Inhibition of Reactive Oxygen Species by Lovastatin Downregulates Vascular Endothelial Growth Factor Expression and Ameliorates Blood-Retinal Barrier Breakdown in db/db Mice

Abstract

OBJECTIVEOxidative stress is a key pathogenic factor in diabetic retinopathy. We previously showed that lovastatin mitigates blood-retinal barrier (BRB) breakdown in db/db mice. The purpose of this study is to determine the mechanisms underlying the salutary effects of lovastatin in diabetic retinopathy.RESEARCH DESIGN AND METHODSExpression of NADPH oxidase (Nox) 4, vascular endothelial growth factor (VEGF), and hypoxia-inducible factor (HIF)-1α; production of reactive oxygen species (ROS); and retinal vascular permeability were measured in cultured retinal capillary endothelial cells (RCECs) and in db/db mice treated with lovastatin.RESULTSExpressions of Nox4 and VEGF were significantly increased in retinas of db/db mice and reduced by lovastatin treatment. In cultured RCECs, hypoxia and high glucose upregulated mRNA and protein expression of Nox4, ROS generation, and VEGF level. These changes were abrogated by pretreatment with lovastatin or NADPH oxidase inhibitor diphenyleneiodonium chloride. Overexpression of Nox4 increased basal level of ROS generation, HIF-1α, and VEGF expression in RCECs. In contrast, blockade of Nox4 activity using adenovirus-expressing dominant-negative Nox4 abolished hypoxia- and high-glucose–induced ROS production and VEGF expression. Moreover, inhibition of Nox4 attenuated hypoxia-induced upregulation of HIF-1α and high-glucose–elicited phosphorylation of STAT3. Finally, depletion of Nox4 by adenovirus-delivered Nox4 small interfering RNA significantly decreased retinal NADPH oxidase activity and VEGF expression and reduced retinal vascular premeability in db/db mice.CONCLUSIONSActivation of Nox4 plays an important role in high-glucose– and hypoxia-mediated VEGF expression and diabetes-induced BRB breakdown. Inhibition of Nox4, at least in part, contributes to the protective effects of lovastatin in diabetic retinopathy.

Genetic Targeting or Pharmacologic Inhibition of NADPH Oxidase Nox4 Provides Renoprotection in Long-Term Diabetic Nephropathy

“…Importantly, this is likely to be Nox4 dependent because downregulation of Nox4 in human podocytes was also associated with attenuation of VEGF expression. Consistent with our findings, other researchers reported a link between VEGF with Nox4 in the retina, where Nox4 was associated with vascular permeability, 39 as well as similar findings in a stroke model. 40 Diabetic nephropathy is considered to be at least in part an inflammatory disorder in which progressive glomerular injury is associated with macrophage infiltration.…”

Endothelial NO synthase and reactive oxygen species mediated effect of simvastatin on vessel structure and function: Pleiotropic and dose-dependent effect on tumor vascular stabilization

“…Accumulating reports clearly showed that bevacizumab, administered by intraocular injection, has a global effect in normalizing the pathologic intraocular environment and changing the immature, fenestrated vessels toward a normalized status by neutralizing VEGF in ocular disease (45,47). Coincidentally, as equally possessed with antagonistic action against VEGF, high-dose statins have been widely used in treating DR and complications both in clinical settings and experimental models, in consideration of its activities in decreasing retinal neovascularization, retaining endothelial barrier integrity and eventually reducing the area of hypoxia (13,48). Thus, it is reasonable to infer that the beneficial effects of high-dose simvastatin are also applicable to neovessels of tumor.…”

“…Briefly, tumor tissues of interest were freshly dissected in 1X PBS and then 60-80 µm thick frozen tumor sections were prepared for incubation with the freshly prepared H 2 DCFDA (10 µM) for 15 min in dark at room temperature. After three washes with PBS, sections were immediately monitored by a confocal microscope and the fluorescent intensity of DCF was semiquantified using Image-Pro Plus 6.0 software (13). To determined alterations after ROS clearance, tumor-bearing mice received an intraperitoneal injection of DPI (a NADPH oxidase inhibitor) or same amount of vehicle for 5 days.…”

“…3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitors, or statins, that have been reported to exert impressive beneficial effects, such as anti-inflammation, antioxidation, protecting against cardiovascular events and DR and anticancer efficacy via lipid-independent mechanisms (10)(11)(12) tions and markedly reduce retinal microvascular permeability through mitigation of oxidative stress and inflammatory status by downregulating reactive oxygen species (ROS) generation, VEGF and intercellular adhesion molecule (ICAM-1) expression in diabetic animals (10,11,13). Oxidative stress and elevated ROS generation have been incriminated in many pathological conditions, including diabetes and cancer (14,15).…”

Neuropeptides, Trophic Factors, and Other Substances Providing Morphofunctional and Metabolic Protection in Experimental Models of Diabetic Retinopathy

“…The citation "Wang et al (2010)" has been changed to " Wang et al (2010)" to match the author name/date in the reference list. Please check here and in subsequent occurrences, and correct if necessary.…”