Effects of Obesity and Weight Loss on the Functional Properties of Early Outgrowth Endothelial

Online Appendix for the following January 26 JACC articleTITLE: Effects of Obesity and Weight Loss on the Functional Properties of Early Outgrowth Endothelial Progenitor CellsAUTHORS: Nana-Maria Heida, MD, Jan-Peter Müller, MD, I-Fen Cheng, MSc, Maren Leifheit,P H D, Vivien Faustin, P H D, Joachim Riggert, MD, Gerd Hasenfuss, MD, Stavros Konstantinides, MD, Katrin Schäfer, MD_____________________________________________________________________ APPENDIXSupplemental Material and MethodsAdhesion to Extracellular Matrix Proteins. EPCs were fluorescently labeled by incubation in 5 mL of serum-free medium containing 2.5 µL/mL CellTracker CM-DiI (Invitrogen). EPCs were gently detached with 5 mmol/L EDTA in PBS, washed, and suspended in adhesion medium (EBM plus 0.5% BSA, 1 mmol/L MgCl2, 1 mmol/L CaCl2, and 1 mmol/L MnCl2). EPCs (2 ´ 105 per mL) were seeded on 24-well plates (500 µL/well), precoated with 1 µg/cm2 of either vitronectin (Promega) or fibronectin, and allowed to adhere for 30 minutes (1). After vigorous washing, bound EPCs were fixed with 2% paraformaldehyde (PFA) in PBS and subsequently stained with DAPI. Experiments were performed in duplicate, and the number of adherent cells was quantified by counting DAPI-positive cells in 5 random 200´ microscope fields.Migration Assay. CM-DiI-labeled EPCs (1 ´ 105) or mature endothelial cells (HUVEC; 1 ´ 105) in 600 µL EBM containing 0.5% FCS were seeded in the upper chamber of transwell cell culture inserts (8 µm pore size; BD Falcon), coated with 10 µg/mL fibronectin and blocked with 0.5% BSA (1). The lower chamber contained 600 µL EBM/0.5% FCS or EPC-derived conditioned medium. After incubation for 12 hours (for EPCs) or 4 hours (for HUVEC) at 37°C, membranes were washed and briefly fixed in 2% PFA, and nonmigrated cells on the upper membrane surface were removed with a cotton swab. Then membranes were excised, inverted, and mounted on glass slides. Migrated cells were quantified by counting the number of fluorescent EPCs or hematoxylin-stained HUVEC per membrane at 200´ magnification.Matrigel Angiogenesis Assay. EPC adhesion to endothelial cell tube-like structures was examined by co-incubation of 3 ´ 104 CM-DiI-labeled EPCs and 1.2 ´ 105 human umbilical vein endothelial cells (HUVEC; PromoCell) for 8 hours in 96-well plates precoated with 50 µL ECMatrixTM (Chemicon) and then photographed using fluorescence microscopy (Zeiss Axiovert 200). The number of CM-DiI-positive cells adherent to tubular structures provided by HUVEC was counted in 8 random microscope fields and expressed per millimeter of tube length.Spheroid Angiogenesis Assay. For these experiments, 8 ´ 103 fluorescence-labeled EPCs and 3.2 ´ 104 HUVEC were mixed and resuspended in 10 mL EBM-MV containing 20% methylcellulose solution (in M199 medium; Gibco) and incubated in round-bottom 96-well plates (100 µL per well) for 24 hours to form spheroids (2). Type I rat tail collagen (BD Biosciences) was diluted 1:1 with 0.1% acetic acid, mixed with 10´ M199 medium, and neutralized with 0.2 N NaOH immediately before use. Spheroids were harvested in methylcellulose solution supplemented with 5% FCS andgently mixed (1:1) with collagen working solution. Spheroid suspensions were distributed in duplicates into pre-warmed 24-well plates (1 mL per well) and incubated at 37°C for 30 minutes. After solidification of the collagen, 500 µL of medium supplemented with 4% FCS was added to each well and incubated for 24 hours at 37°C. In some analyses, spheroids consisting only of HUVEC were incubated with 500 µL of EPC-derived conditioned medium. Pictures of 10 spheroids at random fields were taken on a fluorescence microscope and evaluated using Zeiss AxioVision 3.1 software.In Vivo Murine Hind Limb Ischemia Model. Unilateral hind limb ischemia was induced in athymic nude mice (NMRI-Foxn1nu/nu; Harlan Winkelmann) by ligation of the right femoral artery (immediately distal to the origin of the deep femoral artery) as well as the distal portion of the saphenous artery with 6-0 silk sutures according to (3). One day later, mice received 1 ´ 106 cells CM-DiI-labeled EPCs by intracardiac injection. Ten days later, the gastrocnemius muscle from the left (nonischemic) and right (ischemic) hind leg were carefully excised, positioned vertically on cork plates using small pins, emerged in OCT compound and processed for cryoembedding. Capillary density in the gastrocnemius muscle was assessed on 5 µm-thick, acetone-fixed (at 200-µm intervals) frozen transverse sections after staining with a monoclonal rat anti-mouse antibody against CD31 (sc-18916; SantaCruz), followed by FITC-labeled secondary antibodies (Molecular Probes). Cell nuclei were counterstained with DAPI. The number of CD31-immunopositive cells was manually counted on 7 random microscope fields per section (magnification, 200´) and expressed per square millimeter, excluding any longitudinally cut structures. All animal care and experimental procedures were approved by the Animal ResearchCommittee of the University of Goettingen and complied with national guidelines for the care and use of laboratory animals.Flow Cytometry Analysis. EPCs were detached, washed, and resuspended in 0.5% BSA in PBS at a concentration of 1 ´ 106 cells per milliliter. A volume of 100 µL of cell suspension was incubated with 10 µL of PE-conjugated monoclonal antibodies against human vascular endothelial growth factor receptor 1 (FAB321P), vascular endothelial growth factor receptor 2 (FAB357P), CCR2 (FAB151P), CXCR2 (FAB331P; all R&D Systems). Each analysis included 10,000 gated events. For the quantification of circulating endothelial cells, whole blood was Fc-blocked with 1 µg of human IgG (R&D Systems) per 105 cells and then incubated with 10 µL of PE-conjugated monoclonal antibody against human CD146 (FAB932P, R&D Systems) or 20 µL of FITC-conjugated antibody against human CD31 (555445; BD Pharmingen) per 100 µL of whole blood. Red blood cells were lysed using FACS Lysing Solution (Becton Dickinson). PE- or FITC-conjugated mouse IgG antibodies served as isotype control. Apoptosis was quantified using the Annexin V-FITC Apoptosis Detection kit (BD Biosciences).SDS Gel Electrophoresis and Western Blot Analysis. EPCs were washed with ice-cold PBS, scraped off, and resuspended in 100 µL lysis buffer (1% Triton-X 100, 150 mM NaCl, 50 mM Tris, 5 mM EDTA, pH 7.5) containing fresh protease (4 mg/mL aprotinin, 4 µg/mL leupeptin, 4 µg/mL pepstatin A, 1 mM PMSF), and phosphatase (20 mM NaF, 1 mM Na3VO4, 1 mM Na4O7P2) inhibitors. After incubation for 20 minutes on ice, cell lysates were cleared by centrifugation, and equal amounts of protein were fractionated by SDS gel electrophoresis and then transferred to nitrocellulose membranes. Membranes were blocked in 1% BSA (in TBS/0.1% Tween-20) prior toincubation with rabbit anti-human phospho-p38 or p38 MAPK antibodies (Cell Signaling Technology) overnight at 4ºC. Protein bands were visualized using a HRP-conjugated secondary donkey anti-rabbit IgG antibody (Amersham Biosciences), followed by enhanced chemiluminescent substrate detection and autoradiography.Assessment of Cytokine Secretion in the Conditioned Medium of EPCs. Conditioned medium was collected on day 7 after incubation of 2 ´ 106 cells in 1 mL medium supplemented with 4% FCS for 24 hours, and either immediately used or stored at −80°C pending analysis. For p38 MAPK inhibition, EPCs were incubated with 20 µM SB203580 (Calbiochem) or an equal volume of dimethylsulfoxide (Sigma-Aldrich) for 24 hours before conditioned medium collection.The presence of cytokines and growth factors in conditioned medium from obese and lean subjects was analyzed using specific enzyme-linked immunosorbent assays for the detection of interleukin 6 (RayBiotech), interleukin-8 (Diaclone), monocyte chemoattractant protein 1 (RayBiotech), stromal cell derived factor-1a (RayBiotech), tumor necrosis factor-a (R&D Systems), and vascular endothelial growth factor (R&D Systems) as well as the commercial human cytokine antibody array 5 (RayBio; Hoelzel Diagnostika).References1. Schroeter MR, Leifheit M, Sudholt P, et al. Leptin enhances the recruitment of endothelialprogenitor cells into neointimal lesions after vascular injury by promoting integrin-mediated adhesion. Circ Res 2008;103:536-44.2. Korff T, Augustin HG. Integration of endothelial cells in multicellular spheroids preventsapoptosis and induces differentiation. J Cell Biol 1998;143:1341-52.3. Couffinhal T, Silver M, Zheng LP, et al. Mouse model of angiogenesis. Am J Pathol 1998;152:1667-79.。