Manuela Bartoli

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.

Oxidative stress inactivates VEGF survival signaling in retinal endothelial cells via PI 3-kinase tyrosine nitration

In diabetic retinopathy, endothelial cell apoptosis is paradoxically increased despite upregulation of the potent pro-survival factor VEGF. We tested the hypothesis that elevated glucose levels disrupt VEGFs pro-survival function via peroxynitrite-mediated alteration of the Akt-1/p38 MAP kinase signaling pathway by studies of retinal endothelial cells in vitro. High glucose or exogenous peroxynitrite caused significant increases in apoptosis in the presence or absence of VEGF. Treatment with a peroxynitrite decomposition catalyst blocked these effects, implying a causal role of peroxynitrite. Peroxynitrite or high glucose treatment also increased phosphorylation of p38 MAP kinase, whereas phosphorylation of Akt-1 was significantly decreased in basal or VEGF-stimulated conditions. High glucose- or peroxynitrite-treated cells also showed significant increases in tyrosine nitration on the p85 subunit of PI 3-kinase that blocked PI 3-kinase and Akt-1 kinase activity. Inhibiting peroxynitrite formation or blocking tyrosine nitration of p85 restored the activity of PI 3-kinase and Akt-1 kinase, blocked phosphorylation of p38 MAP kinase and normalized pro-survival function. Transfecting the cells with constitutively active Akt-1 or inhibiting activity of p38 MAP kinase completely masked the pro-apoptotic effects of high glucose and exogenous peroxynitrite, suggesting an interaction between the Akt-1 and p38 MAP kinase pathways. In conclusion, high glucose treatment blocks the pro-survival effect of VEGF and causes accelerated endothelial cell apoptosis via the action of peroxynitrite in causing tyrosine nitration of PI 3-kinase, inhibiting activity of Akt-1 kinase and increasing the activity of p38 MAP kinase.

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.

Role of NADPH Oxidase in Retinal Vascular Inflammation

PURPOSE In another study, it was demonstrated that NADPH oxidase-derived reactive oxygen species (ROS) are important for ischemia-induced increases in vascular endothelial growth factor (VEGF) and retinal neovascularization. Diabetes-induced increases in retinal ROS, VEGF expression, and vascular permeability are accompanied by increases in the NADPH oxidase catalytic subunit NOX2 within the retinal vessels. The goal of this study was to evaluate the potential role of NOX2 and NADPH oxidase activity in the development of retinal vascular inflammation. METHODS Studies were performed in wild-type mice, mice lacking NOX2, and mice treated with the NADPH oxidase inhibitor apocynin in models of endotoxemia and streptozotocin-induced diabetes. Intracellular adhesion molecule (ICAM)-1 expression was determined by Western blot analysis. Leukocyte adhesion was assessed by labeling adherent leukocytes with concanavalin A. Vascular permeability was assessed by extravasation of FITC-conjugated albumin. ROS production was determined by dichlorofluorescein imaging. RESULTS Both endotoxemia- and diabetes-induced increases in ICAM-1 expression and leukostasis were significantly inhibited by deletion of NOX2, indicating that this enzyme is critically involved in both conditions. Moreover, apocynin treatment and deletion of NOX2 were equally effective in preventing diabetes-induced increases in ICAM-1, leukostasis, and breakdown of the blood-retinal barrier, suggesting that NOX2 is primarily responsible for these early signs of diabetic retinopathy. CONCLUSIONS These data suggest that NOX2 activity has a primary role in retinal vascular inflammation during acute and chronic conditions associated with retinal vascular inflammatory reactions. Targeting this enzyme could be a novel therapeutic strategy for treatment of the retinopathies associated with vascular inflammation.

Role of NADPH Oxidase and Stat3 in Statin-Mediated Protection against Diabetic Retinopathy

PURPOSE Inhibitors of 3-hydroxy-3-methylglutaryl CoA reductase (statins) reduce signs of diabetic retinopathy in diabetic patients and animals. Indirect clinical evidence supports the actions of statins in improving cardiovascular function, but the mechanisms of their protective actions in the retina are not understood. Prior studies have implicated oxidative stress and NADPH oxidase-mediated activation of signal transducer and activator of transcription 3 (STAT3) in diabetes-induced increases in expression of vascular endothelial growth factor (VEGF) and intercellular adhesion molecule (ICAM)-1 and breakdown of the blood-retinal barrier (BRB). Because statins are known to be potent antioxidants, the hypothesis for the current study was that the protective effects of statins in preventing diabetic retinopathy involve blockade of diabetes-induced activation of NADPH oxidase and STAT3. METHODS The hypothesis was tested by experiments in which rats with streptozotocin (STZ)-induced diabetes and retinal endothelial cells maintained in high-glucose medium were treated with simvastatin. Blood-retinal barrier (BRB) function was assayed by determining extravasation of albumin. Oxidative stress was assayed by measuring lipid peroxidation, protein nitration of tyrosine, dihydroethidine oxidation, and chemiluminescence. Immunoprobe techniques were used to determine the levels of NADPH oxidase subunit expression and STAT3 activation. RESULTS These studies showed that simvastatin blocks diabetes or high-glucose-induced increases in VEGF and ICAM-1 and preserves the BRB by a process involving blockade of diabetes/high-glucose-induced activation of STAT3 and NADPH oxidase. Statin treatment also prevents diabetes-induced increases in expression of the NADPH oxidase catalytic and subunit NOX2. CONCLUSIONS These results suggest that simvastatin protects against the early signs of diabetic retinopathy by preventing NADPH oxidase-mediated activation of STAT3.

Vascular Endothelial Growth Factor Activates STAT Proteins in Aortic Endothelial Cells

Vascular endothelial growth factor (VEGF) intracellular signaling in endothelial cells is initiated by the activation of distinct tyrosine kinase receptors, VEGFR1 (Flt-1) and VEGFR2 (Flk-1/KDR). Because the tyrosine kinase-dependent transcription factors known as STAT (signal transducers and activators of transcription) proteins are important modulators of cell growth responses induced by other growth factor receptors, we have determined the effects VEGF of on STAT activation in BAEC (bovine aortic endothelial cells). Here, we show that VEGF induces tyrosine phosphorylation and nuclear translocation of STAT1 and STAT6. VEGF also stimulates STAT3 tyrosine phosphorylation, but nuclear translocation does not occur. We found that placenta growth factor, which selectively activates VEGFR1, has no effect on the STATs. However, upon VEGF stimulation, STAT1 associates with the VEGFR2 in a tyrosine kinase-dependent manner, indicating that VEGF-induced STAT1 activation is mediated primarily by VEGFR2. Thus, our study shows for the first time that VEGF activates the STAT pathway through VEGFR2. Because the growth-promoting activity of VEGF depends upon VEGFR2 activation, these findings suggest a role for the STATs in the regulation of gene expression associated with the angiogenic effects of VEGF.

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)