The MRDS has shown that the retina uptakes circulating LDL and distributes the lipids to the different cellular layers. To perform this lipid uptake and transport, the retina expresses the same proteins used by the systemic """"""""reverse cholesterol"""""""" pathway. The retina has adapted this pathway to its own particular needs by controlling the expression and location of the different lipoproteins, transporters and receptors. Monkey retina has been found to express apoA1, apoE, apoB, ABCA1, LDLR, SR-BI and II, CD36, CETP, and LCAT. The particular locations where these molecules are expressed within the retina suggest an internal lipid transport based on HDL-like lipid particles. We hypothesize that the retina requires a high turnover of lipids because of the high susceptibility of this class of molecules to oxidation and particularly to photooxidation. One of the main priorities of the MRDS is to identify the mechanism by which oxidized lipids, which may be highly toxic, are metabolized and excreted from the retina. Recently the MRDS has found that the retina contains significant levels of the highly toxic oxysterol, 7-ketocholesterol. This molecule is of particular interest because it is known to be highly cytotoxic to various cell types and is the major toxic component in atherosclerotic plaques. This oxysterol is formed by copper and/or iron mediated oxidation of cholesterol and cholesterol-esters in lipoprotein deposits. In the monkey retina 7-ketocholesterol is found in association with oxidized lipoprotein deposits in the choriocapillaris and Bruchs membrane. In photodamaged albino rats 7-ketocholesterol is found in areas of high mitochondrial content especially the RPE, photoreceptor inner segments and ganglion cells. Intermediates identified in these rats by LCMS indicate that the 7-ketocholesterol was formed via a free radical mediated mechanism. This mechanism requires a transition metal catalyst which is likely Fe+2. The source of the iron was not conclusively identified but light is known to cause the release of iron from ferritin and possibly cytochrome c. The source of the iron and mechanisms of its release are being actively investigated. In cultured human RPE and vascular endothelial cells the MRDS has found that 7-ketocholesterol is a potent inducer of VEGF and other cytokines including IL-6 and IL-8. The pharmacological properties of 7-ketocholesterol are complex and seem to be dose dependent. At low doses 7-ketocholesterol is pro-inflammatory while at higher doses it can induce cell death by necrosis or apoptosis depending on the cell type. In most cell types 7-ketocholesterol-induced inflammation is dependent on reactive oxygen species (ROS) formation. However, in cultured RPE-derived cells the pro-inflammatory pathway seems to be independent of ROS. 7-ketocholesterol is also known to form micro-crystals in membranes causing destabilization and possible leakage and/or breakdown. The MRDS is actively investigating the 7-ketocholesterol-mediated inflammatory and death pathways in the retina in vivo. In addition, the MRDS is testing the anti-angiogenic properties of certain antagonist to 7-ketocholesterol-mediated VEGF induction using a CNV rat model (laser treated). The metabolism of 7-ketocholesterol by the neural retina and RPE are another topic of interest to the MRDS. The retina expresses significant levels of the mitochondrial CYP27A1, a cytochrome P450 enzyme capable of hydrolylating 7-ketocholesterol at the side chain 27-carbon. This hydroxylated form of 7-ketocholesterol is non-toxic and lacks the pro-inflammatory properties of 7-ketocholesterol. However, repeated attempts at detecting hydroxylated metabolites of 7-ketocholesterol in retinal extracts have been unsuccessful. Two other enzymes capable of metabolizing 7-ketocholesterol have been investigated, the 24-cholesterol hydroxylases (CYP46A1) and the sulfotransferases (SULT2B1). Both of these enzymes are present in the retina but only in trace amounts and their potential metabolites are also undetectable. The retina seems to eliminate 7-ketocholesterol without metabolizing it. Preliminary evidence suggests transporters such as ABCG1 which is present in the basal aspect of the RPE may be involved in the elimination of 7-ketocholesterol. Once in circulation 7-ketocholesterol is known to be quickly metabolized by the liver. Another area of interest to the MRDS are anti-oxidative enzymes specially those that protect the mitochondria. In this regard we have investigated the retinal expression of peroxiredoxin 3 and the methionine sulfoxide reductases (MSRs). These are enzymes capable of reversing the oxidation of methionine in proteins and thus preserving function. There are two major forms of MSRs categorized by their stereospecificity on methionine sulfoxide (MetO). The MSRAs recognize the Met(S)O and the MSRBs recognize the Met(R)O stereoisomers. These enzymes have been shown to be important in preserving the health of cells and in the aging process. The MSRs are highly expressed in the retina especially in the macular region. These enzymes protect cultured cells from multiple forms of oxidative damage including damage caused by 7-ketocholesterol. The MRDS is actively investigating the role of these enzymes in preventing the light-mediated iron release from protein like ferritin. In summary the MRDS is pursuing several basic research projects investigating lipid transport, oxidation and protective mechanisms with the goal of obtaining a better understanding of the processes involved in aging and the pathogenesis mechanisms of diseases like the age-related macular degenerations.
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