Arterioscler Thromb Vasc Biol Increase of Circulating Endothelial Progenitor Cells in Patients with Coronary Artery Disease After Exercise-Induced Ischemia by Volker Adams, Karsten Lenk, Axel Linke, Dominik Lenz, Sandra Erbs, Markus Sandri, Attila Tarnok, Stephan Gielen, Frank Emmrich, Gerhard Schuler, and Rainer Hambrecht Arterioscler Thromb Vasc Biol Volume 24(4):684-690 April 1, 2004 Copyright © American Heart Association, Inc. All rights reserved.

Figure 1. Characterization of attached MNCs by FACS analysis (A), immunofluorescent staining (B, C), and RT-PCR (D). Figure 1. Characterization of attached MNCs by FACS analysis (A), immunofluorescent staining (B, C), and RT-PCR (D). A, Representative light microscopic picture and histograms of FACS analysis of cultured EPCs after 4 days for VE-cadherin, KDR, and CD3 are depicted. Plots show isotype control (gray) versus specific antibody staining (black). B and C, Immunofluorescent staining of attached cells with anti-vWF, anti-CD31, and anti-CD45 staining is shown. D, The RT-PCR analysis of the attached cells revealed a positive amplification of GAPDH (lane 2), eNOS (lane 4), and vWF (lane 5), whereas the negative controls (lane 1,3) were negative. M=100-bp size marker. Volker Adams et al. Arterioscler Thromb Vasc Biol. 2004;24:684-690 Copyright © American Heart Association, Inc. All rights reserved.

Figure 2. Freshly isolated MNCs were grown in endothelium-specific media, and microscopic pictures were taken after 4 days, 10 days, and 4 weeks. Figure 2. Freshly isolated MNCs were grown in endothelium-specific media, and microscopic pictures were taken after 4 days, 10 days, and 4 weeks. After 4 weeks in culture, the morphology changed and cobble-stone cells were visible. This change in morphology toward endothelial cells is accompanied with a significant increase of vWF expression in the cells as detected by RT-PCR. Lane 1 indicates negative control (no template added to the PCR reaction); lane 2, after 4 days; lane 3, after 4 weeks; and lane 4, HUVEC cells as positive control. For quantitative analysis, the ratio of vWF/GAPDH is depicted. Volker Adams et al. Arterioscler Thromb Vasc Biol. 2004;24:684-690 Copyright © American Heart Association, Inc. All rights reserved.

Figure 3. Quantitative evaluation of circulating EPCs by cell culture assay. Figure 3. Quantitative evaluation of circulating EPCs by cell culture assay. MNC were isolated from blood samples of IP (triangle), CP (circles), and HC (squares) after different time points after a single symptom-limited exercise test, and EPCs were quantified as double-positive stained cells (Di-LDL and UEA-1) by LSC. Values are expressed as fold increase versus beginning. Black symbols represent measurements of the whole patient population, whereas white symbols represent measurements from the subgroup of patients, who were followed-up over a longer time period. Volker Adams et al. Arterioscler Thromb Vasc Biol. 2004;24:684-690 Copyright © American Heart Association, Inc. All rights reserved.

Figure 4. Quantitative evaluation of circulating EPCs by FACS analysis. Figure 4. Quantitative evaluation of circulating EPCs by FACS analysis. A representative FACS analysis, in which the CD34positive/CD3negative cells were gated and further analyze for KDR+/CD34+ double-positive cells (EPCs), is shown on top of the graph. The amount of circulating EPCs in blood samples of IP (triangle), CP (circles), and HC (squares) was analyzed after different time points after a single symptom-limited exercise test. Values are expressed as fold increase versus beginning. Black symbols represent measurements of the whole patient population, whereas white symbols represent measurements from the subgroup of patients, who were followed-up over a longer time period. Volker Adams et al. Arterioscler Thromb Vasc Biol. 2004;24:684-690 Copyright © American Heart Association, Inc. All rights reserved.