Extra-hepatic metabolism of 7-ketocholesterol occurs by esterification to fatty acids via cPLA2α and SOAT1 followed by selective efflux to HDL.

Accumulation of 7-ketocholesterol (7KCh) in tissues has been previously associated with various chronic aging diseases. Orally ingested 7KCh is readily metabolized by the liver and does not pose a toxicity threat. However, 7KCh formed in situ, usually associated with lipoprotein deposits, can adversely affect surrounding tissues by causing inflammation and cytotoxicity. In this study we have investigated various mechanisms for extra-hepatic metabolism of 7KCh (e.g. hydroxylation, sulfation) and found only esterification to fatty acids. The esterification of 7KCh to fatty acids involves the combined action of cytosolic phospholipase A2 alpha (cPLA2α) and sterol O-acyltransferase (SOAT1). Inhibition of either one of these enzymes ablates 7KCh-fatty acid ester (7KFAE) formation. The 7KFAEs are not toxic and do not induce inflammatory responses. However, they can be unstable and re-release 7KCh. The higher the degree of unsaturation, the more unstable the 7KFAE (e.g. 18:0>18:1>18:2>18:3≫20:4). Biochemical inhibition and siRNA knockdown of SOAT1 and cPLA2α ablated the 7KFAE synthesis in cultured ARPE19 cells, but had little effect on the 7KCh-induced inflammatory response. Overexpression of SOAT1 reduced the 7KCh-induced inflammatory response and provided some protection from cell death. This effect is likely due to the increased conversion of 7KCh to 7KFAEs, which reduced the intracellular 7KCh levels. Addition of HDL selectively increased the efflux of 7KFAEs and enhanced the effect of SOAT1 overexpression. Our data suggests an additional function for HDL in aiding extra-hepatic tissues to eliminate 7KCh by returning 7KFAEs to the liver for bile acid formation.

7-Ketocholesterol-induced inflammation signals mostly through the TLR4 receptor both in vitro and in vivo.

The cholesterol oxide 7-ketocholesterol (7KCh) has been implicated in numerous age-related diseases such as atherosclerosis, Alzheimer's disease, Parkinson's disease, cancer and age-related macular degeneration. It is formed by the autooxidation of cholesterol and especially cholesterol-fatty acid esters found in lipoprotein deposits. This molecule causes complex and potent inflammatory responses in vitro and in vivo. It is suspected of causing chronic inflammation in tissues exposed to oxidized lipoprotein deposits. In this study we have examined the inflammatory pathways activated by 7KCh both in cultured ARPE19 cells and in vivo using 7KCh-containing implants inserted into the anterior chamber of the rat eye. Our results indicate that 7KCh-induced inflammation is mediated mostly though the TLR4 receptor with some cross-activation of EGFR-related pathways. The majority of the cytokine inductions seem to signal via the TRIF/TRAM side of the TLR4 receptor. The MyD88/TIRAP side only significantly effects IL-1ß inductions. The 7KCh-induced inflammation also seems to involve a robust ER stress response. However, this response does not seem to involve a calcium efflux-mediated UPR. Instead the ER stress response seems to be mediated by yet identified kinases activated through the TLR4 receptor. Some of the kinases identified are the RSKs which seem to mediate the cytokine inductions and the cell death pathway but do not seem to be involved in the ER stress response.

Sterculic acid antagonizes 7-ketocholesterol-mediated inflammation and inhibits choroidal neovascularization.

Sterculic acid is a cyclopropene fatty acid with numerous biological activities. In this study we demonstrate that sterculic acid is a potent inhibitor of endoplasmic reticulum (ER) stress and related inflammation caused by 7-ketocholesterol (7KCh). 7KCh is a highly toxic oxysterol suspected in the pathogenesis of various age-related diseases such as atherosclerosis, Alzheimer's disease and age-related macular degeneration. Sterculic acid demonstrated to be 5-10 times more effective than other anti-inflammatory fatty acids at inhibiting 7KCh-mediated inflammatory responses in cultured cells. In vivo, sterculic acid was effective at inhibiting the formation of choroidal neovascularization (CNV) in the laser-injury rat model. Our data suggests that sterculic acid may be useful in treating CNV in certain forms of age-related macular degeneration.

Passage of low-density lipoproteins through Bruch's membrane and choroid.

Plasma lipoproteins are thought to transport cholesterol, vitamins and carotenoids to the retinal pigment epithelium (RPE) for ultimate use by the photoreceptors. However, to reach the RPE, these lipoprotein particles must cross Bruch's membrane. We examined the reflection coefficient of Bruch's membrane (BrM) to low-density lipoprotein (LDL). Bruch's membrane and choroid were removed from 47 bovine eyes. Specimens were placed in a Ussing chamber and perfused with phosphate-buffered saline (PBS) with (31 specimens) or without (16 specimens) fluorescent low-density lipoproteins (DiI-LDL). The hydraulic conductivity of the tissue was determined for both calf and cow eyes. In the perfusions with DiI-LDL, the fluorescence intensity emitted by DiI-LDL in the efflux was measured and the reflection coefficient of BrM/choroid preparations to DiI-LDL determined. Leakage tests were done to confirm tissue integrity. Several specimens were examined using scanning electron microscopy (SEM) to examine tissue integrity before and after perfusion. Leak testing confirmed that BrM was intact both before and after perfusion. The average hydraulic conductivity of BrM/choroid perfusion of calf eyes with PBS alone was 1.42 ± 0.55 × 10(-9) m/s/Pa (mean ± SD, n = 11). The average hydraulic conductivity of the cow eyes was 4.94 ± 1.48 × 10(-10) m/s/Pa (n = 5), nearly a 3-fold decrease with age. While the flow rate remained constant during the PBS perfusions, it decreased as a function of time during perfusion with DiI-LDLs. Our major finding was of fluorescence in the effluent collected in all perfusions with DiI-LDLs, demonstrating passage of LDL through the tissue. The average reflection coefficient of calf BrM/choroid preparations to DiI-LDL was 0.58 ± 0.25 (n = 23); a similar distribution of reflection coefficients was seen in tissue from cow eyes (0.51 ± 0.33, n = 8). Our data suggested that the DiI-LDL was modestly hindered and/or captured by the tissue. This might explain the progressive decrease of hydraulic conductivity with continued perfusion of DiI-LDL.

The oil spill in ageing Bruch membrane.

Ageing is the largest risk factor for age-related macular degeneration (AMD), and soft drusen and basal linear deposits are lipid-rich extracellular lesions specific to AMD. Oil red O binding neutral lipid represents a major age-related deposition in the Bruch membrane (BrM) and the first identified druse component. Decades after these seminal observations, a natural history of neutral lipid deposition has been articulated and a biochemical model proposed. Results obtained with multiple biochemical, histochemical, and ultrastructural methods, and supported indirectly by epidemiology, suggest that the RPE secretes apolipoprotein B (apoB)-lipoprotein particles of unusual composition into BrM, where they accumulate with age eventually forming a lipid wall, a precursor of basal linear deposit. The authors propose that constituents of these lesions interact with reactive oxygen species to form pro-inflammatory peroxidised lipids that elicit neovascularisation. Here, the authors summarise key evidence supporting both accumulation of BrM lipoproteins leading to lesion formation and lipoprotein production by the RPE. The authors update their model with genetic associations between AMD and genes historically associated with plasma HDL metabolism, and suggest future directions for research and therapeutic strategies based on an oil-spill analogy.

Adequacy of exchanging the content of the anterior chamber.

Perfusion studies of the anterior segment of the eye frequently involve an exchange of the contents of the anterior chamber. We here determined how much fluid was necessary to pass through the upstream tubing and anterior chamber such that the contents of the anterior chamber were adequately exchanged. We used fluorescein dextran (500 kD) to assess the adequacy of exchange of enucleated porcine eyes that were washed out with varying volumes of buffer. The results were compared with two theoretical models, one that accounted for the convective dispersion that occurs in the upstream tubing while in the other, more simple model, it was assumed that the upstream tubing and anterior chamber behave essentially as a well-mixed chamber. We found that the experiment results were bounded by these two models, with the well-mixed model giving a lower bound on the rate at which the anterior chamber can be cleared. We found that exchange of the anterior chamber to reduce the concentration of a drug or tracer by 20-fold required a perfused volume three times the combined volume of the upstream tubing and anterior chamber.

7-ketocholesterol-induced inflammation: involvement of multiple kinase signaling pathways via NFκB but independently of reactive oxygen species formation.

PURPOSE: 7-Ketocholesterol (7KCh) accumulates in oxidized lipoprotein deposits and is known to be involved in macrophage foam cell formation and atherosclerosis. 7-KCh is present in the primate retina and is associated with oxidized lipoprotein deposits located in the choriocapillaris, Bruch's membrane, and retinal pigment epithelium (RPE). 7-KCh can also be formed in the retina as a consequence of light-induced iron release. The purpose of this study was to examine the signaling pathways involved in the 7KCh-mediated inflammatory response focusing on three cytokines, VEGF, IL-6, and IL-8. METHODS: ARPE-19 cells were treated with 7KCh solubilized in hydroxypropyl-ß-cyclodextrin. Cytokines were quantified by qRT-PCR (mRNA) and ELISA (protein) using commercially available products. NFκB activation was determined by IκBα mRNA induction. RESULTS: Treatment of ARPE-19 cells with 15 µM 7KCh markedly induced the expression of VEGF, IL-6, and IL-8. No increase in NOX-4 expression or ROS formation was detected. 7KCh induced the phosphorylation of ERK1/2 and p38MAPK, and inhibitors to these kinases markedly reduced the cytokine expression but did not affect the IκBα mRNA expression. By contrast, inhibition of PI3K and PKCζ significantly decreased the cytokine and IκBα mRNA expression. Inhibition of the IκB kinase complex essentially ablated all cytokine induction. CONCLUSIONS: 7KCh induces cytokines via three kinase signaling pathways, AKT-PKCζ-NFκB, p38 MAPK, and ERK. The MAPK/ERK pathways seem to preferentially enhance cytokine induction downstream from NFκB activation. The results of this study suggest that 7KCh activates these pathways through interactions in the plasma membrane, but the mechanism(s) remains unknown.

Apolipoprotein B-containing lipoproteins in retinal aging and age-related macular degeneration.

The largest risk factor for age-related macular degeneration (ARMD) is advanced age. With aging, there is a striking accumulation of neutral lipids in Bruch's membrane (BrM) of normal eye that continues through adulthood. This accumulation has the potential to significantly impact the physiology of the retinal pigment epithelium (RPE). It also ultimately leads to the creation of a lipid wall at the same locations where drusen and basal linear deposit, the pathognomonic extracellular, lipid-containing lesions of ARMD, subsequently form. Here, we summarize evidence obtained from light microscopy, ultrastructural studies, lipid histochemistry, assay of isolated lipoproteins, and gene expression analysis. These studies suggest that lipid deposition in BrM is at least partially due to accumulation of esterified cholesterol-rich, apolipoprotein B-containing lipoprotein particles produced by the RPE. Furthermore, we suggest that the formation of ARMD lesions and their aftermath may be a pathological response to the retention of a sub-endothelial apolipoprotein B lipoprotein, similar to a widely accepted model of atherosclerotic coronary artery disease (Tabas, I., K. J. Williams, and J. Borén. 2007. Subendothelial lipoprotein retention as the initiating process in atherosclerosis: update and therapeutic implications. Circulation. 116:1832-1844). This view provides a conceptual basis for the development of novel treatments that may benefit ARMD patients in the future.

Aging, age-related macular degeneration, and the response-to-retention of apolipoprotein B-containing lipoproteins.

The largest risk factor for age-related macular degeneration (ARMD) is advanced age. A prominent age-related change in the human retina is the accumulation of histochemically detectable neutral lipid in normal Bruch's membrane (BrM) throughout adulthood. This change has the potential to have a major impact on physiology of the retinal pigment epithelium (RPE). It occurs in the same compartment as drusen and basal linear deposit, the pathognomonic extracellular, lipid-containing lesions of ARMD. Here we present evidence from light microscopic histochemistry, ultrastructure, lipid profiling of tissues and isolated lipoproteins, and gene expression analysis that this deposition can be accounted for by esterified cholesterol-rich, apolipoprotein B-containing lipoprotein particles constitutively produced by the RPE. This work collectively allows ARMD lesion formation and its aftermath to be conceptualized as a response to the retention of a sub-endothelial apolipoprotein B lipoprotein, similar to a widely accepted model of atherosclerotic coronary artery disease (CAD) (Tabas et al., 2007). This approach provides a wide knowledge base and sophisticated clinical armamentarium that can be readily exploited for the development of new model systems and the future benefit of ARMD patients.

Morphometric analysis of lipoprotein-like particle accumulation in aging human macular Bruch's membrane.

PURPOSE: To determine the size and regional distribution of lipoprotein-like particles (LLPs) that accumulate with age in Bruch's membrane (BrM). METHODS: The quick-freeze/deep-etch method was used to prepare specimens of human BrM (age range, 27-78) for electron microscopic examination. Stereologic methods were used to analyze the resultant micrographs and determine the age-related changes of the LLP volume fraction and diameter distribution in various locations in BrM. RESULTS: The volume fraction occupied by LLPs was found to increase monotonically with age in both the inner collagenous layer (ICL) and elastic layer (EL), but not in the outer collagenous layer (OCL). The mass of total LLP-associated lipids in BrM also increased with age. There was no significant increase in LLP size with age, but there was a modest increase in size with increased volume fraction of LLPs in BrM. CONCLUSIONS: The pattern of accumulation of particles was consistent with a retinal pigment epithelium (RPE) source for the LLPs, which explains why once the EL and ICL were filled with particles, LLPs continued to accumulate near the RPE, but no further accumulation was found in the OCL. The quantity of LLP-associated lipids found in BrM accounts for a large portion of the accumulated lipids measured in this tissue.

Comparison of morphology of human macular and peripheral Bruch's membrane in older eyes.

Deposits in macular human Bruch's membrane (BrM) increase with age and have been postulated to be associated with age-related maculopathy. We used two ultrastructural methods to compare these deposits by electron microscopy in macular and peripheral BrM of eight eyes from donors 63-86 years of age. Quick-freeze/deep-etch (QFDE) was used to prepare replicas that showed the ultrastructure of deposits, and osmium-tannic acid-paraphenylenediamine (OTAP) was used to preserve small extracellular lipid particles. We found that an accumulation of lipoprotein-like particles (LLPs) occurred in the peripheral BrM just as it does in the macular region, but with perhaps a somewhat slower time course. The "lipid wall," reported in macular BrM, was also found occasionally in the peripheral regions. The same processes that lead to age-related accumulation of LLPs in macular BrM appear to also occur in the peripheral regions.

Age-related changes in human macular Bruch's membrane as seen by quick-freeze/deep-etch.

Lipid-containing inclusions have been observed in human Bruch's membrane (BrM) and are postulated to be associated with age-related maculopathy (ARM), a major cause of legal blindness in developed countries. The dehydration associated with specimen preparation for thin-section transmission electron microscopy causes loss of these inclusions. Better preservation of the ultrastructure of the inclusions and tissue is achieved by using a quick-freeze/deep-etch preparation. We use this technique to examine normal human macular BrM in order to characterize the deposition of the lipid-rich inclusions and their age-related accumulation within different layers of the tissue. We find that various inclusions mentioned in other studies can be formed by combinations of three basic structures: lipoprotein-like particles (LLPs), small granules (SGs) and membrane-like structures. These inclusions are associated with collagen and elastic fibrils by fine filaments. In younger eyes, these inclusions are found mostly in the elastic (EL) and outer collageneous layer (OCL) and occupy a small fraction of the interfibrillar spacing. As age increases, LLPs and SGs gradually fill the interfibrillar spacing of the EL and inner collageneous layer (ICL) of the tissue, and later form a new sublayer, the lipid wall, within the boundary region between the basal lamina of retinal pigment epithelium (RPE) and ICL. Because the formation of the lipid wall only occurs after these inclusions fill the ICL, and it seems unlikely that the LLPs can pass through the packed layer, this result suggests a possible RPE origin of the LLPs that make up the lipid wall.

Quick-freeze/deep-etch visualization of age-related lipid accumulation in Bruch's membrane.

PURPOSE: To examine age-related changes in the ultrastructure of Bruch's membrane with quick-freeze/deep-etch (QFDE) and conventional thin-section transmission electron microscopy (TEM). METHODS: Four eyes from human donors aged 27, 41, 76, and 78 years were preserved within 4 hours of death. Full-thickness tissue blocks from the macula were prepared for TEM or for QFDE. RESULTS: Ultrastructure seen by conventional TEM was revealed in greater detail by QFDE. Cholesterol-containing particles (mean diameter, 80 nm) formed a thin densely packed layer external to the basal lamina of the retinal pigment epithelium (RPE) only in older eyes. The mesh size of the RPE basal lamina was smaller than the particles, and it appeared larger in older eyes. QFDE also revealed less decorated collagen fibrils in older eyes. CONCLUSIONS: The data suggest that the predilection of a extremely thin sublayer of inner Bruch's membrane for accumulating lipid particles may eventually lead to a confluent lipid wall capable of isolating the retina from its blood supply. If these lipids originate in the retinal pigment epithelium, then they are unlikely to have passed through the basal lamina in this form. The age-related increase in lipid particles corresponds with an age-related increase in hydraulic resistance determined in excised Bruch's membrane/choroid by others. QFDE will be useful for future modeling studies of Bruch's membrane transport and to identify those moieties responsible for deleterious age-related transport changes in Bruch's membrane.