Daniel H. Platt

Diabetes-induced Coronary Vascular Dysfunction Involves Increased Arginase Activity

Increases in arginase activity have been reported in a variety of disease conditions characterized by vascular dysfunction. Arginase competes with NO synthase for their common substrate arginine, suggesting a cause and effect relationship. We tested this concept by experiments with streptozotocin diabetic rats and high glucose (HG)-treated bovine coronary endothelial cells (BCECs). Our studies showed that diabetes-induced impairment of vasorelaxation to acetylcholine was correlated with increases in reactive oxygen species and arginase activity and arginase I expression in aorta and liver. Treatment of diabetic rats with simvastatin (5 mg/kg per day, subcutaneously) or l-citrulline (50 mg/kg per day, orally) blunted these effects. Acute treatment of diabetic coronary arteries with arginase inhibitors also reversed the impaired vasodilation to acetylcholine. Treatment of BCECs with HG (25 mmol/L, 24 hours) also increased arginase activity. This effect was blocked by treatment with simvastatin (0.1 &mgr;mol/L), the Rho kinase inhibitor Y-27632 (10 &mgr;mol/L), or l-citrulline (1 mmol/L). Superoxide and active RhoA levels also were elevated in HG-treated BCECs. Furthermore, HG significantly diminished NO production in BCECs. Transfection of BCECs with arginase I small interfering RNA prevented the rise in arginase activity in HG-treated cells and normalized NO production, suggesting a role for arginase I in reduced NO production with HG. These results indicate that increased arginase activity in diabetes contributes to vascular endothelial dysfunction by decreasing l-arginine availability to NO synthase.

Vascular endothelial growth factor and diabetic retinopathy: pathophysiological mechanisms and treatment perspectives.

Retinal neovascularization and macular edema are central features of diabetic retinopathy, the major cause of blindness in the developed world. Current treatments are limited in their efficacy and are associated with significant adverse effects. Characterization of the molecular and cellular processes involved in vascular growth and permeability has led to the recognition that the angiogenic growth factor and vascular permeability factor vascular endothelial growth factor (VEGF) plays a pivotal role in the retinal microvascular complications of diabetes. Therefore, VEGF represents an exciting target for therapeutic intervention in diabetic retinopathy. This review highlights the current understanding of the mechanisms that regulate VEGF gene expression and mediate its biological effects and how these processes may become altered during diabetes. The cellular and molecular alterations that characterize experimental models of diabetes are considered in relation to the influence of high glucose‐mediated oxidative stress on VEGF expression and on the mechanisms of VEGFs actions under hyperglycemic induction. Finally, potential therapeutic strategies for preventing VEGF overexpression or blocking its pathological effects in the diabetic retina are considered. Copyright

Vascular Endothelial Growth Factor and Diabetic Retinopathy: Role of Oxidative Stress

Retinal neovascularization and macular edema are central features of diabetic retinopathy, a major cause of blindness in working age adults. The currently established treatment for diabetic retinopathy targets the vascular pathology by laser photocoagulation. This approach is associated with significant adverse effects due the destruction of neural tissue and is not always effective. Characterization of the molecular and cellular processes involved in vascular growth and hyperpermeability has led to the recognition that the angiogenic growth factor and vascular permeability factor VEGF (vascular endothelial growth factor) play a pivotal role in the retinal microvascular complications of diabetes. Thus, VEGF represents an important target for therapeutic intervention in diabetic retinopathy. Agents that directly inhibit the actions of VEGF and its receptors show considerable promise, but have not proven to be completely effective in blocking pathological angiogenesis. Therefore, a better understanding of the molecular events that control VEGF expression and mediate its downstream actions is important to define more precise therapeutic targets for intervention in diabetic retinopathy. This review highlights the current understanding of the process by which VEGF gene expression is regulated and how VEGFs biological effects are altered during diabetes. In particular, cellular and molecular alterations seen in diabetic models are considered in the context of high glucose-mediated oxidative stress effects on VEGF expression and action. Potential therapeutic strategies for preventing VEGF overexpression or blocking its pathological actions in the diabetic retina are considered.

Inhibition of NAD(P)H Oxidase Activity Blocks Vascular Endothelial Growth Factor Overexpression and Neovascularization during Ischemic Retinopathy

Because oxidative stress has been strongly implicated in up-regulation of vascular endothelial growth factor (VEGF) expression in ischemic retinopathy, we evaluated the role of NAD(P)H oxidase in causing VEGF overexpression and retinal neovascularization. Dihydroethidium imaging analyses showed increased superoxide formation in areas of retinal neovascularization associated with relative retinal hypoxia in a mouse model for oxygen-induced retinopathy. The effect of hypoxia in stimulating superoxide formation in retinal vascular endothelial cells was confirmed by in vitro chemiluminescence assays. The superoxide formation was blocked by specific inhibitors of NAD(P)H oxidase activity (apocynin, gp91ds-tat) indicating that NAD(P)H oxidase is a major source of superoxide formation. Western blot and immunolocalization analyses showed that retinal ischemia increased expression of the NAD(P)H oxidase catalytic subunit gp91phox, which localized primarily within vascular endothelial cells. Treatment of mice with apocynin blocked ischemia-induced increases in oxidative stress, normalized VEGF expression, and prevented retinal neovascularization. Apocynin and gp91ds-tat also blocked the action of hypoxia in causing increased VEGF expression in vitro, confirming the specific role of NAD(P)H oxidase in hypoxia-induced increases in VEGF expression. In conclusion, NAD(P)H oxidase activity is required for hypoxia-stimulated increases in VEGF expression and retinal neovascularization. Inhibition of NAD(P)H oxidase offers a new therapeutic target for the treatment of retinopathy.

VEGF differentially activates STAT3 in microvascular endothelial cells

Increased VEGF expression is found in several pathologies characterized by abnormal angiogenesis. Previous studies have shown that the transcription factor STAT3 mediates VEGF gene transcription and its activation. In this study, Western analysis and confocal immunocytochemistry were used to examine STAT3 activation in retinal microvascular endothelial cells (BREC). We found that VEGF rapidly induces STAT3 tyrosine phosphorylation and nuclear translocation. Immunoprecipitation studies also showed that VEGF forms a complex with VEGFR2 only in BREC and not in aortic macrovascular endothelial cells (BAEC). In addition, quantitative real‐time RT‐PCR analysis of VEGF‐induced VEGF expression showed a significant increase in specific mRNA formation only in BREC and not in BAEC, and this effect was significantly reduced by antisense‐mediated reduction of STAT3 expression. Furthermore, studies conducted in human dermal microvascular endothelial cells (HDMEC) showed that, in this endothelial cell type, VEGF autocrine expression is also accompanied by STAT3 activation as in BREC. In this study we showed that VEGF can differentially induce STAT3 activation in micro‐ versus macro‐vascular endothelial cells and that this effect is linked to VEGFR2/STAT3 complex formation, which correlates with VEGF autocrine ability to stimulate its own gene expression.

Peroxynitrite mediates VEGF's angiogenic signal and function via a nitration-independent mechanism in endothelial cells

The modulation of angiogenic signaling by reactive oxygen species (ROS) is an emerging area of interest in cellular and vascular biology research. We provide evidence here that peroxynitrite, the powerful oxidizing and nitrating free radical, is critically involved in transduction of the VEGF signal. We tested the hypothesis that VEGF induces peroxynitrite formation, which causes tyrosine phosphorylation and mediates endothelial cell migration and tube formation, by stud‐ ies of vascular endothelial cells in vitro and in a model of hypoxia‐induced neovascularization in vivo. The specific peroxynitrite decomposition catalyst FeTPPs blocked VEGF‐induced phosphorylation of VEGFR2 and c‐Src and inhibited endothelial cell migration and tube formation. Furthermore, exogenous peroxynitrite mimicked VEGF activity in causing phosphorylation of VEGFR2 and stimulating endothelial cell growth and tube formation in vitro and new blood vessel growth in vivo. The selective nitration inhibitor epicatechin en‐ hanced VEGFs angiogenic function in activating VEGFR2, c‐Src, and promoting endothelial cell growth, migration, and tube formation in vitro and retinal neovascularization in vivo. Decomposing peroxynitrite with FeTPPs or blocking oxidation using the thiol donor NAC blocked VEGFs angiogenic functions in vitro and in vivo. In conclusion, peroxynitrite is critically involved in transducing VEGFs angiogenic signal via nitration‐independent and oxidation‐mediated tyrosine phosphorylation.—El‐Remessy, A. B., Al‐Shabrawey, M., Platt, D. H., Bartoli, M., Behzadian, M. A., Ghaly, N., Tsai, N., Motamed, K., Caldwell, R. B. Peroxynitrite mediates VEGFs angiogenic signal and function via a nitration‐independent mechanism in endothelial cells. FASEB J. 21, 2528–2539 (2007)

Diabetes-induced vascular dysfunction involves arginase I.

Arginase can cause vascular dysfunction by competing with nitric oxide synthase for l-arginine and by increasing cell proliferation and collagen formation, which promote vascular fibrosis/stiffening. We have shown that increased arginase expression/activity contribute to vascular endothelial cell (EC) dysfunction. Here, we examined the roles of the two arginase isoforms, arginase I and II (AI and AII, respectively), in this process. Experiments were performed using streptozotocin-induced diabetic mice: wild-type (WT) mice and knockout mice lacking the AII isoform alone (AI(+/+)AII(-/-)) or in combination with partial deletion of AI (AI(+/-)AII (-/-)). EC-dependent vasorelaxation of aortic rings and arterial fibrosis and stiffness were assessed in relation to arginase activity and expression. Diabetes reduced mean EC-dependent vasorelaxation markedly in diabetic WT and AI(+/+)AII(-/-) aortas (53% and 44% vs. controls, respectively) compared with a 27% decrease in AI(+/-)AII (-/-) vessels. Coronary fibrosis was also increased in diabetic WT and AI(+/+)AII(-/-) mice (1.9- and 1.7-fold vs. controls, respectively) but was not altered in AI(+/-)AII (-/-) diabetic mice. Carotid stiffness was increased by 142% in WT diabetic mice compared with 51% in AI(+/+)AII(-/-) mice and 19% in AI(+/-)AII (-/-) mice. In diabetic WT and AI(+/+)AII(-/-) mice, aortic arginase activity and AI expression were significantly increased compared with control mice, but neither parameter was altered in AI(+/-)AII (-/-) mice. In summary, AI(+/-)AII (-/-) mice exhibit better EC-dependent vasodilation and less vascular stiffness and coronary fibrosis compared with diabetic WT and AI(+/+)AII(-/-) mice. These data indicate a major involvement of AI in diabetes-induced vascular dysfunction.