Celiprolol reduces the intimal thickening of autogenous vein grafts via an enhancement of nitric oxide function through an inhibition of superoxide production

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Celiprolol reduces the intimal thickening of autogenous vein grafts via an enhancement of nitric oxide function through an inhibition of superoxide production Keisuke Hattori, MD, Dai Yamanouchi, MD, PhD, Hiroshi Banno, MD, PhD, Masayoshi Kobayashi, MD, PhD, Kiyoto Yamamoto, MD, PhD, Junko Kajikuri, BS, Takeo Itoh, PhD, Kimihiro Komori, MD, PhD Journal of Vascular Surgery Volume 46, Issue 1, Pages 116-123 (July 2007) DOI: 10.1016/j.jvs.2007.03.044 Copyright © 2007 The Society for Vascular Surgery Terms and Conditions

Fig 1 Effect of celiprolol on the development of intimal hyperplasia of the vein graft 4 weeks after operation. A, Microscopic findings of the middle portion of an autologous vein graft from the control group fed with normal rabbit chow and the celiprolol group fed daily with special chow containing 100 mg/body celiprolol. Arrowheads indicate the internal elastic lamina (elastica van Gieson staining; original magnification ×400). B, Quantitative analysis of intimal hyperplasia of vein grafts by random eight-point measurement. The intimal thickness was significantly smaller in the celiprolol group (n = 5) than in the control group (n = 8). C, The intima/media index was also significantly smaller in the celiprolol group (n = 5) than in the control group (n = 8). Results are expressed as the mean ± SEM,*P < .05 vs control. Journal of Vascular Surgery 2007 46, 116-123DOI: (10.1016/j.jvs.2007.03.044) Copyright © 2007 The Society for Vascular Surgery Terms and Conditions

Fig 2 Effects of celiprolol on proliferative activity of the vein graft neointima. The proliferative cell nuclear antigen (PCNA) index was measured by using vein grafts harvested 2 weeks after implantation. Counterstaining was done lightly with hematoxylin. A, Microscopic findings with PCNA staining of the middle portion of an autologous vein graft from the control group and the celiprolol group. Arrowheads show PCNA-positive cells in the neointima (original magnification ×400). B, Quantitative analysis of the frequency of PCNA-positive cells. The PCNA index, the number of PCNA-positive cells divided by the number of total cells, was significantly suppressed in the celiprolol group (n = 6) compared with the control group (n = 8). Results are expressed as the mean ± SEM, *P < .05 vs control. Journal of Vascular Surgery 2007 46, 116-123DOI: (10.1016/j.jvs.2007.03.044) Copyright © 2007 The Society for Vascular Surgery Terms and Conditions

Fig 3 Effects of celiprolol on proliferative activity of the vein graft neointima. The Ki-67 index was measured by using vein grafts harvested 2 weeks after implantation. Counterstaining was done lightly with hematoxylin. A, Microscopic findings with Ki-67 staining of the middle portion of an autologous vein graft from control group and the celiprolol group. Arrowheads show Ki-67-positive cells in the neointima (original magnification ×400). B, Quantitative analysis of the frequency of Ki-67-positive cells. The Ki-67 index, the number of Ki-67-positive cells divided by the number of total cells, was significantly suppressed in the celiprolol group (n = 6) compared with the control group (n = 8). Results are expressed as the mean ± SEM, *P < .05 vs control. Journal of Vascular Surgery 2007 46, 116-123DOI: (10.1016/j.jvs.2007.03.044) Copyright © 2007 The Society for Vascular Surgery Terms and Conditions

Fig 4 Effects of celiprolol on the contraction induced high K+ (128 mM) and PGF2α in vein graft strips. High K+- induced contraction (control rabbit: 8.8 ± 2.1 mg, n = 7, celiprolol rabbit: 6.8 ± 2.3 mg, n = 8; P = 0.40) and PGF2α- induced contraction (control group: 5.1 ± 2.3 mg, n = 7, celiprolol group: 5.6 ± 2.7 mg, n = 8; P = 0.66) were comparable between the two groups. There were no statistically significant differences between high K+-induced contraction and the PGF2α- induced contraction in each group (P = .25 in control group, P = .74 in celiprolol group). Data are shown as means and SEM. Journal of Vascular Surgery 2007 46, 116-123DOI: (10.1016/j.jvs.2007.03.044) Copyright © 2007 The Society for Vascular Surgery Terms and Conditions

Fig 5 Effects of celiprolol on endothelium-dependent relaxation in response to ACh in the strip of vein graft harvested 4 weeks after implantation from control group (n = 7) and celiprolol group (n = 8). A, ACh (10-8 to 10-6 mol/l) were cumulatively applied during a contraction induced by PGF2α (5 x 10-6 mol/l). The amplitude of maintained tonic contraction induced by PGF2α before application of ACh was normalized as a relative tension of 1.0 for strip. The vein graft in control group showed no response, whereas a dose-dependent relaxation in response to ACh was observed in the celiprolol group (P < .001). B, The application of L-NAME completely abolished endothelium- dependent relaxation in the celiprolol group (n = 4) (P < .001). Vein grafts without endothelium showed no response to acetylcholine in the celiprolol group (n = 4) (P < .001). The PGF2α (5 μM)- induced contraction with endothelium before L-NAME application (1.4 ± 0.4 mg, n = 4) and the PGF2α (1 μM)- induced contraction with endothelium in the presence of L-NAME (1.9 ± 0.7 mg, n = 4) were comparable (P = 0.58). The PGF2α (5 μM)- induced contraction with endothelium before L-NAME application and the PGF2α (3 μM)- induced contraction without endothelium (3.9 ± 1.3 mg, n = 4) were comparable (P = 0.12). Data are shown as means and SEM. Asterisks denote a statistically significant difference (P < .05) between the two groups. Journal of Vascular Surgery 2007 46, 116-123DOI: (10.1016/j.jvs.2007.03.044) Copyright © 2007 The Society for Vascular Surgery Terms and Conditions

Fig 6 Effects of celiprolol on eNOS expression in endothelial layer of vein graft from rabbits. A, Immunofluorescence against eNOS can be seen in endothelial layers of two groups. B, Summary of the effect of celiprolol on eNOS expression. As for fluorescence intensities (arbitrary units), there are no significant differences between the control and the celiprolol groups. The mean of data from four different sections (from each animal) with SEM, P = .48 vs control. Journal of Vascular Surgery 2007 46, 116-123DOI: (10.1016/j.jvs.2007.03.044) Copyright © 2007 The Society for Vascular Surgery Terms and Conditions

Fig 7 Effects of celiprolol on superoxide production in endothelial layer of vein graft from rabbits. A, Immunofluorescence estimated superoxide can be seen in the endothelial layers of two groups. B, Summary of the effect of celiprolol on superoxide production. Superoxide production is significantly decreased in the celiprolol group. The mean of data from four different sections (from each animal) with SEM, *P < .05 vs control. Journal of Vascular Surgery 2007 46, 116-123DOI: (10.1016/j.jvs.2007.03.044) Copyright © 2007 The Society for Vascular Surgery Terms and Conditions