cells per well or 315 cells/cm2 [9]. The following day, cells were either left untreated (control) or treated with pravastatin (100 nM to 10 mM, Calbiochem, Catalog #524403), simvastatin (100 nM to 10 mM, Calbiochem, Catalog #567021) or Akt inhibitor IV (Calbiochem, Catalog #124015). Seven days after plating, cell colonies were enumerated by phase contrast microscopy at 40x magnification, followed by trypsinization and enumeration of total cells per well. Western Blot Analysis Cells were harvested in homogenization buffer and total protein content was quantified using Bio-Rad’s DC Protein Assay. Samples were separated on 10% SDS-PAGE gels, and transferred to PVDF membranes. Blots were probed with the following primary antibodies: Akt (rabbit polyclonal antibody, Cell Signaling Technology Catalog #9272; 1:1000 dilution, overnight at 4uC), Phospho-Akt (Ser473,736E11, rabbit monoclonal antibody, Cell Signaling Technology Catalog #3787; 1:1000 dilution, overnight at 4uC), GAPDH (V-18, goat polyclonal antibody, Santa Cruz Biotechnology Catalog #sc-20357; 1:300 dilution overnight at 4uC). Protein bands were detected by chemiluminescence using horseradish perox- idase conjugated secondary antibodies (WestPico kit, Pierce Biotechnology, 1:5000 dilution for 1 hour at room tempera- ture), and quantiated by densitometry (Bio-Rad, Quantity One software). Matrigel Cord Formation Assay LOEPCs were plated at a density of 5,000 cells per well in a 96 well tissue culture plates coated with 50 mL of Matrigel (BD Biosciences) per well, and observed up to five days for capillary tube formation. Wells were imaged 48 hours after cells were plated to Matrigel using a Nikon phase contrast microscope and a 2x objective. Images were captured using a Canon EOS digital camera and associated software. Total vessel length and the number of branch points were determined from the captured images using ImageJ. Experiments were performed in triplicate. Significant differences in vessel length and number of branch points with each treatment was determined using an one-way ANOVA with a Holm-Sidak post-hoc test, p,0.05. Hibernating Myocardium Model Hibernating myocardium was produced as previously described [10]. Briefly, pigs were sedated (Telazol; tiletamine 50 mg/mL and zolazepam 50 mg/mL)/xylazine (100 mg/mL, 0.022 mg/kg i.m.), intubated and ventilated with 0.5–2% isoflurane-oxygen mixture. Through a limited pericardiotomy, the proximal LAD was instrumented with a Deltrin occluder (1.5 mm i.d.). Antibiotics (cefazolin, 25 mg/kg and gentamicin, 3 mg/kg i.m.) were given 1-hour before surgery and repeated after closing the chest. Following surgery analgesia was perfomed by intercostal nerve block (0.5% Marcaine), intramuscular doses of butorphanol (2.2 mg/kg q6 h) and flenixin (1–2 mg/kg q.d.). Quantitative Immunohistochemistry of Capillary Density Samples adjacent to LAD (hibernating) and posterior descend- ing arteries (normal) were obtained for immunohistochemical studies. To identify effects on myocardial capillary density, frozen tissue sections (5 mm) were incubated with anti-PECAM1 (clone LCI-4, 1:100 dilution, AbD Serotec). Images were acquired with a confocal microscope (Bio-Rad MRC 1024). The number of CD31+ cells was determined in 478643.1 microscopic fields 2 (73.863.7 mm ). Statins, EPCs, and Capillary Density Statistical Analysis Data are expressed as means 6 SEM unless otherwise specified. Statistical comparison between PECAM (+) cell growth from porcine peripheral blood before and after treatment with pravastatin in vivo was performed using paired t-test. All other comparisons were performed using one-way ANOVA; p,0.05 is considered as statistically significant. Results Derivation and Characterization of Late Outgrowth Endothelial Progenitor Cells from Pigs We derived LOEPCs from porcine blood mononuclear cells. LOEPC grew as colonies or clones upon initial outgrowth from blood mononuclear cells (Figure 1A), and were passaged serially. Porcine LOEPCs expressed antigens found in endothelial cells, and described in LOEPCs from human blood [7] including PECAM (CD31), VE-Cadherin (CD144), VEGFR2, c-kit (CD117), endothelial specific nitric oxide synthase (eNOS), and Tie 2 (Figure 1B). Additionally, live porcine LOEPCs retained cell surface expression of the endothelial markers CD31, CD105, and absorbed acetylated LDL, but were negative for the early outgrowth EPC marker CD133, and hematopoietic cell marker CD45 (Figure 2). Collectively, porcine LOEPCs have an antigen profile and activities consistent with features found in human LOEPCs. Pravastatin Mobilizes LOEPCs into the Blood Stream Given the positive effects of statins on the quantity and activity of early outgrowth EPCs shown previously, we hypothesized that statin therapy similarly mobilizes LOEPCs into the bloodstream when given to animals in vivo [12]. To test this hypothesis, we collected blood samples from pigs at baseline, and after treatment with pravastatin 160 mg daily for three days in vivo. Blood mononuclear cells were obtained and cultured under conditions that support growth of LOEPCs for 14 days in the absence of further statin treatment. We then determined the quantity of CD31+ LOEPCs per 16107 MNCs plated. We found that short- term therapy with pravastatin efficiently mobilizes LOEPCs, increasing the number of LOEPCs greater than two-fold (37,78865,738 pravastatin vs. 17,05062,517 untreated, p#0.05- Figure 3). Statins Enhance Derivation of LOEPCs from Mononuclear Cells In Vitro We hypothesized that statin treatment may augment the growth or survival of LOEPCs in culture. To determine the effect of statin therapy on the derivation of LOEPCs, both pravastatin and simvastatin were added to MNCs from normal pigs in vitro, and the quantity of CD31+ cells per 16107 cells plated was determined by flow cytometry after 14 days of growth. Both pravastatin and simvastatin treatment of normal porcine MNCs in vitro increased the number of LOEPCs obtained greater than three-fold (62,24769,684 CD31+ cells/16107 MNCs for pravastatin- 100 nM, 59,83366,999 CD31+ cells/16107 MNCs for simvasta- tin-100 nM, 17,05062,518 CD31+ cells/16107 MNCs untreat- ed). While higher doses of pravastatin (1 mM to 10 mM) did not have a statistically significant effect on the quantity of LOEPCs obtained, simvastatin at high doses (10 mM) resulted in cytotoxicity (Figure 4). The concentration range of statins added to LOEPCs in our study is also clinically relevant. In normal human subjects, 40 mg of pravastatin taken daily provides a steady state level of pravastatin ,100 nanomolar in the bloodstream [11]. PLoS ONE | www.plosone.org 3 September 2011 | Volume 6 | Issue 9 | e24868