Ablation of angiotensin type 2 receptor prevents endothelial nitric oxide synthase glutathionylation and nitration in ischaemic abductor muscle of diabetic mice

Figures

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  Figure 1.. Blood flow reperfusion and vascular density are improved in diabetic Agtr2-/Y mice: (a) blood flow reperfusion images of nondiabetic and diabetic Agtr2+/Y and Agtr2−/Y mice before, after, and 2 weeks following femoral artery ligature, (b) quantification of the percentage of blood flow reperfusion using the laser Doppler imaging system, (c) immunofluorescence of α-smooth muscle actin (green) and endothelial cells (CD31-red) in the ischaemic abductor muscle of nondiabetic and diabetic Agtr2+/Y and Agtr2−/Y mice and (d) quantification of vascular density (number of vessels smaller than 35 µm). Results are shown as mean ± SEM of 12 mice per group (A and B) and 6 mice per group (C and D).*p = 0.0025 versus nondiabetic Agtr2+/Y, †p = 0.0023 versus diabetic Agtr2−/Y.

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  Figure 2.. Ablation of AT2R in diabetes preserved muscle structure integrity and vascular apoptosis: (a) structural analysis of the ischaemic muscle after hematoxylin and eosin staining of nondiabetic and diabetic Agtr2+/Y and Agtr2−/Y mice, (b) immunofluorescence of vascular endothelial cells (CD31-green) and positive apoptotic cells (red) and (c) quantification of the percentage of vascular endothelial apoptotic cells (white arrows) per mm2 was determined by counting positive apoptotic cells which colocalized with CD31 per total count of CD31 positive cells. Results are shown as mean ± SEM of ischaemic sections of 6 mice per group (A) and 7 mice per group (B and C).

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  Figure 3.. VEGF and VEGFR, but not PGDF mRNA expression were enhanced in the muscle of diabetic Agtr2−/Y mice: (a) VEGF, (b) VEGFR Flk-1, (c) PDGF and (d) PDGFR expression were measured in the abductor ischaemic muscle of nondiabetic and diabetic Agtr2+/Y and Agtr2−/Y mice. Results are shown as mean ± SEM of 6–8 mice per group.

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  Figure 4.. AT2R-deficient mice were protected from diabetes-induced reduction of eNOS expression, and increased glutathionylation and nitration of eNOS. Immunoblot analysis of (a) eNOS, (b) phospho-eNOS, (c) VEGF, (d) nitrotyrosine and (e) S-glutathione expression following the immunoprecititation of eNOS, (f) immunofluorescence of endothelial cell marker CD31 in green and 3-nitrotyrosine expression (in red) in the abductor ischaemic muscle of nondiabetic and diabetic Agtr2+/Y and Agtr2−/Y mice and (g) immunoblot analysis of S-glutathione following the immunoprecipitation of eNOS of endothelial cells treated with or without CGP 42112A (10 nM), Nox inhibitor (200 nM) and NAC (500 µM). Results are shown as mean ± SEM of 6 mice per group (A–E, G) and 8 mice per group (F).