Prohibitin as the Molecular Binding Switch in the Retinal Pigment Epithelium.

Previously, our molecular binding study showed that prohibitin interacts with phospholipids, including phosphatidylinositide and cardiolipin. Under stress conditions, prohibitin interacts with cardiolipin as a retrograde response to activate mitochondrial proliferation. The lipid-binding switch mechanism of prohibitin with phosphatidylinositol-3,4,5-triphosphate and cardiolipin may suggest the role of prohibitin effects on energy metabolism and age-related diseases. The current study examined the region-specific expressions of prohibitin with respect to the retina and retinal pigment epithelium (RPE) in age-related macular degeneration (AMD). A detailed understanding of prohibitin binding with lipids, nucleotides, and proteins shown in the current study may suggest how molecular interactions control apoptosis and how we can intervene against the apoptotic pathway in AMD. Our data imply that decreased prohibitin in the peripheral RPE is a significant step leading to mitochondrial dysfunction that may promote AMD progression.

Increased Oxidative and Nitrative Stress Accelerates Aging of the Retinal Vasculature in the Diabetic Retina.

Hyperglycemia-induced retinal oxidative and nitrative stress can accelerate vascular cell aging, which may lead to vascular dysfunction as seen in diabetes. There is no information on whether this may contribute to the progression of diabetic retinopathy (DR). In this study, we have assessed the occurrence of senescence-associated markers in retinas of streptozotocin-induced diabetic rats at 8 and 12 weeks of hyperglycemia as compared to normoglycemic aging (12 and 14 months) and adult (4.5 months) rat retinas. We have found that in the diabetic retinas there was an up-regulation of senescence-associated markers SA-ß-Gal, p16INK4a and miR34a, which correlated with decreased expression of SIRT1, a target of miR34a. Expression of senescence-associated factors primarily found in retinal microvasculature of diabetic rats exceeded levels measured in adult and aging rat retinas. In aging rats, retinal expression of senescence associated-factors was mainly localized at the level of the retinal pigmented epithelium and only minimally in the retinal microvasculature. The expression of oxidative/nitrative stress markers such as 4-hydroxynonenal and nitrotyrosine was more pronounced in the retinal vasculature of diabetic rats as compared to normoglycemic aging and adult rat retinas. Treatments of STZ-rats with the anti-nitrating drug FeTPPS (10mg/Kg/day) significantly reduced the appearance of senescence markers in the retinal microvasculature. Our results demonstrate that hyperglycemia accelerates retinal microvascular cell aging whereas physiological aging affects primarily cells of the retinal pigmented epithelium. In conclusion, hyperglycemia-induced retinal vessel dysfunction and DR progression involve vascular cell senescence due to increased oxidative/nitrative stress.

Amyloid ß peptide-induced inhibition of endothelial nitric oxide production involves oxidative stress-mediated constitutive eNOS/HSP90 interaction and disruption of agonist-mediated Akt activation.

BACKGROUND: Amyloid ß (Aß)-induced vascular dysfunction significantly contributes to the pathogenesis of Alzheimer's disease (AD). Aß is known to impair endothelial nitric oxide synthase (eNOS) activity, thus inhibiting endothelial nitric oxide production (NO). METHOD: In this study, we investigated Aß-effects on heat shock protein 90 (HSP90) interaction with eNOS and Akt in cultured vascular endothelial cells and also explored the role of oxidative stress in this process. RESULTS: Treatments of endothelial cells (EC) with Aß promoted the constitutive association of HSP90 with eNOS but abrogated agonist (vascular endothelial growth factor (VEGF))-mediated HSP90 interaction with Akt. This effect resulted in blockade of agonist-mediated phosphorylation of Akt and eNOS at serine 1179. Furthermore, Aß stimulated the production of reactive oxygen species in endothelial cells and concomitant treatments of the cells with the antioxidant N-acetyl-cysteine (NAC) prevented Aß effects in promoting HSP90/eNOS interaction and rescued agonist-mediated Akt and eNOS phosphorylation. CONCLUSIONS: The obtained data support the hypothesis that oxidative damage caused by Aß results in altered interaction of HSP90 with Akt and eNOS, therefore promoting vascular dysfunction. This mechanism, by contributing to Aß-mediated blockade of nitric oxide production, may significantly contribute to the cognitive impairment seen in AD patients.

Dysplastic hepatocytes develop nuclear inclusions in a mouse model of viral hepatitis.

Viral hepatitis resulting in chronic liver disease is an important clinical challenge and insight into the cellular processes that drive pathogenesis will be critical in order to develop new diagnostic and therapeutic options. Nuclear inclusions in viral and non-viral hepatitis are well documented and have diagnostic significance in some disease contexts. However, the origins and functional consequences of these nuclear inclusions remain elusive. To date the clinical observation of nuclear inclusions in viral and non-viral hepatitis has not been explored at depth in murine models of liver disease. Herein, we report that in a transgenic model of hepatitis B surface antigen mediated hepatitis, murine hepatocytes exhibit nuclear inclusions. Cells bearing nuclear inclusions were more likely to express markers of cell proliferation. We also established a correlation between these inclusions and oxidative stress. N-acetyl cysteine treatment effectively reduced oxidative stress levels, relieved endoplasmic reticulum (ER) stress, and the number of nuclear inclusions we observed in the transgenic mice. Our results suggest that the presence of nuclear inclusions in hepatocytes correlates with oxidative stress and cellular proliferation in a model of antigen mediated hepatitis.

The role of indoleamine 2,3 dioxygenase in beneficial effects of stem cells in hind limb ischemia reperfusion injury.

Ischemia-Reperfusion (IR) injury of limb remains a significant clinical problem causing secondary complications and restricting clinical recovery, despite rapid restoration of blood flow and successful surgery. In an attempt to further improve post ischemic tissue repair, we investigated the effect of a local administration of bone marrow derived stem cells (BMDSCs) in the presence or absence of immune-regulatory enzyme, IDO, in a murine model. A whole limb warm ischemia-reperfusion model was developed using IDO sufficient (WT) and deficient (KO) mice with C57/BL6 background. Twenty-four hours after injury, 5 × 105 cells (5×105 cells/200 µL of PBS solution) BMDSCs (Sca1 + cells) were injected intramuscularly while the control group received just the vehicle buffer (PBS). Forty-eight to seventy-two hours after limb BMDSC injection, recovery status including the ratio of intrinsic paw function between affected and normal paws, general mobility, and inflammatory responses were measured using video micrometery, flow cytometry, and immunohistochemistry techniques. Additionally, MRI/MRA studies were performed to further study the inflammatory response between groups and to confirm reconstitution of blood flow after ischemia. For the first time, our data, showed that IDO may potentially represent a partial role in triggering the beneficial effects of BMDSCs in faster recovery and protection against structural changes and cellular damage in a hind limb IR injury setting (P = 0.00058).

Loss of thioredoxin function in retinas of mice overexpressing amyloid ß.

Amyloid ß peptides (Aß) have been implicated in the pathogenesis of age-related macular degeneration (ARMD) and glaucoma. In this study, retinas of mice overexpressing Aß (Tg) were compared to those of wild-type mice (Wt) and analyzed for oxidative stress parameters. We observed a progressive decrease in all retinal cell layers, which was significantly greater in Tg mice at 14 months and culminated in loss of the outer retina at 18 months of age. We also observed higher levels of reactive oxygen species, glial fibrillary acidic protein, and hydroperoxide in Tg versus Wt mice (14 months). These effects were associated with phosphorylation/activation of the apoptosis signal kinase 1 and the p38 mitogen-activated kinase. Western blotting analysis revealed progressive increases in the levels of thioredoxin 1 and thioredoxin inhibitory protein in Tg compared to Wt mice. No changes were observed in the levels of thioredoxin reductase 1 (TrxR1); however, measurements of TrxR1 activity showed a 42.7±8% reduction in Tg mice versus Wt at 14 months of age. Our data suggest that Aß-mediated retinal neurotoxicity involves impairment of the thioredoxin system and enhanced oxidative stress, potentially implicating this mechanism in the pathogenesis of ARMD and glaucoma.

Trans-Chalcone prevents VEGF expression and retinal neovascularization in the ischemic retina.

Retinal neovascularization (RNV) is a critical pathological event and a major cause of blindness. Vascular inflammation and oxidative stress have been shown to play a key role in the induction and progression of RNV. Trans-Chalcone-derived flavonoids have been previously shown to be negative modulators of oxidative stress and inflammatory responses as well as tumor angiogenesis. In this study, we characterized the effects of the flavonoid trans-Chalcone in preventing RNV in a model of ischemic retinopathy. Ischemic retinopathy was induced in neonatal mice subjected to oxygen-induced retinopathy. Trans-Chalcone was administered intra-peritoneum at the dose of 25 mg/kg/day. Vascular density was assessed by morphometric analysis of flat mounted retinas stained with Texas red-Isolectin B4. Western blotting analysis was conducted to determine protein levels of vascular endothelial growth factor (VEGF), inter-cellular adhesion molecule 1 (ICAM-1) and the transcriptional activators' signal transducer and activator of transcription 3 (STAT3) and nuclear factor kappa beta (NF-κB). Treatment with trans-Chalcone significantly inhibited RNV in the ischemic retina, as shown by decreased number of neovascular tufts. Trans-Chalcone also blocked ischemia-induced VEGF and ICAM-1 expression and this effect correlated with inhibition of activated STAT3 and NF-κB. Our results show that trans-Chalcone effectively prevents RNV in the murine retina thus suggesting that Chalcone-derived flavonoids may be beneficial in preventing pathological neovascularization in the ischemic retina.

Cleavage of the retinal pigment epithelium-specific protein RPE65 under oxidative stress.

The regeneration of the 11-cis-retinyl imine chromophore of rhodopsin during the visual cycle and mechanisms that control this process are central questions in the field of vision research. The retinal pigment epithelium (RPE)-specific protein RPE65 is centrally involved in the isomerization and hydrolysis of all-trans-retinyl esters. In this study, we investigated RPE65 cleavage and potential regulatory mechanisms under oxidative stress conditions. The D407 RPE cell cultures were exposed to H(2)O(2) (100-1000 microM). Changes in the levels of RPE65 and proteins related to apoptosis were investigated using gel electrophoresis and western blotting. Mass spectrometry was used to confirm the identity of RPE65. C57BL/6J (M450) and C3HeB/FeJ (L450) mice were used for in vivo experiments. We found that a novel 45kDa truncated fragment of the RPE65 protein, designated RPE45, appears in RPE cells upon light exposure or oxidative stress. RPE45 is generated in vitro by recombinant caspases via an ubiquitination-dependent mechanism. Collectively, our results indicate that oxidative stress during the visual cycle results in cleavage of RPE65.

Neuroprotective role of erythropoietin by antiapoptosis in the retina.

Erythropoietin (EPO) stimulates red blood cell production, in part by inhibiting apoptosis of the red blood cell precursors. The erythropoietic effects of EPO are circadian stage dependent. Retinal injury due to light occurs through oxidative mechanisms and is manifest by retinal and retinal pigment epithelium (RPE) cells apoptosis. The visual cycle might be circadian coordinated as a means of effectively protecting the retina from the detrimental effects of light-induced, oxygen-dependent, free radical-mediated damage, especially at the times of day when light is more intense. We show that the retinal expression of EPO and its receptor (EPOR), as well as subsequent Janus kinase 2 (Jak2) phosphorylations, are each tightly linked to a specific time after oxidative stress and in anticipation of daily light onset. This is consistent with physiological protection against daily light-induced, oxidatively mediated retinal apoptosis. In vitro, we verify that EPO protects RPE cells from light, hyperoxia, and hydrogen peroxide-induced retinal cell apoptosis, and that these stimuli increase EPO and EPOR expression in cultured RPE cells. Together, these data support the premise that EPO and its EPOR interactions represent an important retinal shield from physiologic and pathologic light-induced oxidative injury.