The MRDS has shown that the retina internalizes circulating LDL and distributes the lipids to the different cellular layers. This lipid uptake is performed by the RPE and Muller cells by two distinct mechanisms. The LDL receptor is highly expressed in RPE cells and this seems to be the main mechanism for lipid uptake in the back of the retina. In the neural retina vascular endothelial cells the mechanism seems to involve some form of transcytosis transport across the capillary endothelial cells into the surrounding Muller cells. The retina expresses the same proteins used by the systemic """"""""reverse cholesterol"""""""" pathway to uptake and transport lipids internally. 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 autooxidation and 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. The MRDS has found that the retina contains significant levels of the highly inflammatory and 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 has not been conclusively identified but light is known to cause the release of iron from ferritin and possibly cytochrome c. 7-Ketocholesterol can reach concentrations in excess of 100 micromolar in lipoprotein deposits. The MRDS is actively investigating the 7-ketocholesterol-mediated inflammatory and death pathways in the retina, both in vitro and in vivo. Recently, three inflammatory pathways have been found to be involved in mediating the 7-ketocholesterol-mediated inflammation, MAPK/ERK, p38MAPK and PKC which work via NFkappaB. In cultured human RPE and vascular endothelial cells 7-ketocholesterol has been found to be a very potent inducer of VEGF and other cytokines including 1L-1, IL-6, IL-8 and TNF. 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 cultured RPE-derived cells and vascular endothelial cells the the 7-ketocholesterol-mediated inflammation works independently of reactive oxygen species formation. In vivo, 7-ketocholesterol has been found to be a potent pro-angiogenic agent. Waffers (0.5 mm) containing 7-ketocholesterol or oxidized LDL induced extensive neovascularization when inserted into corneal pockets in rats. Retinas of laser-injured rats were found to contain levels of 7-ketocholesterol 7-10 times greater than normal retinas before the formation of the choroidal neovascularization (CNV) at lesion sites. These two neovascularization rat models are being used to test agents that were found to attenuate the 7-ketocholesterol-mediated inflammation in vitro. In the laser-injury model preliminary results indicate that one of these agents is able to prevent and reverse the CNV when applied in topical drops. Work on the corneal pocket model is in progress. Recent work has also shown that 7-ketocholesterol is not well metabolized by cultured RPE cells. Instead 7-ketocholesterol is eliminated by the cells into the media via lipoprotein acceptors. HLD is particularly efficient at removing 7-ketocholesterol from cells but the precise mechanism has not been elucidated. The ATP-binding cassette transporters ABCA1 and ABCG1 do not seem to be involved in the process. Knockdown of the expression of these two transporters with siRNAs had no effect on the 7-ketocholesterol efflux. In summary, the MRDS has made considerable progress in determining how 7-ketocholesterol forms in the retina and the consequences of its pro-inflammatory and angiogenic properties. 7-Ketocholesterol seems to be an important age-related risk factor in age-related macular degeneration.
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