L participants for ex vivo EPC assessment, and measurement of plasma
L participants for ex vivo EPC assessment, and measurement of plasma glucose and HbA1c levels.Isolation, cultivation and characterization of EPCs EPCs were isolated cultured and characterized, as described previously [14]. Peripheral blood mononuclear cells (MNCs) were isolated by density gradient centrifugation using LymphoprepTM (Axis-Shield, Oslo, Norway), and then grown in endothelial cell basal medium-2 (EBM-2) (PromoCell GmbH, Heidelberg, Germany) for five days. The EPCs in the cultures were identified as adherent cells that stained double positive for acetylated LDL (acLDL) uptake and the binding of FITC-labeled lectin under a laser scanning confocal microscope. For this purpose, the adherent cells from the EPC cultures were first incubated with 2.2 g/mL 1,1′-dioctadecyl-3,3,3′,3’tetramethylindocarbocyanine-labeled acLDL (Biomedical Technologies, Inc., MA, USA) for two hours at 37 . After two hours, the cells were fixed in 2 paraformaldehyde, and then counterstained with 10 g/mL fluorescein isothiocyanate-labeled lectin from Ulex europaeus agglutinin (UEA-1) (Sigma Aldrich, MO, USA). The putative PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26024392 EPCs were stained also for CD34 antigen (CHEMICON Inc., CA, USA), kinase-insert domain receptor (KDR), and eNOS (R D Systems, MN, USA) (Fig. 1a). Determination of EPC count by flow cytometry Circulating EPCs were analyzed for the expression of the surface antigens CD34 and kinase insert domain receptor (KDR) by two-color direct immunofluorescence flow cytometry [15]. Isolated MNCs were stained with an FITCconjugated monoclonal antibody against human CD34 (MACS, Bergisch Gladbach, Germany) and a PE-conjugated PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27532042 monoclonal antibody against human KDR (R D Systems). Identical IgG isotype served as negative controls (R D Systems). The frequency of double-positive peripheral MNCs was determined by forward and side-scatter fluorescence dot-plot analysis of a 5 ?105 cell sample using a FACS Calibur analyzer (Becton Dickinson, NJ, USA). Data were processed using the Macintosh CELLQuest software program (Becton Dickinson). Experimental protocols Isolated EPCs from healthy volunteers were maintained in EBM-2 with either 5.5 mmol/L D-glucose (NG) or 25 mmol/L D-glucose (HG) for five days before they wereFigure 1 Endothelial progenitor cell characterization Endothelial progenitor cell characterization. Endothelial progenitor cells (EPCs) were cultured for five days. (a) From left to right, Panel 1: acetylated LDL uptake by adherent spindle-shaped EPCs, FITC-conjugated lectin UEA-1 binding to the surface of EPCs, and double-positive stained EPCs for acetylated LDL uptake and lectin binding. Panel 2: Immunofluorescence detection of the CD34 antigen (red), and KDR (green) on the EPC surface. Panel 3: Immunofluorescence detection of eNOS in a single non-stained EPC (green). Panel 4: Immunofluorescence detection of the CD34 antigen on the EPC surface (red), and eNOS (green). The EPC nuclei were stained with the blue fluorescent DNA dye DRAQ5TM. Scale bare 50 m. (b) A representative colony of EPCs with a central core of round cells that is surrounded by elongated spindle-shaped cells. Scale bare 100 m.used in the following two experimental protocols. High Lglucose (25 mmol/L) was used as an osmolarity control.Protocol 1 In order to establish whether NO EPZ-5676 structure production and O2- generation were impaired in glucose-stressed EPCs, their NO production and O2- generation were compared to that measured in non-stressed EPCs in the presence and absence of 100 M/mL L-NAME.
