Accumulation of glycosphingolipids in human atherosclerotic plaque and unaffected aorta tissues

Subroto B.Chatterjee 0 1 Srabani Dey 0 1 Wan Yang Shi 0 1 Karl Thomas 0 1 Grover M.Hutchins 0 1 0 Hopkins Hospital , Baltimore, MD 21287-3654 , USA 1 Department of Pedialrics, Lipid Research Arteriosclerosis Unit and 'Department of Pathology, The Johns Hopkins University, School of Medicine , Baltimore, MD 21287-3654 , USA We have measured the levels of glycosphingolipids and the activity of glycosphingolipid glycosyltransferases in human aortic intima and media from patients who died of atherosclerosis. The effects of lactosylceramide (LacCer) and glucosylceramide (GlcCer) from plaque intima on smooth muscle cell proliferation were assessed. When the GlcCer data was expressed as (ug GlcCer/mg cholesterol and/mg total phospholipid, a 28-fold and 7-fold increase in plaque intima compared to normal intima was observed. Similarly, the level of LacCer was elevated 5-fold and 4-fold, respectively, compared to unaffected intima. The activity of UDPGlcCer: ceramide pi->4 glucosyltransferase (GlcT-1) was similar in unaffected tissue, fatty streaks, and plaques. However, the activity of UDP-galactose: GlcCer, p i - 4 galactosyltransferase (GalT-2) activity was moderately higher in plaque than in unaffected tissue. LacCer, but not GlcCer derived from plaque intima exerted a -2.8-fold increase in the proliferation of human aortic smooth muscle cells grown in tissue culture compared to control presumably due to a marked increase in LacCer molecular species containing C16:0, C22:l, and C24:0 fatty acids in plaque intima compared to control. In sum, our findings provide an interesting and novel pathogenic mechanism of lactosylceramide mediated plaque formation via stimulation of aortic smooth muscle cell proliferation. Introduction Atherosclerosis is the number one killer of mankind in developed and rapidly developing countries (Ross, 1993). Although, this is a multifactorial disease involving gross changes in the biology of the vascular wall, the proliferation of aortic smooth muscle cells is considered a hallmark in the pathogenesis of atherosclerosis. Our laboratory has recently shown that exogeneously added oxidized human plasma low density lipoproteins (Ox-LDL) and lactosylceramide both can independently stimulate the proliferation of aortic smooth muscle cells grown in tissue culture (Chatterjee, 1991, 1992). Moreover, our studies indicate that Ox-LDL can exert a time and concentrationdependent stimulation of the activity of a galactosyltransferase implicated in the synthesis of lactosylceramide (Chatterjee and Ghosh, 1996). Accordingly, it was deemed reasonable to investigate the glycosphingolipids (GSLs) in affected and unaffected human aorta tissue of individuals who died with atherosclerosis at The Johns Hopkins Hospital. To assess the pathophysiological role of intima GSLs, we have measured their effects on the proliferation of cultured human aortic smooth muscle cells. This investigation of a limited number of samples suggests that glycosphingolipid levels, specifically glucosylceramide and lactosylceramide, are markedly higher in regions of intima tissue with visible plaque development than in areas free of plaque. Moreover, LacCer from affected intima stimulated the proliferation of cultured human aortic smooth muscle cell. However, the stimulation by plaque LacCer was several fold higher. Results Comparison of content of glycosphingolipids in intima and media in unaffected (normal), and plaques from patients with atherosclerosis on the basis of cholesterol and phospholipids Since cholesterol and phospholipids are integral components of membranes as well as plaque, we have expressed our GSL data on the basis of levels of these lipids in affected and unaffected aortic tissues. The levels of GlcCer in plaque intima was elevated 28-fold (Figure 1) and 7-fold (Figure 2), respectively, compared to unaffected intima. The level of LacCer was elevated in plaque intima 5-fold (Figure 1) and 4-fold (Figure 2), respectively, compared to unaffected intima. The levels of GbOs^Cer were decreased in plaque intima compared to unaffected intima when data was expressed as p-g/mg cholesterol (Figure 1), but increased when expressed as (ig/mg phospholipid (Figure 2). Interestingly, when the GbOse4Cer data was expressed/mg cholesterol or phospholipid, none was found in the intima, but a marked increase was found in plaque media as compared to unaffected media. Free cholesterol and phospholipid contents of affected and unaffected aortic intima, media tissues are presented in Table I. Glycosphingolipid composition of human platelets, monocytes, human aortic smooth muscle cells, and oxidized LDL Human platelets contained GlcCer, LacCer, GbOsejCer, and GbOse4Cer. The level of these GSLs were in descending order (Figure 3). In contrast, in monocytes GlcCer was the predominant GSL and the levels of LacCer and GbOsejCer were almost similar. However, in monocytes the level of GbOse4Cer was about 2-fold higher than LacCer and G b O ^ C e r (Figure 3). In normal human aortic smooth muscle cells, GlcCer, LacCer and GbOse3Cer were the predominant GSL. The level of CALCIFIED PLAQUE CALCIFIED PLAQUE 1 1 II 1 1 ^ GbOSe4Cer CALCIFIED PLAQUE LacCer in these cells was about 14-fold lower than GlcCer. Barely detectable level of GbOse4Cer was found in smooth muscle cells (Figure 3). Human plasma Ox-LDL had about 3-fold higher levels of GlcCer than LacCer. The level of GbOsejCer was very low and GbOse4Cer was not detectable in Ox-LDL (Figure 3). Analysis of TMSi-sugars by GC-mass spectrometry GC-mass spectrometric analysis of sugars derived from lactosylceramide revealed a ratio of approximately 1:1 for galactose and glucose. The mass chromatograms were similar to standard TMSi-glucose and TMSi galactose. They contained ion m/z 204 and common ions representative of all sugars (data not shown). Distribution of fatty acids in LacCer derived from normal (unaffected) intima, plaque and calcified plaque intima As shown in Figure 4 (top and bottom panels), we found that compared to LacCer derived from normal (unaffected tissue) intima, the LacCer from plaque intima contained a higher level of C16:0, C22:0, C22:l, and C24:0 fatty acids. In addition, the levels of fatty acids such as C18:0 and C18:l were markedly CALCIFIED PLAQUE CALCIFIED PLAQUE GbOse4Cer CALCIFIED PLAQUE CALCIFIED PLAQUE INTIMA MEDIA decreased in LacCer from plaque intima compared to unaffected intima LacCer. Moreover, several fatty acids present in LacCer of normal tissue intima were absent in LacCer derived from plaque intima. These fatty acids were C16:l, C18:2, C20: 1, and C17:0 (Figure 4, top and bottom panels). Distribution offatty acids in LacCer derived from platelets, monocytes, and oxidized LDL The predominant fatty acids in LacCer of these samples were C16:0, C18:0 and C20:4 and the minor fatty acids were C14:0, C17:0, C18:l, C18:2, and C24:0 (Figure 5). In general, the fatty acid composition of LacCer derived (...truncated)