Lysosomal alterations during coronary atherosclerosis in the pigeon: correlative cytochemical and three-dimensional HVEM/IVEM observations

Lysosomal changes have been implicated as one of the major factors contributing to the progression and complications of atherosclerosis, and recently foam cell formation has been correlated with increases in several acid hydrolases. To explore at the subcellular level relationships among lesion progression, cellular lipid accumulation, and lysosomal change, atherosclerotic lesions from hypercholesterolemic White Carneau pigeons have been studied through combined ultrastructural cytochemistry and stereo (three-dimensional) high-voltage electron microscopy. Lysosomal enzyme activity in the prelesion intima and in foam cells of early lesions was in discrete lysosomes of macrophage foam cells. Foam cell lipid at the early stages was primarily (72%) in cytoplasmic droplets, which formed a three-dimensional network with the small (0.25-0.8 microM in diameter), reaction-positive lysosomes suspended at the vertices of a cytoplasmic lattice that delineated individual lipid pools. Concomitant with lesion progression and increasing complexity, foam cell lysosome number, size, and complexity increased. The complexity was characterized by lysosome lipid accumulation (60% of cell lipid) and the fusion of lysosomes to form multilobulated organelles in which the acid phosphatase reaction product typically was circumferential to the lysosomal lipid core. The involvement of lysosomes climaxed in the more advanced region of lesions with foam cells in which the bulk of cytoplasmic volume was occupied by large (15-20 microM in diameter), multicompartmental, lipid-containing lysosomes. It is suggested that this progressive involvement of lysosomes is responsible for cell and tissue necroses characteristic of advanced lesions.     

Early atherogenesis in the White Carneau pigeon. III. Lipid accumulation in nascent foam cells.

The role of lysosomes in aortic atherogenesis in White Carneau pigeons was examined by means of acid phosphatase cytochemistry. Foam cells were the major constituent of nascent atherosclerotic lesions in pigeons fed a 0.5% cholesterol diet for either 5 or 10 weeks. Seventy-four percent of foam cell lipid from animals at 5 weeks was in cytoplasmic droplets. The remaining lipid appeared in secondary lysosomes. After 10 weeks of cholesterol feeding, lysosomal lipid accounted for 73% of the lipid volume. The lipid accumulation correlated with increases in both size and number of lysosomes. An average of 2.4 lysosomes per 10(4) cu mu of cytoplasm was observed at 5 weeks. This value doubled by 10 weeks. The average lysosome diameter also increased between 5 and 10 weeks from 2.2 mu to 5.75 mu. Concomitantly, the complexity of lysosomes increased from simple, spherical organelles at 5 weeks to complex, multichambered organelles at 10 weeks. In contrast, lipid storage within cytoplasmic lipid droplets did not change either in size or in number. These observations suggest that by 5 weeks lipid storage within cytoplasmic droplets was maximized, and continued increases in lipid stores occurred predominantly through lysosomal loading.     

Early extracellular and cellular lipid deposits in aorta of cholesterol-fed rabbits.

Subendothelial accumulation of extracellular liposomes rich in unesterified cholesterol has been described as an early feature of atherosclerosis induced by cholesterol feeding in rabbits. Beta-very-low-density lipoproteins, however, the presumed source of atherogenic lipid in this animal model, contain mostly esterified cholesterol. The purpose of this study was to test for the presence of extracellular neutral lipid deposits consistent with esterified cholesterol, by employing new electron microscopic techniques. Rabbits were fed 0.5% cholesterol, 5% butter for 0, 1, 2, and 4 weeks. The lipid-preserving ultrastructural techniques showed, in control and atherosclerotic rabbit arteries, neutral lipid droplets adherent to the endothelial luminal surface. After 1 to 2 weeks, subendothelial extracellular deposits of mostly membranous lipid appeared; these deposits contained variable amounts of neutral lipid. At the same time, cytoplasmic neutral lipid droplets appeared in smooth muscle cells and in a small number of subendothelial macrophagelike cells. After 4 weeks, monocytic infiltration and macrophage foam cell development were prominent, but abundant extracellular lipid deposits also were found. Therefore, in arteries of cholesterol-fed rabbits, deposition of membranous and neutral lipid in the extracellular space and neutral lipid accumulation in resident arterial cells are early and probably independent events, both occurring before monocytic infiltration of the arterial intima.     

Lysosomal lipid accumulation in vascular smooth muscle cells

Using an inverted culture technique, the accumulation of lipid within vascular smooth muscle cells incubated with lipid droplets was studied. Initially, lipid was found exclusively within cytoplasmic inclusions but, as accumulation continued, lysosomes became the predominant site of lipid storage. After 3 hr of incubation, 84% of lipid was within lysosomes. This lysosomal lipid accumulation produced a tripling of the average size of lysosomes and resulted in lysosomes with complex, multilobed shapes. In contrast, although the number of cytoplasmic inclusions increased with lipid loading, individual inclusions maintained a spherical shape and a consistent diameter of 1-1.3 microns. Concomitant with changes in cellular lipid storage, incubation with lipid droplets induced development of an anastomosing network of acid phosphatase-containing tubules which were spatially related to sites of lysosomal lipid accumulation. Thus lipid accumulation produced ultrastructural alterations in a number of metabolic compartments. Similar alterations in the intracellular compartmentalization of acquired lipid have been demonstrated in foam cells during atherogenesis and have been hypothesized to have profound effects on lipid metabolism and disease progression.     

Early atherogenesis in White Carneau pigeons. II. Ultrastructural and cytochemical observations.

The addition of cholesterol (0.5%) to the diet of White Carneau pigeons induces site specific, temporally predictable, atherosclerotic lesions. The earliest lesions, which occurred after 3 weeks, were small (less than 2500 sq mu in surface area) and were composed primarily of macrophage foam cells (94% of lesion volume). With a prolonged time on the diet the lesions expanded due to increases in the number and size of foam cells, increases in the amount of extracellular space, and influx of smooth muscle cells. Macrophage foam cells in advanced lesions composed 61% of the lesion volume, smooth muscle cells 25%, and extracellular space 14%. Concurrent with the alteration in the constituency of the lesion, redistribution of lipid within foam cells was noted. Lipid in small lesions was primarily cytoplasmic (88%), with the remaining 12% in acid-phosphatase-positive secondary lysosomes. In more advanced lesions, 34% of the lipid was cytoplastic and 66% was lysosomal. The changes in large lesions appeared to be a function of lesion age, because at the growing edge of large lesions both composition and lipid distribution resembled those of small early lesions.     

Pigeon monocyte/macrophage lysosomes during beta VLDL uptake. Induction of acid phosphatase activity. A model for complex arterial lysosomes.

Lysosomes have long been implicated as a factor contributing to the progression and complication of atherosclerosis. The authors' laboratory previously has shown that lysosomal ultrastructure in arterial macrophage foam cells is altered as primary lysosomes give rise to large pleiomorphic organelles on lipid accumulation during lesion progression. To further explore the subcellular alterations in lysosomes and associated organelles during foam cell formation, three-dimensional (3D) intermediate voltage electron microscopy was used to examine monocyte-derived macrophages (monocyte/macrophages) during early in vitro uptake of beta migrating very-low-density lipoproteins (beta VLDL). Lysosomes were identified using acid phosphatase cytochemistry, and in control cells these organelles constituted 3.5% of the total cytoplasmic volume. Both primary and secondary lysosomes were observed. Upon beta VLDL uptake, the total volume of acid-phosphatase-positive organelles increased threefold over 30 minutes, and the reaction product was found in three additional morphologically distinct structures: tubular lysosomes, membrane stacks, and endoplasmic reticulum with widened cisternae. The proportion of the cell occupied by each of the five acid-phosphatase-positive organelles was quantitated at 10 minutes, 30 minutes, 1 hour, and 4 hours of beta VLDL incubation, and their relative abundance was compared with controls that were processed either with no lipoprotein challenge or albumin incubation for 1 hour. Secondary lysosomes compartment volume peaked at 30 minutes; over the ensuing 3.5 hours, however, the reaction progressively shifted to three new membrane-limited locations. Our observations document the complex 3D organization and spacial relationships among the acid-phosphatase-positive structures induced by lipoprotein uptake. The 3D organization patterns for acid-phosphatase-positive lysosomes in lipoprotein-stimulated pigeon monocyte/macrophages were similar in several aspects to the complex lysosomes previously observed in the macrophages of pigeon arterial lesions.     

Regression of Early Cholesterol-Induced Aortic Lesions in Rhesus Monkeys

The reversibility of early uncomplicated cholesterol-induced aortic lesions in rhesus monkeys was investigated. Three groups of Rhesus monkeys were used: the control group was fed a chow diet for 8 weeks; the progression group was fed an atherogenic diet for 8 weeks and the regression group was fed an atherogenic diet for 8 weeks and returned to the chow diet for 16 weeks. The lesions produced in the progression animals characteristically contained many lipid-laden monocytes immediately beneath the endothelium, abundant lipid droplets in intimal smooth muscle cells and moderate amounts of lipid in the extracellular spaces. Lesions in regression animals contained few lipid-laden monocytes, less lipid in smooth muscle cells and larger and more numerous lipid particles in the extracellular spaces. The results indicate that aortic lesions can be produced predictably after 8 weeks of feeding a high-cholesterol diet and that qualitative changes in the lesions occur 16 weeks after withdrawal from the diet.     

Stage-specific remodeling of atherosclerotic lesions upon cholesterol lowering in LDL receptor knockout mice

Reducing the concentration of circulating lipids leads to decreased cardiovascular morbidity and mortality, but the dynamic remodeling that established atherosclerotic lesions undergo upon lipid lowering is poorly understood. Early or advanced lesions in the aortic root were induced by feeding LDL receptor knockout mice a high-fat, high-cholesterol Western-type diet for 5 or 9 weeks, respectively. In the first week after switching to a chow diet, plasma total cholesterol levels dropped 70%, but both early and advanced lesions increased in size. Early lesions grew because of an increase in smooth muscle cells; advanced lesions had an enlargement of absolute macrophage area. From 1 to 3 weeks after the diet switch, plasma total cholesterol levels were completely normalized, but the size of early lesions remained stable; however, advanced lesions became smaller due to a reduction of the absolute macrophage area. From 3 to 6 weeks, both early and advanced lesions progressed further, as a result of expansion of the absolute collagen and necrotic core area. In contrast, early lesions became proinflammatory, as evidenced by the increased infiltration of neutrophils and increased oxidative stress, probably caused by the activation of mast cells in the adventitia. Thus, the severity of atherosclerotic lesions affects their dynamic response to lipid lowering, indicating the importance of establishing stage-specific therapeutic protocols for the treatment of atherosclerosis.     

Intralysosomal lipid in long-term maintenance transplant atherosclerosis.

Intralysosomal accumulation of lipid has been implicated as an important mechanism in the pathogenesis of atherosclerosis. Although atherosclerosis develops frequently in organ transplants maintained on a long-term basis, to our knowledge no studies to date have demonstrated the intracellular localization of the lipid in this setting. Light and electron microscopic study of a renal artery branch from a transplanted kidney maintained for 3 1/2 years demonstrates that the lipid is sequestered within intimal smooth muscle cell lysosomes. The features of the atherosclerotic plaque in long-term transplantation appear to be identical to spontaneous lesions or those induced experimentally.     

Atherosclerosis and the protective role played by different proteins in apolipoprotein E-deficient mice

The aim of the present study was to analyze the early events in atherogenesis and the role of pro- or anti-atherosclerotic proteins in the development of atherosclerotic lesions. We used apolipoprotein E-deficient (E(0)) mice that spontaneously develop hypercholesterolemia and atherosclerotic lesions in the aorta in a time-dependent manner. Aortas of mice aged 6, 8, 10 and 12 weeks were examined to determine histopathological changes. In mice aged 8-12 weeks, developing atherosclerotic lesions were present in different regions of the aortas. These lesions protruded into the lumen of the vessel and showed lipid deposits, lipid-filled macrophages and extensive accumulation of collagen and elastic fibers throughout the entire arterial wall. A parallel immunohistochemical study included analysis of three proteins known to be involved in atherosclerosis, i.e. inducible nitric oxide synthase (iNOS, NOS2), vascular endothelial growth factor (VEGF) and matrix metalloproteinase-2 (MMP2). Increased immunolabelling of iNOS and VEGF accompanied atherosclerosis development in E(0) mice aged 8, 10 and 12 weeks. On the contrary, immunolabelling for MMP2 was negative in E(0) mice aged 10 and 12 weeks. Our results indicate morphological alterations in the Tunica intima and Tunica media of atherosclerotic aortas and possible protective roles for iNOS and VEGF proteins against atherosclerosis development. These data may be relevant for developing therapeutic strategies for atherosclerosis development.     

A mouse model for human atherosclerosis: long-term histopathological study of lesion development in the aortic arch of apolipoprotein E-deficient (E0) mice

Apolipoprotein E-deficient mice, since their introduction in the early 1990s, have proved to be a very popular model for studying spontaneous hypercholesterolemia and the subsequent development of atherosclerotic lesions. The pathogenesis of atherosclerotic lesions in these mice mimics that found in humans on a very short time-scale. Atherosclerotic lesion development is especially prominent in the aortic arch. We have followed the progressive histopathological development of atherosclerotic lesions in the aortic arch of apolipoprotein E-deficient mice aged from 6 weeks to 18 months in 1 μm epoxy-resin sections stained with alkaline toluidine blue, which gives greatly improved resolution over wax sections. During the early stages of lesion formation, lipid-filled macrophages appear in the subendothelium, and accumulate leading to “fatty streaks”. Macrophage degeneration and the formation of lipid pools are accompanied by accumulation of cholesterol deposits. Disruptions of elastic laminae of the Tunica media are accompanied by structural changes in the myocytes. More advanced lesions involve fibrous cap development, calcification of the vessel wall and progressive occlusion of the lumen. Unstable plaque may also be found. Various approaches for quantitative determination of lesion size are considered. The study provides a histopathological baseline for spontaneous atherosclerosis associated with hypercholesterolemia, which can be used in connection with experimental interventional studies on the efficacy of drugs or foodstuffs in retardation of atherosclerosis.     

Lysosomes of the Arterial Wall: IV. Cytochemical Localization of Acid Phosphatase and Catalase in Smooth Muscle Cells and Foam Cells from Rabbit Atheromatous Aorta

Cytochemical methods for acid phosphatase and catalase were applied to atheromatous aortas from cholesterol-fed rabbits. Whole tissue, partially digested aortic slices and isolated cells were used for the study. Present in the atheromatous lesions were smooth muscle cells in all stages of foamy transformation, from virtually normal appearing smooth muscle cells to severely altered cells with pronounced lipid accumulation. The results with the acid phosphatase method show that lysosomes increase both in size and in number as the smooth muscle cells become foam cells. In normal appearing smooth muscle cells, acid phosphatase reaction product was found in stacked cisternae of the Golgi apparatus and in small vesicles located in the Golgi region and distributed throughout the cytoplasm. In foam cells, reaction product was found in membrane-limited vacuoles of varying size which typically contained membranous debris or myelin-like figures together with massive lipid deposits. No reaction was seen in “free” cytoplasmic lipid droplets lacking a surrounding membrane. These results confirm and extend previous biochemical findings indicating that, in the cholesterol-fed rabbit, the change from normal smooth muscle cell to foam cell is accompanied by marked physical and chemical changes of the lysosomes, including their progressive overloading with cholesteryl ester. Small diaminobenzidine-positive particles were present in normal smooth muscle cells and in those at all stages of foamy transformation. These particles were more frequent in foam cells, in agreement with the marked increase in catalase activity detected biochemically in these cells.     

Morphological effects of moderate diet and clofibrate on swine atherosclerosis

Early proliferative coronary atherosclerosis was produced in swine by feeding them a high-fat, high-cholesterol diet for 17 months, at which time one group of animals was killed (reference group), while the remainder was transferred for 12 months to a moderate diet that resulted in serum cholesterol levels of about 190 mg/100 ml. The moderate diet only did not decrease the size of coronary lesions, but prevented their progression. The addition of clofibrate therapy caused regression that involved a significant decrease in size, gross sudanophilia, and extent of calcification and the disappearance of foam-cell lesions. Resultant serum cholesterol levels appear to be more important than the amount of dietary cholesterol in the progression, prevention, and regression of swine coronary atherosclerosis.     

Non-invasive MRI of mouse models of atherosclerosis

Early detection and characterization of atherosclerotic lesions susceptible to sudden rupture and thrombosis may decrease morbidity and mortality. Plaque development has been extensively studied using MRI in animal models of rapidly progressing atherosclerosis. These transgenic mice develop atherosclerotic plaques in the aortic root by 10 weeks of age and throughout the vasculature thereafter. Transplantation of lesion-containing segments of the thoracic aorta into wild-type mice results in nearly total reversal of atherosclerosis, making it possible to study both progression and regression of plaques in this model. MRI permits the non-invasive accurate assessment of atherosclerotic plaque burden and the differentiation between the lipid and fibrous content of individual plaques, thus providing a non-invasive approach to serially monitor the evolution of individual plaques in the mouse models. Emergence of novel contrast agents that target a diverse set of molecules within the plaque are now helping to elucidate the changes at the cellular and molecular levels during plaque progression and regression.     

Characterization of lipid-laden aortic cells from cholesterol-fed rabbits. III. Intracellular localization of cholesterol and cholesteryl ester.

The subcellular sites of accumulation of cholesterol and cholesteryl esters in rabbit atheromatous cells, were investigated by morphologic and biochemical techniques. Electron microscopy of lipid-filled cells in situ in atheromatous aortas of cholesterol-fed rabbits revealed lipid accumulation in the cytoplasm as lipid droplets and within lysosomes in the form of lipid globules, membranous whorls, and crystals. When such cells were isolated from the rabbit aortas by enzymic digestion, and then treated with Flickinger's aldehyde fixative containing 0.2 per cent digitonin, characteristic digitonide-lipid complexes ("spicules") were observed in discrete sites of the cytoplasm distinct from the cytoplasmic droplets. If these cells were first stained cytochemically for acid phosphatase and then treated with digitonin-aldehyde fixative, enzyme reaction product was found associated with the spicules indicating that the lysosomes of the atheromatous cells possess digitonin-reactive lipid. Subcellular fractionation of isolated rabbit aortic foam cells by sucrose density gradient centrifugation demonstrated the coequilibration of most of the intracellular unesterified cholesterol with low density lysosomes. Some cholesteryl ester was also associated with low density lysosomes, although most was found in a lipid droplet fraction of very low density. Together the results indicate that in rabbit atheromatous cells, lysosomes are the site of accumulation of intracellular cholesterol in excess of that structurally associated with membranes and that both cytoplasmic droplets and lysosomes are depot sites for cholesteryl esters.     

The effects of COX-2 selective and non-selective NSAIDs on the initiation and progression of atherosclerosis in ApoE−/− mice

In this study, we investigated the effects of prolonged administration of the selective COX-2 inhibitors celecoxib and rofecoxib and the non-selective NSAID naproxen on the initiation and progression of atherosclerosis. ApoE(-/-) mice, as well as corresponding wild-type mice, were fed either a normal chow or a high fat Western diet with or without addition of the respective drugs over a period of 16 weeks. Thereafter, aortic lesion size, plasma lipid levels, and COX-2 expression in the plaques were determined. The results showed that neither the COX-2 selective inhibitors nor naproxen had a significant impact on the initiation and progression of atherosclerosis in diet-fed ApoE(-/-) mice, although both celecoxib and rofecoxib showed a tendency to reduce plaque size. This slight effect may be due to selective inhibition of COX-2 activity because the COX-2 expression was not altered in the plaque. Plasma lipid levels were also not significantly influenced by these drugs. Interestingly, in ApoE(-/-) mice that have been fed with normal chow, we found an increased incidence of plaque formation after treatment with celecoxib and rofecoxib, indicating that coxibs may promote the initiation of atherosclerosis. This effect was probably masked in diet-fed mice by the more pronounced effects of the high cholesterol diet. In conclusion, the reduction in diet-induced plaque size in animals fed a high fat diet and the promotion of atherosclerosis in mice on a normal diet indicate a dual role of the coxibs. In advanced stages of atherosclerosis, they may exert antithrombotic properties due to their COX-2 inhibiting activity, whereas in very early stages they may favor the initiation of atherogenesis. However, because these results were only observed in ApoE(-/-) and not in wild-type animals, coxibs may increase the risk of thrombosis in patients with a predisposition for thrombotic complications.     

Cellular pathology of homozygous familial hypercholesterolemia.

Tissues were studied from four subjects with homozygous familial hypercholesterolemia (FH). The specimens consisted of tissues obtained from a 20-week-old fetus at autopsy, samples from a 9-year-old girl during open-heart surgery, and biopsies of cutaneous xanthomas from a 13-year-old girl and a 21-year-old man. The FH fetus, but not the 3 control fetuses, exhibited multifocal lipid deposition particularly involving the stromal cells of the thymus, spleen, and skin and both the stromal and parenchymal cells of the kidney. Only one minute focus of intimal lipid accumulation was found in the aorta and coronary arteries of the FH fetus. A segment of the ascending aorta from the 9-year-old girl showed: 1) foam-cell transformation of many medial smooth-muscle cells, 2) abnormal vascularization of the inner media and intima, and 3) intimal involvement by a typical artherosclerotic plaque with lipid deposits in thin, elongated cells that showed some myocytic features and in foam cells that lacked such features. The mitral and aortic valves of this patient also contained numerous foam cells and showed mild to moderate fibrous thickening. A segment of the saphenous vein, however, contained no lipid deposits. The three xanthomas from two FH homozygotes exhibited marked lipid accumulation in histiocytic foam cells but no lipid deposits in the endothelium of blood vessels in the lesions. The findings in this study, in conjunction with those reported in studies of other FH homozygotes, indicate that homozygous FH is characterized by accelerated atherosclerosis and prominent lipid accumulation in macrophages and other stromal cells of the aortic and mitral valves, skin, tendon, and, varibly, in other extravascular sites. Since most of the intracellular lipid was in the form of non-membrane-bound neutral lipid droplets, it appears that the cytoplasm is the major site of lipid storage in this disease.     

Monocytes and focal glomerulosclerosis

It has been suggested that focal glomerulosclerosis (FGS) is analogous to atherosclerosis. Because monocytes and their derivatives are involved in the latter, these cells may be involved in the development of the former. To investigate this possibility a combined histochemical and ultrastructural study of FGS was done. Sections from 13 biopsies showing FGS were stained for either nonspecific esterase or lysozyme to detect monocytes and their derivatives. These include foam cells (lipid-containing macrophages) in which there was positive cytoplasmic staining for both nonspecific esterase and lysozyme. Twenty-one of 29 glomeruli (72%) with segmental sclerotic lesions contained monocytes and/or foam cells, whereas only 18 of 251 glomeruli (7%) without the lesions demonstrated these cells (p less than 0.0001). The mean number of monocytes and/or foam cells in segmentally sclerotic glomeruli was 2.0 +/- 1.7 compared with 0.2 +/- 0.3 for uninvolved glomeruli (p less than 0.01). In glomeruli with sclerotic lesions foam cells predominated over monocytes. Neutral lipid was observed focally and segmentally in 29 of 35 biopsies with FGS. Electron microscopy in 23 biopsies consistently demonstrated intracapillary cells with monocytic features but few foam cells in very early lesions characterized by epithelial cell changes but no or minor glomerular tuft alterations. With progression, the relative number of monocytes declined but foam cells were observed more frequently. These results suggest that monocytes and their derivatives are involved in the development of FGS.     

Lipid profile in the evolution of experimental atherosclerotic plaques from thrombus.

Nonocclusive white mural thrombosis was induced in the abdominal aortae of normolipidemic rabbits by insertion of a polyethylene catheter into the abdominal aortae. The thrombus subsequently organized into intimal thickenings which resembled fibrofatty type atherosclerosis seen in man, showing large numbers of foam cells containing stainable lipid, fatty necrotic centers, cholesterol clefts, calcification, and fibrous caps. The lipid composition of the thrombus and lesions was followed at serial time intervals from 4 hours to 60 weeks. Lipid analysis showed significant concentrations of lipid in the early lesions and with time these lipid concentrations increased and later decreased. These studies demonstrate that the fibrofatty lesions derived from a white thrombus have significant amounts of the same lipids that characterize the atherosclerotic lesions of man, however, there is a lower proportion of cholesterol to the other constituents and a higher proportion of phospholipids. The free cholesterol and cholesteryl ester of the 4-day lesions were much greater than that of platelets alone indicating that significant amounts of plasma are trapped in a thrombus when it forms.     

Apolipoprotein B in cholesterol-containing drusen and basal deposits of human eyes with age-related maculopathy

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. Fewer macular drusen were stained by traditional lipid stains and apolipoprotein antibodies than peripheral drusen. RPE contained apo B and apo E mRNA and protein. Finding cholesterol and apo B in sub-RPE deposits links ARM with important molecules and mechanisms in atherosclerosis initiation and progression. The combination of apo B mRNA and protein in RPE raises the possibility that intraocular assembly of apo B-containing lipoproteins is a pathway involved in forming cholesterol-enriched lesions in ARM.