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Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:1607-1615

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:1607-1615.)
© 1995 American Heart Association, Inc.

Articles

Glycosphingolipid Accumulation in the Aortic Wall Is Another Feature of Human Atherosclerosis

Dmitry N. Mukhin; Fei-Fei Chao; Howard S. Kruth

From the Institute of Experimental Cardiology, Cardiology Research Center, Russian Academy of Medical Sciences, Moscow, Russia (D.N.M.), and the Section of Experimental Atherosclerosis, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Md (F.-F.C., H.S.K.).

Correspondence to Dr Howard S. Kruth, Section of Experimental Atherosclerosis, NHLBI, NIH, Bldg 10, Rm 5N113, 10 Center Dr MSC 1422, Bethesda, MD 20892-1422.


*    Abstract
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 *Abstract
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Abstract 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. The findings suggest that the GSL concentration and distribution within the normal and atherosclerotic aorta reflects a number of factors that include (1) synthesis of GSLs within the vessel wall, (2) deposition of GSLs within the vessel wall from plasma-derived lipoproteins, (3) the degree of association of the various GSLs with intimal cells as well as extracellular lipid particles, and (4) metabolic relationships between cholesterol and GSL accumulation.


Key Words: atherosclerosis • aorta • glycosphingolipids • extracellular lipid particles • gangliosides


*    Introduction
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 *Introduction
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High accumulation of lipids in the intima of the major arteries is a typical feature of atherosclerotic lesions. Lipid accumulation occurs both in cells and in the extracellular spaces of the intima. Cholesterol has long been known to be the major lipid component accumulating in atherosclerotic lesions,1 and many studies have been devoted to the role of cholesterol in the formation of atherosclerotic lesions. However, the mechanism of initiation and progression of atherosclerotic lesions is still unclear. Much less attention has been paid to the accumulation of minor lipid components in atherosclerotic lesions. These lipids may influence processes that contribute to the development of atherosclerotic lesions.2 3 Some previous studies specifically focused on GSL accumulation in arteries during human atherosclerosis and experimental atherosclerosis in animals.4 5 6 7 Earlier studies identified the chemical structure of the major GSLs of human aorta8 9 and showed that GSLs accumulate in the intimal cells of aortic atherosclerotic lesions.10 11 However, quantification of the absolute concentration of GSL species in unlesioned and atherosclerotic aortas was not carried out.

We have now undertaken a more detailed study to investigate changes in GSL composition in human atherosclerotic aorta. In this study, for the first time we have quantified the concentration of GSLs accumulated in atherosclerotic tissues and compared the GSL composition of the media and the elastic-hyperplastic and musculoelastic layers of the intima. Comparison of the degree of GSL accumulation in the whole tissue and enzymatically isolated aortic cells suggested to us that much GSL accumulated extracellularly in the intima. Early stages of atherogenesis are characterized by the appearance of extracellular lipid particles that are known to be a locus of extracellular cholesterol accumulation.12 13 14 15 16 17 18 19 20 Two types of these particles have been described: esterified cholesterol–rich droplets and unesterified cholesterol–rich multilamellar liposomes.21 We have explored the possibility that these lipid particles are a locus not only of extracellular cholesterol accumulation but also of extracellular GSL accumulation in human atherosclerotic lesions.


*    Methods
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Preparation of Aortic Tissue Samples for GSL Analysis
Aorta samples were taken aseptically 1.5 to 3 hours after death from 40- to 60-year-old subjects who had died suddenly of myocardial infarction or some other cause. The samples were kept frozen until analysis. Upon being thawed, the vessels were cut longitudinally and the types of atherosclerotic lesions were assessed according to the classification suggested by Smith.22 Two types of lesions were analyzed in the study: (1) fatty streaks, strips 2 to 3 mm wide and 5 to 30 mm long, slightly elevated over the vessel surface, and (2) atherosclerotic plaques, lesions highly elevated over the luminal surface, usually of rounded shape, light yellow or pearl in color. Only uncomplicated plaques that were not ulcerated, thrombosed, or calcified were used for the study. Regions of aorta that were without macroscopic lesions and that did not stain with Sudan IV were regarded as normal unaffected aortic tissue. Regions of normal aortic tissue and areas affected by fatty streaks or atherosclerotic plaques were excised, and tissue of each type of lesion (each aorta contained a mixture of fatty streaks and plaques) was pooled. Tissue was processed further as described below.

Human adult aortic intima, as well as coronary artery intima, is known to consist of two major layers.23 24 25 The musculoelastic layer adjacent to the media is separated from it by an internal elastic lamina, and is separated from the layer adjacent to the lumen (ie, the elastic-hyperplastic layer) by a secondary elastic lamina. To extend our previous work10 11 25 we examined the GSL composition in each of the two intimal layers separately. For each type of aortic tissue (normal or lesioned), the intima was separated along the main elastic membranes from the media, and the intimal musculoelastic layer was separated from the intimal elastic-hyperplastic layer with the aid of forceps. The accuracy of the procedure was verified for each specimen by light microscopy according to Smith et al15 with partially stripped tissue samples that were stained for Verhoeff's elastic, as illustrated previously.25 The detachment of the intima from the media and of the elastic-hyperplastic layer from the musculoelastic layer was considered faulty if it occurred above or beneath these membranes. Such tissue preparations were discarded. In some cases, the elastic-hyperplastic layer contained a third, external connective tissue layer. However, it was usually difficult to establish the marginal line between these two layers, and therefore this connective tissue layer was left intact in the preparation of the elastic-hyperplastic layer. For convenience, we shall henceforth refer to the musculoelastic intimal layer as muscular and to the layer that includes the elastic-hyperplastic and connective tissue layers as hyperplastic. The lipid core (if any) localized within the hyperplastic layer, leaving intact the muscular layer. Only in complicated plaques did the necrotic zone involve the muscular layer of intima. In such plaques it was impossible to separate the two intimal layers correctly, and these preparations were discarded.

We performed three independent experiments, each time taking tissue from 5 to 8 aortas.

Isolation and Purification of Aortic Lipid Particles
Aortic tissue specimens from three different aortas for lipid particle isolation were obtained 5 to 10 hours after death, stored in Leibovitz 15 medium26 (Hazleton Laboratory) for transport to the laboratory, and frozen in this medium within 24 hours of autopsy. Tissues from each aorta contained a mixture of fatty streaks and plaques. Aortic lipid particles were isolated from the entire intima as described previously.21 The aortic tissues were thawed at 4°C, rinsed several times in chilled D-PBS, and dissected free of adventitia and media. The intimal tissues were then rinsed in D-PBS, blotted dry, and weighed. The tissues were minced for approximately 30 minutes with scissors in 0.15 mol/L NaCl (pH 7.4) containing 0.1% EDTA, 0.05% glutathione, and 0.02% NaN3 (1 g tissue per 8 mL solution). The majority of the minced pieces were less than 1 mm3 in size. These and all subsequent procedures were carried out at 4°C.

The lipid-containing supernatant was collected after a low-speed centrifugation (1500g for 15 minutes) of the minced tissue. This sample was then passed through low-protein–binding polysulfone 0.45-µm (pore size) filters (Acrodisc 4184, Gelman Sciences) to remove crystalline lipid and lipid droplets. The filtrate was concentrated to 2 to 3 mL in an Amicon stirred cell by use of a cellulosic filter with a molecular weight cutoff of 10 000 (YM-10, Amicon). Next, the filtrate was dialyzed overnight against 4 L of 1.5 mol/L NaCl (pH 7.4) containing 0.1% EDTA, 0.05% glutathione, and 0.02% NaN3. The dialysate was subjected to gel-filtration column chromatography (Bio-Gel, A-50m) with a 1.6x50-cm column eluted at a flow rate of 7 mL · hr-1 · cm-2 with the same solution that was used for dialysis.

Density gradient centrifugation of the pooled void volume fractions was carried out in a Beckman Ultraclear tube centrifuged in a Beckman SW-41 rotor. A discontinuous gradient was constructed from bottom to top with NaCl solutions: 3 mL at d=1.100 g/mL, 3 mL of void volume fraction at d=1.063 g/mL, 3 mL at d=1.019 g/mL, and 2.5 mL at d=1.006 g/mL. All salt solutions were at pH 7.0 and contained 0.1% EDTA and 0.01% thimerosal. The gradient was centrifuged for 22 hours at 170 000g. During the centrifugal procedure, a continuous gradient was formed as described by Redgrave et al.27 After centrifugation, an esterified cholesterol–rich lipid fraction was collected at d<1.01 g/mL and an unesterified cholesterol–rich lipid fraction was collected at density limits between 1.03 and 1.05 g/mL.

Isolation of Plasma LDL
LDLs (d=1.019 to 1.065 g/mL) were isolated from the plasma of three male patients (40 to 60 years old) with coronary heart disease and angiographically documented stenosis of the coronary arteries. LDLs were isolated by sequential flotational ultracentrifugation according to Lindgren.28 Each ultracentrifugation was performed at 10°C in a 60 Ti rotor (Beckman) for 18 to 20 hours at 105 000g. The final LDL preparation from each patient was washed once at its higher limiting (solvent) density and was then exhaustively dialyzed at 4°C against a solution containing 0.15 mol/L NaCl, 0.01% NaN3, and 0.01% EDTA at pH 7.4.

Isolation of Aortic Cells
Cells were isolated from human aortic hyperplastic intimal layers of grossly unlesioned or atherosclerotic plaque tissue by digestion with Medium 199 containing 0.1% collagenase (Type II, Worthington Diagnostic System Inc), 10% fetal calf serum, 100 g/mL streptomycin, and 2.5 g/mL fungizone (all purchased from Grand Island Biological Co), as described previously.10 11 25 29 30 The viability of the isolated cells determined with trypan blue was 85% to 90%. The isolated cells were extracted immediately after the isolation. Cells isolated from 10 aortas were pooled. The pooled cell samples contained about 2.0x107 cells.

Chemical Analysis of Cholesterol
Lipids of the aortic tissue and isolated aortic lipid particles, aortic intimal cells, and LDLs were extracted with chloroform/methanol (2:1, vol/vol).31 Unesterified and esterified cholesterol were determined enzymatically with a fluorometric method32 for tissue and lipid particles and with a calorimetric method by use of the Monotest kit (Boehringer Mannheim GmbH) for aortic cells and LDLs.

Isolation and Purification of GSLs
Aortic tissue samples (1 to 32 g wet tissue) were homogenized in chloroform/methanol (2:1, vol/vol) and extracted successively in chloroform/methanol (2:1 and 1:2, vol/vol). Total lipids were treated with 0.1 mol/L NaOH in methanol for 2 hours at 40°C, neutralized with 0.35 mol/L acetic acid, and evaporated. The dry residue was dissolved in water and dialyzed against distilled water for 48 hours at 4°C. After evaporation, lipids were dissolved in 50 mL chloroform/methanol/water (30:60:8, vol/vol/vol) and applied to a column (1.5x20 cm) packed with DEAE–Sephadex A-25 (40 to 100 mesh, Pharmacia Fine Chemicals) in acetate form.33 Neutral GSLs were eluted with the same solvent mixture as the one the lipids were in when applied to the column. Gangliosides were eluted with chloroform/methanol/0.8 mol/L ammonium acetate (30:60:8, vol/vol/vol). After evaporation of elution solvent, gangliosides were dissolved in water and dialyzed against distilled water for 48 hours at 4°C. Neutral GSLs were subsequently purified through Silica Gel L 40/100 (Chemapol) column chromatography as follows: The GSL samples were loaded on a silica gel column (6x150 mm, 3 g) in chloroform. The column was washed with chloroform, and neutral GSLs were eluted with chloroform/methanol/water (65:25:4, vol/vol/vol).

GSLs were isolated from cells as described earlier.10 11 Washed cell pellets were resuspended in 2 mL chloroform/methanol/water (5:5:1, vol/vol/vol) and sonicated. Lipid extraction was carried out for 2 hours at 22°C. The residue was pelleted, and the extraction was repeated twice more. Extracts were combined, evaporated, dissolved in 1 mL chloroform/methanol (2:1, vol/vol), and hydrolyzed for 20 minutes at 22°C after addition of 10 µL 10N HCl.34 Two milliliters of 0.5N NaOH dissolved in methanol was added to the hydrolyzate and incubated for 2 hours at 40°C. After evaporation, the precipitate was dissolved in a small volume of water, neutralized with 1N HCl, and desalted by use of a Sep-Pak C18 cartridge (Waters Associates).35 GSLs were then fractionated into acidic and neutral fractions on a 10x50-mm DEAE–Sephadex A-25 column, and neutral GSLs were subsequently purified through Silica Gel L 40/100 as described above.

Quantification of GSLs
Gangliosides were separated on high-performance thin-layer chromatography plates of Silica Gel 60 (E. Merck). A mixture of chloroform/methanol/0.2% CaCl2 (55:45:10, vol/vol/vol) was used for ganglioside separation. Either chloroform/methanol/water (65:25:4, vol/vol/vol) or chloroform/methanol/0.2% CaCl2 (50:40:6, vol/vol/vol) was used to separate neutral GSLs. Quantitative analysis of GSLs was carried out on chromatography plates (after visualization with resorcinol reagent for gangliosides and orcinol reagent for neutral GSLs) with scanning densitometry on a PMQ-3 microspectrophotometer (Carl Zeiss) at a wavelength of 580 nm for gangliosides36 and 515 nm for neutral GSLs.37 GSLs isolated from human aorta and previously characterized8 9 were used as standards.

Statistical Analysis
Multivariate comparison of GSL content in normal and atherosclerotic tissues was performed with MANOVA. Probability values of less than .05 were considered significant.


*    Results
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 *Results
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Ganglioside Content in the Aortic Tissue
GM3 was a major ganglioside in all aortic layers, accounting for 70% to 95% of the ganglioside content (Figs 1ADown and 2Down). GM3 content in the hyperplastic layer of the plaque intima was 12-fold higher than in normal regions (P<.05). Also, the level of GM3 in the muscular layer of plaque was 2.5-fold higher than in normal tissue. GM1 and GD1a gangliosides were present in the hyperplastic layer of plaque but were present at lower levels in unaffected intimal hyperplastic regions. However, GM1 and GD1a gangliosides, which were low in normal hyperplastic intima, were more abundant in normal muscular intima and normal media. The difference between the media and the muscular layer of normal intima was that the muscular layer had a higher level of GD3. At the same time, the muscular layer of fatty streaks had an unexpectedly low content of all gangliosides compared with unlesioned regions. The media underlying lesioned regions showed a twofold to threefold increase in the content of gangliosides GD3 (P<.05) and GD1a compared with the media of unlesioned aorta.



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  		Figure 1. Photographs show thin-layer chromatograms of human aortic glycosphingolipids. A, Gangliosides; B, neutral GSLs. Lane 1, standard GSLs; lane 2, media under normal region; lane 3, media under fatty streak; lane 4, media under atherosclerotic plaque; lane 5, intimal muscular layer of normal region; lane 6, intimal muscular layer of fatty streak; lane 7, intimal muscular layer of atherosclerotic plaque; lane 8, intimal hyperplastic layer of normal region; lane 9, intimal hyperplastic layer of fatty streak; and lane 10, intimal hyperplastic layer of atherosclerotic plaque. The plates were developed with chloroform/methanol/0.2% CaCl2 (55:45:10, vol/vol/vol) for gangliosides and with chloroform/methanol/0.2% CaCl2 (50:40:6, vol/vol/vol) for neutral GSLs.



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  		Figure 2. Bar graphs show ganglioside content of aortic tissue. Open bar, normal tissue; shaded bar, fatty streak; solid bar, atherosclerotic plaque. Each value is the mean±SD of three independent experiments carried out on different pooled samples. *P<.05 compared with the normal tissue (MANOVA).

The changes in the relative concentration of gangliosides in both intimal layers show an increase in the percentage of GM3 and a decrease in the percentage of more complex gangliosides in the direction from normal to fatty streak to plaque (Table 1Down). In contrast, the relative concentrations of gangliosides in the media show a decrease in the percentage of GM3 and an increase in the percentage of GD3 and GD1a in the direction from normal to fatty streak to plaque. Such changes probably reflect the changes in function of these tissues in the course of development of atherosclerotic lesions.


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  		Table 1. Percentage of Gangliosides in Aorta Tissue

The media and intimal muscular layer contained two additional gangliosides. The first had a chromatographic mobility corresponding to the mobility of ganglioside GD1b from bovine brain. The second was visualized lower than GM3 and higher than GM1 on the chromatogram in Fig 1AUp. This is likely to be the ganglioside sialylparagloboside, and its presence in smooth muscle cells and leukocytes has been shown elsewhere.38 39 Unfortunately, we were not able to isolate the above-mentioned gangliosides from the human aorta in sufficient quantity to confirm their chemical structure.

Neutral GSL Content in Aortic Tissue
GbOse3Cer and GbOse4Cer were the main neutral GSLs in normal intima and media (Figs 1BUp and 3Down). Both the media and muscular layer of intima differed from the hyperplastic layer of intima by their high levels of GbOse3Cer and GbOse4Cer. However, much higher amounts of GlcCer and LacCer were detected in muscular and hyperplastic layers of atherosclerotic intimal tissues compared with normal intimal tissues (significantly different for hyperplastic layer, P<.05). The differences in the GbOse3Cer and GbOse4Cer content of normal and lesioned intimal tissues were not significant. The changes in the relative concentrations of neutral GSLs in intimal layers show a general tendency of increase in the concentration of simple GSLs (GlcCer and LacCer) from normal to fatty streak to plaque and a decrease in the concentration of complex GSLs (GbOse3Cer and GbOse4Cer) in these same aortic tissues (Table 2Down). Neutral GSL content was virtually unchanged in lesioned and normal regions of the media.



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  		Figure 3. Bar graphs show neutral GSL content of aortic tissue. Open bar, normal tissue; shaded bar, fatty streak; solid bar, atherosclerotic plaque. Each value is the mean±SD of three independent experiments carried out on different pooled samples. *P<.05 compared with normal tissue (MANOVA).


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  		Table 2. Percentage of Neutral Glycosphingolipids in Aorta Tissue

Comparative Analysis of GSL Content in Aortic Cells and Tissues
Table 3Down shows the contents of GlcCer, LacCer, and ganglioside GM3, the major GSLs that accumulate in the cells and tissue of the intimal hyperplastic layer of atherosclerotic plaques. Because normal intima cells contain little GlcCer and LacCer, we did not previously succeed in quantifying GlcCer and LacCer in these cells because of the insufficient numbers of cells used for the analysis.11 In the present study we isolated 10 times more cells for analysis, which made it possible to quantify GlcCer and LacCer in cells isolated from normal regions of aorta. Table 3Down shows that the increase in GlcCer, LacCer, and GM3 content in the atherosclerotic plaque tissue compared with unaffected intimal tissue was considerably greater than the increase in these GSLs in the cells isolated from similar atherosclerotic plaque and normal intimal tissues. Thus, it can be concluded that the rate of extracellular accumulation of GSLs is higher than the rate of intracellular accumulation of GSLs.


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  		Table 3. Glucosylceramide, Lactosylceramide, and Ganglioside GM3 Content in Cells and Tissues of Hyperplastic Layer of Human Aortic Atherosclerotic Plaque

GSL Composition of Extracellular Aortic Lipid Particles
To elucidate a possible location of extracellular GSLs, we have studied the GSL content of the two types of extracellular lipid particles (one enriched in unesterified cholesterol, the other enriched in esterified cholesterol) that can be isolated from atherosclerotic aortic intimal tissue, as reported earlier.21 More than 80% of cholesterol was unesterified in unesterified cholesterol–rich lipid particles and more than 80% of cholesterol was esterified in esterified cholesterol–rich lipid particles.

Tables 4Down and 5Down show the GSL content of aortic lipid particles, plasma LDLs isolated from patients with documented coronary artery disease, and cells isolated from the hyperplastic layer of atherosclerotic plaques. Unesterified cholesterol–rich lipid particles exhibited a higher content of neutral GSLs and GM3 compared with LDLs and a higher content of GlcCer, LacCer, and GM3 compared with intimal cells. A high content of GlcCer and a lower content of GM3 differentiated the GSL composition of the esterified cholesterol–rich lipid particles from that of LDLs. The GlcCer content of the esterified cholesterol–rich lipid particles was greater than the level observed in intimal cells. The content of most other GSLs in esterified cholesterol–rich lipid particles approximated that in LDLs. The molar composition of GSLs in each type of aortic lipid particles was constant for the samples of lipid particles prepared from the three different aortas (data not shown). The molar composition showed that GlcCer was relatively enriched in the aortic lipid particles compared with aortic intimal cells. The opposite was true for GbOse4Cer.


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  		Table 4. Neutral Glycosphingolipid Content and Composition of Esterified and Unesterified Cholesterol–Rich Particles Isolated From Human Aortic Intimal Tissue, Human LDLs, and Cells Isolated From Intimas of Human Aortas


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  		Table 5. Ganglioside Content and Composition of Esterified and Unesterified Cholesterol–Rich Particles Isolated From Human Aortic Intimal Tissue, Human LDLs, and Cells Isolated From Intimas of Human Aortas


*    Discussion
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 *Discussion
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Only a few studies have previously focused on elevated GSL content in atherosclerotic lesions, and these studies did not examine GSL content in defined layers of lesions.4 5 6 7 Therefore, the present investigation, together with our previous studies,8 9 10 11 can be regarded as the first detailed quantitative study on GSL accumulation in atherosclerotic aorta. The first finding is that ganglioside compositions of media and the hyperplastic (most luminal) layer of intima in grossly unaffected regions are different. The media contains much more GM1 and GD1a than the hyperplastic layer of intima. These gangliosides are more typical of nervous tissue, and their presence in the media may reflect electrical conduction within the media. This difference in ganglioside composition also raises the question as to whether intimal SMCs change their ganglioside composition when migrating from the media into the intima during the thickening of aorta vessel wall. Alternatively, possibly most intimal cells within the hyperplastic layer do not originate from medial SMCs. At the same time, the similarity of ganglioside composition of the normal media to that in the normal muscular layer but not that in the normal hyperplastic layer suggests that the muscular layer is occupied by SMCs less dedifferentiated than the SMCs in the hyperplastic layer.

GlcCer and LacCer are very low in comparison to other GSLs in normal intima but increase in atherosclerotic intima. We have observed a great variability of the content of all GSLs with the exception of GlcCer, LacCer, and GM3 in intimal tissue. This could be due to either individual variation or peculiarities of the atherosclerosis development process. Nevertheless, the tendency of GlcCer and LacCer as well as GM3 to accumulate in atherosclerotic lesions is evident. There is a decreased content of total gangliosides in the muscular layer of fatty streaks compared with unlesioned tissue and atherosclerotic plaque. This result agrees with our previous findings of ganglioside content for cells of fatty streaks compared with cells of unlesioned regions and atherosclerotic plaque.10 However, whether loss of gangliosides reflects atherosclerosis development or some other factors needs to be clarified. The increased amount of GD3 with unchanged GM3 in the media underlying atherosclerotic lesions is interesting because of recently published data on stimulation of angiogenesis by a change in the GD3:GM3 ratio.40 41 Perhaps this change in the GD3:GM3 ratio reflects the process of angiogenesis that occurs in atherosclerotic lesions.42

Another question arising in connection with the data obtained is whether GSL accumulation in intimal atherosclerotic lesions reflects passive accumulation of LDLs in the aortic wall, as suggested by Hara and Taketomi,7 or is an active process reflecting both influx and metabolism of GSLs in the aortic wall. On one hand, some findings support the possibility that GSL accumulation only reflects accumulation of LDLs in the aortic wall. First, GSL and LDL accumulation occurs in intima rather than in media. Second, GlcCer, LacCer, and GM3 are the major GSLs of LDLs,43 44 45 46 and accumulation of these particular GSLs takes place in the intima. On the other hand, there are some findings inconsistent with this possibility. First, LDLs also contain a number of other GSLs (including GbOse3Cer, GbOse4Cer, GD3, and GT1b43 44 45 46 ) whose content did not increase in atherosclerotic lesions (in particular, gangliosides GD3 and GT1b). Second, aorta samples from donors about 50 years old were used in the experiments. The percentage of the total phospholipids and neutral lipid classes in such aortas is close to that of LDL lipids.47 Despite the rise in the lipid level in atherosclerotic lesions, the percentage of phospholipids and neutral lipid classes remains the same.25 In other words, accumulation of total phospholipids, triglycerides, cholesterol, and cholesteryl esters in the aortic wall is consistent with progressive accumulation of LDL lipid. At the same time, the GSL composition of normal and atherosclerotic regions of the aorta varies greatly. Therefore, the data suggest that if LDLs are the source of GSLs in atherosclerotic lesions, there is a selective accumulation of GlcCer, LacCer, and GM3 from the LDLs. If the GSL profile results from aortic wall metabolism, it is possible that aortic cells either should have synthesized those GSL species that accumulate in the aortic wall or should have excreted various glycosidases, which are able to convert more complex GSLs entering the wall together with LDLs into more simple GSLs such as GlcCer, LacCer, and GM3.

The secondary elastic lamina that separates hyperplastic and muscular layers of the intima has been demonstrated to be important in limiting the progression of atherosclerotic changes.48 Cells that occupy the hyperplastic layer differ from cells composing the muscular layer.49 We have shown earlier that the changes in the composition of neutral lipids and in the thickness of the intima are mainly confined to the hyperplastic layer.25 In contrast to those findings, the changes in ganglioside composition shown in the present study are more general, affecting to different degrees both layers of the intima and the media. However, the uneven distribution of GSLs in the aortic layers indicates a different involvement of GSL metabolism during atherogenesis in these layers.

A comparative analysis of the GSL content in the hyperplastic layer and cells isolated from the hyperplastic layer revealed that most GSLs accumulate in the extracellular space of the aorta. GSLs, like other lipids in a water phase, should tend either to form vesicles or to incorporate into vesicles or liposomes formed by other lipids in the extracellular space of the aorta. The results of the present study demonstrate that extracellular aortic lipid particles, identified by a number of investigators,12 13 14 15 16 17 18 19 20 21 are one locus of this extracellular GSL accumulation (another possible locus for the accumulation of GSLs could be the plasma membranes of cells surrounding extracellular lipid particles). As demonstrated previously, aortic lipid particles appear in the aorta in a prelesional stage of atherogenesis.16 17 18 Thus, the accumulation of GSLs in the aorta, in addition to the accumulation of cholesterol, may prove to be an early biochemical event in the development of the atherosclerotic lesion.

Both types of lipid particles that can be isolated from the aorta, unesterified cholesterol–rich multilamellar liposomes and esterified cholesterol–rich droplets,21 contained GSLs. Previous chemical analysis indicates that the extracellular esterified cholesterol–rich lipid particles could be derived from extracellular deposition of LDLs.15 Chao et al50 have shown that unesterified cholesterol–rich multilamellar liposomes can be produced from LDLs with enzymatic hydrolysis of their cholesteryl ester core. It is possible that unesterified cholesterol–rich multilamellar particles are derived from LDLs, but only after LDLs have been processed by aortic cells. Schmitz, Robenek, and coworkers51 52 have shown that cholesterol-rich particles of this type can be secreted by cholesterol-rich macrophages after uptake of acetylated LDLs. However, the generally high content of GlcCer and GM3 detected in these particles in comparison to that in LDLs suggests that if these particles are derived from LDLs they must also acquire GlcCer from some other source. The concentration of GSLs in the cholesterol-rich aortic lipid particles may be determined by the availability of different GSLs through local synthesis within the blood vessel wall. There is a factor, in addition to synthesis of GSLs by vascular cells, that could influence the GSL profiles found associated with the isolated aortic lipid particles. The distribution of GSLs in these particles may reflect differences in association of various GSLs with other types of lipids depending on their hydrophobicity.

GSL accumulation in the aortic wall is very likely to be related to the phenomenon of sphingomyelin accumulation in the aortic wall during atherosclerosis.53 In addition to aortic lipid particles' content of cholesterol and GlcCer, sphingomyelin is the predominant phospholipid in these particles.21 Thus, these aortic lipid particles provide another example of the coaccumulation of cholesterol, sphingomyelin, and GSLs. There are some data available demonstrating that sphingolipid biosynthesis is closely associated with intracellular cholesterol metabolism.54 55 56 57 An association between the accumulation of cholesterol and GSLs has been observed in tissues of patients afflicted with the various genetic variants of Niemann-Pick disease.58 In this disease, unesterified cholesterol, sphingomyelin, and mainly GlcCer accumulate in tissues. However, further investigation is needed to elucidate the relationship between GSL and cholesterol accumulation during atherosclerosis.

The present study demonstrates the accumulation of GSLs in the atherosclerotic human aorta. The majority of GSL accumulates in the extracellular space of intima, and extracellular lipid particles are one locus of this extracellular GSL accumulation. The absolute level and relative distribution of GSLs within atherosclerotic and nonatherosclerotic regions and within different layers, as well as within isolated aortic lipid particles, probably reflect a number of factors that include (1) synthesis of GSLs within the vessel wall, (2) deposition of GSLs within the vessel wall from plasma-derived lipoproteins, (3) the degree of association of the various GSLs with intimal cells as well as extracellular lipid particles, and (4) metabolic relationships between cholesterol and GSL accumulation. GSLs present in the extracellular space could interact with vessel wall cells either by means of exchange between the lipid particles and intimal cell membranes59 or by uptake of the extracellular lipid particles by the intimal cells. As a result of such interactions, GSLs could potentially influence processes important in the development of atherosclerotic lesions. For example, exogenous LacCer stimulates the proliferation of SMCs,60 and can, like gangliosides, activate alternative complement pathways.61 62 Exogenous gangliosides, in turn, could interact with LDL particles, influencing their structure and resulting in their aggregation.63 Exogenous GM3 has been shown to influence the uptake of LDLs by peritoneal mouse macrophages and to enhance cholesterol accumulation in these macrophages.64 Gangliosides can favor the adherence, spreading, and aggregation of platelets.65 GSLs are known to influence cell growth and differentiation, transmembrane signaling, and cellular interactions.66 In addition, enhanced expression of GM3 in human aorta was recently shown to correlate with the process of calcification.67 One may assume that these properties of GSLs should influence atherosclerotic lesion development. Further investigation should be followed to study the relationship between changes in GSL composition in the aortic wall and other features of atherosclerotic lesion development such as modifications of LDLs within the aortic wall, intracellular lipid accumulation, cell proliferation, angiogenesis, attraction of blood-borne cells, and changes in SMC phenotype from contractile to synthetic.


*    Selected Abbreviations and Acronyms
 
GM3 = ganglioside GM3
GbOse3Cer = globotriaosylceramide
GbOse4Cer = globotetraosylceramide
GlcCer = glucosylceramide
GSL = glycosphingolipid
LacCer = lactosylceramide
SMC = smooth muscle cell


*    Acknowledgments
 
We thank Dr James Resau for providing some of the aortic tissue specimens and the USA-Russia Exchange Program in Cardiovascular Research for support of this work.

Received October 21, 1994; accepted July 13, 1995.


*    References
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