Kinase insert domain receptor kdr

PBMNCs were isolated by Ficoll density gradient centrifugation from healthy subjects and CAD patients as described previously, and late EPCs were cultured and characterized by following the protocol described [15 (link), 16 (link)]. In short, PBMCs were cultured on fibronectin-coated 6-well plates in EBM-2 (contents: ascorbic acid 0.5 ml, rhFGF-B 2.0 ml, heparin 0.5 ml, GA-1000 0.5 ml, rhEGF 0.5 ml, hydrocortisone 0.2 ml, VEGF 0.5 ml, R3-IGF-1 0.5 ml) supplemented with endothelial growth medium-SingleQuots (Clonetics, San Diego, CA, USA). For all assays, late EPCs were used at passage 3 (approximately 28 days) after being removed by complete washing with culture medium. After 28 days, endothelial markers of cultured EPCs were examined by flow cytometry analysis using CD31 (BD Biosciences, San Jose, CA, USA), Tie-2 receptor (BD Biosciences), and kinase-insert domain receptor (KDR) (R&D Systems, Inc., Minneapolis, MN, USA). EPCs isolated from healthy and CAD patients separately were both cultured at 37 °C and 5% CO₂ in EBM-2, and the medium was changed every 48 h.

The expression of endothelial marker proteins was examined in the cultured EPCs by using flow cytometric analysis with phycoerythrin (PE)-labeled monoclonal mouse anti-human antibodies recognizing CD31 (BD Pharmingen, San Diego, CA, USA), von Willebrand factor (vWF) (BD Pharmingen), kinase-insert domain receptor (KDR) (R&D Systems, Minneapolis, MN, USA), and CD14 (BD Pharmingen). To identify the cells that expressed these surface antigens, the EPCs were incubated for 40 min at 4 °C in a volume of 100 μl of solution containing an appropriate amount of PE-labeled antibody or corresponding IgG isotype control. At least 1 × 10⁵ EPCs were acquired by using a flow cytometer (Beckman-Coulter, Fullerton, CA, USA).