Mohamed Al-Shabrawey

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.

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.

Inflammation and diabetic retinal microvascular complications

Diabetic retinopathy (DR) is one of the most common complications of diabetes and is a leading cause of blindness in people of the working age in Western countries. A major pathology of DR is microvascular complications such as non-perfused vessels, microaneurysms, dot/blot hemorrhages, cotton-wool spots, venous beading, vascular loops, vascular leakage and neovascularization. Multiple mechanisms are involved in these alternations. This review will focus on the role of inflammation in diabetic retinal microvascular complications and discuss the potential therapies by targeting inflammation.

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)

Suppression of retinal peroxisome proliferator-activated receptor γ in experimental diabetes and oxygen-induced retinopathy: role of NADPH oxidase.

PURPOSE Recently, the authors have shown that NADPH oxidase is positively correlated with increased leukocyte adhesion and vascular leakage in diabetes and neovascularization in oxygen-induced retinopathy (OIR). Peroxisome proliferator-activated receptor gamma (PPARgamma) agonists have been shown to prevent vascular inflammation and leakage in an experimental model of diabetes. The goal of this study was to investigate whether there is a link between NADPH oxidase and PPARgamma that leads to vascular dysfunction in diabetic retina or OIR. METHODS Diabetes was induced with streptozotocin in wild-type mice or NOX2 knockout mice. One group of wild-type mice was treated with apocynin. Bovine retinal endothelial cells (BRECs) were treated with normal glucose (5 mM) or high glucose (25 mM) in the presence or absence of superoxide dismutase (SOD) or NADPH oxidase inhibitors (apocynin or diphenyleneiodonium [DPI]). Western blotting and immunofluorescence were used to evaluate PPARgamma expression. Activation of nuclear factor (NF)kappaB was measured using the transcription factor assay kit and Western blot analysis of phospho-NFkappaB. PPARgamma expression was also tested in OIR and lipopolysaccharide-induced retinal inflammation. RESULTS Retinal expression of PPARgamma was suppressed in experimental models of diabetes, OIR, and retinal inflammation. This was associated with the activation of NFkappaB in the diabetic retina. These effects were prevented by apocynin or deletion of NOX2. PPARgamma expression was also suppressed in endothelial cells treated with high glucose, and this was prevented by apocynin, DPI, and SOD. CONCLUSIONS Suppression of PPARgamma is involved in the pathogenesis of diabetic retinopathy and OIR. NADPH oxidase could be an upstream mediator of these changes.

Increased Expression and Activity of 12-Lipoxygenase in Oxygen-Induced Ischemic Retinopathy and Proliferative Diabetic Retinopathy: Implications in Retinal Neovascularization

OBJECTIVE Arachidonic acid is metabolized by 12-lipoxygenase (12-LOX) to 12-hydroxyeicosatetraenoic acid (12-HETE) and has an important role in the regulation of angiogenesis and endothelial cell proliferation and migration. The goal of this study was to investigate whether 12-LOX plays a role in retinal neovascularization (NV). RESEARCH DESIGN AND METHODS Experiments were performed using retinas from a murine model of oxygen-induced ischemic retinopathy (OIR) that was treated with and without the LOX pathway inhibitor, baicalein, or lacking 12-LOX. We also analyzed vitreous samples from patients with and without proliferative diabetic retinopathy (PDR). Western blotting and RT-PCR were used to assess the expression of 12-LOX, vascular endothelial growth factor (VEGF), and pigment epithelium–derived factor (PEDF). Liquid chromatography–mass spectrometry was used to assess the amounts of HETEs in the murine retina and human vitreous samples. The effects of 12-HETE on VEGF and PEDF expression were evaluated in Müller cells (rMCs), primary mouse retinal pigment epithelial cells, and astrocytes. RESULTS Retinal NV during OIR was associated with increased 12-LOX expression and 12-, 15-, and 5-HETE production. The amounts of HETEs also were significantly higher in the vitreous of diabetic patients with PDR. Retinal NV was markedly abrogated in mice treated with baicalein or mice lacking 12-LOX. This was associated with decreased VEGF expression and restoration of PEDF levels. PEDF expression was reduced in 12-HETE–treated rMCs, astrocytes, and the retinal pigment epithelium. Only rMCs and astrocytes showed increased VEGF expression by 12-HETE. CONCLUSIONS 12-LOX and its product HETE are important regulators of retinal NV through modulation of VEGF and PEDF expression and could provide a new therapeutic target to prevent and treat ischemic retinopathy.

12/15-Lipoxygenase-Derived Lipid Metabolites Induce Retinal Endothelial Cell Barrier Dysfunction: Contribution of NADPH Oxidase

The purpose of the current study was to evaluate the effect of 12/15- lipoxygenase (12/15-LOX) metabolites on retinal endothelial cell (REC) barrier function. FITC-dextran flux across the REC monolayers and electrical cell-substrate impedance sensing (ECIS) were used to evaluate the effect of 12- and 15-hydroxyeicosatetreanoic acids (HETE) on REC permeability and transcellular electrical resistance (TER). Effect of 12- or 15-HETE on the levels of zonula occludens protein 1 (ZO-1), reactive oxygen species (ROS), NOX2, pVEGF-R2 and pSHP1 was examined in the presence or absence of inhibitors of NADPH oxidase. In vivo studies were performed using Ins2Akita mice treated with or without the 12/15-LOX inhibitor baicalein. Levels of HETE and inflammatory mediators were examined by LC/MS and Multiplex Immunoassay respectively. ROS generation and NOX2 expression were also measured in mice retinas. 12- and 15- HETE significantly increased permeability and reduced TER and ZO-1expression in REC. VEGF-R2 inhibitor reduced the permeability effect of 12-HETE. Treatment of REC with HETE also increased ROS generation and expression of NOX2 and pVEGF-R2 and decreased pSHP1 expression. Treatment of diabetic mice with baicalein significantly decreased retinal HETE, ICAM-1, VCAM-1, IL-6, ROS generation, and NOX2 expression. Baicalein also reduced pVEGF-R2 while restored pSHP1 levels in diabetic retina. Our findings suggest that 12/15-LOX contributes to vascular hyperpermeability during DR via NADPH oxidase dependent mechanism which involves suppression of protein tyrosine phosphatase and activation of VEGF-R2 signal pathway.