ASAXS study on the formation of core-shell Ag/Au nanoparticles in glass.

Nanosized metal particles of various configurations embedded in surface regions of glass have great potential as nonlinear optical materials for photonic devices. We have prepared Ag/Au nanoparticles in core-shell configuration in soda-lime silicate glass by double-ion implantation and investigated their structural characteristics by anomalous small-angle x-ray scattering (ASAXS) and transmission electron microscopy. Measurements at x-ray energies slightly below the Au L(3) edge indicate the formation of bimetallic Ag/Au shells in some of the nanoparticles for high-dose ion implantation. An element-specific analysis of the ASAXS results allowed us not only to validate and quantify the core-shell structure, but simultaneously also the composition of the shells. Hollow nanoparticles were found for an Au-Ag implantation sequence, whereas an Ag-Au sequence generates a diluted core composition. The shift of the maximum position of optical absorption of the samples due to surface plasmon resonance of bimetallic nanoparticles, as monitored by optical spectroscopy, revealed the considerable influence of the respective particle configuration.

Monoclonal antibody detection of plasma membrane cholesterol microdomains responsive to cholesterol trafficking.

The hypothesis of lipid domains in cellular plasma membranes is well established. However, direct visualization of the domains has been difficult. Here we report direct visualization of plasma membrane cholesterol microdomains modulated by agents that affect cholesterol trafficking to and from the plasma membrane. The cholesterol microdomains were visualized with a monoclonal antibody that specifically detects ordered cholesterol arrays. These unique cholesterol microdomains were induced on macrophages and fibroblasts when they were enriched with cholesterol in the presence of an ACAT inhibitor, to block esterification of excess cellular cholesterol. Induction of the plasma membrane cholesterol microdomains could be blocked by agents that inhibit trafficking of cholesterol to the plasma membrane and by cholesterol acceptors that remove cholesterol from the plasma membrane. In addition, plasma membrane cholesterol microdomains did not develop in mutant Niemann-Pick type C fibroblasts, consistent with the defect in cholesterol trafficking reported for these cells. The induction of plasma membrane cholesterol microdomains on inhibition of ACAT helps explain how ACAT inhibition promotes cholesterol efflux from cells in the presence of cholesterol acceptors such as HDL. The anti-cholesterol monoclonal antibody also detected extracellular cholesterol-containing particles that accumulated most prominently during cholesterol enrichment of less differentiated human monocyte-macrophages. For the first time, cholesterol microdomains have been visualized that function in cholesterol trafficking to and from the plasma membrane.

Macrophage foam cells and atherosclerosis.

Focal buildup of cholesterol in arteries is the process that produces atherosclerotic plaques, the cause of most coronary artery disease and strokes. Monocyte-derived macrophages are central cells that accumulate this cholesterol in atherosclerotic lesions, a manifestation of the scavenging function of the macrophage. Different types of cholesterol-containing lipid particles found in atherosclerotic lesions may enter macrophages by a variety of endocytic pathways. The fate of cholesterol that enters macrophages determines whether macrophages help or hinder cholesterol removal from the vessel wall. Macrophages may function to carry cholesterol out of lesions, or to process the cholesterol for excretion in association with small protein-phospholipid complexes. Alternatively, macrophages that do not efficiently function to remove cholesterol from lesions may ultimately undergo cell death. Some cytokines, hormones, and pharmacologic agents show potential to modulate these processes and may be useful in directing macrophage function in atherosclerotic lesions towards beneficial rather than harmful effects.

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.

Lipoprotein cholesterol and atherosclerosis.

Progressive accumulation of cholesterol in the arterial wall causes atherosclerosis, the pathologic process underlying most heart attacks and strokes. Low density lipoprotein (LDL), the major carrier of blood cholesterol, has been implicated in the buildup of cholesterol in atherosclerotic plaques. Endothelial cells that line arteries function to transport LDL into the vessel wall. Models for the mechanism of cholesterol accumulation in atherosclerotic plaques emphasize increased LDL uptake into the vessel wall or increased retention of LDL that has entered the vessel wall. This article reviews the pathways of cholesterol entry and removal, the metabolism, and the physical changes of cholesterol in the vessel wall. How these processes are believed to contribute to cholesterol buildup in atherosclerotic plaques is discussed.

Dominant late-onset retinal degeneration with regional variation of sub-retinal pigment epithelium deposits, retinal function, and photoreceptor degeneration.

PURPOSE: To clarify the pathogenesis of late-onset retinal degeneration (L-ORD), an autosomal dominant disorder characterized by thick deposits of lipid-rich material between the retinal pigment epithelium (RPE) and Bruch's membrane. STUDY DESIGN: Comparative clinicopathologic case report and case series. TISSUES: Eyes of an 82-year-old L-ORD eye donor and an age-matched control. SUBJECTS: Five descendants of the eye donor and his affected sister. METHODS: The eyes were processed for histopathologic examination, including electron microscopy and immunohistochemistry. Family members were examined clinically and with retinal function tests. RESULTS: The L-ORD eye had sub-RPE deposits that were positive for lipid, including esterified and unesterified cholesterol. The deposits were thinnest in the macula, which retained the highest percentage of photoreceptors. In the periphery, RPE thinning and photoreceptor loss correlated with thickness of the sub-RPE deposits. The eye donor was asymptomatic until his late 50s, when he developed problems with adapting to darkness. At age 68, the eye donor had normal acuity but a midperipheral scotoma and subnormal electroretinograms (ERGs); visual loss was progressive. The five descendants (at the time of examination ages 44-58) of the eye donor and his affected sister, who were at 50/50 risk of inheriting L-ORD, had normal ERGs, but four showed defects in dark adaptation. The dark adaptation abnormalities had a distribution similar to the thickness of the sub-RPE deposits in the eye donor, with slow kinetics in the midperiphery and normal kinetics centrally. CONCLUSIONS: The L-ORD donor eye differed from a previous case in the regional distribution of sub-RPE deposits and photoreceptors. In the next generation of this L-ORD family, the first expression of disease, abnormal dark adaptation, mirrored the regional distribution of the deposits in the donor eye. The fine structure and staining characteristics of the sub-RPE deposits in L-ORD resemble those in age-related macular degeneration and Sorsby fundus dystrophy.

Plasmin-mediated macrophage reversal of low density lipoprotein aggregation.

Evidence suggests that aggregated low density lipoprotein (AgLDL) accumulates in atherosclerotic lesions. Previously, we showed that AgLDL induces and enters surface-connected compartments (SCC) in human monocyte-derived macrophages by a process we have named patocytosis. Most AgLDL taken up by these macrophages in the absence of serum is stored in SCC and remains undegraded. We now show that macrophages released AgLDL (prepared by vortexing or treatment with phospholipase C or sphingomyelinase) from their SCC when exposed to 10% human lipoprotein-deficient serum (LPDS). Macrophages also took up AgLDL in the presence of LPDS, but subsequently released it. In both cases, the released AgLDL was disaggregated. Although the AgLDL that macrophages took up could not pass through a 0.45-micrometer filter, >60% of AgLDL could pass this filter after release from the macrophages. Disaggregation of AgLDL was verified by gel-filtration chromatography and electron microscopy that also showed particles larger than LDL, reflecting fusion of LDL that aggregates. The factor in serum that mediated AgLDL release and disaggregation was plasmin generated from plasminogen by macrophage urokinase plasminogen activator. AgLDL release was decreased >90% by inhibitors of plasmin (epsilon-amino caproic acid and anti-plasminogen mAb), and also by inhibitors of urokinase plasminogen activator (plasminogen activator inhibitor-1 and anti-urokinase plasminogen activator mAb). Moreover, plasminogen could substitute for LPDS and produce similar macrophage release and disaggregation of AgLDL. Because only plasmin bound to the macrophage surface is protected from serum plasmin inhibitors, interaction of AgLDL with macrophages was necessary for reversal of its aggregation by LPDS. The released disaggregated LDL particles were competent to stimulate LDL receptor-mediated endocytosis in cultured fibroblasts. Macrophage-mediated disaggregation of aggregated and fused LDL is a mechanism for transforming LDL into lipoprotein structures size-consistent with lipid particles found in atherosclerotic lesions.

Characterization of patocytosis: endocytosis into macrophage surface-connected compartments.

Previously, we described a unique macrophage endocytosis pathway in which aggregated low density lipoproteins and microcrystalline cholesterol induce and enter a labyrinth of membrane-bound compartments that remain connected to the cell surface. We now show that certain types of non-lipid particles such as polystyrene microspheres and colloidal gold also induce and enter macrophage surface-connected compartments (SCC), a process we call patocytosis. A common property among particles that stimulate patocytosis is their hydrophobic nature. Both aggregated LDL and microcrystalline cholesterol that we showed previously to stimulate patocytosis are hydrophobic. We now show that hydrophobic polystyrene microspheres and gold particles but not their hydrophilic counterparts triggered patocytosis. Uptake by patocytosis was limited to hydrophobic polystyrene microsphere particles less than 0.5 micron in diameter. Hydrophobic polystyrene microspheres greater than this size entered macrophages by phagocytosis. Actin-independent capping of hydrophobic polystyrene microspheres on the plasma membrane preceded actin-dependent uptake of the microspheres into SCC. Sequential rounds of microsphere uptake into SCC over two successive days could occur. There was some mixing of initial and subsequently accumulated microspheres in SCC. SCC formed from plasma membrane invaginations that connected with spaces created by unfolding of stacks of internal microvilli. Microsphere transport from plasma membrane invaginations into these spaces was inhibited by primaquine. Patocytosis is a unique endocytic process in macrophages triggered by small hydrophobic particles that provides a mechanism to sequester large amounts of these materials within a labyrinth of SCC.

Apolipoprotein B stimulates formation of monocyte-macrophage surface-connected compartments and mediates uptake of low density lipoprotein-derived liposomes into these compartments.

Much of the cholesterol that accumulates in atherosclerotic plaques is found within monocyte-macrophages transforming these cells into "foam cells." Native low density lipoprotein (LDL) does not cause foam cell formation. Treatment of LDL with cholesterol esterase converts LDL into cholesterol-rich liposomes having >90% cholesterol in unesterified form. Similar cholesterol-rich liposomes are found in early developing atherosclerotic plaques surrounding foam cells. We now show that cholesterol-rich liposomes produced from cholesterol esterase-treated LDL can cause human monocyte-macrophage foam cell formation inducing a 3-5-fold increase in macrophage cholesterol content of which >60% is esterified. Although cytochalasin D inhibited LDL liposome-induced macrophage cholesteryl ester accumulation, LDL liposomes did not enter macrophages by phagocytosis. Rather, the LDL liposomes induced and entered surface-connected compartments within the macrophages, a unique endocytic pathway in these cells that we call patocytosis. LDL liposome apoB rather than LDL liposome lipid mediated LDL liposome uptake by macrophages. This was shown by the findings that: 1) protease treatment of the LDL liposomes prevented macrophage cholesterol accumulation; 2) liposomes prepared from LDL lipid extracts did not cause macrophage cholesterol accumulation; and 3) purified apoB induced and accumulated within macrophage surface-connected compartments. Although apoB mediated the macrophage uptake of LDL liposomes, this uptake did not occur through LDL, LDL receptor-related protein, or scavenger receptors. Also, LDL liposome uptake was not sensitive to treatment of macrophages with trypsin or heparinase. Cholesterol esterase-mediated transformation of LDL into cholesterol-rich liposomes is an LDL modification that: 1) stimulates uptake of LDL cholesterol by apoB-dependent endocytosis into surface-connected compartments, and 2) causes human monocyte-macrophage foam cell formation.

Inhibition of aortic wall calcification in bioprosthetic heart valves by ethanol pretreatment: biochemical and biophysical mechanisms.

The effectiveness of ethanol pretreatment on preventing calcification of glutaraldehyde-fixed porcine aortic bioprosthetic heart valve (BPHV) cusps was previously demonstrated, and the mechanism of action of ethanol was attributed in part to both lipid removal and a specific collagen conformational change. In the present work, the effect of ethanol pretreatment on BPHV aortic wall calcification was investigated using both rat subdermal and sheep circulatory implants. Ethanol pretreatment significantly inhibited calcification of BPHV aortic wall, but with less than complete inhibition. The maximum inhibition of calcification of BPHV aortic wall was achieved using an 80% ethanol pretreatment; calcium levels were 71.80+/-8.45 microg/mg with 80% ethanol pretreatment compared to the control calcium level of 129.90+/-7.24 microg/mg (p = 0.001). Increasing the duration of ethanol exposure did not significantly improve the inhibitory effect of ethanol on aortic wall calcification. In the sheep circulatory implants, ethanol pretreatment partly prevented BPHV aortic wall calcification with a calcium level of 28.02+/-4.42 microg/mg compared to the control calcium level of 56.35+/-6.14 microg/mg (p = 0.004). Infrared spectroscopy (ATR-FTIR) studies of ethanol-pretreated BPHV aortic wall (vs. control) demonstrated a significant change in protein structure due to ethanol pretreatment. The water content of the aortic wall tissue and the spin-lattice relaxation times (T1) as assessed by proton nuclear magnetic resonance spectroscopy did not change significantly owing to ethanol pretreatment. The optimum condition of 80% ethanol pretreatment almost completely extracted both phospholipids and cholesterol from the aortic wall; despite this, significant calcification occurred. In conclusion, these results clearly demonstrate that ethanol pretreatment is significantly but only partially effective for inhibition of calcification of BPHV aortic wall and this effect may be due in part to lipid extraction and protein structure changes caused by ethanol. It is hypothesized that ethanol pretreatment may be of benefit for preventing bioprosthetic aortic wall calcification only in synergistic combination with another agent.

The fate of lipoprotein cholesterol entering the arterial wall.

Recent findings have helped to explain the fate of cholesterol entering the arterial wall. LDL can undergo both fusion and aggregation. These changes may cause increased retention of LDL in lesion connective tissue matrix and LDL uptake by macrophages. In the cornea, apparent fusion of LDL occurs in the absence of macrophages. Mast cells may be important in LDL fusion, as mast cell-derived proteases can induce fusion of LDL through proteolysis of apolipoprotein B. LDL in arterial wall atherosclerotic lesions was found to be sialic acid-poor and ceramide-enriched. These chemical changes promote LDL aggregation. Processes that may function to remove cholesterol from the arterial wall have been reported. Macrophage-produced apolipoprotein E can mediate macrophage cholesterol efflux and macrophages can convert cholesterol to 27-oxygenated products that macrophages excrete. Alternately, another oxygenated sterol, 7-ketocholesterol, impairs macrophage cholesterol efflux. In addition, mast-cell derived chymase proteolyses HDL and reduces its capacity to stimulate cholesterol efflux.

Prevention of bioprosthetic heart valve calcification by ethanol preincubation. Efficacy and mechanisms.

BACKGROUND: Calcification of the cusps of bioprosthetic heart valves fabricated from either glutaraldehyde cross-linked porcine aortic valves or bovine pericardium frequently causes the clinical failure of these devices. Our investigations studied ethanol pretreatment of glutaraldehyde cross-linked porcine aortic valves as a new approach to prevent cuspal calcification. The hypothesis governing this approach holds that ethanol pretreatment inhibits calcification resulting from protein structural alterations and lipid extraction. METHODS AND RESULTS: Results demonstrated complete inhibition of calcification of glutaraldehyde-pretreated porcine bioprosthetic aortic valve cusps by 80.0% ethanol in rat subdermal implants (60-day ethanol-pretreated calcium level, 1.87 +/- 0.29 micrograms/mg tissue compared with control calcium level, 236.00 +/- 6.10 micrograms/mg tissue) and in sheep mitral valve replacements (ethanol-pretreated calcium level, 5.22 +/- 2.94 micrograms/mg tissue; control calcium level, 32.50 +/- 11.50 micrograms/mg tissue). The mechanism of ethanol inhibition may be explained by several observations: ethanol pretreatment resulted in an irreversible alteration in the amide I band noted in the infrared spectra for both purified type I collagen and glutaraldehyde cross-linked porcine aortic leaflets. Ethanol pretreatment also resulted in nearly complete extraction of leaflet cholesterol and phospholipid. CONCLUSIONS: Ethanol pretreatment of glutaraldehyde cross-linked porcine aortic valve bioprostheses represents a highly efficacious and mechanistically based approach and may prevent calcific bioprosthetic heart valve failure.

Aggregated low density lipoprotein induces and enters surface-connected compartments of human monocyte-macrophages. Uptake occurs independently of the low density lipoprotein receptor.

Aggregation of low density lipoprotein (LDL) stimulates its uptake by macrophages. We have now shown by electron microscopic and chemical experiments that aggregated LDL (produced by vortexing (VxLDL) or treatment with phospholipase C) induced and became sequestered in large amounts within surface-connected compartments (SCC) of human monocyte-derived macrophages. This occurred through a process different from phagocytosis. Formation of SCC and accumulation of aggregated LDL in SCC are cell-mediated processes that were temperature-dependent (10 x greater cell association at 37 degrees C than at 4 degrees C) and blocked by cytochalasin D but not by nocodazole. Because of the surface connections of SCC, trypsin could release aggregated LDL from SCC. Degradation of 125I-VxLDL through the SCC pathway showed delayed and a lower rate of degradation (10-55%) compared with nonaggregated 125I-acetylated LDL that did not enter SCC. However, similar to 125I-acetylated LDL degradation, 125I-VxLDL degradation occurred through a chloroquine-sensitive pathway. Uptake of VxLDL into SCC was not mediated by the LDL receptor. Methylation of LDL prevents its binding to the LDL receptor. However, methylated LDL still entered SCC after it was aggregated by vortexing. On the other hand, degradation of 125I-VxLDL was substantially decreased by methylation of LDL and by cholesterol enrichment of macrophages, which decreases macrophage LDL receptor expression. The results suggest that whereas uptake of aggregated LDL into SCC occurs independently of the LDL receptor, movement of aggregated LDL from SCC to lysosomes may depend in part on LDL receptor function. Sequestration into SCC is a novel endocytosis pathway for uptake of aggregated LDL that allows the macrophage to store large amounts of this lipoprotein before it is further processed.

Apolipoprotein E produced by human monocyte-derived macrophages mediates cholesterol efflux that occurs in the absence of added cholesterol acceptors.

Human monocyte-derived macrophages can efflux accumulated cholesterol without exogenously added cholesterol acceptors (Kruth, H. S., Skarlatos, S. I., Gaynor, P. M., and Gamble, W. (1994) J. Biol. Chem. 269, 24511-24518). Most of the effluxed cholesterol accumulates in the medium as apolipoprotein E-discoidal lipid particles. In the current study, we determined whether and to what degree cholesterol efflux from human monocyte-macrophages depended on apolipoprotein E secretion. Unexpectedly, 2-week-old differentiated monocyte-macrophages secreted similar amounts of apolipoprotein E without or with cholesterol enrichment. Apolipoprotein E mRNA levels in these macrophages were not increased by cholesterol enrichment and were comparable with levels in HepG2 cells. Without cholesterol enrichment, monocyte-macrophages secreted lipid-poor apolipoprotein E with a density >1.21 g/ml. By contrast, cholesterol enrichment of monocyte-macrophages induced the association of apoE with phospholipid and cholesterol to form discoidal particles that floated at densities of 1.08-1.10 g/ml. An anti-apolipoprotein E monoclonal antibody added to the culture medium significantly inhibited cholesterol and phospholipid efflux from the monocyte-macrophages. This showed that apolipoprotein E was required for most of the cholesterol efflux, and that apolipoprotein E did not leave macrophages with lipid but rather associated with lipid after it was secreted. Thus, 1) apolipoprotein E was constitutively secreted by differentiated human monocyte-macrophages, 2) apolipoprotein E only formed discoidal particles following macrophage cholesterol enrichment, 3) apolipoprotein E was necessary for cholesterol efflux to occur in the absence of added cholesterol acceptors and, in addition 4) the level of macrophage unesterified cholesterol was not rate-limiting for this cholesterol efflux, and 5) net phospholipid synthesis occurred in macrophages secondary to apoE-mediated loss of macrophage phospholipid. In conclusion, apolipoprotein E functions in an autocrine pathway that mediates cholesterol efflux from human monocyte-derived macrophages.

Cholesterol accumulation in human cornea: evidence that extracellular cholesteryl ester-rich lipid particles deposit independently of foam cells.

The cornea is a connective tissue site where lipid accumulates as a peripheral arcus lipoides. We found that cholesterol, in predominantly esterified form, progressively accumulated with age in the peripheral corneas of 20- to 90-yr-old individuals. Ultrastructural studies showed extracellular solid spherical lipid particles (< 200 nm in diameter) enmeshed between collagen fibers. Immunostaining showed significant apoE and apoA-I, but very little apoB in the peripheral cornea. Lipid particles were extracted from minced corneas into a buffer and subjected to isopycnic density gradient centrifugation. The lipid particles had a density < 1.02 g/ml, contained > 75% of their cholesterol in esterified form, and were distributed in two populations with average diameters of 22 +/- 5 nm (SD) and 79 +/- 26 nm. Gel-filtration chromatographic analysis of the corneal lipid particles showed that most cholesterol eluted with the larger particles and these larger particles lacked apoB. ApoA-I was associated with lipid particles the size of HDL. Most apoE was associated with lipid particles larger than the apoA-I-containing lipid particles and smaller than the large lipid particles that carried most of the corneal cholesterol. Thus, the cholesteryl ester-rich lipid particles that accumulate in the cornea are 1) similar to lipid particles previously localized within and isolated from human atherosclerotic lesions, 2) accumulate without foam cells, and 3) may be derived from low density lipoproteins that have lost their apoB and fused.

Inhibition of atherosclerosis development in cholesterol-fed human apolipoprotein A-I-transgenic rabbits.

BACKGROUND: Prospective epidemiological studies support the hypothesis that high levels of high-density lipoprotein (HDL) cholesterol and apolipoprotein (apo) A-I limit atherosclerosis development. However, more data from studies with animal models of atherosclerosis that resemble the human disease are required to demonstrate the effect of apo A-I in the inhibition of atherogenesis. The rabbit is a good animal model for human atherosclerosis. METHODS AND RESULTS: Human apo A-I-transgenic rabbits have been produced, and we have evaluated the effect of apo A-I on the development of atherosclerosis in transgenic rabbits fed a cholesterol-rich diet for 14 weeks. Plasma cholesterol levels of atherogenic apo B-containing lipoproteins were similar for transgenic and control rabbits (> 1000 mg/dL), while plasma levels of HDL cholesterol in the transgenic group were always about twice that of the control group (68 +/- 11 versus 37 +/- 3 mg/dL at 14 weeks; P < .001). At the end of the experiment, the amount of aortic surface area covered by lesions as well as the amount of lipid accumulation in the aorta were significantly less in transgenic rabbits compared with the control group (15 +/- 12% versus 30 +/- 8%, P < .0027 for the surface area of the thoracic aorta; 116 +/- 31 versus 247 +/- 39 mumol/g aorta, P < .0068 for cholesterol content in total aorta). CONCLUSIONS: Overexpression of human apo A-I in rabbits inhibits the development of atherosclerosis in this animal model that resembles, in many respects, human atherosclerosis.

Accumulation of HDL apolipoproteins accompanies abnormal cholesterol accumulation in Schnyder's corneal dystrophy.

Schnyder's corneal dystrophy is an autosomal dominant disorder that results in clouding of the central cornea and premature development of peripheral arcus in the cornea. Previous studies showed that abnormal lipid accumulation is the basis for the corneal clouding. We examined whether apolipoproteins are involved in this disorder and characterized the lipid accumulation in the central portion of corneas removed from patients with Schnyder's dystrophy. Our findings show that cholesterol and phospholipid contents increased greater than 10-fold and 5-fold, respectively, in affected compared with normal corneas. In addition, the percentage of cholesterol that was unesterified (63% versus 50%) and the molar ratio of unesterified cholesterol to phospholipid (1.5 versus 0.5) were higher in affected compared with normal corneas. Large multilamellar vesicles and electron-dense granules (100 to 300 nm in diameter) as well as cholesterol crystals accumulated in the extracellular matrix of affected corneas. Immunohistochemical analysis showed that apolipoprotein constituents of HDL (apoA-I, apoA-II, and apoE), but not apoB, a marker of LDL, accumulated in the affected cornea. Western blot analysis confirmed the increased amounts of these HDL apolipoproteins in affected corneas and showed that the apparent molecular weights of the apolipoproteins were normal. Our findings show for the first time that HDL apolipoproteins accumulate in the corneas of patients with Schnyder's corneal dystrophy. Thus, this disorder influences the metabolism of HDL in the corneas of these patients.

Glycosphingolipid accumulation in the aortic wall is another feature of human atherosclerosis.

High accumulation of lipids is a typical feature of an atherosclerotic lesion. We have previously identified the chemical structure of the major glycosphingolipids (GSLs) of human aorta; however, quantification of the absolute concentration of GSLs was not carried out. In the present study, for the first time we have performed a quantitative comparative analysis of GSL composition in the media and two sublayers of the intima taken from normal regions, fatty streaks, and atherosclerotic plaques of the human aorta. The intimal tissue containing fatty streaks and atherosclerotic plaques accumulated GSLs, predominantly glucosylceramide (GlcCer), lactosylceramide (LacCer), and ganglioside GM3. GSL levels in plaques were highest: GlcCer was 18- and 8-fold, LacCer was 8- and 7-fold, and GM3 was 2.5- and 12-fold higher than in musculoelastic and elastic-hyperplastic intimal layers of normal regions, respectively. We did not observe a significant increase in other GSLs. An increase in the content of gangliosides GD3 and GD1a was detected in the media underlying atherosclerotic lesions. On the basis of an analysis of the ratio of GlcCer, LacCer, and GM3 accumulated in the tissue and cells of the elastic-hyperplastic layer of intima, we have concluded that the accumulation of the above-mentioned GSLs occurs mainly in the extracellular space of the intima. In this study, we have also demonstrated that extracellular lipid liposomes, which appear in the early stages of atherogenesis, are one locus of GSL accumulation in the extracellular space of the intima.(ABSTRACT TRUNCATED AT 250 WORDS)

Sequestration of acetylated LDL and cholesterol crystals by human monocyte-derived macrophages.

Monocyte-derived macrophages accumulate and process cholesterol in atherosclerotic lesions. Because of the importance of this process, we examined the interaction of cholesterol crystals and acetylated low density lipoprotein (AcLDL) with human monocyte-macrophages in a combined chemical and morphological study. These two forms of cholesterol induced extensive compartmentalization of the macrophage cytoplasm. Unexpectedly, the compartments maintained a physical connection to the extracellular space as demonstrated with ruthenium red staining. The compartments formed through invagination of the top surface of the macrophage plasma membrane. Some cholesterol crystals and AcLDL were sequestered within these surface-connected compartments for up to five days in the case of the crystals and for one day in the case of AcLDL. Pulse-chase studies of fractionated macrophages indicated that [3H]cholesterol redistributed from the surface-connected compartments into lysosomes (where the cholesterol remained unesterified) and into lipid droplets (where the cholesterol was stored as cholesteryl ester). Intracellular uptake and esterification of cholesterol was blocked by cytochalasin D. However, once cholesterol was sequestered in the surface-connected compartments, subsequent esterification of the cholesterol could not be inhibited by cytochalasin D. Apolipoprotein E was localized within the surface-connected compartments by immunogold labeling suggesting a possible function for this protein in the processing of lipid taken up through the sequestration pathway. Removal of microcrystalline cholesterol from the medium resulted in release of most of the accumulated cholesterol microcrystals from the macrophages, as well as disappearance of the surface-connected compartments. Thus, sequestration is a novel endocytic mechanism in which endocytic compartments remain connected to the extracellular space. This differs from phagocytosis where endocytic vacuoles rapidly pinch off from the plasma membrane. Sequestration provides a means for macrophages to remove substances from the extracellular space and later release them.