Interaction between NO and COX pathways in retinal cells exposed to elevated glucose and retina of diabetic rats

A nonselective inhibitor of cyclooxygenase (COX; high-dose aspirin) and a relatively selective inhibitor of inducible nitric oxide synthase (iNOS; aminoguanidine) have been found to inhibit development of diabetic retinopathy in animals, raising a possibility that NOS and COX play important roles in the development of retinopathy. In this study, the effects of hyperglycemia on retinal nitric oxide (NO) production and the COX-2 pathway, and the interrelationship of the NOS and COX-2 pathways in retina and retinal cells, were investigated using a general inhibitor of NOS [N(G)-nitro-l-arginine methyl ester (l-NAME)], specific inhibitors of iNOS [l-N(6)-(1-iminoethyl)lysine (l-NIL)] and COX-2 (NS-398), and aspirin and aminoguanidine. In vitro studies used a transformed retinal Müller (glial) cell line (rMC-1) and primary bovine retinal endothelial cells (BREC) incubated in 5 and 25 mM glucose with and without these inhibitors, and in vivo studies utilized retinas from experimentally diabetic rats (2 mo) treated or without aminoguanidine or aspirin. Retinal rMC-1 cells cultured in high glucose increased production of NO and prostaglandin E(2) (PGE(2)) and expression of iNOS and COX-2. Inhibition of NO production with l-NAME or l-NIL inhibited all of these abnormalities, as did aminoguanidine and aspirin. In contrast, inhibition of COX-2 with NS-398 blocked PGE(2) production but had no effect on NO or iNOS. In BREC, elevated glucose increased NO and PGE(2) significantly, whereas expression of iNOS and COX-2 was unchanged. Viability of rMC-1 cells or BREC in 25 mM glucose was significantly less than at 5 mM glucose, and this cell death was inhibited by l-NAME or NS-398 in both cell types and also by l-NIL in rMC-1 cells. Retinal homogenates from diabetic animals produced significantly greater than normal amounts of NO and PGE(2) and of iNOS and COX-2. Oral aminoguanidine and aspirin significantly inhibited all of these increases. The in vitro results suggest that the hyperglycemia-induced increase in NO in retinal Müller cells and endothelial cells increases production of cytotoxic prostaglandins via COX-2. iNOS seems to account for the increased production of NO in Müller cells but not in endothelial cells. We postulate that NOS and COX-2 act together to contribute to retinal cell death in diabetes and to the development of diabetic retinopathy and that inhibition of retinopathy by aminoguanidine or aspirin is due at least in part to inhibition of this NO/COX-2 axis.     

Hyperglycemia increases mitochondrial superoxide in retina and retinal cells

Oxidative stress is believed to play a significant role in the development of diabetic retinopathy. In this study, we have investigated the effects of elevated glucose concentration on the production of superoxide anion by retina and retinal cells, the cellular source of the superoxide, the effect of therapies that are known to inhibit diabetic retinopathy on the superoxide production, and the role of the superoxide in cell death in elevated glucose concentration. Superoxide release was measured from retinas collected from streptozotocin-diabetic rats (2 months) treated with or without aminoguanidine, aspirin, or vitamin E, and from transformed retinal Müller cells (rMC-1) and bovine retinal endothelial cells (BREC) incubated in normal (5 mM) and high (25 mM) glucose. Diabetes (retina) or incubation in elevated glucose concentration (rMC-1 and BREC cells) significantly increased superoxide production, primarily from mitochondria, because an inhibitor of mitochondrial electron transport chain complex II normalized superoxide production. Inhibition of reduced nicotinamine adenine dinucleotide phosphate (NADPH) oxidase or nitric oxide synthase had little or no effect on the glucose-induced increase in superoxide. Treatment of diabetic animals with aminoguanidine, aspirin, or vitamin E for 2 months significantly inhibited the diabetes-induced increase in production of superoxide in the retinas. Despite the increased production of superoxide, no increase in protein carbonyls was detected in retinal proteins from animals diabetic for 2-6 months or rMC-1 cells incubated in 25 mM glucose for 5 d unless the activities of calpain or the proteosome were inhibited. Addition of copper/zinc-containing superoxide dismutase to the media of rMC-1 and BREC cells inhibited the apoptotic death caused by elevated glucose. Diabetes-like glucose concentration increases superoxide production in retinal cells, and the superoxide contributes to impaired viability and increased cell death under those circumstances. Three therapies that inhibit the development of diabetic retinopathy all inhibit superoxide production, raising a possibility that these therapies inhibit retinopathy in part by inhibiting a hyperglycemia-induced increase in superoxide production.     

Increased production of nitrotyrosine in lung tissue of rats with radiation-induced acute lung injury

The purposes of this study were 1) to identify the nitric oxide (NO) synthase (NOS) isoform responsible for NO-mediated radiation-induced lung injury, 2) to examine the formation of nitrotyrosine, and 3) to see whether nitrotyrosine formation and lung injury are reduced by an inducible NOS (iNOS) inhibitor, aminoguanidine. The left hemithorax of rats was irradiated (20 Gy), and the degree of lung injury, the expression of NOS isoforms, and the formation of nitrotyrosine and superoxide were examined after 2 wk. iNOS mRNA was induced, and endothelial NOS mRNA was markedly increased in the irradiated lung. Nitrotyrosine was detected biochemically and immunohistochemically. Aminoguanidine prevented acute lung injury as indicated by decreased protein concentration and lactate dehydrogenase activity in bronchoalveolar lavage fluid and improved NMR parameters and histology. Furthermore, the formation of nitrotyrosine was significantly reduced in the aminoguanidine group. We conclude that iNOS induction is a major factor in radiation-induced lung injury and that nitrotyrosine formation may participate in the NO-induced pathogenesis.     

Evidence of oxidative stress in human corneal diseases.

This study localized malondialdehyde (MDA, a toxic byproduct of lipid peroxidation), nitrotyrosine [NT, a cytotoxic byproduct of nitric oxide (NO)], and nitric oxide synthase isomers (NOS) in normal and diseased human corneas. Normal corneas (n=11) and those with clinical and histopathological diagnoses of keratoconus (n=26), bullous keratopathy (n=17), and Fuchs' endothelial dystrophy (n=12) were examined with antibodies specific for MDA, NT, eNOS (constitutive NOS), and iNOS (inducible NOS). Normal corneas showed little or no staining for MDA, NT, or iNOS, whereas eNOS was detected in the epithelium and endothelium. MDA was present in all disease groups, with each group displaying a distinct pattern of staining. NT was detected in all keratoconus and approximately one half of Fuchs' dystrophy corneas. iNOS and eNOS were evident in all the diseased corneas. Keratoconus corneas showed evidence of oxidative damage from cytotoxic byproducts generated by lipid peroxidation and the NO pathway. Bullous keratopathy corneas displayed byproducts of lipid peroxidation but not peroxynitrite (MDA but not NT). Conversely, Fuchs' dystrophy corneas displayed byproducts of peroxynitrite with little lipid peroxidation (NT > MDA). These data suggest that oxidative damage occurs within each group of diseased corneas. However, each disease exhibits a distinctive profile, with only keratoconus showing prominent staining for both nitrotyrosine and MDA. These results suggest that keratoconus corneas do not process reactive oxygen species in a normal manner, which may play a major role in the pathogenesis of this disease.     

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 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.     

Expression of inducible nitric oxide synthase in human umbilical vein endothelial cells during primary culture

The adaptive response of endothelial cells to stress may lead to the upregulation of nitric oxide (NO) production. Herein, we report inducible nitric oxide synthase (iNOS) induction in primary cultures of human umbilical vein endothelial cells (HUVEC). The enzyme expression was earlier observed in 12-h cultures, reaching maximal levels after 3 days and decreasing when cells become confluent. The time course of NO production by HUVEC paralleled iNOS expression during the whole culture period, indicating that enzyme was functionally active. Conversely, iNOS induction could not be further detected in HUVEC subcultures passed once from cells presenting maximal levels of iNOS expression in the primary culture. Induction of iNOS in HUVEC was not related to lipopolysaccharide contamination, since the enzyme expression was not affected in the presence of polymyxin B added to primary cultures. Further analysis showed that aminoguanidine, a specific iNOS inhibitor, did not affect cell proliferation, suggesting that the NO produced by HUVEC may not be directly related to cell growth. Platelet endothelial cell adhesion molecule-1 expression was upregulated during cell confluence, in contrast to the decrease of iNOS expression and activity. The data suggest that iNOS expression may be a molecular mechanism mediating the adaptive response of endothelial cells to culture environment.