Simultaneous application of basic fibroblast growth factor and hepatocyte growth factor to enhance the blood vessels formation Akira Marui, MD, Akihiro.

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Date of download: 7/7/2016 Copyright © The American College of Cardiology. All rights reserved. From: Sustained Release of Erythropoietin Using Biodegradable.

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Simultaneous application of basic fibroblast growth factor and hepatocyte growth factor to enhance the blood vessels formation Akira Marui, MD, Akihiro Kanematsu, MD, PhD, Kenichi Yamahara, MD, PhD, Kazuhiko Doi, MD, Toshihiro Kushibiki, MS, Masaya Yamamoto, PhD, Hiroshi Itoh, MD, PhD, Tadashi Ikeda, MD, PhD, Yasuhiko Tabata, PhD, DMSc, DPharm, Masashi Komeda, MD, PhD Journal of Vascular Surgery Volume 41, Issue 1, Pages 82-90 (January 2005) DOI: 10.1016/j.jvs.2004.10.029 Copyright © 2005 The Society for Vascular Surgery Terms and Conditions

Fig 1 Capillary network formation assay with human umbilical vein endothelial cells (HUVECs). Capillary network formation was assessed on Matrigel in the presence of basic fibroblast growth factor (bFGF) or hepatocyte growth factor (HGF), or bFGF and HGF together. A, Dose-response of capillary network formation area (%) of HUVECs in the presence of bFGF (closed bar) or HGF (open bar). †P < .05 vs HGF 50 ng/mL; #P < .01vs HGF 500 ng/mL. B, Capillary network formation area in the presence of a fixed concentration of bFGF (50 ng/mL) and the indicated concentrations of HGF (0.5 to 500 ng/mL). †P < .05, #P < .01 vs 0 ng/mL of HGF. C, Enhancement of capillary network formation of HUVEC in Matrigel by the combination of bFGF and HGF. HUVECs were cultured in Matrigel in the presence of (a) control (no growth factor added); (b) HGF alone (50 ng/mL); (c) bFGF alone (50 ng/mL); (d) or combination of bFGF and HGF (50 ng/mL each) (×10). Journal of Vascular Surgery 2005 41, 82-90DOI: (10.1016/j.jvs.2004.10.029) Copyright © 2005 The Society for Vascular Surgery Terms and Conditions

Fig 2 In vivo release profile of basic fibroblast growth factor (bFGF) or hepatocyte growth factor (HGF) from collagen. In vivo release profile of the radioactivity remaining after subcutaneous implantation of the collagen sheet incorporating 125I-labeled bFGF or HGF (n = 6 each). The lines on the graphs are approximately growth factor/collagen (solid line), collagen degradation (dashed line), and growth factor/solution (dotted line). A, Sustained release of bFGF (square) from collagen was observed over 4 weeks, and the time profile correlates with that of the collagen degradation (circle). R2 = 0.974, P < .0005, simple regression analysis. The bolus injection of bFGF solution (triangle) disappeared within 3 days. B, Sustained release of HGF (square) was observed over 4 weeks. The bolus injection of HGF solution (triangle) disappeared within 3 days. Journal of Vascular Surgery 2005 41, 82-90DOI: (10.1016/j.jvs.2004.10.029) Copyright © 2005 The Society for Vascular Surgery Terms and Conditions

Fig 3 Restoration of the blood perfusion with basic fibroblast growth factor (bFGF) or hepatocyte growth factor (HGF) by sustained release vs bolus injection. Restoration of the blood perfusion in the ischemic limb with bFGF or HGF by sustained release or bolus injection was evaluated. Hindlimb blood perfusion was assessed by a ratio (%) of ischemic (right) to normal (left) hindlimb blood perfusion measured by laser Doppler perfusion imaging (LDPI) analyzer (n = 7 to 8). A, Serial LDPI measurements in ischemic limb treated with 80 μg of bFGF/CM, 80 μg of bFGF/solution, collagen microspheres (CM), and the control (no treatment) (#P < .01 vs. control). B, Serial LDPI measurements in ischemic limb treated with 80 μg of HGF/CM, 80 μg of HGF/solution, CM, and the control. #P < .01 vs. control. Journal of Vascular Surgery 2005 41, 82-90DOI: (10.1016/j.jvs.2004.10.029) Copyright © 2005 The Society for Vascular Surgery Terms and Conditions

Fig 4 Restoration of the blood perfusion by single or dual release of basic fibroblast growth factor (bFGF) or hepatocyte growth factor (HGF). Quantitative analysis of ischemic/normal perfusion ratio (%) in ischemic hindlimb measured by laser Doppler perfusion image (LDPI) analyzer (n = 7 to 8). Serial LDPI measurements in ischemic limb treated with indicated doses. A, Threshold of restoration of the blood perfusion of bFGF/CM (1, 5, 20, and 80 μg) and the control. #P < .01 vs control. B, Threshold of restoration of the blood perfusion of HGF/CM (1, 5, 20, and 80 μg) and the control (#P < .01 vs control). C, Restoration of the blood perfusion with dual/CM (CM incorporating 5 μg of bFGF and 20 μg of HGF), 20 μg of HGF/CM alone, 5 μg of bFGF/CM alone, and the control. #P < .01 vs every other group. D, Restoration of the blood perfusion with dual/CM (CM incorporating 5 μg of bFGF and 20 μg of HGF), 80 μg of bFGF/CM, 80 μg of HGF/CM, and the control. CM, collagen microspheres; NS, not significant. Journal of Vascular Surgery 2005 41, 82-90DOI: (10.1016/j.jvs.2004.10.029) Copyright © 2005 The Society for Vascular Surgery Terms and Conditions

Fig 5 Immunohisotchemical analysis in the ischemic limb after treatments. A, Capillary density: quantitative analysis of capillary density (anti-vWF-positive vessels/mm2) in the ischemic limb. *P < .01 vs control, †P < .05 vs (d) 20 μg of bFGF/CM. Table, a-m, Several treatments in the ischemic hindlimb: a, control; b-e, sustained single release of 1, 5, 20, and 80 μg of bFGF; f-1, sustained single release of 1, 5, 20, and 80 μg of HGF; j, sustained dual release of 5 μg of bFGF and 20 μg of HGF; k-m, bolus injection of (k) 80 μg of bFGF, (l) 80 μg of HGF, and (m) 80 μg of bFGF and HGF. B, Immunostaining of the ischemic hindlimb tissue with anti-vWF 4 weeks after the treatment: a, control; b, 20 μg of HGF/CM; c, 5 μg of bFGF/CM; d, dual/CM (CM incorporating 5 μg of bFGF and 20 μg HGF 5) (×100; scale bars, 100 μm). C, Maturation index: maturity of newly formed vessels was evaluated by a percentage of α-SMA–positive vessels compared with total vessels counted by anti-vWF antibody staining. *P < .01 vs a, †P < .05 vs d, ‡P < .01 vs i, and P < .05 vs e. D, Immunostaining of the ischemic hindlimb tissue with α-SMA 4 weeks after the treatment: (a) Control, (b) 20 μg of HGF/CM, (c) 5 μg of bFGF/CM, (d) dual/CM (CM incorporating 5 μg of bFGF and 20 μg of HGF) (×100; scale bars, 100 μm). E, Focal inflammation: Number of infiltrating leukocytes in ischemic limb obtained from immunostaining with anti-CD45 antibody 3 days after the treatment (n = 4 to 5).*P < .01, **P = .072, ***P = .83 vs control, vWF, von Willebrand factor; bFGF, basic fibroblast growth factor; HGF, hepatocyte growth factor; CM, collagen microspheres; SMA, smooth muscle actin. Journal of Vascular Surgery 2005 41, 82-90DOI: (10.1016/j.jvs.2004.10.029) Copyright © 2005 The Society for Vascular Surgery Terms and Conditions

Fig 5 Immunohisotchemical analysis in the ischemic limb after treatments. A, Capillary density: quantitative analysis of capillary density (anti-vWF-positive vessels/mm2) in the ischemic limb. *P < .01 vs control, †P < .05 vs (d) 20 μg of bFGF/CM. Table, a-m, Several treatments in the ischemic hindlimb: a, control; b-e, sustained single release of 1, 5, 20, and 80 μg of bFGF; f-1, sustained single release of 1, 5, 20, and 80 μg of HGF; j, sustained dual release of 5 μg of bFGF and 20 μg of HGF; k-m, bolus injection of (k) 80 μg of bFGF, (l) 80 μg of HGF, and (m) 80 μg of bFGF and HGF. B, Immunostaining of the ischemic hindlimb tissue with anti-vWF 4 weeks after the treatment: a, control; b, 20 μg of HGF/CM; c, 5 μg of bFGF/CM; d, dual/CM (CM incorporating 5 μg of bFGF and 20 μg HGF 5) (×100; scale bars, 100 μm). C, Maturation index: maturity of newly formed vessels was evaluated by a percentage of α-SMA–positive vessels compared with total vessels counted by anti-vWF antibody staining. *P < .01 vs a, †P < .05 vs d, ‡P < .01 vs i, and P < .05 vs e. D, Immunostaining of the ischemic hindlimb tissue with α-SMA 4 weeks after the treatment: (a) Control, (b) 20 μg of HGF/CM, (c) 5 μg of bFGF/CM, (d) dual/CM (CM incorporating 5 μg of bFGF and 20 μg of HGF) (×100; scale bars, 100 μm). E, Focal inflammation: Number of infiltrating leukocytes in ischemic limb obtained from immunostaining with anti-CD45 antibody 3 days after the treatment (n = 4 to 5).*P < .01, **P = .072, ***P = .83 vs control, vWF, von Willebrand factor; bFGF, basic fibroblast growth factor; HGF, hepatocyte growth factor; CM, collagen microspheres; SMA, smooth muscle actin. Journal of Vascular Surgery 2005 41, 82-90DOI: (10.1016/j.jvs.2004.10.029) Copyright © 2005 The Society for Vascular Surgery Terms and Conditions