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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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Downregulation of HIF-1α and VEGF expression by NADPH oxidase inhibitor or lovastatin in RCECs. Bovine RCECs were pretreated with DPI (A and B) for 4 h or lovastatin (C and D) for 24 h followed by exposure to hypoxia for 16 h. Expression of VEGF (A and C) and HIF-1α (B and D) was determined by Western blot analysis and quantified by densitometry. Results were expressed as means ± SE. (n = 3). *P < 0.05; **P < 0.01 vs. normoxia; †P < 0.05; ‡P < 0.01 vs. hypoxia.
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Figure 4: Downregulation of HIF-1α and VEGF expression by NADPH oxidase inhibitor or lovastatin in RCECs. Bovine RCECs were pretreated with DPI (A and B) for 4 h or lovastatin (C and D) for 24 h followed by exposure to hypoxia for 16 h. Expression of VEGF (A and C) and HIF-1α (B and D) was determined by Western blot analysis and quantified by densitometry. Results were expressed as means ± SE. (n = 3). *P < 0.05; **P < 0.01 vs. normoxia; †P < 0.05; ‡P < 0.01 vs. hypoxia.

Mentions: To determine whether increased Nox4 activity is a potential mediator of VEGF expression in RCECs in a hypoxic condition, cells were pretreated with DPI for 4 h, followed by exposure to hypoxia for 16 h. The results showed that a low dose (100 nmol/l) of DPI was sufficient to decrease VEGF expression induced by hypoxia (Fig. 4A). Moreover, this effect is associated with the downregulation of HIF-1α, a master transcription factor responsible for hypoxia-induced VEGF expression (Fig. 4B). In addition, pretreatment of cells with lovastatin significantly attenuated cellular VEGF expression and decreased HIF-1α level in a dose-dependent manner (Fig. 4C and D).


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)

Downregulation of HIF-1α and VEGF expression by NADPH oxidase inhibitor or lovastatin in RCECs. Bovine RCECs were pretreated with DPI (A and B) for 4 h or lovastatin (C and D) for 24 h followed by exposure to hypoxia for 16 h. Expression of VEGF (A and C) and HIF-1α (B and D) was determined by Western blot analysis and quantified by densitometry. Results were expressed as means ± SE. (n = 3). *P < 0.05; **P < 0.01 vs. normoxia; †P < 0.05; ‡P < 0.01 vs. hypoxia.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2874715&req=5

Figure 4: Downregulation of HIF-1α and VEGF expression by NADPH oxidase inhibitor or lovastatin in RCECs. Bovine RCECs were pretreated with DPI (A and B) for 4 h or lovastatin (C and D) for 24 h followed by exposure to hypoxia for 16 h. Expression of VEGF (A and C) and HIF-1α (B and D) was determined by Western blot analysis and quantified by densitometry. Results were expressed as means ± SE. (n = 3). *P < 0.05; **P < 0.01 vs. normoxia; †P < 0.05; ‡P < 0.01 vs. hypoxia.
Mentions: To determine whether increased Nox4 activity is a potential mediator of VEGF expression in RCECs in a hypoxic condition, cells were pretreated with DPI for 4 h, followed by exposure to hypoxia for 16 h. The results showed that a low dose (100 nmol/l) of DPI was sufficient to decrease VEGF expression induced by hypoxia (Fig. 4A). Moreover, this effect is associated with the downregulation of HIF-1α, a master transcription factor responsible for hypoxia-induced VEGF expression (Fig. 4B). In addition, pretreatment of cells with lovastatin significantly attenuated cellular VEGF expression and decreased HIF-1α level in a dose-dependent manner (Fig. 4C and D).

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