Research performed at the MRDS has demonstrated that the retina uptakes much of its lipids from circulating low density lipoproteins (LDL) via LDL-receptors in the RPE and choriocapillaris. Inside the RPE the lipids are processed and delivered as HDL-like particles to HDL-receptors (SR-BI and SR-BII) in the photoreceptor cells and other cells of the inner retina. We have demonstrated that the retina expresses all of the main genes involved in the well-known systemic reverse cholesterol transport 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. 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. ? Another related area of interest is the formation of oxidized lipids and their cytotoxicity. One particular molecule, 7-ketocholesterol, 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-esters in lipoprotein deposits. It can also be made by photooxidation of cholesterol in the presence of a suitable photosensitizing agent. The mechanism(s) of toxicity of 7-ketocholesterol is complex. 7-ketocholesterol is known to form micro-crystals in membranes causing destabilization and possible leakage. However, 7-ketocholesterol also has potent pharmacological properties causing the induction of VEGF, IL-6 and IL-8. This seems to occur via the liver X receptors (LXRs) and the transcription factor Sp1. Our studies have found small amounts of 7-ketocholesterol in normal primate retina mostly associated with oxidized LDL deposits in Bruchs membrane and choriocapillaris. In light-damaged albino rats 7-ketocholesterol levels greatly increase throughout the retina especially in the ganglion cells, RPE and photoreceptor inner segments. This suggests that photooxidation is a plausible mechanism for generating 7-ketocholesterol in the retina. Moreover, this also suggests that chronic mitochondrial damage due to 7-ketocholesterol formation could be a factor in aging diseases of the retina. The chronic deposition of oxidized LDL in the choriocapillaris and Bruchs membrane may be a potential mechanism for choroidal neovascularization and the forms of AMD associated with choroidal hemorrhage. ? ? Our experiments have also shown that lipoproteins like LDL can be readily photoxidized forming a mixture of oxidized lipids of similar composition to those found in atherosclerotic plaques. The polyunsaturated fatty acids are also of interest because they are components of numerous lipid classes and are highly oxidizable at their double bonds. Docosahexaenoic acid (DHA) is of particular interest since it comprises approximately 50% of the lipids in the outer segment membranes. This fatty acid is highly susceptible to photooxidation and its transport and metabolism in the retina is not fully understood.? ? Enzymes that protect the mitochondria from oxidative damage or from 7-ketocholesterol toxicity are also a major area of interest. We have found that either sulfation or hydroxylation can neutralize the toxicity of 7-ketocholesterol in vitro. The MRDS has focused particularly on the cytochrome P450s, CYP27A1 and CYP46A1. CYP27A1 is abundant in the photoreceptor and RPE mitochondria and hydroxylates 7-ketocholesterol mostly at the 26-27 carbon positions. CYP46A1 is present only in trace amounts in the retina and is unlikely to play any significant role in 7-ketocholesterol hydroxylation. Moreover, after extensively searching for hydroxylated forms of 7-ketocholesterol by LCMS, we have only found traces in both in vivo and in vitro experiments. This suggests that hydroxylation is an unlikely form of 7-ketocholesterol detoxification. Sulfation by the sulfotransferase enzyme SULT2B1 was also investigated but this enzyme is only present in trace amounts in the retina and 7-ketocholesterol sulfate is not detectable in retinal extracts. There are several other sulfotransferases in the retina but the MRDS is not actively investigating their function at this time. ? Another group of protective enzymes of interest to the MRDS are the methionine sulfoxide reductases (MSRs). These groups of enzymes (MSRAs and MSRBs) convert oxidized methionines in proteins back to methionine often resulting in the restoration of lost function. Previous studies on MSRA suggest that it plays a key role in aging and age-related disease. We have found that MSRA is highly expressed in the macular RPE. The retina contains high levels of both MSRA and MSRB activities. We also discovered that MSRA is controlled by two distinct promoters. One promoter (P1) makes an mRNA whose product is targeted to the photoreceptor synaptic mitochondria and the other promoter (P2) makes two mRNAs whose products are targeted to the cytosol and nucleus. The P2 promoter is highly expressed in RPE cells. We have partially characterized the P2 promoter and have identified several relevant transcription factors involved in regulating its expression. ? ? 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.
