In FY14 the Mechanisms of Retinal Diseases Section (MRDS) was involved in three major projects. 1) Investigating the molecular mechanism of extra-hepatic metabolism of 7-ketocholesterol (7KCh) as well as the mechanism of 7KCh-induced cytoxicity and inflammation. 2) Further development of the 7KCh-induced anterior chamber angiogenesis rat model. This model has proven useful in testing anti-inflammatory drugs as well as in achieving the goals of project 1, by confirming that the inflammatory pathways found in vitro where also involved in vivo. 3) LCMS analyses to determine levels of 7KCh, Cholesterol (Ch) and various other related lipids in lens, neural retina and RPE/Choroid tissues in monkey ranging in age from 2 to 25 years. Also in this study neural retina, RPE and drusen samples from elderly human donors were analyzed. 7KCh is an oxidized derivative of cholesterol found in very high concentrations in atherosclerotic plaques (as much as 25% weight) and in the back of the retina in deposits in Bruchs membrane (BM) and the choriocapillaris (CH). 7KCh forms by the autooxidation of cholesterol and especially cholesterol-fatty acid esters (CEs) in the presence of oxygen and a transition metal such as Cu+2 or Fe+2. This is the main reason why the CE-rich LDL deposits are so readily oxidized. The chronic inflammation induced by 7KCh is suspected in the pathogenesis of most human chronic aging diseases such as atherosclerosis, Alzheimers Disease, some forms of cancer, diabetes type II, and age-related macular degeneration. The precise mechanism of action of 7KCh is not well understood. Studies performed by the MRDS and other investigators have shown that 7KCh activates numerous inflammatory pathways mostly mediated by NFkB. Using sterculic acid (a potent 7KCh antagonist) and a variety of other inhibitors and siRNAs we were able to determine that most of the 7KCh-induced inflammatory and cell death responses are mediated by the TLR4 receptor. The activation of TLR4 can also cause an almost immediate activation of the epidermal growth factor receptor (EGFR) which in turn sets off multiple inflammatory pathways that lead to NFkB activation. This was demonstrated both in vitro (ARPE19 culture cells) and in vivo (anterior chamber 7KCh-implant model). Sterculic acid is a kinase inhibitor capable of inhibiting 16 different kinases downstream of the Toll-like receptors (TLRs). Sterculic acid inhibits all 4 known ribosomal S90 kinases (RSKs). Using other specific inhibitors to the RSKs we were able to determine that RSK1&2 are likely involved in the 7KCh-induced cell death while RSK4&5 are mostly involved in the inflammatory responses and angiogenesis. The complete inhibition of NFkB can significantly attenuated but not ablated the 7KCh-induced inflammatory responses. This suggests that 7KCh is activating other inflammatory pathways that are independent of NFkB. The identity of these other pathways was not determined. Our finding regarding the TLR4 receptor and the inflammation pathways involved both in vitro and in vivo were recently published (Huang et al. PlosOne, 2014). Orally administered 7KCh is known to be quickly metabolized by the liver and excreted as bile acids. This is mostly due to the liver specific enzyme CYP7A1. However, the metabolism of 7KCh outside of the liver is not well understood. Various investigators have reported hydroxylation by the cytochrome P450s, mainly CYP27A or sulfation by SULT2B1b. We examined the mRNA expression of most enzymes with the potential to metabolize 7KCh by qRTPCR. We found that most of these enzymes are either not expressed at all in the retina or RPE/CH (e.g. SULT2B1b) or express at low levels (e.g. CYP27A1 and CYP46A1). These enzymes are able to use 7KCh as substrate in vitro, but we have found no evidence of any activity on 7KCh in vivo. LCMS analyses of monkey and human RPE/CH tissues with high levels of 7KCh found no trace of any hydroxylated or sulfated forms of 7KCh. Small amounts of 24- and 27-hydroxycholesterol were found in RPE/CH but these were likely contaminants from the blood since the animals were not perfused and blood contamination is otherwise inevitable. Our in vitro experiments using ARPE-19 cells indicate that 7KCh metabolism occurs through esterification to membrane fatty acids. We have found that as much as 25% of the 7KCh that enters the cells is converted into 7KCh-fatty acid esters (7KFAEs). Two enzymes are essential to this process, the cytosolic phospholipase C (cPLA2α) and the Sterol O-acyltransferase (acyl-Coenzyme A: cholesterol acyltransferase) 1 (SOAT1). Inhibition of either one of these enzymes, with specific inhibitors or with siRNA knockdown ablates the 7KFAEs formation. Overexpression of SOAT1 significantly reduced intracellular 7KCh levels and increased the rate of 7KFAE formation. This resulted in a moderate but statistically significant protection from 7KCh-induced cell death and inflammation. While performing lipid analyses on commercially available LDL and HDL samples we found that HDL contains significant amounts of 7KFAEs and small amounts of 7KCh while LDL did not contain any oxidized components. These esters cannot be formed by autooxidation of CEs since autooxidation would affect both the cholesteryl and fatty acid moieties. This suggests that HDL selectively effluxes 7KFAEs. This was tested in ARPE19 cells exposed to 7KCh with and without SOAT1 overexpression. Even very low concentrations of HDL were able to protect the cells from 7KCh-induced inflammation and cell death. This effect was enhanced with SOAT1 overexpression. Thus, it seems we may have found a novel function for HDL, which is to selectively efflux 7KFAEs formed in cells in contact with 7KCh-containing deposits and return the 7KFAEs to the liver for proper metabolism. The results of our findings regarding the 7KCh metabolism and HDL have been submitted for publication (Lee et al. submitted) The MRDS had previously developed an angiogenesis model in rats (Amaral et al. PlosOne, 2013). We have been using this model to test anti-angiogenic and anti-inflammatory drugs. As mentioned above, this model was used to confirm the involvement of the various inflammatory pathways induced by 7KCh. This was done by incorporating the desired test compound into the 7% 7KCh-containing implant in concentration up to 12% (w/w). The aqueous humor was also tested for the presence of various cytokines. Interestingly, IL-18 was found to be constitutively expressed in aqueous humor and further induced by 7KCh. Also various inflammatory miRNAs were also induced by 7KCh. We have also successfully incorporated proteins into the implant without significant loss of biological activity. The results demonstrating the usefulness of this model for drug testing will be submitted for publication soon (Amaral et al. in preparation). Our third study consisted of analyzing lens, neural retina and RPE/CH of monkeys ranging in age from 2-25 yrs. A clear trend of increasing 7KCh concentration was observed in all tissues. This was particularly pronounced in the RPE/CH which also contained considerable amount of CEs. CEs which are particularly susceptible to autooxidation are likely the reason for the higher levels of 7KCh found in the RPE/CH. The difference in 7KCh levels between young and old monkeys was 20-50 fold greater. Samples of drusen, neural retina and RPE from fixed tissues of elderly human (72-96 yr) were also analyzed. As with the monkey tissues human RPE samples contained the highest levels of 7KCh. Drusen also contains very high levels of 7KCh. The levels in the human samples varied greatly from 3 fold to over 100 fold greater than the highest monkey. This work has been submitted for publication (Rodriguez et al. EER, in press).
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