Impairment of human cell–based vasculogenesis in rats by hypercholesterolemia- induced endothelial dysfunction and rescue with l-arginine supplementation

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Impairment of human cell–based vasculogenesis in rats by hypercholesterolemia- induced endothelial dysfunction and rescue with l-arginine supplementation Erik J. Suuronen, PhD, Samir Hazra, BSc, Pingchuan Zhang, MD, PhD, Renaud Vincent, PhD, Premkumari Kumarathasan, PhD, Yan Zhang, MD, MSc, Joel Price, MD, Vincent Chan, MD, Frank W. Sellke, MD, Thierry G. Mesana, MD, PhD, John P. Veinot, MD, Marc Ruel, MD, MPH The Journal of Thoracic and Cardiovascular Surgery Volume 139, Issue 1, Pages 209-216.e2 (January 2010) DOI: 10.1016/j.jtcvs.2009.04.055 Copyright © 2010 The American Association for Thoracic Surgery Terms and Conditions

Figure 1 Immunohistochemical analysis of tissue endothelial nitric oxide synthase expression. Representative images of endothelial nitric oxide synthase levels in ischemic muscle of rats in groups A (A), B, (B), and C (C). Scale bar represents 150μm. The Journal of Thoracic and Cardiovascular Surgery 2010 139, 209-216.e2DOI: (10.1016/j.jtcvs.2009.04.055) Copyright © 2010 The American Association for Thoracic Surgery Terms and Conditions

Figure 2 Biochemical analysis of endothelial function. Ratio of endothelin 1 (ET1) to nitrite (A), ratio of endothelin 1 (ET1) to big endothelin 1 (bET1) (B), and nitrotyrosine level (C) were all significantly increased in serum of group B (CHOL) relative to groups A (NORM) and C (CHOL-ARG). Asterisk indicates P < .05 versus other groups. The Journal of Thoracic and Cardiovascular Surgery 2010 139, 209-216.e2DOI: (10.1016/j.jtcvs.2009.04.055) Copyright © 2010 The American Association for Thoracic Surgery Terms and Conditions

Figure 3 Inflammatory index. Ratio of proinflammatory to anti-inflammatory cytokine expression was greater in group B (CHOL) than in groups A (NORM) and C (CHOL-ARG). The Journal of Thoracic and Cardiovascular Surgery 2010 139, 209-216.e2DOI: (10.1016/j.jtcvs.2009.04.055) Copyright © 2010 The American Association for Thoracic Surgery Terms and Conditions

Figure 4 Arteriolar density. Representative images of α-smooth muscle actin–stained arterioles in ischemic muscle of rats in groups A (A, NORM), B (B, CHOL), and C (C, CHOL-ARG). Both arteriolar density (D) and ratio of ischemic to nonischemic arteriolar count (E) were significantly reduced in group B relative to groups A and C. Scale bar represents 250 μm. Asterisk indicates P < .04 versus other groups; double asterisk indicates P < .01 versus other groups. The Journal of Thoracic and Cardiovascular Surgery 2010 139, 209-216.e2DOI: (10.1016/j.jtcvs.2009.04.055) Copyright © 2010 The American Association for Thoracic Surgery Terms and Conditions

Figure 5 Capillary density. Representative images of CD31-stained capillaries (arrowheads) in ischemic muscle of rats in groups A (A, NORM), B (B, CHOL), and C (C, CHOL-ARG). D, Capillary density was significantly reduced in group B relative to groups A and C. Scale bar represents 75 μm. Asterisk indicates P < .02 versus other groups. The Journal of Thoracic and Cardiovascular Surgery 2010 139, 209-216.e2DOI: (10.1016/j.jtcvs.2009.04.055) Copyright © 2010 The American Association for Thoracic Surgery Terms and Conditions

Figure 6 Hind-limb perfusion. Laser Doppler analysis was used to measure perfusion before (Pre-op) and after (Post-op) operation and at 7, 14, and 21 postoperative days. Data are presented as average ratio of ischemic to nonischemic limb blood flow. Asterisk indicates P < .001 between groups B (CHOL) and C (CHOL-ARG). NORM, Group A. The Journal of Thoracic and Cardiovascular Surgery 2010 139, 209-216.e2DOI: (10.1016/j.jtcvs.2009.04.055) Copyright © 2010 The American Association for Thoracic Surgery Terms and Conditions

Figure 7 Cell engraftment at 3 weeks after cell transplant. A, Representative image of transplanted human circulating progenitor cell (arrowhead), stained with human-specific pancentromeric probe (purple nuclei), found engrafted in ischemic hind-limb muscle of rat 3 weeks after injection. All cell nuclei are stained with 4′,6-diamidino-2-phenylindole (blue). Inset shows higher magnification of stained human cell. Scale bar represents 75 μm. B, Number of transplanted human cells found engrafted in ischemic hind limb after 2 weeks was not different (P = .9) between groups A (NORM), B (CHOL), and C (CHOL-ARG). The Journal of Thoracic and Cardiovascular Surgery 2010 139, 209-216.e2DOI: (10.1016/j.jtcvs.2009.04.055) Copyright © 2010 The American Association for Thoracic Surgery Terms and Conditions