Abundant Lipid and Protein Components of Drusen

Abstract

BackgroundDrusen are extracellular lesions characteristic of aging and age-related maculopathy, a major retinal disease of the elderly. We determined the relative proportions of lipids and proteins in drusen capped with retinal pigment epithelium (RPE) and in RPE isolated from non-macular regions of 36 human retinas with grossly normal maculas obtained <6 hr after death.Methodology/Principal FindingsDruse pellets were examined by light and electron microscopy. Component proteins were extracted using novel methods for preserved tissues, separated, subjected to tryptic digestion and LC-MS(MS)2 analysis using an ion trap mass spectrometer, and identified with reference to databases. Lipid classes were separated using thin layer chromatography and quantified by densitometry. Major druse components were esterified cholesterol (EC), phosphatidylcholine (PC), and protein (37.5±13.7, 36.9±12.9, and 43.0±11.5 ng/druse, respectively). Lipid-containing particles (median diameter, 77 nm) occupied 37–44% of druse volume. Major proteins include vitronectin, complement component 9, apoE, and clusterin, previously seen in drusen, and ATP synthase subunit β, scavenger receptor B2, and retinol dehydrogenase 5, previously seen in RPE. Drusen and RPE had similar protein profiles, with higher intensities and greater variability in drusen. C8, part of the complement membrane attack complex, was localized in drusen by immunofluorescence.Conclusions/SignificanceAt least 40% of druse content is comprised by lipids dominated by EC and PC, 2 components that are potentially accounted for by just one pathway, the secretion of lipoproteins by RPE. Manipulating genes encoding apolipoprotein pathways would be a fruitful approach to producing drusen with high EC content in laboratory animals. Therapies that directly mitigate drusen should prepare for the substantial volume of neutral lipids. The catalog of major druse proteins is nearing completion.

Identification of hydroxyapatite spherules provides new insight into subretinal pigment epithelial deposit formation in the aging eye

“…The ionic signatures for other phospholipids and/or triglycerides were not detected at this spatial resolution. However, the spherules in sub-RPE deposits clearly contain calcium phosphate (these experiments did not distinguish the different calcium phosphates), and they are also associated with cholesterol and/or cholesteryl esters in accord with previous observations on the composition of extracellular lipid droplets in Bruch's membrane and sub-RPE deposits (11,30).…”

HTRA1, an age‐related macular degeneration protease, processes extracellular matrix proteins EFEMP1 and TSP1

“…Histologically, patients with early stages of AMD are characterized by the presence of drusen, extracellular debris that accumulates between the retinal pigment epithelium (RPE) and Bruch's membrane. Studies of the composition of drusen revealed extracellular matrix proteins as well as inflammatory molecules as the major components of drusen (Crabb et al., 2002; Wang et al., 2010). Many of the extracellular matrix proteins with increased expression in AMD high‐risk RPE cells from our data were also found in proteomic analyses of drusen.…”

“…Thus, if the CFH Y402H risk mutation lowers its affinity for CRP, then the lack of inhibition might be expected to trigger overactive inflammation in the extracellular space. The CFH risk allele is associated with increased soft drusen in the eye, and immunohistochemical and proteomic studies of drusen composition revealed several complement proteins in addition to CFH (Crabb et al., 2002; Magnusson et al., 2005; Wang et al., 2010). Several clinical trials have tried using anti‐inflammatory agents to target the complement system and systemic inflammation, but these had limited success (Ambati, Atkinson & Gelfand, 2013).…”

Smoke Exposure Causes Endoplasmic Reticulum Stress and Lipid Accumulation in Retinal Pigment Epithelium through Oxidative Stress and Complement Activation

“…The analysis of lipid composition in BrM has suggested that the lipoprotein particles are different from those found in plasma. They contain free and esterified cholesterol (37), phosphatidylcholine, and apolipoprotein B100 (38), and differ in composition such as their enrichment for esterified cholesterol and the percent phospholipid being comprised of phosphatidylcholine (39). It is suggested that the lipoproteins in BrM may be derived in part from the photoreceptor outer segments, but are processed significantly by the RPE (40).…”