Enhanced Cerebral but Not Peripheral Angiogenesis in the Goto-Kakizaki Model of Type 2 Diabetes Involves VEGF and Peroxynitrite Signaling
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Figures
- FIG. 1.
Comparison of cerebral vascularization and its spatial distribution in control and diabetic GK rats. Different brain regions (A) and ROIs (B) where angiogenic parameters were assessed. C: Representative FITC-perfused cerebrovascular images from control and diabetic rats showing differences in angiogenesis in the cerebral cortex and striatum. D–F: Significant differences in vascular density (D), volume (E), and surface area (F) were observed between the cortex and striatum in both control and diabetic groups; however, diabetic rats exhibited a more than twofold increase in vascular density in the ROI in C and D. E and F: Vascular volume and surface area were markedly increased in both the cortex and striatum of the diabetic group compared with controls. *P < 0.01 cortex vs. striatum; #P < 0.05 diabetes vs. control. Data are means ± SEM, n = 4–11. (A high-quality digital representation of this figure is available in the online issue.)
- FIG. 2.
Immature cerebral microvessels are more abundant in diabetes. A: Diabetic group had significantly increased cortical and striatal nonperfused vessels compared with controls. B: Pericyte-to-endothelial ratio was decreased in the diabetic group. C: There was a visual but not statistically significant increase in retinal vasculature, whereas peripheral vasculature was decreased in diabetes. *P = 0.0016 diabetes vs. control; ψP = 0.01; **P = 0.029 diabetes vs. control. Data are means ± SEM, n = 3–4 (exact Wilcoxon test).
- FIG. 3.
Increased VEGF expression and tyrosine nitration status of micro- and macrovasculature in diabetes. A: VEGF levels were significantly increased in the cerebral microvasculature but not the macrovasculature in diabetes. B: Both diabetic micro- and macrovasculature had significantly increased tyrosine nitration compared with controls. *P = 0.001; **P = 0.05 diabetes vs. control. Data are means ± SEM, n = 4 (exact Wilcoxon test).
- FIG. 4.
Diabetic BMECs show significant increases in tubologenesis over time. A: Representative images of BMECs showing more tube formation in the diabetic group after 24 and 48 h of plating on reduced matrigel. B: Diabetic BMECs have significantly increased tube formation properties after 24 and 48 h of plating on reduced matrigel than in control cells. C: Diabetic BMECs have a significantly higher percentage increase in proliferation after 24 and 48 h. D and E: Diabetic BMECs have significantly smaller cell mean diameter and volume compared with control cells, respectively. *P < 0.01 diabetes vs. control; #P < 0.01 24 vs. 48 h; **P = 0.0022 diabetes vs. control. Data are means ± SEM, n = 3–6.
- FIG. 5.
Diabetic BMECs have increased cell migration that is mediated by VEGF in an autocrine manner. A: Representative images of BMECs showing increased spontaneous cell migration. B: Representative images of control cells treated with conditioned medium from diabetic cells in the presence and absence of VEGF-neutralizing antibody. Control BMECs treated with diabetic BMEC-conditioned media show increased cell migration and anti-VEGF antibody inhibits this effect significantly. C: Quantitative analysis of data shown in A and B. Diabetic BMECs plated on fibronectin show significantly increased spontaneous cell migration after 24 h. Control cells showed enhanced migratory properties when treated with diabetic endothelial cell–conditioned media (ECM), and VEGF-neutralizing antibody significantly abrogated this response. *P = 0.0026 across control groups; **P < 0.05 vs. other control groups by Tukey post hoc analysis. Data are means ± SEM, n = 5–7. D and E: Diabetic endothelial cells secrete relatively higher levels of native and dimerized VEGF-A. #P = 0.016 diabetes vs. control. Data are means ± SEM, n = 3–8 (exact Wilcoxon test). (A high-quality color representation of this figure is available in the online issue.)
- FIG. 6.
Effect of diabetes on basal expression and phosphorylation status of angiogenesis mediators. A: Native and phosphorylated VEGF-R2 levels were determined by immunoblotting. Phospho-VEGF was increased in diabetic BMECs. Data are means ± SEM, n = 3. B: In parallel with increased VEGF-R2 activation, c-src phosphorylation also was increased in diabetes. Data are means ± SEM, n = 4. C: Diabetic endothelial cells have elevated protein tyrosine nitration compared with controls. Data are means ± SEM, n = 6–11. D and E: MMP-2 activity was assessed by gelatin zymography. Although there was no difference in secreted latent and active MMP-2, cell-associated MMP-2 was significantly increased. F: MT1-MMP levels determined by immunoblotting were greater in diabetes. Data are means ± SEM, n = 3–4. *P = 0.0079 diabetes vs. control; #P = 0.029 diabetes vs. control; ψP = 0.015 diabetes vs. control; **P = 0.057 diabetes vs. control (exact Wilcoxon test).
- FIG. 7.
Evidence for involvement of endogenous VEGF signaling in increased migration in diabetes. The role of various angiogenic proteins were assessed by using respective inhibitors on cell migration assays. Two- or 24-h pretreatment with anti-VEGF antibody (A), peroxynitrite decomposition catalyst FeTPPs (B), src inhibitor PP2 (C), or MMP inhibitor minocyline (D) significantly reduced migration of BMECs in diabetic but not control endothelial cells. #P < 0.05 vs. untreated diabetes; *P < 0.01 untreated diabetes vs. control. Data are means ± SEM, n = 4–7.
- FIG. 8.
The differential effect of exogenous VEGF on VEGF signaling in control and diabetic cells. Cells were treated with vehicle, peroxynitrite decomposition catalyst FeTPPs alone, VEGF alone, or VEGF plus FeTPPs. Phosphorylated VEGF receptor activation (p-VEGF-R2–to–VEGF-R2 ratio), peroxynitrite formation, and phosphorylated c-src activation (p-c-src–to–c-src ratio) were determined by immunoblotting, as shown in A–C, respectively. Exogenous VEGF treatment stimulated VEGF-R2 activation, c-src activation, and tyrosine nitration in control cells, and cotreatment with FeTPPs prevented this activation. Diabetic cells that show increased basal VEGF-R2, c-src, and nitrotyrosine activation do not show further elevation in response to exogenous VEGF but respond to FeTPPs treatment, indicating that peroxynitrite sustains VEGF-R2 phosphorylation and also mediates downstream signaling in these cells. D: Schematic representation of the role peroxynitrite in modulation of the angiogenic signal in brain microvascular endothelial cells. *P < 0.05 vs. other control groups; **P < 0.01 vs. untreated control; #P < 0.05 vs. untreated diabetes. (A high-quality color representation of this figure is available in the online issue.)
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