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PubMed1986

Specialized membrane areas in non-activated and thrombin-activated platelets.

Filipin, a complex of polyene antibiotics, forms morphologically distinctive complexes with cholesterol in cell membranes under proper experimental conditions. When applied to non-activated, discoid platelets, filipin-induced lesions (FIL) occurred in rows at the platelet equator, suggesting a specialized membrane organization at the platelets' largest circumference. In some thrombin-activated platelets we observed surface membrane blebbing and release of lipid vesicles that predominantly originated from the plasma membrane proper, but some originated from (unidentified) platelet granules. FIL were initially present in high numbers over the entire bleb, they accumulated later at the neck of blebs, while the released vesicle was free of FIL. Absence of intramembrane protein particles (IMP) from the membranes of blebs and vesicles suggests that released vesicles are essentially without cholesterol and intrinsic membrane proteins and may consist predominantly of phospholipids. Membrane blebbing and vesicle release may represent unmasking and release of procoagulant platelet factor 3 activity.

Blood Platelets↗
PubMed1977

In vitro activating properties of polyene antibiotics for murine lymphocytes.

The effect of four polyene antibiotics, Candicidin, Etruscomycin, Filipin and Pimaricin upon mouse lymphocytes was studied. Polyene antibiotics are known to form aqueous pores in the cell membranes inducing known anion or cation selective fluxes. Candicidin was capable of inducing marked DNA-synthesis and polyclonal antibody production when added to normal spleen cells. Etruscomycin and Pimaricin showed a weak inconsistent DNA synthetic stimulatory effect, whereas Filipin was found to be totally uneffective. The stimulating property of Candicidin was also demonstrated on spleen cells from nude mice whereas there was no effect on cortisone resistant thymocytes or spleen cells passed through a nylon fibre column. Thus we conclude that Candicidin is a PBA for mouse lymphocytes. We have previously reported that the two anionselective polyenes, Nystatin and Amphotericin B, are polyclonal B-cell activators for mouse lymphocytes and in this paper the possible mechanism of triggering is further discussed.

Animals↗
PubMed1980

Effect of fatty acids on action of polyene antibiotics.

Fatty acids cause a decrease in the absorption spectra of the antifungal polyene macrolide antibiotics nystatin, filipin, candicidin, and amphotericin B. For nystatin, filipin, and candicidin, this decrease in absorption could be correlated with the activity of the fatty acids in protecting the yeast Saccharomyces cerevisiae against the action of these antibiotics. With amphotericin B a correlation was observed between the decrease in absorption caused by certain derivatives of fatty acids and the protective action of these derivatives against the activity of amphotericin B on yeast. It is concluded that, like the sterols, fatty acids also interact with the polyene antibiotics and thereby reduce their effective concentrations.

Amphotericin B↗
PubMed2004

Lipid raft polarization contributes to hyphal growth in Candida albicans.

The polarization of sterol- and sphingolipid-enriched domains (lipid rafts) has been linked to morphogenesis and cell movement in diverse cell types. In the yeast Saccharomyces cerevisiae, a dramatic polarization of sterol-rich domains to the shmoo tip was observed in pheromone-induced cells (M. Bagnat and K. Simons, Proc. Natl. Acad. Sci. USA 99:14183-14188, 2002). We therefore examined whether plasma membrane lipid polarization contributes to the ability of the fungal pathogen Candida albicans to grow in a highly polarized manner to form hyphae. Interestingly, staining with filipin revealed that membrane sterols were highly polarized to the leading edge of growth during all stages of hyphal growth. Budding and pseudohyphal cells did not display polarized staining. Filipin staining was also enriched at septation sites in hyphae, where colocalization with septin proteins was observed, suggesting a role for the septins in forming a boundary domain. Actin appeared to play a role in sterol polarization and hyphal morphogenesis in that both were disrupted by low concentrations of latrunculin A that did not prevent budding. Furthermore, blocking either sphingolipid biosynthesis with myriocin or sterol biosynthesis with ketoconazole resulted in a loss of ergosterol polarization and caused abnormal hyphal morphogenesis, suggesting that lipid rafts are involved. Since hyphal growth is required for the full virulence of C. albicans, these results suggest that membrane polarization may contribute to the pathogenesis of this organism.

Antifungal Agents↗
PubMed1963

INHIBITION OF THE PLEUROPNEUMONIA-LIKE ORGANISM MYCOPLASMA GALLISEPTICUM BY CERTAIN POLYENE ANTIFUNGAL ANTIBIOTICS.

Lampen, J. Oliver (Rutgers, The State University, New Brunswick, N.J.), James W. Gill, Peter M. Arnow, and I. Magana-Plaza. Inhibition of the pleuropneumonia-like organism Mycoplasma gallisepticum by certain polyene antifungal antibiotics. J. Bacteriol. 86:945-949. 1963.-The growth of Mycoplasma gallisepticum, a sterol-requiring pleuropneumonia-like organism (PPLO), was inhibited by certain polyene antifungal antibiotics at the following concentrations: filipin, 0.5 mug/ml; amphotericin B, 3 mug/ml; and fungichromin, 10 mug/ml. The inhibition of the organism was due to fungicidal action of the polyenes rather than to decreased availability of the sterol. The PPLO rapidly bound nystatin (NY), but was relatively insensitive to this antifungal agent or to N-acetyl-candidin (NAC). Even with a comparatively small inoculum, 100 mug of NY per ml or 50 mug of NAC per ml, were required to inhibit growth. In contrast, Saccharomyces cerevisiae, growing under similar conditions, was completely inhibited by 3 mug of NY or NAC per ml. Filipin, however, was effective against both organisms, inhibiting PPLO and yeast at 0.5 mug/ml and 3 mug/ml, respectively. The relative insensitivity of PPLO to NY was not the result of protection by the sterol present in the growth medium. These organisms, incubated for 2 hr in sterol-free buffer in the presence of 20 mug of NY per ml showed little, if any, inhibition of growth when transferred to growth medium. Under the same conditions, more than 99% of a yeast suspension was killed. It is suggested that the PPLO are primarily sensitive to the disruptive action on the cell membrane which is characteristic of the smaller polyenes (34 to 37 carbon atoms).

Amphotericin B↗
PubMed1987

Increase in cholesterol in the apical plasma membrane of uterine epithelial cells during early pregnancy in the rat.

Freeze-fracture cytochemistry with the cholesterol-binding antibiotic filipin has been used to examine the plasma membrane of uterine epithelial cells at different stages of pregnancy in the rat. We find many more filipin-induced lesions on day 6 of pregnancy than on day 1 and suggest that this indicates a higher cholesterol content at this time. Since day 6 of pregnancy is the time at which blastocysts implant in the rat uterus, we consider the possible significance of an increased cholesterol content for implantation.

Animals↗
PubMed1988

Cholesterol-rich microdomains in rat and porcine thyroid membranes involved in TSH-induced endocytotic processes.

Filipin, a polyene antibiotic, was used to detect cholesterol in thyroid membranes in vivo and in culture during TSH stimulation. We found that apical and basolateral plasma membranes were heterogeneously modified by filipin which induced abundant lesions in apical membranes, whereas Golgi apparatus, endoplasmic reticulum, nuclear membranes were unmodified. Small apical vesicles and colloid droplets were generally highly enriched in these complexes, suggesting a high cholesterol concentration in their membranes. Pseudopod membranes, known to be highly specialized domains in the apical plasma membrane, appeared enriched in cholesterol. Consequently, we suggest that an increased cholesterol content may be involved in the stabilization of thyroid membranes during endocytotic processes.

Animals↗
PubMed2006

Regulation of Pseudomonas aeruginosa internalization after contact lens wear in vivo and in serum-free culture by ocular surface cells.

PURPOSE: To determine the effects of contact lenses (CLs) and Pseudomonas aeruginosa (PA) infection on localization of cystic fibrosis transmembrane conductance regulator (CFTR) on corneal surface epithelial cells and the association between lipid raft formation and CFTR in mediating PA binding and internalization in ocular surface epithelium. METHODS: CFTR immunolocalization was evaluated in vivo in rabbit corneal-conjunctival epithelium (with/without CL wear) before and after PA exposure and in serum-free human corneal epithelial cell culture (hTCEpi). Lipid raft formation was visualized with Alexa555-conjugated cholera toxin beta-subunit. Lipid raft involvement in PA internalization was assayed in vivo by gentamicin survival assays after topical filipin pretreatment. Involvement of CFTR in PA binding and internalization was evaluated by blockade with CFTR peptides or LPS. RESULTS: CL wear in vivo enhanced anti-CFTR staining, but CFTR localization did not correlate with the PA binding by ocular surface cells. Conjunctival epithelial cells stained for CFTR but did not bind or internalize PA. Corneal epithelial cells in vivo did not stain for CFTR unless challenged by contact lens-induced hypoxia. PA internalization by hTCEpi was significantly inhibited by LPS (P < 0.01), but not by CFTR peptides. Remarkably, normal conjunctival epithelial cells showed lipid raft formation and CFTR staining but did not bind PA. Inhibition of raft formation by filipin blocked PA internalization in vivo after CL wear. CONCLUSIONS: CFTR is not the predominant receptor for ocular surface PA infection, and after hypoxic CL challenge, neither lipid rafts nor CFTR localization alone predicts PA binding; however, lipid rafts are critical to CL-mediated PA internalization.

Animals↗
PubMed1989

Alignment of cholesterol in the membrane bilayer.

Freeze-fracture replicas of filipin-treated samples of guinea pig colon mucosa reveal areas in the membrane of the goblet cell granules labeled by filipin-cholesterol complexes (FCC) intermingled with regions patterned by "lines." The FCC and "lines" are arranged in an approximately rhombic pattern. Other membranes of the same cell or of other cells display either FCC only, aligned and occasionally ordered in "rhombs," "lines" only, with a similar pattern, or randomly distributed FCC. Optical diffraction was used to analyze and compare replicas of membranes with ordered FCC and "lines", as well as randomly distributed FCC. The results demonstrate that all these structures are reciprocally related through a common distribution pattern in the membrane. This observation supports the assumption that cholesterol has a preferential ordered distribution within the membrane bilayer.

Animals↗
PubMed2003

Membrane cholesterol regulates LFA-1 function and lipid raft heterogeneity.

Many surface receptors and signaling molecules are thought to associate with unique membrane microdomains termed lipid rafts. We examined the involvement of lipid rafts in the activation of leukocyte function-associated antigen-1 (LFA-1). Depletion or sequestration of cholesterol with methyl-beta-cyclodextrin (MCD) or filipin, respectively, strongly inhibited LFA-1-mediated adhesion of T-cell lines and primary T cells. This inhibition was reversed by cholesterol reconstitution. LFA-1 on T-cell lines was detected in cold Triton X-100-insoluble lipid rafts, which were disrupted by MCD or filipin treatment. However, no LFA-1 on primary T cells was detected in lipid rafts isolated by the same procedures, and these rafts were resistant to cholesterol depletion or sequestration. Association of LFA-1 with lipid rafts of primary T cells could be detected only when they were isolated with another nonionic detergent, Brij 35. Upon treatment with MCD, LFA-1 in Brij 35-insoluble lipid rafts partially shifted to nonraft fractions. T-cell lines were found to have a high level of cholesterol and a low level of ganglioside GM1, a common marker for lipid rafts, whereas primary T cells have a much lower level of cholesterol and a very high amount of GM1. Cross-linking of LFA-1 on primary T cells induced cocapping of cholesterol but not GM1. These results suggest that lipid rafts of T cells are heterogenous, and LFA-1 associates with a subset of lipid rafts containing a high level of cholesterol. This association seems to regulate LFA-1 functions, possibly by facilitating LFA-1 clustering.

Animals↗
PubMed2002

Uptake of long-chain fatty acids in HepG2 cells involves caveolae: analysis of a novel pathway.

We investigated the role of caveolae in uptake and intracellular trafficking of long chain fatty acids (LCFA) in HepG2 human hepatoma cells. The uptake of [(3)H]oleic acid and [(3)H]stearic acid into HepG2 cells was measured by radioactive assays and internalization of the non-metabolizable fluorescent fatty acid 12-(N-methyl)-N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino] (12-NBD) stearate into single HepG2 cells was semi-quantitatively assessed by laser scanning microscopy. The initial rate of [(3)H]oleic acid uptake (V(0)) in HepG2 cells exhibited saturable transport kinetics with increasing concentrations of free oleic acid (V(max) 854 +/- 46 pmol mg protein(-1) min(-1), K(m) 100 +/- 14 nmol/l). While inhibition of clathrin coated pits did not influence LCFA uptake in HepG2, inhibition of caveolae formation by filipin III, cyclodextrin, and caveolin-1 antisense oligonucleotides resulted in reduction of [(3)H]oleic acid uptake by 54%, 45%, and 23%, respectively. Furthermore, filipin III inhibited the uptake of [(3)H]stearic acid and its fluorescent derivative 12-NBD stearate by 44% and 50%, respectively. Transfection studies with alpha-caveolin-1/cyanofluorescent protein chimeras showed significant colocalization of caveolae and internalized 12-NBD stearate. In conclusion, these data suggest a significant role for caveolae mediated uptake and intracellular trafficking of LCFA in HepG2 cells.

Biological Transport↗
PubMed2003

Involvement of cholesterol-rich lipid rafts in interleukin-6-induced neuroendocrine differentiation of LNCaP prostate cancer cells.

IL-6 is an inflammatory cytokine that has been linked to aggressive prostate cancer (PCa). Previous studies have demonstrated that IL-6 can enhance the differentiation of PCa cells toward a neuroendocrine (NE) phenotype, a possible indicator of hormone-refractory disease. In this report, we present evidence that the mechanism of IL-6-stimulated NE differentiation employs a detergent-resistant (lipid raft) membrane compartment for signal transduction in LNCaP PCa cells. Signal transducer and activator of transcription (STAT)3, a mediator of IL-6 signaling, was rapidly phosphorylated and translocated to the nucleus in LNCaP cells treated with IL-6. Both processes were inhibited by filipin, a cholesterol-binding compound that disrupts plasma membrane lipid rafts. Isolation of Triton X-100-insoluble raft fractions from LNCaP cells by discontinuous sucrose gradient centrifugation demonstrated that the 80-kDa IL-6 receptor localized almost exclusively to the raft compartment. Although STAT3 was located predominantly in the Triton X-100-soluble subcellular fraction in exponentially growing cells, abundant phosphorylated STAT3 was detected in the raft fraction after stimulation with IL-6. Increases in expression of the NE marker, neuron-specific enolase, and neuron-specific enolase promoter activity after IL-6 treatment were reduced after membrane rafts were disrupted by filipin treatment. LNCaP cells expressed the raft-resident proteins flotillin-2 and G(ialpha2), but notably not caveolins, the predominant structural protein present in caveolar membrane rafts in many tissues and tumor cells. These results are the first to define a role for lipid raft membrane microdomains in signal transduction mechanisms capable of promoting the NE phenotype in PCa cells, and they demonstrate that the raft compartment is capable of mediating such signals in the absence of caveolins. Our results also suggest a mechanistic role for membrane cholesterol in cell signaling events relevant to PCa progression.

Cell Differentiation↗
PubMed2000

Intracellular distribution and mobilization of unesterified cholesterol in adipocytes: triglyceride droplets are surrounded by cholesterol-rich ER-like surface layer structures.

In addition to their central role in triglyceride storage, fat cells are a primary depot of unesterified cholesterol (FC) in the body. In comparison, peripheral cells contain very little FC. This difference in adipocytes versus peripheral tissues is inconsistent with the current theory of cholesterol homeostasis. Attempting to resolve this discrepancy, we examined intracellular storage sites of FC in murine 3T3-F442A adipocytes. Using the cholesterol-binding antibiotic, filipin, in combination with high resolution fluorescence microscopy, intense fluorescent staining characteristically decorated the periphery of triglyceride droplets (TGD) as well as the plasma membrane (PM) of fat cells. Filipin-staining was not visible inside the lipid droplets. Purification of TGD by subcellular fractionation demonstrated that the rise in total FC content of adipocytes upon differentiation was attributable to an increase in TGD-FC, which contributed up to one third of the total cellular FC. The protein component of purified TGD from cultured adipocytes as well as from murine adipocytes obtained from fresh tissues contained the lumenal endoplasmic reticulum (ER) immunoglobulin binding protein (BiP) and the integral ER membrane protein calnexin. Efflux experiments using the extracellular FC acceptors (&bgr;)-cyclodextrin or apolipoprotein A-I demonstrated that TGD-associated FC was releasable from TGD. Whereas FC efflux from adipocytes was unaffected in the presence of brefeldin A or monensin, the secretion of a control protein, lipoprotein lipase, was effectively reduced. In summary, our findings identify the TGD surface layer as primary intracellular storage site for FC within adipocytes. We suggest that the structural role of ER-resident proteins in this adipocyte TGD envelope has been previously neglected. Our findings support the suggestion that an ER-like structure, albeit of modified lipid composition, constitutes the lipid droplets' surface layer. Finally, the efflux process of FC from adipocytes upon extracellular stimulation with (beta)-cyclodextrin provides evidence for an energy-dependent intracellular trafficking route between the TGD-FC pool and the PM-FC sites which is distinct from the secretory pathway of proteins.

Adipocytes↗
PubMed2001

Bicarbonate stimulated phospholipid scrambling induces cholesterol redistribution and enables cholesterol depletion in the sperm plasma membrane.

Mammalian sperm cells are activated prior to fertilization by high bicarbonate levels, which facilitate lipoprotein-mediated cholesterol efflux. The role of bicarbonate and cholesterol acceptors on the cholesterol organization in the sperm plasma membrane was tested. Bicarbonate induced an albumin-independent change in lipid architecture that was detectable by an increase in merocyanine staining (due to protein kinase A-mediated phospholipid scrambling). The response was limited to a subpopulation of viable sperm cells that were sorted from the non-responding subpopulation by flow cytometry. The responding cells had reduced cholesterol levels (30% reduction) compared with non-responding cells. The subpopulation differences were caused by variable efficiencies in epididymal maturation as judged by cell morphology. Membrane cholesterol organization was observed with filipin, which labeled the entire sperm surface of non-stimulated and non-responding cells, but labeled only the apical surface area of bicarbonate-responding cells. Addition of albumin caused cholesterol efflux, but only in bicarbonate-responding cells that exhibited virtually no filipin labeling in the sperm head area. Albumin had no effect on other lipid components, and no affinity for cholesterol in the absence of bicarbonate. Therefore, bicarbonate induces first a lateral redistribution in the low cholesterol containing spermatozoa, which in turn facilitates cholesterol extraction by albumin. A model is proposed in which phospholipid scrambling induces the formation of an apical membrane raft in the sperm head surface that enables albumin mediated efflux of cholesterol.

Acrosome Reaction↗
PubMed2001

Internalization of cholera toxin by different endocytic mechanisms.

The mechanism of cholera toxin (CT) internalization has been investigated using Caco-2 cells transfected with caveolin to induce formation of caveolae, HeLa cells with inducible synthesis of mutant dynamin (K44A) and BHK cells in which antisense mRNA to clathrin heavy chain can be induced. Here we show that endocytosis and the ability of CT to increase the level of cAMP were unaltered in caveolin-transfected cells grown either in a non-polarized or polarized manner. Treatment of Caco-2 cells with filipin reduced CT-uptake by less than 20%, suggesting that caveolae do not play a major role in the uptake. Extraction of cholesterol by methyl-beta-cyclodextrin, which removes caveolae and inhibits uptake from clathrin-coated pits, gave 30-40% reduction of CT-endocytosis. Also, CT-uptake in HeLa K44A cells was reduced by 50-70% after induction of mutant dynamin, which inhibits both caveolae- and clathrin-dependent endocytosis. These cells contain few caveolae, and nystatin and filipin had no effect on CT-uptake, indicating major involvement of clathrin-coated pits in CT-internalization. Similarly, in BHK cells, where clathrin-dependent endocytosis is blocked by induction of antisense clathrin heavy chain, the CT-uptake was reduced by 50% in induced cells. In conclusion, a large fraction of CT can be endocytosed by clathrin-dependent as well as by caveolae- and clathrin-independent endocytosis in different cell types.

Animals↗
PubMed1983

Ultrastructural analysis of crystalloid endoplasmic reticulum in UT-1 cells and its disappearance in response to cholesterol.

The crystalloid endoplasmic reticulum (ER) consists of hexagonally packed membrane tubules that contain 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA reductase), an intrinsic membrane protein that catalyses the rate-limiting step in cholesterol synthesis. The crystalloid ER appears in a clone of Chinese hamster ovary cells, designated UT-1, that contain high levels of HMG CoA reductase as a result of growth in the presence of compactin, a competitive inhibitor of the reductase. In the present studies, we have used ultrastructural morphometry to estimate that the crystalloid ER: (1) occupies about 15% of the volume of UT-1 cells; (2) contains 3.4-fold more membrane area than the plasma membrane; and (3) contains less than 700 subunits of HMG CoA reductase per micrometer2 of membrane surface. The crystalloid ER tubules contain 2000 intramembrane particles per micrometer2 with a mean diameter of 10.4 nm, as determined by freeze-fracture. The crystalloid ER membranes are low in cholesterol, as indicated by the small number of filipin-cholesterol complexes in freeze-fracture images after treatment with filipin. The addition of cholesterol or related sterols to UT-1 cells promoted a rapid and stepwise disappearance of the crystalloid ER. Initially, the crystalloid ER fragmented into randomly arranged vesicles and tubules. Subsequently, membrane-bound structures disappeared from the cell so that after incubation with cholesterol for 24-72 h, the cells appeared completely normal. We found no morphological evidence that autophagic vacuoles participate in the degradation. We conclude: (1) that the crystalloid ER is more extensive than necessary merely to support HMG CoA reductase; and (2) that upon exposure to cholesterol the crystalloid ER is degraded by a process that does not involve autophagy.

Animals↗
PubMed2002

Lipid microdomains are required sites for the selective endocytosis and nuclear translocation of IFN-gamma, its receptor chain IFN-gamma receptor-1, and the phosphorylation and nuclear translocation of STAT1alpha.

IFN-gamma contains a nuclear localization sequence that may play a role in the nuclear transport of activated STAT1alpha via a complex of IFN-gamma/IFN-gamma receptor (IFNGR)-1/STAT1alpha with the nuclear importer nucleoprotein interactor 1. In this study, we examine the mechanism of endocytosis of IFNGR-1 and the relationship of its nuclear translocation to that of STAT1alpha. In untreated WISH cells, both IFNGR-1 and IFNGR-2 were constitutively localized within caveolae-like microdomains isolated from plasma membrane. However, treatment of cells with IFN-gamma resulted in rapid migration of IFNGR-1, but not IFNGR-2, from these microdomains. Filipin pretreatment, which specifically inhibits endocytosis from caveolae-like microdomains, inhibited the nuclear translocation of IFN-gamma and IFNGR-1 as well as the tyrosine phosphorylation and nuclear translocation of STAT1alpha, but did not affect the binding of IFN-gamma to these cells. In the Jurkat T lymphocyte cell line, which does not express caveolin-1, nuclear translocation of IFNGR-1 and STAT1alpha were similarly inhibited by filipin pretreatment. Isolation of lipid microdomains from Jurkat cells showed that both IFNGR-1 and IFNGR-2 were associated with lipid microdomains only after stimulation with IFN-gamma, suggesting that the IFNGR subunits are recruited to lipid microdomains by IFN-gamma binding in lymphocytes (Jurkat) in contrast to their constitutive presence in epithelial (WISH) cells. In contrast, treatments that block clathrin-dependent endocytosis did not inhibit either activation or nuclear translocation of STAT1alpha or the nuclear translocation of IFN-gamma or IFNGR-1. Thus, membrane lipid microdomains play an important role in IFN-gamma-initiated endocytic events involving IFNGR-1, and the nuclear translocation of IFN-gamma, IFNGR-1, and STAT1alpha.

Active Transport, Cell Nucleus↗
PubMed2001

Accumulation of cholesterol with age in human Bruch's membrane.

PURPOSE: To determine the cholesterol composition of normal human Bruch's membrane and choroid as a function of age and retinal location. METHODS: Human eyes with grossly normal maculas were preserved <4 hours after donor death. Cryosections of retina and choroid from the macula and temporal equator were stained with filipin to reveal esterified (EC) or unesterified (UC) cholesterol (n = 20, 17-92 years). Filipin fluorescence in Bruch's membrane was quantified with digital microscopy. Maculas were prepared for lipid-preserving electron microscopy (n = 18, 16-87 years) and for ultrastructural analysis after lipid extraction (n = 2, 85 and 89 years). Punches of macular Bruch's membrane, 8 mm in diameter, were assayed for cholesterol content by enzymatic fluorometry (n = 10, >70 years). RESULTS: EC and UC in Bruch's membrane increased with age in the macula. EC was sevenfold higher in macula than in periphery. Sixty percent of total cholesterol was esterified, and Bruch's membrane EC was 16- to 40-fold enriched relative to plasma. Solid, 100-nm-diameter particles occupied >30% of the inner collagenous layer in eyes >60 years. Cholesterol accumulated in choroidal arteries and in small age-related drusen. CONCLUSIONS: Human Bruch's membrane ages like arterial intima and other connective tissues for which plasma lipoproteins are the known source of extracellular cholesterol. Age-related maculopathy and atherosclerotic cardiovascular disease may share common pathogenic mechanisms.

Adolescent↗