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.

Diabetes leads to dysfunction of the neural retina before and independent of classical microvascular diabetic retinopathy, but previous studies have failed to demonstrate which neurons and circuits are affected at the earliest stages. Here, using patch-clamp recording and two-photon Ca -permeable receptors in A17 amacrine cells mediate synaptic release of GABA, the reduced Ca 2ϩ permeability of these receptors in diabetic animals leads to reduced release of GABA, followed by disinhibition and increased release of glutamate from rod bipolar cells. This perturbation of neuron and microcircuit dynamics can explain the decreased dynamic range and sensitivity of scotopic vision that has been observed in diabetes.

Diabetic retinopathy (DR) is a leading cause of vision loss among working-age adults. Retinal endothelial cell apoptosis is an early event in DR, and oxidative stress is known to play an important role in this pathology. Recently, we found that high glucose induces apoptosis in retinal neural cells by a caspase-independent mechanism. Here, we investigated the mechanisms underlying retinal endothelial cell apoptosis induced by high glucose and oxidative/nitrosative stress conditions. Endothelial cells (TR-iBRB2 rat retinal endothelial cell line) were exposed to high glucose (long-term exposure, 7 days), or to NOC-18 (nitric oxide donor; 250microM) or H(2)O(2) (100microM) for 24h. Cell viability was assessed by the MTT assay and cell proliferation by [methyl-(3)H]-thymidine incorporation into DNA. Apoptotic cells were detected with Hoechst or Annexin V staining. Active caspases were detected by an apoptosis detection kit. Active caspase-3 and apoptosis-inducing factor (AIF) protein levels were assessed by Western blot or immunohistochemistry. High glucose, NOC-18 and H(2)O(2) increased apoptosis in retinal endothelial cells. High glucose and mannitol decreased cell proliferation, but mannitol did not induce apoptosis. Caspase activation did not increase in high glucose- or NOC-18-treated cells, but it increased in cells exposed to H(2)O(2). However, the protein levels of AIF decreased in mitochondrial fractions and increased in nuclear fractions, in all conditions. These results are the first demonstrating that retinal endothelial cell apoptosis induced by high glucose is independent of caspase activation, and is correlated with AIF translocation to the nucleus. NOC-18 and H(2)O(2) also activate a caspase-independent apoptotic pathway, although H(2)O(2) can also induce caspase-mediated apoptosis.

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