1371/journal.pone.0024868.g002 Figure 3. Oral Pravastatin Promotes the Outgrowth of LOEPCs from Blood Mononuclear Cells. Pigs were treated with a placebo or pravastatin (4 mg/kg orally) daily for three consecutive days. Blood mononuclear cells were obtained on day three, and cultured on type I collagen coated dishes to derive LOEPCs. The quantity of LOEPCs per 36107MNCs plated was determined using flow cytometry to determine the number of PECAM positive cells after 14 days in culture. The data are presented as the means 6 SEM from eight different animals. * p,0.01 vs. control using a paired t-test. doi:10.1371/journal.pone.0024868.g003 the presence of AKT inhibitor significantly augmented growth relative to untreated controls. These results suggest that statins may act via an AKT independent pathway to promote clonal growth of LOEPCs. The findings support a differential, pleiotropic effect of statins to support the growth and survival of LOEPCs, which may be reflective of statin effects in vivo. Effects of HMG-CoA Reductase Inhibitors on Matrigel Tube Formation in Porcine LOEPCs The preceding experiments show that both the HMG-CoA reductase inhibitors Pravastatin and Simvastatin increase the clonal growth of LOEPCs in culture. We next determined the effects of HMG-CoA reductase inhibitors on a different function of endothelial cell biology, formation of tubules in Matrigel. Prior studies have shown that little or no cell proliferation occurs in the Matrigel tube formation assay, but reliably models the process of angiogenic tube formation of endothelial cells in vitro [15]. Therefore the goal of these experiments was to determine if statins differentially affected endothelial cell differentiation and angio- genesis independent of the growth promoting effects of statins [16]. LOEPCs were plated to solidified Matrigel in the absence or presence of Pravastatin, Simvastatin or Atorvastatin (100 nM each). Cells were imaged with phase contrast microscopy 24 hours after plating to Matrigel. Vessel segment length and the number of PLoS ONE | www.plosone.org 5 September 2011 | Volume 6 | Issue 9 | e24868