or sprouting angiogenesis of existing blood vessels. Endothelial progenitor cells (EPCs) have been defined in various ways based on cell surface marker phenotypes including CD133 and VEGFR2 expression, or by cultured based assays [5]. However, recent evidence indicates that that an additional cell type contained in blood mononuclear cells known as late outgrowth endothelial progenitor cells (LOEPCs) have distinct growth properties from other EPC cell types, including adherence to collagen coated substrates, the ability to undergo serial replating, clonogenic growth in culture, and the ability to form functional vessels in vivo [6]. Our goal was to determine how HMG-CoA reductase inhibitors affected the mobilization, derivation, and clonogenic growth of LOEPCs as a surrogate for effects of statins on angiogenesis, and determine how statins affect capillary density in animals with chronically ischemic myocardium. Materials and Methods Procedures and protocols conformed to institutional guidelines for the care and use of animals in research. All our animal work on pigs was performed according to a standing protocol approved by the SUNY at Buffalo School of Medicine and Biomedical Sciences IACUC (Protocol Number MED022011Y). Briefly, all studies where blood was to obtained, or studies on pigs with hibernating myocardium were performed on animals that were sedated using tiletamine (50 mg/mL) and zolazepam (50 mg/mL), followed by intubation and sedation with 0.5–2% isoflurane-oxygen mixture. Terminal procedures were performed using a deep surgical plane of anesthesia prior to euthanasia. Preparation of Mononuclear Cells from Porcine Peripheral Blood Porcine mononuclear cells (MNCs) were isolated as described [7,8] with minor modifications. Briefly, peripheral blood was collected into cell preparation tubes for density gradient centrifugation (BD Vacutainer CPT, Catalog number 362761), centrifuged for 30 minutes at room temperature, 1500 6 g. After centrifugation, the supernatant was discarded, and the mononu- clear fraction was retained, washed twice in PBS and resuspended in EBM-2 medium (Lonza, Walkersvile,MD) supplemented with 10% fetal bovine serum (FBS; Hyclone,Logen,UT) (complete EGM-2 medium). Culturing of MNCs for Late Outgrowth Endothelial Progenitor Cells (LOEPCs) MNCs in complete EGM-2 medium were seeded onto six-well tissue culture plates (36107cells per well) coated with type I rat tail collagen (BD Biosciences, Bedford, MA), and incubated at 37uC with 5% CO2 overnight. Nonadherent cells and debris were discarded and adherent cells were washed once with EGM-2 medium, and grown in EGM-2 medium. Medium was changed daily for 7 days and then every other day until first passage. After 7–10 days, monolayers of cobblestone-appearing cells appeared. Immunoflurescence Microscopy Cells were grown on 24 well tissue culture dishes to 80% confluence, and fixed with 4% paraformaldehyde (Electron Microscopy Services Inc.). Cells were blocked in 5% normal donkey serum (Chemicon) for 1 hour. Primary antibody was added (16100 dilution) in PBS, and incubated with primary antibodies 3 hours to overnight at room temperature. The following primary anti- bodies were used: Mouse, goat, and rabbit IgG (R&D Systems), CD31; mouse monoclonal, clone LCI-4, Santa Cruz Biotech- nology Catalog #SC-80912), CD105; mouse monoclonal, clone Statins, EPCs, and Capillary Density MEM-229, Santa Cruz Biotechnology Catalog #SC-51594), CD117; rabbit polyclonal, Santa Cruz Biotechnology Catalog #SC-168), CD144; goat polyclonal, Santa Cruz Biotechnology Catalog # SC-6458), VEGFR2 (Flk-1; mouse monoclonal, clone A- 3, Santa Cruz Biotechnology Catalog # SC-6251), eNOS (rabbit polyclonal, Santa Cruz Biotechnology Catalog #SC-654), CD133 (goat polyclonal, Santa Cruz Biotechnology Catalog # SC-19365). Alexa Fluor conjugated 488 or 555 donkey anti-mouse, goat or rabbit secondary antibody (Molecular Probes) was added at a dilution of 1:500 for 1 hour at room temperature. Nuclei were stained with 49,6-diamidino-2-phenylindole (DAPI). Immunofluo- rescent images were obtained using a Zeiss Axiovert 200 inverted fluorescence microscope and Zeiss LD Achroplan 106 and 206 objectives using identical exposure times between antibodies. Images were merged using Zeiss Axiovision software. Immunophenotyping and Acetylated LDL Uptake by Flow cytometry analysis Cells were dissociated to a single cell suspension using 0.05% Trypsin-EDTA, resuspended in DMEM/F12 with HEPES supplemented with 10% FBS (Staining Medium), and pelleted by centrifugation at 30 x g. Cells were incubated with primary antibodies for 1 h at 4uC. Porcine specific antibodies used for flow cytometry analysis included the following: CD31 (PECAM) (mouse monoclonal, clone LCI-4, AbD Serotec. Oxford, UK), CD45 (mouse monoclonal, clone K252.1E4, Santa Cruz Biotech- nology Catalog # SC-59065), CD105 (Endoglin); mouse mono- clonal, clone MEM-229, Santa Cruz Biotechnology Catalog #SC-51594), CD133 (mouse monoclonal, clone AC133, Miltenyi Biotec). Alexa Fluor conjugated 488 donkey anti-mouse antibody (Molecular Probes) was used to detect primary antibodies. Primary antibodies were incubated at 1:10 dilution, secondary antibodies were incubated at 1:200 dilution. Uptake of acetylated LDL was determined by incubation of LOEPCs with 10 mg/ml Alexa Fluor conjugated 488 Dil-Ac-LDL (Molecular Probes) in EGM-2 medium 3 hours at 37uC. Flow cytometry analysis was performed on a BD FACSCalibur. 100,000 events per condition were collected. Flow cytometry data was gated in the FSC vs. SSC plots to exclude dead or clumped cells. Cytometry data were analyzed using FCS Express (DeNovo Software). Effects of Statins on In Vivo Mobilization and In Vitro Derivation of LOEPCs from Porcine Peripheral Blood Peripheral blood was obtained from eight healthy pigs (11–14 weeks old, 4069 kg, five males, three females) before and after treatment with pravastatin for three days (4 mg/kg orally). Mononuclear cells were isolated using the methods described above. Mononuclear cells were plated at the density of 36107 cells per well on six-well plates. After culture in EGM-2 medium for 14 days, cells were trypsinized and total cell number per well was determined using a hemocytometer. The percentage of PE- CAM(+) cells per well were determined by flow cytometry using the protocol described above. The number of PECAM(+) cells per 16107 blood MNCs plated was determined as the product of total cells per well 6 percentage of PECAM(+) cells per well. The effect of statin treatment on the in vitro derivation of LOEPCs was determined in MNCs obtained from normal pigs (not treated with pravastatin), and treated with pravastatin or simvastatin (10 nM to 10 mM) added 24 hours after plating. LOEPCs Colony-Forming Assays Early passage LOEPCs (passages 3-5) were seeded onto six-well plates precoated with type I rat tail collagen at clonal density, 500 PLoS ONE | www.plosone.org 2 September 2011 | Volume 6 | Issue 9 | e24868