Heme oxygenase-1 protects retinal endothelial cells against high glucose- and oxidative/nitrosative stress-induced toxicity

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 hem...

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Published in:	PloS one Vol. 7; no. 8; p. e42428
Main Authors:	Castilho, Áurea F, Aveleira, Célia A, Leal, Ermelindo C, Simões, Núria F, Fernandes, Carolina R, Meirinhos, Rita I, Baptista, Filipa I, Ambrósio, António F
Format:	Journal Article
Language:	English
Published:	United States Public Library of Science 03-08-2012 Public Library of Science (PLoS)
Subjects:	Animals Annexin V Antioxidants Antioxidants (Nutrients) Apoptosis Bilirubin Biology Blindness Carbon monoxide Cell death Cell Survival - drug effects Cytokines Cytoprotection - drug effects Diabetes Diabetes mellitus Diabetic retinopathy Endothelial cells Endothelial Cells - drug effects Endothelial Cells - enzymology Endothelial Cells - pathology Endothelium Enzymes Exposure Glucose Glucose - toxicity Heme Heme oxygenase (decyclizing) Heme Oxygenase-1 - antagonists & inhibitors Heme Oxygenase-1 - metabolism Human Umbilical Vein Endothelial Cells - drug effects Human Umbilical Vein Endothelial Cells - pathology Humans Hydrogen Hydrogen peroxide Hydrogen Peroxide - toxicity Hyperglycemia Hyperglycemia - pathology Immunofluorescence Inhibition Intracellular Space - drug effects Intracellular Space - metabolism Medicine Metabolism Microvasculature Mortality Nitric oxide Nitrosation - drug effects Nitroso Compounds - toxicity Oxidation Oxidative stress Oxidative Stress - drug effects Oxygen Oxygenase Rats Reactive oxygen species Reactive Oxygen Species - metabolism Retina Retina - pathology Retinopathy Rodents Stresses Time Factors Tin protoporphyrin Toxicity Portugal
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Summary:	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 (H(2)O(2)). 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 H(2)O(2) and NOC-18 (NO donor). Overexpression of HO-1 prevented the toxic effect induced by H(2)O(2) and NOC-18. In conclusion, HO-1 exerts a protective effect in retinal endothelial cells exposed to hyperglycemic and oxidative/nitrosative stress conditions.
Bibliography:	Competing Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: AFC CAA ECL NFS CRF RIM FIB AFA. Performed the experiments: AFC CAA ECL NFS CRF RIM FIB. Analyzed the data: AFA. Wrote the paper: AFC AFA.
ISSN:	1932-6203 1932-6203
DOI:	10.1371/journal.pone.0042428