Peroxisome Proliferator-Activated Receptor and Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is the leading cause of new blindness in the western world and is becoming more of a socio-medical problem as the proportion of the aged population increases. There are multiple efforts underway to better understand this disease process. AMD involves the abnormal retinal pigment epithelium (RPE), drusen formation, photoreceptor atrophy, and choroidal neovascularization. Peroxisome proliferator-activated receptors (PPARs) play an important role in lipid degeneration, immune regulation, regulation of reactive oxygen species (ROSs), as well as regulation of vascular endothelial growth factor (VEGF), matrix metalloproteinase-9 (MMP-9), and docosahexaenoic acid (DHA). These molecules have all been implicated in the pathogenesis of AMD. In addition, PPAR gamma is expressed in RPE, an essential cell in photoreceptor regeneration and vision maintenance. This review summarizes the interactions between PPAR, AMD-related molecules, and AMD-related disease processes

Enhanced HtrA2/Omi expression in oxidative injury to retinal pigment epithelial cells and murine models of neurodegeneration. Invest Ophthalmol Vis Sci. 2009; 50:4957–4966. [PubMed: 19443712

PURPOSE. To investigate the role of HtrA2/Omi, a nuclear-encoded mitochondrial serine protease with a proapoptosis function, under H 2 O 2 -induced oxidative stress in human RPE, in the Ccl2 Ϫ/Ϫ Cx3cr1 Ϫ/Ϫ double-knockout (DKO) mouse retina, and the HtrA2/Omi-deficient mice. METHODS. Oxidative stress was induced in ARPE-19 cells by 1 mM H 2 O 2 for 2 hours. HtrA2/Omi and caspase-3 expression was evaluated using RQ-PCR, immunohistochemistry, or Western blot. Cell viability was detected by MTT assay. HtrA2/Omi expression in the subcellular components and activated caspase-3 were measured. These processes were also evaluated in cells treated with UCF-101, an HtrA2/Omi inhibitor or in cells subjected to RNAi against HtrA2/Omi. Oxidative stress was assayed and compared in retinas of DKO and wild-type (WT) mice by determining serum NADPH oxidase subunits and nitrite levels. Transmission electron microscopy was used to view the retinal ultrastructure of the HtrA2/Omi-deficient mice. A ge-related macular degeneration (AMD), a neurodegenerative disease of the central retina, is the leading cause of blindness in industrialized countries 1 and among the elderly throughout the world. RESULTS. 2 Although the exact pathogenesis of AMD remains unclear, the current pathophysiologic concept points to the critical role of cumulative oxidative damage to the retinal pigment epithelium (RPE). 3,4 The RPE not only participates in photoreceptor metabolism by transporting nutrients from the choroid into the retina, but also removes waste products from the retina, thereby acting as a metabolic gatekeeper between the photoreceptors and choriocapillaris. 5 These cells are involved in maintaining retinal homeostasis by phagocytizing the discs shed from the photoreceptor outer segments. 3,4 High oxygen tension in the macula, exposure to light, and the RPE-mediated processes of phagocytosis and lipid peroxidation increase ROS generation in the RPE. 10 Accordingly, oxidative stress may promote AMD pathogenesis by interfering with RPE function, decreasing RPE junctional integrity, enhancing RPE expression of proinflammatory and proangiogenic cytokines, and/or promoting RPE apoptosis. 13 HtrA2/Omi belongs to the family of high temperature requirement protein A (HtrA) serine proteases conserved from bacteria to humans

Protocol for vital dye staining of corneal endothelial cells

To describe a step-by-step methodology to establish a reproducible staining protocol for the evaluation of human corneal endothelial cells. Four procedures were performed to determine the best protocol. (1) To determine the optimal trypan blue staining method, goat corneas were stained with 4 dilutions of trypan blue (0.4%, 0.2%, 0.1%, and 0.05%) and 1% alizarin red. (2) To determine the optimal alizarin red staining method, goat corneas were stained with 2 dilutions of alizarin red (1% and 0.5%) and 0.2% trypan blue. (3) To ensure that trypan blue truly stains damaged cells, goat corneas were exposed to either 3% hydrogen peroxide or to balanced salt solution, and then stained with 0.2% trypan blue and 0.5% alizarin red. (4) Finally, fresh human corneal buttons were examined; 1 group was stained with 0.2% trypan blue and another group with 0.4% trypan blue. For the 4 procedures performed, the results are as follows: (1) trypan blue staining was not observed in any of the normal corneal samples; (2) 0.5% alizarin red demonstrated sharper cell borders than 1% alizarin red; (3) positive trypan blue staining was observed in the hydrogen peroxide exposed tissue in damaged areas; (4) 0.4% trypan blue showed more distinct positive staining than 0.2% trypan blue. We were able to determine the optimal vital dye staining conditions for human corneal endothelial cells using 0.4% trypan blue and 0.5% alizarin red

A High Omega-3 Fatty Acid Diet Reduces Retinal Lesions in a Murine Model of Macular Degeneration

Age-related macular degeneration (AMD) is one of the leading cause of blindness among the elderly; however, current therapy options are limited. Epidemiological studies have shown that a diet that is high in ω-3 polyunsaturated (n-3) fatty acids can slow disease progression in patients with advanced AMD. In this study, we evaluated the effect of such a diet on the retinas of Ccl2−/−/Cx3cr1−/− mice, a model that develops AMD-like retinal lesions that include focal deep retinal lesions, abnormal retinal pigment epithelium, photoreceptor degeneration, and A2E accumulation. Ccl2−/−/Cx3cr1−/− mice that ingested a high n-3 fatty acid diet showed a slower progression of retinal lesions compared with the low n-3 fatty acids group. Some mice that were given high levels of n-3 fatty acids had lesion reversion. We found a shunted arachidonic acid metabolism that resulted in decreased pro-inflammatory derivatives (prostaglandin E2 and leukotriene B4) and an increased anti-inflammatory derivative (prostaglandin D2). We also measured lower ocular TNF-α and IL-6 transcript levels in the mice fed a diet of high n-3 fatty acids. Our findings in these mice are in line with human studies of AMD risk reduction by long-chain n-3 fatty acids. This murine model provides a useful tool to evaluate therapies that might delay the development of AMD