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Inhibition of reactive oxygen species by Lovastatin downregulates vascular endothelial growth factor expression and ameliorates blood-retinal barrier breakdown in db/db mice: role of NADPH oxidase 4.

Li J, Wang JJ, Yu Q, Chen K, Mahadev K, Zhang SX - Diabetes (2010)

Bottom Line: Moreover, inhibition of Nox4 attenuated hypoxia-induced upregulation of HIF-1alpha 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.Activation of Nox4 plays an important role in high-glucose- and hypoxia-mediated VEGF expression and diabetes-induced BRB breakdown.

View Article: PubMed Central - PubMed

Affiliation: Harold Hamm Oklahoma Diabetes Center and Section of Endocrinology and Diabetes, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA.

ABSTRACT

Objective: Oxidative 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 methods: Expression of NADPH oxidase (Nox) 4, vascular endothelial growth factor (VEGF), and hypoxia-inducible factor (HIF)-1alpha; 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.

Results: Expressions 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-1alpha, 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-1alpha 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.

Conclusions: Activation 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.

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Genetic depletion of Nox4 downregulate retinal VEGF expression and ameliorated vascular leakage in db/db mice. Db/db mice were randomly selected to receive an intravitreal injection of Ad-Nox4i or Ad-Ctrli. Nondiabetic littermate mice received same treatment as control. Three weeks after injection, retinal levels of Nox4 and VEGF, NADPH-dependent ROS generation, and vascular permeability were evaluated. A and B: Expression of Nox4 (A) and VEGF (B) was determined by Western blot analysis and quantified by densitometry (means ± SE, n = 5). *P < 0.05 vs. Ad-Ctrli. C and D: In situ NADPH-dependent ROS generation was measured in freshly prepared retinal sections. When compared with nondiabetic littermate controls (C, a), NADPH-dependent ROS generation was markedly increased in retinas of db/db mice (C, b), and dramatically decreased after Ad-Nox4i treatment (C, c). The fluorescent intensity of DCF was semiquantified (D) (means ± SE, n = 4). *P < 0.05 vs. nondiabetic control; †P < 0.05 vs. db/db + Ad-Ctrli. E: Retinal vascular permeability was measured by the Evans blue method. Results were expressed as micrograms per milligram total retinal protein (means ± SE, n = 6). Db/db mice showed retinal vascular hyperpermeability compared with nondiabetic littermate controls. Intravitreal injection of Ad-Nox4i significantly decreased retinal vascular permeability in db/db mice. *P < 0.05 vs. nondiabetic control; †P < 0.05 vs. db/db + Ad-Ctrli. (A high-quality digital representation of this figure is available in the online issue.)
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Figure 7: Genetic depletion of Nox4 downregulate retinal VEGF expression and ameliorated vascular leakage in db/db mice. Db/db mice were randomly selected to receive an intravitreal injection of Ad-Nox4i or Ad-Ctrli. Nondiabetic littermate mice received same treatment as control. Three weeks after injection, retinal levels of Nox4 and VEGF, NADPH-dependent ROS generation, and vascular permeability were evaluated. A and B: Expression of Nox4 (A) and VEGF (B) was determined by Western blot analysis and quantified by densitometry (means ± SE, n = 5). *P < 0.05 vs. Ad-Ctrli. C and D: In situ NADPH-dependent ROS generation was measured in freshly prepared retinal sections. When compared with nondiabetic littermate controls (C, a), NADPH-dependent ROS generation was markedly increased in retinas of db/db mice (C, b), and dramatically decreased after Ad-Nox4i treatment (C, c). The fluorescent intensity of DCF was semiquantified (D) (means ± SE, n = 4). *P < 0.05 vs. nondiabetic control; †P < 0.05 vs. db/db + Ad-Ctrli. E: Retinal vascular permeability was measured by the Evans blue method. Results were expressed as micrograms per milligram total retinal protein (means ± SE, n = 6). Db/db mice showed retinal vascular hyperpermeability compared with nondiabetic littermate controls. Intravitreal injection of Ad-Nox4i significantly decreased retinal vascular permeability in db/db mice. *P < 0.05 vs. nondiabetic control; †P < 0.05 vs. db/db + Ad-Ctrli. (A high-quality digital representation of this figure is available in the online issue.)

Mentions: To establish the causative role of Nox4 in retinal VEGF expression and vascular leakage in db/db mice, we downregulated Nox4 expression in the retina using an adenovirus-delivered siRNA against mouse Nox4. Three weeks after an intravitreal injection of Ad-Nox4i or Ad-Ctrli, mice were humanely killed, and retinal expression of Nox4 and VEGF was determined by Western blot analysis. As shown in Fig. 7A, intravitreal injection of Ad-Nox4i led to a significant decrease of Nox4 expression in the retina by approximately 60%. Moreover, Ad-Nox4i significantly decreased retinal VEGF expression in db/db mice, when compared with Ad-Ctrli treatment (Fig. 7B). In parallel, NADPH-dependent ROS generation was markedly increased by eightfold in retinas of db/db mice, indicating enhanced NADPH oxidase activity in the diabetic retina, which was significantly blunted in Ad-Nox4i–treated eyes (Fig. 7C and D). In addition, intravitreal injection of Ad-Nox4i almost completely abolished the increase in retinal vascular permeability in db/db mice (Fig. 7E). These results strongly suggest a causal role of Nox4 in vascular hyperpermeability in diabetic retinopathy, further supporting the notion that lovastatin inhibits retinal vascular leakage through suppression of Nox4 expression.


Inhibition of reactive oxygen species by Lovastatin downregulates vascular endothelial growth factor expression and ameliorates blood-retinal barrier breakdown in db/db mice: role of NADPH oxidase 4.

Li J, Wang JJ, Yu Q, Chen K, Mahadev K, Zhang SX - Diabetes (2010)

Genetic depletion of Nox4 downregulate retinal VEGF expression and ameliorated vascular leakage in db/db mice. Db/db mice were randomly selected to receive an intravitreal injection of Ad-Nox4i or Ad-Ctrli. Nondiabetic littermate mice received same treatment as control. Three weeks after injection, retinal levels of Nox4 and VEGF, NADPH-dependent ROS generation, and vascular permeability were evaluated. A and B: Expression of Nox4 (A) and VEGF (B) was determined by Western blot analysis and quantified by densitometry (means ± SE, n = 5). *P < 0.05 vs. Ad-Ctrli. C and D: In situ NADPH-dependent ROS generation was measured in freshly prepared retinal sections. When compared with nondiabetic littermate controls (C, a), NADPH-dependent ROS generation was markedly increased in retinas of db/db mice (C, b), and dramatically decreased after Ad-Nox4i treatment (C, c). The fluorescent intensity of DCF was semiquantified (D) (means ± SE, n = 4). *P < 0.05 vs. nondiabetic control; †P < 0.05 vs. db/db + Ad-Ctrli. E: Retinal vascular permeability was measured by the Evans blue method. Results were expressed as micrograms per milligram total retinal protein (means ± SE, n = 6). Db/db mice showed retinal vascular hyperpermeability compared with nondiabetic littermate controls. Intravitreal injection of Ad-Nox4i significantly decreased retinal vascular permeability in db/db mice. *P < 0.05 vs. nondiabetic control; †P < 0.05 vs. db/db + Ad-Ctrli. (A high-quality digital representation of this figure is available in the online issue.)
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Figure 7: Genetic depletion of Nox4 downregulate retinal VEGF expression and ameliorated vascular leakage in db/db mice. Db/db mice were randomly selected to receive an intravitreal injection of Ad-Nox4i or Ad-Ctrli. Nondiabetic littermate mice received same treatment as control. Three weeks after injection, retinal levels of Nox4 and VEGF, NADPH-dependent ROS generation, and vascular permeability were evaluated. A and B: Expression of Nox4 (A) and VEGF (B) was determined by Western blot analysis and quantified by densitometry (means ± SE, n = 5). *P < 0.05 vs. Ad-Ctrli. C and D: In situ NADPH-dependent ROS generation was measured in freshly prepared retinal sections. When compared with nondiabetic littermate controls (C, a), NADPH-dependent ROS generation was markedly increased in retinas of db/db mice (C, b), and dramatically decreased after Ad-Nox4i treatment (C, c). The fluorescent intensity of DCF was semiquantified (D) (means ± SE, n = 4). *P < 0.05 vs. nondiabetic control; †P < 0.05 vs. db/db + Ad-Ctrli. E: Retinal vascular permeability was measured by the Evans blue method. Results were expressed as micrograms per milligram total retinal protein (means ± SE, n = 6). Db/db mice showed retinal vascular hyperpermeability compared with nondiabetic littermate controls. Intravitreal injection of Ad-Nox4i significantly decreased retinal vascular permeability in db/db mice. *P < 0.05 vs. nondiabetic control; †P < 0.05 vs. db/db + Ad-Ctrli. (A high-quality digital representation of this figure is available in the online issue.)
Mentions: To establish the causative role of Nox4 in retinal VEGF expression and vascular leakage in db/db mice, we downregulated Nox4 expression in the retina using an adenovirus-delivered siRNA against mouse Nox4. Three weeks after an intravitreal injection of Ad-Nox4i or Ad-Ctrli, mice were humanely killed, and retinal expression of Nox4 and VEGF was determined by Western blot analysis. As shown in Fig. 7A, intravitreal injection of Ad-Nox4i led to a significant decrease of Nox4 expression in the retina by approximately 60%. Moreover, Ad-Nox4i significantly decreased retinal VEGF expression in db/db mice, when compared with Ad-Ctrli treatment (Fig. 7B). In parallel, NADPH-dependent ROS generation was markedly increased by eightfold in retinas of db/db mice, indicating enhanced NADPH oxidase activity in the diabetic retina, which was significantly blunted in Ad-Nox4i–treated eyes (Fig. 7C and D). In addition, intravitreal injection of Ad-Nox4i almost completely abolished the increase in retinal vascular permeability in db/db mice (Fig. 7E). These results strongly suggest a causal role of Nox4 in vascular hyperpermeability in diabetic retinopathy, further supporting the notion that lovastatin inhibits retinal vascular leakage through suppression of Nox4 expression.

Bottom Line: Moreover, inhibition of Nox4 attenuated hypoxia-induced upregulation of HIF-1alpha 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.Activation of Nox4 plays an important role in high-glucose- and hypoxia-mediated VEGF expression and diabetes-induced BRB breakdown.

View Article: PubMed Central - PubMed

Affiliation: Harold Hamm Oklahoma Diabetes Center and Section of Endocrinology and Diabetes, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA.

ABSTRACT

Objective: Oxidative 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 methods: Expression of NADPH oxidase (Nox) 4, vascular endothelial growth factor (VEGF), and hypoxia-inducible factor (HIF)-1alpha; 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.

Results: Expressions 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-1alpha, 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-1alpha 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.

Conclusions: Activation 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.

Show MeSH
Related in: MedlinePlus