Abstract
Diabetic retinopathy is a leading cause of visual loss and blindness, characterized by microvascular dysfunction. Hyperglycemia is considered the major pathogenic factor for the development of diabetic retinopathy and is associated with increased oxidative/nitrosative stress in the retina. Since heme oxygenase-1 (HO-1) is an enzyme with antioxidant and protective properties, we investigated the potential protective role of HO-1 in retinal endothelial cells exposed to high glucose and oxidative/nitrosative stress conditions. Retinal endothelial cells were exposed to elevated glucose, nitric oxide (NO) and hydrogen peroxide (H2O2). Cell viability and apoptosis were assessed by MTT assay, Hoechst staining, TUNEL assay and Annexin V labeling. The production of reactive oxygen species (ROS) was detected by the oxidation of 2′,7′-dichlorodihydrofluorescein diacetate. The content of HO-1 was assessed by immunobloting and immunofluorescence. HO activity was determined by bilirubin production. Long-term exposure (7 days) of retinal endothelial cells to elevated glucose decreased cell viability and had no effect on HO-1 content. However, a short-time exposure (24 h) to elevated glucose did not alter cell viability, but increased both the levels of intracellular ROS and HO-1 content. Moreover, the inhibition of HO with SnPPIX unmasked the toxic effect of high glucose and revealed the protection conferred by HO-1. Oxidative/nitrosative stress conditions increased cell death and HO-1 protein levels. These effects of elevated glucose and HO inhibition on cell death were confirmed in primary endothelial cells (HUVECs). When cells were exposed to oxidative/nitrosative stress conditions there was also an increase in retinal endothelial cell death and HO-1 content. The inhibition of HO enhanced ROS production and the toxic effect induced by exposure to H2O2 and NOC-18 (NO donor). Overexpression of HO-1 prevented the toxic effect induced by H2O2 and NOC-18. In conclusion, HO-1 exerts a protective effect in retinal endothelial cells exposed to hyperglycemic and oxidative/nitrosative stress conditions.
Highlights
Diabetic retinopathy is a common complication of diabetes mellitus and is a leading cause of vision loss and blindness in working-age adults in developed countries [1]
Since heme oxygenase-1 (HO-1), an inducible enzyme, has strong antioxidant properties, we evaluated whether heme oxygenase (HO)-1 immunoreactivity could be affected by the longterm exposure to elevated glucose or mannitol, by western blotting and immunocytochemistry
Accompanying the rise in HO-1 levels, there was an increase in the enzymatic activity of HO in cells exposed to high glucose, H2O2 or NOC-18 for 24 h (Figure 4)
Summary
Diabetic retinopathy is a common complication of diabetes mellitus and is a leading cause of vision loss and blindness in working-age adults in developed countries [1]. Diabetic retinopathy is considered a microvascular disease. Hyperglycemia is considered the main factor for the development of vascular complications in diabetes [2], triggering the cascade of metabolic and biochemical changes occurring in this pathology. Biochemical alterations induced by hyperglycemia lead to an increase in oxidative and nitrosative stress [5,6]. A large body of evidence supports the idea that the increase in oxidative stress in retinal microvasculature is a key factor for the development of diabetic retinopathy [7,8,9]
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