The oil spill in ageing Bruch membrane

Abstract

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.

Figure 1

Parallel processes in AMD and atherosclerotic coronary artery disease. Both diseases involve lipoprotein retention in vessel walls. In atherosclerosis, apolipoprotein B (apoB) lipoproteins (low-density lipoprotein (LDL), chylomicron remnants) cross the vascular endothelium of large arteries, bind to proteoglycans, become modified via oxidative and non-oxidative processes, and launch numerous downstream deleterious events, including macrophage recruitment and differentiation into foam cells, cytokine release, plaque instability, neovascularisation, haemorrhage and thrombosis (the ‘Response-to-Retention’ hypothesis). Coronary artery disease is a price humans pay for the apoB lipoprotein system, which transports and delivers energy-rich lipids and lipophilic nutrients (exogenous via dietary chylomicrons, endogenous via hepatic very-low-density lipoprotein (VLDL)) to body tissues. Statins work largely by reducing plasma levels of atherogenic apoB-containing lipoprotein particles. In AMD, lipoprotein-like particles with abundant esterified cholesterol accumulate with age in the macular Bruch membrane (BrM), especially in the plane between the retinal pigment epithelium (RPE) basal lamina and the inner collagenous layer where type I choroidal neovascularisation dissects in wet AMD. In situ modifications of these lipoproteins could enable complement activation and multiple pro-inflammatory, pro-angiogenic processes. Strong evidence supports the RPE as an intraocular source of BrM lipoproteins, without definitely excluding plasma sources. Biological processes driving RPE lipoprotein assembly and secretion are presently uncertain, but lipid compositional data reviewed herein suggest that lipophilic nutrient delivery via plasma lipoproteins could be important. Details in Curcio et al.

Figure 2

The Lipid Wall and basal linear deposits (BlinD). The Lipid Wall is a precursor to BlinD, a specific lesion of AMD. (A, B) Thin-section transmission electron micrograph showing lipoproteins as vesicles; osmium postfixation, vertical plane, bars=1 µm. (C, D) Quick freeze deep etch showing modified lipoproteins as solid particles; oblique plane, bars=200 nm. In (A), lipoproteins (spherical vesicles of uniform diameter) accumulate 3–4 deep between the retinal pigment epithelium (RPE) basal lamina (black arrowheads) and the inner collagenous layer of the Bruch membrane (BrM) (lipid wall, white arrowheads). L, lipofuscin. In (B), BlinD (white arrowheads) appears as membrane-bounded vesicles in the same plane as the lipid wall in (A). The high electron density at the vesicular borders is associated with increased unesterified cholesterol content. Black arrowheads, RPE basal lamina. M, mitochondrion. In (C), tightly packed BrM lipoproteins in the lipid wall display classic core and surface of lipoproteins. In (D), in BlinD from a 78-year-old donor with geographic atrophy, lipoproteins have more heterogeneous sizes and shapes. Pooled lipid is also apparent, consistent with a model of surface degradation and particle fusion.

Figure 3

Lipoprotein basics. Lipoproteins (upper left) are multimolecular assemblies that solubilise oil droplets rich in esterified cholesterol (EC) and triglyceride (TG) for transport through an aqueous environment within a thin surface of phospholipid, unesterified cholesterol (UC), and apolipoproteins (like apoB or apoA-I). Plasma lipoproteins are the best characterised (CM, very-low-density lipoprotein (VLDL), low-density lipoprotein (LDL), high-density lipoprotein (HDL)) but lipoproteins are also secreted by brain, heart, placenta and kidney. Of the lipoprotein components, cholesterol is either unesterified (free) in membranes (UC) or esterified to a fatty acid for storage and transport (EC). UC is released from cells via several mechanisms, such as release to circulating HDL and enzymatic conversion to oxysterols. EC is released directly from cells only as part of apoB-containing lipoproteins. The neutral lipids EC and TG comprise long-chain fatty acids esterified to backbone molecules UC and glycerol, respectively. Two fatty acids of interest are linoleate (18:2n6), the most abundant in the body, and docosahexaenoate (22:6n3), especially abundant in brain and retina. Linoleate (18:2n-6) and alpha-linoleate (18:3n-3, precursor to docosahexaenoate) are not synthesised by humans and are considered dietary essential fatty acids. ApoB, a member of the Large Lipid Transfer Protein family, is found in a full-length form associated with hepatic VLDL (apoB-100, 512 kDa) or a shorter form associated with intestinal chylomicrons (apoB-48). VLDL is the precursor particle to LDL, popularly known as the bad cholesterol. After Vance and Vance.

Figure 4

Bruch membrane (BrM) lipoprotein. (A) Lipoprotein particles isolated from BrM. The particles are large, spherical and electron-lucent. Bar=50 nm. (B) BrM lipoprotein composition data from direct assay and by inference from druse composition and retinal pigment epithelium gene expression. Apo, apolipoproteins; EC, esterified cholesterol; PL, phospholipid; TG, triglyceride; UC, unesterified cholesterol. The question mark indicates that not all apolipoproteins are known.

Figure 5

Oil spill in the Bruch membrane (BrM) from biology to pathobiology. See text for references at each step of this hypothesis. From top to bottom, photoreceptor outer segments, retinal pigment epithelium (RPE), BrM and choriocapillaris are depicted. RPE cells contain nuclei and mitochondria (magenta). Lipofuscin is omitted for clarity. (1) Plasma low-density lipoprotein (LDL) and high-density lipoprotein (HDL) delivering lipophilic nutrients are taken up at LDL and scavenger receptor class B type I (SR-BI) receptors on the basolateral RPE. (2) Proteins classically associated with plasma HDL metabolism are expressed in the subretinal space and may be involved in rapid turnover of unesterified cholesterol (UC) in the neurosensory retina. Docosahexaenoate (DHA) is cycled between RPE and retina. (3) A basolaterally secreted apolipoprotein (apo)B lipoprotein is assembled from multiple sources, including uptake of plasma lipoproteins, endogenously synthesised lipids and/or photoreceptor degradation products. Fatty acids (FA), especially linoleate (18:2n6), come largely from taken-up plasma lipoproteins. UC, from as-yet undetermined sources, is re-esterified (not shown). BrM lipoproteins also contain retinyl ester. (4) RPE expresses both apoB and microsomal triglyceride transfer protein and secretes esterified cholesterol (EC)-rich particles into BrM (gold circles), where they are retained and eventually cleared through the choriocapillary endothelium. (5) Lipoprotein particles begin to accumulate during adulthood for unknown reasons, building up a layer 3–4 deep external to the RPE basal lamina (the lipid wall). Other outflow pathways for UC include apoE, ATP-binding cassette A1 (ABCA1)-mediated transfer to circulating HDL, apoE secretion and enzymatic conversion to oxysterols (27-COOH). (6) Over time, perhaps due to reactive oxygen species from neighbouring mitochondria, pro-inflammatory/toxic species such as linoleate hydroperoxide and 7-ketocholesterol appear. Particles could fuse to form lipoprotein-derived debris, the principal component of basal linear deposits and soft drusen. Translocation of necessary compounds from plasma is impeded (blocked arrows). (7) Inflammation is elicited in the inner BrM. (8) Neovascularisation (type I) ensues. CETP, cholesteryl ester transfer protein; 7-KCh, 7-ketocholesterol; LIPC, hepatic lipase.