title: Heme oxygenase-1 protects retinal endothelial cells against high glucose- and oxidative/nitrosative stress-induced toxicity.
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 (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.
authors.affiliation: Centre of Ophthalmology and Vision Sciences, IBILI, Faculty of Medicine, University of Coimbra, Coimbra, Portugal.
authors.forename: Áurea  F
authors.initials: Á
authors.lastname: Castilho
authors.forename: Célia A
authors.initials: CA
authors.lastname: Aveleira
authors.forename: Ermelindo C
authors.initials: EC
authors.lastname: Leal
authors.forename: Núria F
authors.initials: NF
authors.lastname: Simões
authors.forename: Carolina R
authors.initials: CR
authors.lastname: Fernandes
authors.forename: Rita I
authors.initials: RI
authors.lastname: Meirinhos
authors.forename: Filipa I
authors.initials: FI
authors.lastname: Baptista
authors.forename: António F
authors.initials: AF
authors.lastname: Ambrósio
mesh_terms: D000818:Animals; D002470:Cell Survival / Q000187:drug effects; D019610:Cytoprotection / Q000187:drug effects*; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology*; D005947:Glucose / Q000633:toxicity*; D051547:Heme Oxygenase-1 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects / Q000473:pathology; D006801:Humans; D006861:Hydrogen Peroxide / Q000633:toxicity; D006943:Hyperglycemia / Q000473:pathology; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D015538:Nitrosation / Q000187:drug effects; D009603:Nitroso Compounds / Q000633:toxicity; D018384:Oxidative Stress / Q000187:drug effects*; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D012160:Retina / Q000473:pathology*; D013997:Time Factors
chemical_list: C093285:NOC 18; D009603:Nitroso Compounds; D017382:Reactive Oxygen Species; D006861:Hydrogen Peroxide; D051547:Heme Oxygenase-1; D005947:Glucose