Lipids accumulate in Bruch's membrane (BrM), a specialized vascular intima of the eye, and in extracellular lesions associated with aging and age-related maculopathy (ARM). We tested the hypothesis that ARM and atherosclerotic cardiovascular disease share molecules and mechanisms pertaining to extracellular lipid accumulation by localizing cholesterol and apolipoprotein B (apo B) in BrM, basal deposits, and drusen. Human donor eyes were preserved <4 hours postmortem and cryosectioned. Sections were stained with traditional lipid stains and filipin for esterified and unesterified cholesterol or probed with antibodies to apo B, apo E, and apo C-III. Normal adult retinal pigment epithelium (RPE) was subjected to RT-PCR and Western blot analysis for apolipoprotein mRNA and protein. Esterified and unesterified cholesterol was present in all drusen and basal deposits of ARM and normal eyes. Both apo B and apo E but not apo C-III were found in BrM, drusen, and basal deposits. Age-related maculopathy (ARM) is the leading cause of new, untreatable vision loss in the elderly in Western societies. [1][2][3] As shown in Figure 1A, ARM involves the retinal pigment epithelium (RPE, cells dedicated to sustaining photoreceptor health), the choriocapillaris (the blood supply to photoreceptors and the RPE), and Bruch's membrane (BrM, a thin vascular intima between the RPE and choriocapillaris). 4,5 Early ARM is characterized by moderate vision loss associated with characteristic extracellular lesions. Lesions between the RPE basal lamina and BrM can be focal (drusen) or diffuse (basal linear deposits). A diffuse lesion between the RPE and its basal lamina is basal laminar deposit. The term "sub-RPE deposits" is used for the combination of drusen and basal deposits and "basal deposits" for the combination of basal laminar deposit and basal linear deposit. Late ARM is characterized by severe vision loss associated with extensive RPE atrophy with or without the sequelae of choroidal neovascularization, ie, in-growth of choriocapillaries through BrM and under the RPE in the plane of drusen and basal linear deposits. Because the causes of ARM are obscure, recent studies have sought molecules present in the affected tissues and characteristic lesions to identify biochemical pathways perturbed by disease. 6 An important but incompletely characterized component of BrM and sub-RPE deposits is lipids. Normal BrM accumulates lipids with age, and the accumulation of esterified and unesterified cholesterol (EC and UC)-containing particles is especially prominent in the macula. 7-10 Drusen and basal deposits in aged eyes without ARM contain lipids, including cholesterol, 9 -13 and current evidence suggests that individual sub-RPE deposits are preferentially enriched in either neutral lipids or polar lipids. 13 The source of lipids and mechanisms of deposition are unknown. Analyses of BrM/ choroid lipid composition have implicated both local cells and plasma. 8,9 Atherosclerotic cardiovascular disease (CVD), the leading cause of death in Wester...

PurposeExtracellular deposits containing hydroxyapatite, lipids, proteins, and trace metals that form between the basal lamina of the RPE and the inner collagenous layer of Bruch's membrane are hallmarks of early AMD. We examined whether cultured RPE cells could produce extracellular deposits containing all of these molecular components.MethodsRetinal pigment epithelium cells isolated from freshly enucleated porcine eyes were cultured on Transwell membranes for up to 6 months. Deposit composition and structure were characterized using light, fluorescence, and electron microscopy; synchrotron x-ray diffraction and x-ray fluorescence; secondary ion mass spectroscopy; and immunohistochemistry.ResultsApparently functional primary RPE cells, when cultured on 10-μm-thick inserts with 0.4-μm-diameter pores, can produce sub-RPE deposits that contain hydroxyapatite, lipids, proteins, and trace elements, without outer segment supplementation, by 12 weeks.ConclusionsThe data suggest that sub-RPE deposit formation is initiated, and probably regulated, by the RPE, as well as the loss of permeability of the Bruch's membrane and choriocapillaris complex associated with age and early AMD. This cell culture model of early AMD lesions provides a novel system for testing new therapeutic interventions against sub-RPE deposit formation, an event occurring well in advance of the onset of vision loss.

The principal extracellular lesions of age-related maculopathy (ARM), the leading cause of vision loss in the elderly, involve Bruch's membrane (BrM), a thin vascular intima between the retinal pigment epithelium (RPE) and its blood supply. With age, 80-100 nm solid particles containing esterified cholesterol (EC) accumulate in normal BrM, and apolipoprotein B (apoB) immunoreactivity is detectable in BrM-and ARM-associated lesions. Yet little evidence indicates that increased plasma cholesterol is a risk factor for ARM. To determine if RPE is capable of assembling its own apoB-containing lipoprotein, we examined RPE for the expression of microsomal triglyceride transfer protein (MTP), which is required for this process. Embryologically part of the central nervous system, the retina ( Fig. 1A ) converts light energy to electrochemical signals for transmission to the brain. The photoreceptors are supported by the retinal pigment epithelium (RPE), a monolayer with diverse functions including daily phagocytosis of the distal tips of photoreceptor outer segments (OS), and the choroid, a vascular bed with the body's highest blood flow. The choriocapillaris is a dense capillary plexus in the innermost choroid, and Bruch's membrane (BrM) is a thin vascular intima between the RPE and the choriocapillaris (Fig. 1B, arrowheads). In human retina, the macula subserves high-acuity vision and is vulnerable to age-related maculopathy (ARM), the major cause of vision loss among the elderly of industrialized societies. The most prominent histopathologic and clinical signs of ARM are extracellular lesions [drusen (Fig. 1E, F) and basal linear deposits (not shown)] in the RPE/BrM complex that ultimately impact vision by the photoreceptors (1, 2). Choroidal neovascularization, an invasion of choriocapillaries across BrM and lateral spread within the plane of drusen and basal linear deposit (see 3), is the principal sight-threatening complication of ARM's obscure underlying degeneration.Recent findings highlight a role for lipids and lipoproteins in this degeneration. These include a protective effect of the apolipoprotein E4 (apoE4) genotype in populations and the presence of apoB and apoE and histochemically identified lipids in aging-and ARM-associated drusen and deposits in human tissues (4-8) (Fig. 1E, F). The best established risk factor for early ARM is advanced age (9). A Abbreviations: ABL, abetalipoproteinemia; apoB, apolipoprotein B; apoBEC-1, apolipoprotein B-editing complex-1; ARM, age-related maculopathy; BrM, Bruch's membrane; EC, esterified cholesterol; ESI/MS, electrospray ionization mass spectrometry; INL, inner nuclear layer; MCT3, monocarboxylate transporter 3; MTP, microsomal triglyceride transfer protein; OS, outer segments of photoreceptors; RGC, retinal ganglion cell; RPE, retinal pigment epithelium; TC, total cholesterol; TG, triglyceride; UC, unesterified cholesterol.

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Abstract. We studied the effects of extracellular matrix components on fibroblast contraction of hydrated collagen gels. After 4-h incubations, heparincontaining collagen gels contracted only 10% compared with 50% contraction of control gels. Contraction was not affected by hyaluronic acid, dermatan sulfate, or fibronectin, implying that the activity of heparin was specific. The possibility that heparin inhibited attachment of the cells to the gels was ruled out. Also, addition of heparin to the incubation medium had no effect on contraction. Microscopic examination showed that control collagen gels were composed of a uniform network of interlocking fibrils of similar sizes. Heparin-containing gels, on the other hand, were highly variable with some collagen bundles containing 5-6 collagen fibrils and other regions conmining amorphous material. Unlike the control gels, the fibrils of heparin-containing gels were not continuously interconnected. Based on the results, we propose that fibroblasts attach normally to the collagen fibrils of heparin-containing gels and attempt to contract the gels, but the mechanical forces exerted by fibroblasts on individual collagen fibrils cannot be propagated throughout the gels.

Human Müller cells acquire the capacity to generate tractional forces in vitro and the contraction-promoting growth factors insulin-like growth factor (IGF)-I and platelet-derived growth factor (PDGF) are potent stimuli. Generation of tractional force by Müller cells primarily involves integrin receptors containing alpha2 and beta1 subunits.

. Endothelial 1 (El) is identified as an endothelial cell secreted factor that stimulates collagen gel contraction by fibroblasts . This identification is based on (a) co-localization of stimulatory activity in endothelial cell conditioned media with synthetic El in reversed phase analysis ; (b) removal of the activity from conditioned media with antiserum directed against El; and (c) the activity of synthetic El. Treatment of endothelial cell conditioned media with immobilized anti-El antibodies removed 59% of the activity from the pool suggesting that El is the major contraction promoter in endothelial cell conditioned F IBROBLASTS incubated on gels formed of native type I collagen develop a morphology typical of cells in dermis and tendon (7, 31) . Additionally, over time the fibroblasts will reorganize the collagen fibrils into a structure which closely resembles dense connective tissue (2) . Collagen matrices, condensed in this fashion, have been successfully transplanted and integrated as dermal replacements (15). This in vitro phenomenon resembles a number of pathologic as well as normal processes including dermal and tendonous contracture during wound healing (2), the development of contractile connective tissue membranes above and below the retina in proliferative vitreoretinopathy (19), and the matrix reorganization observed during connective tissue development (29) .Collagen gel contraction by fibroblasts involves the actin cytoskeleton and can be inhibited by agents such as cytochalasins (28,11) . It can involve cell surface integrintype receptors since an antibody to the beta-1 subunit can inhibit this process when stimulated by PDGF (14) . Gel contraction is not dependent upon de novo synthesis of collagens and apparently does not require enzymatic cross-linking, degradation or other covalent modifications of the collagens (11). Heparin can modulate the contraction process by altering the tensile strength or continuity of the collagen matrix, though this effect is on the matrix rather than the cells (12). Gel contraction is dependent upon the presence of exogenous factors that stimulate cells to exert contractile forces on the matrix .We have examined fibroblast-collagen interactions with the aim ofdefining the exogenous factors that stimulate colla-