Abstract
Purpose: :
Hyperglycemia–induced oxidative stress has been shown to be implicated in the pathogenesis of Diabetic Retinopathy. Since the expression of heme oxygenase–1 (HO–1), which is considered an anti–oxidant enzyme, may be induced by stress conditions, we investigated the potential protective role of HO–1 in retinal endothelial cells exposed to high glucose and oxidative–nitrosative stress conditions.
Methods: :
Rat retinal capillary endothelial cells (TR–iBRB2 cell line) were exposed to D–glucose (30 mM), mannitol (25 mM; osmotic control), H2O2 (100 µM) or NOC–18 (250 µM) for different periods of time, in the absence or in the presence of Tin protoporphyrin IX – SnPPIX (10 µM), an inhibitor of HO–1. The immunoreactivity of HO–1 was evaluated by Western Blotting and immunocytochemistry, the enzymatic activity of HO–1 was estimated measuring bilirubin production and cell viability was assessed by MTT assay.
Results: :
Chronic exposure to high glucose or mannitol (7 days) slightly increased the immunoreactivity of HO–1 in retinal endothelial cells. The protein levels of HO–1 also significantly increased, in a time–dependent manner, in endothelial cells exposed to high glucose, H2O2 or NOC–18. In addition, in the presence of SnPPIX, the protein levels of HO–1 were higher in cells exposed to H2O2 or NOC–18 than those observed in cells exposed to H2O2 or NOC–18 alone. The enzymatic activity of HO–1 also increased in cells exposed to high glucose, H2O2 or NOC18 for 24 h. Moreover, oxidative–nitrosative stress conditions decreased cell viability, but the viability of endothelial cells exposed to H2O2 or NOC–18, in the presence of SnPPIX, was lower than in cells exposed to H2O2 or NOC–18 alone, suggesting that HO–1 was protecting endothelial cells.
Conclusions: :
These results demonstrate that high glucose and oxidative–nitrosative stress increase the protein levels and enzymatic activity of HO–1 in retinal endothelial cells. The results also suggest that HO–1 exerts a protective effect against oxidative–nitrosative stress in retinal endothelial cells, and that these cells may compensate the inhibition of HO–1 activity by over expressing this enzyme.
Keywords: diabetic retinopathy • oxidation/oxidative or free radical damage • antioxidants