Sphingolipids in Atherosclerosis and Vascular Biology

From the Lipid Research Atherosclerosis Division, Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, Md.

Correspondence to Subroto Chatterjee, Lipid Research Atherosclerosis Division, Department of Pediatrics, Johns Hopkins University School of Medicine, 600 N Wolfe St, CMSC 6-124, Baltimore, MD 21287-3654. E-mail chatter{at}welchlink.welch.jhu.edu

Abstract

Abstract—Sphingolipids and their metabolic products are now known to have second-messenger functions in a variety of cellular signaling pathways. Lactosylceramide (LacCer), a glycosphingolipid (GSL) present in vascular cells such as endothelial cells, smooth muscle cells, macrophages, neutrophils, platelets, and monocytes, contributes to atherosclerosis. Large amounts of LacCer accumulate in fatty streaks, intimal plaque, and calcified intimal plaque, along with oxidized low density lipoproteins (Ox-LDLs), growth factors, and proinflammatory cytokines. A possible role for LacCer in vascular cell biology was suggested when this GSL was found to stimulate the proliferation in vitro of aortic smooth muscle cells (ASMCs). A further link of LacCer in atherosclerosis was uncovered by the finding that Ox-LDLs stimulated specifically the biosynthesis of LacCer. Ox-LDL–stimulated endogenous synthesis of LacCer by activation of UDP-Gal:GlcCer,ß1-4galtransferase (GalT-2) is an early step in this signaling pathway. In turn, LacCer serves as a lipid second messenger that orchestrates a signal transduction pathway, ultimately leading to cell proliferation. This signaling pathway includes LacCer-mediated activation of NADPH oxidase that produces superoxide. Such superoxide molecules stimulate the GTP loading of p21^ras. Subsequently, the kinase cascade (Raf-1, Mek2, and p⁴⁴MAPK [mitogen-activated protein kinase]) is activated. The phosphorylated form of p⁴⁴MAPK translocates from the cytoplasm to the nucleus and engages in c-fos expression, proliferating cell nuclear antigen (PCNA) such as cyclin activation, and cell proliferation takes place. Interestingly, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), an inhibitor of GalT-2, can abrogate the Ox-LDL–mediated activation of GalT-2, the signal kinase cascade noted above, as well as cell proliferation. Additional studies have revealed that LacCer mediates the tumor necrosis factor- (TNF-)–induced nuclear factor-B expression and intercellular adhesion molecule (ICAM-1) expression in vascular endothelial cells via the redox-dependent transcriptional pathway. LacCer also stimulates the expression of CD11/CD8, or Mac-1, on the surface of human neutrophils. Collectively, this phenomenon may contribute to the adhesion of neutrophils or monocytes to the endothelial cell surface and thus initiate the process of atherosclerosis. In addition, the LacCer–mediated proliferation of ASMCs may contribute to the progression of atherosclerosis. On the other hand, programmed cell death (apoptosis) by proinflammatory cytokines such as TNF-, interleukin-1, and high concentrations of Ox-LDL occur via activation of a cell membrane–associated neutral sphingomyelinase (N-SMase). N-SMase hydrolyzes sphingomyelin into ceramide and phosphocholine. In turn, ceramide or a homologue serves as an important stress-signaling molecule. Interestingly, an antibody against N-SMase can abrogate Ox-LDL– and TNF-–induced apoptosis and therefore may be useful for in vivo studies of apoptosis in experimental animals. Because plaque stability is an integral aspect of atherosclerosis management, activation of N-SMase and subsequent apoptosis may be vital events in the onset of plaque rupture, stroke, or heart failure. Interestingly, in human liver cells, N-SMase action mediates the TNF-–induced maturation of the sterol regulatory-element binding protein. Moreover, a cell-permeable ceramide can reconstitute the phenomenon above in a sterol-independent fashion. Such findings may provide new avenues for therapy for patients with atherosclerosis. The findings described here indicate an important role for sphingolipids in vascular biology and provide an exciting opportunity for further research in vascular disease and atherosclerosis.

Key Words: neutral sphingomyelinase • lipoproteins • cell proliferation • signal transduction • apoptosis

Thudichum¹ first discovered cerebroside from the human brain in 1884. A characteristic component of this and all glycosphingolipids (GSLs) is an aliphatic amino alcohol, sphingosine. Thudichum called this compound sphingosine because of its enigmatic chemical nature (containing both amine and alcohol groups, but insoluble in water), referring to the Sphinx of Greek mythology who posed riddles. Since that time, most of the studies on sphingoglycolipids have been pursued in regard to determining their structure, biosynthesis, and degradation. These compounds might have been forgotten had it not been shown that the inability to catabolize certain GSLs resulted in their accumulation in various tissues, leading to metabolic diseases,² collectively called the "glycosphingolipidoses." Previous studies suggested that because most of the sphingoglycolipids are localized on the cell surface, they might serve as cell surface markers and antigens, eg, blood group ABO, P, and Lewis systems heterophile Forsmann antigen and tumor-associated antigens. Because ceramide confers relatively more structural rigidity than diacylglycerol, the GSLs may impart rigidity to the cell membrane as well as interact with exogenous ligands via carbohydrate components.³ Today, GSLs have been implicated in regulating various aspects of cell phenomena.

In this article, I will focus primarily on the potential role of lactosylceramide (LacCer) in atherosclerosis and vascular biology. Moreover, the possible role of sphingomyelin hydrolysis via neutral sphingomyelinase (N-SMase), the subsequent generation of ceramide, and the potential implication of this pathway in various signal transduction reactions, particularly apoptosis, will be addressed. The role of more complex sphingolipids and complex GSLs has been reviewed recently by others.^{3 4 5 6 7} This review is written to point out the potential role of sphingolipids in atherosclerosis and vascular biology.

Sphingolipids: Structure, Biosynthesis, Degradation, and Localization

Sphingoglycolipids are compounds that contain a long-chain fatty alcohol amine called sphingosine. Sphingosine and dehydrosphingosine are typical mammalian sphingols. The NH₂ group in sphingosine is usually acylated by a fatty acid or a 2-hydroxy fatty acid to form ceramide. The most common fatty acid in GSLs varies, depending on the type of GSL and source. For example, brain cerebroside is C20-C24 whereas, brain ganglioside is C16-C18; erythrocyte globoside is C20-C22. In contrast, the most common fatty acids in sphingomyelin are C16-C18. The addition of sugar residues to ceramide forms GSLs. The GSLs may be divided into at least 3 subclasses: (1) neutral GSLs that contain ceramide and at least 1 sugar residue (glucosylceramide and LacCer are among the most commonly occurring neutral glycolipids present in almost all tissues); (2) gangliosides that contain ceramide and sugars, as well as a sialic acid (N-acetylneuraminic acid) and/or N-glycolylneuraminic acid; and (3) sulfatides that contain ceramide, sugars, and a sulfate. The general classification of GSLs is also based on differences in core structure, such as the lactoseries (R-GlcNAcß1GlcCer); the globoseries (R-Gal14GlcCer); and the gangliosides (R-GalNAcß14GlcCer). The structure of various GSLs found in vascular wall cells is presented in Table 1. The detailed structure of LacCer and sphingomyelin is shown in Figure 1. Altogether, the structure of 60 such GSLs have been published, and it is anticipated that, given the possibility of isomerization, >2000 of such sphingoglycolipids may exist in nature.

View larger version (15K): [in this window] [in a new window]   Figure 1. A, Structure of lactosylceramide, a glycosphingolipid. B, Structure of sphingomyelin, a sphingolipid.

The biosynthesis of GSLs is initiated by the condensation of a common amino acid, serine, with a fatty acyl coenzyme (CoA), usually palmitoyl-CoA, to form 3-ketodihdyrosphingosine (Figure 2). The enzyme that catalyzes this reaction is called serine fatty acyltransferase (EC 2.3.1.5.0). The product formed is called 3-ketosphingananine. On the reduction of 3-ketosphinganine in the presence of NADPH, D-erythro-3-ketodihydrosphingosine is generated. Next, dehydrosphingosine is acylated in the presence of fatty acyl CoA to form D-erythro-dihydroceramide. Next, D-erythro-dihydroceramide is oxidized to form D-erythroceramide.^{8 9} The double bond in the D-erythroceramide molecule is critical to its function in initiating apoptosis, as recent studies have revealed that D-erythrodihydroceramide that does not contain the double bond failed to induce apoptosis in cultured mammalian cells.¹⁰

View larger version (31K): [in this window] [in a new window]   Figure 2. Pathway of sphingolipid metabolism.

Ceramide has several fates. Most commonly, ceramide is utilized for the synthesis of sphingomyelin from phosphatidylcholine (PC) via the enzyme choline phosphotransferase. Second, ceramide may be phosphorylated to form ceramide-1-phosphate. Similarly, sphingosine may be phosphorylated to form sphingosine-1-phosphate and subsequently cleaved to hexadecanol and hydrolyzed to a fatty acid and ethanolamine phosphate. Also, sphingosine may be methylated to form compounds such as N,N'-dimethylsphingosine. Sphingosine can also be glycosylated to form galactosylsphingosine, which is present in brain tissue. Third, the sequential transfer of sugar residues from nucleotide sugars to ceramide is an essential step in the GSL biosynthesis.¹¹ For example, the transfer of glucose from UDP-glucose via UDP-glucose:Cer,ß1-4glucosyltransferase (GlcT-1) results in the synthesis of glucosylceramide (GlcCer). The addition of a galactose residue from UDP-galactose to GlcCer results in the synthesis of LacCer. The enzyme UDP-galactose:GlcCer,ß1-4galtransferase (GalT-2) and/or LacCer synthase catalyzes the synthesis of LacCer. These GSL glycosyltransferases are localized within the Golgi apparatus.¹¹ Increased activity of GalT-2 and increased levels of LacCer in familial hypercholesterolemia and in atherosclerosis has been reported.^{12 13} The levels of other GSLs in fatty streaks and plaques have also been reported.^{13 14} Nevertheless, up to now, a human metabolic disease due to the abnormal biosynthesis of GSL has not been shown. In fact, the biochemical basis of several inherited sphingoglycolipidoses is due to the inability of these patients to hydrolyze various GSLs and sphingomyelin.^{15 16 17}

The catabolism of GSLs occurs predominantly by lysosomally localized enzymes. LacCer and GlcCer are hydrolyzed sequentially by the removal of sugar residues, such as galactose and glucose, respectively, via specific exoglycosidases that are lysosomal in origin.^{2 15} The lack of ß-glucocerebrosidase activity in Gaucher's disease results in the accumulation of GlcCer in the spleen in these patients.¹⁵ The deficiency/absence of ceramidase results in the accumulation of ceramide in neuronal and visceral organs in Farber's disease.¹⁶ Recently, the en bloc hydrolysis of the oligosaccharides from GSLs via enzymes called ceramide glycanases has been reported.^{18 19} Metabolic disorders in mammals due to the lack of this enzyme have not been found. Sphingomyelin is hydrolyzed by a lysosomal SMase called acid SMase as well as a cell membrane–associated Mg²⁺-active SMase, termed N-SMase, because it prefers a neutral pH for optimal activity.^{20 21} Deficiency and/or a lack of acid SMase activity in Neimann-Pick disease results in marked neuropathy and death.¹⁷ Sphingomyelin may also be hydrolyzed by a fatty acyl hydrolase (phospholipase D) to generate lysosphingomyelin. Thus, all sphingolipids are catabolized to ceramide. Ceramide is hydrolyzed by ceramidase to sphingosine and fatty acid. Various structural analogues of sphingoglycolipids available recently have been used to study the role of these compounds (Figure 1; see below).

Although the general belief is that sphingoglycolipids are predominantly localized on the outer leaflet of cell membranes, more evidence is needed to support this notion. GSL appears to occur in a cluster form, as judged by electron microscopy of ferritin-conjugated antibodies and gold-conjugated lectins applied to freeze-fractured cell surface and artificial liposome membranes.²² In studies by us²³ and others,²⁴ LacCer was predominantly found localized within cytoplasmic vesicles inside the cells, although some LacCer is always available on the surface of cells. This has important implications for the various biological phenomena that LacCer may impart. On the other hand, 90% of the sphingomyelin is localized on the outer leaflet of the cell membrane.²⁵ Phosphocholine, the polar head group of sphingomyelin, probably projects out toward the exterior of the cell, and the nonpolar component ceramide of sphingomyelin is "buried" in the lipid bilayer.

Distribution of GSLs in Lipoproteins and Vascular Cells

Earlier studies revealed that several of the GSLs shown in Table 1 were found in plasma.²⁶ Subsequent studies revealed that GSLs do not exist in a free form in the plasma; rather, they are associated with lipoproteins.^{27 28} The total amounts of GSLs (in nanomoles per deciliter) in plasma lipoproteins in normal subjects were as follows: VLDL, trace to 0.46; LDL, 1.08 to 1.48; HDL₂, 0.62 to 0.85; and HDL₃, trace to 2.28. Interestingly, in subjects with high Lp(a) lipoprotein, HDL₂ and HDL₃ contained most of the GSLs in HDL. When the data were expressed in regard to GSLs (nanomoles of glucose per milligram of lipoprotein cholesterol), the values were as follows: VLDL, trace to 21.2; LDL, 11.7 to 15.3; HDL₂, 8.5 to 9.1; and HDL₃, 3.1. GSLs were not detected in the lipoprotein-deficient plasma.²⁹ Elevated levels of GSLs (2- to 4-fold) in homozygous familial hypercholesterolemic subjects have been reported. It would appear that most, if not all, GSLs are associated with LDL in normal individuals as well as in patients with familial hypercholesterolemia and in glycosphingolipidoses.^{27 28} GSLs are not covalently bound²⁹ but are associated to lipoproteins, as they are secreted from the liver. Thus, GSLs may be exchanged among lipoproteins, between cells and lipoproteins, or vice versa, via shedding and/or receptor-mediated endocytosis, respectively. The distribution of GSLs in vascular cells, fatty streaks, and calcified plaques are summarized in Table 1.

Regulation of Sphingoglycolipid Metabolism by Lipoproteins

An interesting feature regarding the regulation of GSLs was the finding that lipoproteins such as LDL exerted a time- and concentration-dependent inhibition of LacCer synthesis,³⁰ such as [³H]serine, [³H]galactose, [³H]glucose, and [³H]palmitate that are precursors of GSL biosynthesis. For example, LDL inhibited the incorporation of several precursors into LacCer. Furthermore, when LDL (10 µg/mL) was added to cultured human kidney cells, fibroblasts, Chinese hamster ovary cells, and ASMCs, the activities of the various enzymes in the biosynthetic pathway (Figure 2) of simple and complex GSLs were analyzed, LDL specifically suppressed the activity of GalT-2.^{31 32 33 34} LDL also decreased the level of LacCer in normal human kidney cells.³⁰ Immunocytochemical studies using anti-LacCer antibodies conjugated with FITC, revealed diminished staining in LDL-treated normal cells compared with controls.³³

Because the kinetics of inhibition of GalT-2 activity by LDL mimicked the inhibition of 3-hydroxy-3-methylglutaryl (HMG) CoA reductase activity by LDL in normal cultured skin fibroblasts, coordinate regulation of LacCer synthesis and cholesterol synthesis by LDL³⁵ was postulated.^{34 35 36} The regulation of LacCer biosynthesis by LDL was also made in a number of other diploid cells, such as ASMCs, renal tubular cells, and Chinese hamster ovary cells.³⁰ Furthermore, LDL binding, internalization, and degradation were essential to LDL-mediated downregulation of GalT-2 activity, implying an important role for LDL receptors in this process.³⁴ The effect of LDL on LacCer metabolism was not simply related to the fact that LDL was the major carrier in blood of LacCer, since HDL, which also has significant amounts of LacCer, did not inhibit the cellular synthesis of LacCer. The lipid components in LDL, including cholesterol, serine, fatty acids, and several phospholipids, failed to suppress the activity of GalT-2 in cultured cells.³⁷ LDL from familial hypercholesterolemic homozygotes contained 2- to 4-fold higher levels of GSLs, including LacCer, suppressed the activity of GalT-2 (in normal cells having functional LDL receptors) to a similar extent as normal LDL. These studies together suggested that the apoprotein B moiety, through its interaction with the LDL receptors, was a necessary step for the suppressive effect of LDL on the activity of GalT-2.

While the presence of LDL receptors in normal cells is required for the downregulation of LacCer biosynthesis by LDL, in sharp contrast, the absence of LDL receptors results in the lack of regulation of LacCer biosynthesis. Several lines of evidence support this tenet. When fibroblasts from LDL receptor–negative fibroblasts from familial hypercholesterolemic homozygotes were incubated with LDL, the activity of GalT-2 increased in vitro and in urinary epithelial cells derived from these patients.^{31 34} In human kidney tumor cells (that lack LDL receptors), LDL markedly stimulated the activity of GalT-2 and LacCer biosynthesis.³⁸ In contrast, LDL downregulated GalT-2 activity and LacCer synthesis in normal kidney proximal tubule cells.^{30 36} When the lysine residues of apolipoprotein B of LDL are blocked by reductive methylation, LDL is unable to enter the cells through the LDL receptor. Thus, methylated LDL internalized via an LDL receptor–independent pathway (scavenger pathway) markedly stimulated the activity of GalT-2, indicating that the entry of modified LDL into the cells through the scavenger pathway leads to an upregulation rather than a downregulation of LacCer production.³⁴

Consequences of LDL Oxidation on Sphingoglycolipid Metabolism, Cell Proliferation, and Cell Death in Atherosclerosis

Previous studies have shown that while a major role of LDL involves the normal delivery of cholesterol to extrahepatic tissue via the LDL (B,E) receptor, elevated levels of plasma and LDL cholesterol promote atherosclerosis via an increased uptake of LDL and/or Ox-LDL via the scavenger pathway.³⁵ Studies in human subjects and in experimental animal models have postulated the role of Ox-LDL in atherosclerosis. Ox-LDL has been found in atherosclerotic plaque.³⁹ Probucol, a potent antioxidant, abrogated LDL oxidation and plaque formation in experimental animals.⁴⁰ After interaction of LDL with endothelial cells⁴¹ and smooth muscle cells,⁴² LDL was oxidized. Such modified LDL promoted foam cell formation by stimulating cholesteryl ester accumulation in macrophages and in ASMCs.

However, these studies did not elaborate the biochemical mechanisms involved in the oxidation of LDL. Furthermore, knowledge on the potential role of Ox-LDL in ASMC proliferation, a "hallmark in the pathophysiology in atherosclerosis," was lacking. Studies in our laboratory with human plasma Ox-LDL (modified by copper sulfate) revealed a dual physiological effect of Ox-LDL in cultured ASMCs. At low concentrations (5 to 10 µg/mL), Ox-LDL exerted a 2- to 3-fold stimulation of cell proliferation, as indicated by viable cell count and [³H]thymidine incorporation into DNA. Ox-LDL had little or no effect on the release of LDH activity, indicating that Ox-LDL at low concentrations was not toxic to the cells. In contrast, at high concentrations (50 to 100 µg/mL of Ox-LDL), there was a marked increase in LDH release in the culture medium, indicating that membrane integrity had been compromised.⁴³ This was substantiated by a marked decrease in [³H]thymidine incorporation and a total loss of cell viability. These preliminary experiments set the stage indicating that Ox-LDL may have a dual role in atherosclerosis. First, low levels of circulating Ox-LDL may be involved in the proliferation of ASMCs during the early stages of atherosclerosis. On the other hand, in advanced stages of atherosclerosis, when Ox-LDL is in abundance in plaques, it may induce programmed cell death (apoptosis) in ASMCs that in turn may lead to plaque rupture and the clinical sequelae of atherosclerosis (see below).

Role of Glycosphingolipids in Cell Proliferation

Effect of Ox-LDL on GalT-2 and LacCer
An important link between Ox-LDL, LacCer, and cell proliferation was revealed when it was found that Ox-LDL exerted a time- and concentration-dependent stimulation both on the activity of GalT-2 and on endogenous LacCer biosynthesis (Figure 3).⁴⁵ This appeared to be a specific effect of Ox-LDL, since Ox-LDL did not stimulate the activity of GlcT-1 and/or the activities of other relevant catabolic enzymes in GSL metabolism (Figure 1). Preincubation of ASMCs with an antibody against either Ox-LDL or GalT-2 compromised Ox-LDL–mediated activation of GalT-2 and cell proliferation.⁴⁵ Next, it was shown that LacCer itself markedly stimulated the proliferation of ASMCs.⁴⁶ To substantiate these studies further, we used an inhibitor D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) (Figure 2). Although, D-PDMP was suggested to be a specific target for GlcT-1,⁴⁴ subsequent studies have revealed that it is a nonspecific inhibitor of a number of GSL glycosyltransferases. Accordingly, it was not surprising to observe that D-PDMP inhibited the activity of both GlcT-1 and GalT-2 in ASMCs. Moreover, D-PDMP abrogated the Ox-LDL–mediated stimulation of cell proliferation in ASMCs.⁴⁷ The inhibition of this pathway was bypassed by the exogenous addition of LacCer, but not GlcCer. On the other hand, L-PDMP activated GalT-2 and stimulated ASMC proliferation. These observations suggest that GalT-2 is the target for Ox-LDL action. LacCer produced as a consequence of GalT-2 activation is recruited as a lipid second messenger in inducing cell proliferation.⁴⁷ A hypothetical model depicting such reactions is presented in Figure 4.

View larger version (14K): [in this window] [in a new window]   Figure 3. Time-activity plot for GalT-2.

View larger version (17K): [in this window] [in a new window]   Figure 4. Hypothetical model depicting how oxidized LDL Ox-LDL utilizes lactosylceramide (LacCer), as a lipid second messenger in the proliferation of human aortic smooth muscle cells.

LacCer, Superoxide, and Second-Messenger Pathways
The studies above suggested a potential role of LacCer as a lipid second messenger in mediating Ox-LDL–induced ASMC proliferation. However, whether LacCer itself or whether some additional molecule recruited by LacCer to transduce the signaling phenomena was not discerned from these studies. A class of highly diffusible and ubiquitous molecules, termed reactive oxygen species (ROS), has recently been recognized to act as signaling intermediates for proinflammatory cytokines including interleukin-1 and tumor necrosis factor- (TNF-).⁴⁸ ROS encompasses species such as superoxide (O₂^-), H₂O₂, NO, and hydroxyl radicals.⁴⁹ Oxidative stress, which is an excess production of ROS, plays a role in different pathological conditions, such as atherosclerosis and cancer.^{50 51} In addition, O₂^- has numerous effects on cell function, including induction of growth, regulation of kinase activity, and inactivation of endothelium-derived relaxation factor, NO.^{52 53} Thus, superoxide and its metabolites can function as intracellular and intercellular second messengers, transducing receptor stimulation into biochemical response. Because they are very small, rapidly diffusible, and highly reactive, free radical and redox stresses are now thought to participate in cellular signaling.^{52 54 55} Current evidence indicates that different stimuli use ROS as signaling messengers to activate transcription factors and induce gene expression.^{56 57}

Our studies revealed that incubation of ASMCs with LacCer stimulated the activity of NADPH oxidase. This, in turn, resulted in a 4-fold stimulation in the production of O₂- (Figure 5), but not of NO. Such superoxide radicals in turn stimulated Ras GTP loading, that in turn stimulated the kinase cascade involving Raf, Mek-2, and p⁴⁴ mitogen-activated protein kinase (p⁴⁴MAPK) activity (References 58 and 59^{58 59} and Figure 4). The activation/phosphorylation of p⁴⁴MAPK by LacCer resulted in the transition of p⁴⁴MAPK from the cytoplasm to the nucleus. On reaching the nucleus, the phosphorylated form of p⁴⁴MAPK stimulated the expression of c-fos, a proto-oncogene intimately involved in cell proliferation (References 58 and 59^{58 59} and Figure 4). Furthermore, additional studies revealed that both Ox-LDL, LacCer, or both could stimulate the expression of proliferating cell nuclear antigen (PCNA), also known as cyclin, in cultured ASMCs. Moreover, D-PDMP inhibited the Ox-LDL–mediated stimulation in the expression of PCNA in these cells.⁶⁰

View larger version (19K): [in this window] [in a new window]   Figure 5. Bar graph of superoxide production.

The involvement of LacCer in superoxide generation, Ras GTP loading, and cell proliferation was substantiated by the judicious use of a variety of inhibitors known to abrogate these reactions. For example, the LacCer-induced NADPH oxidase activity was abrogated by preincubation of cells with diphenylene iodonium (DPI), an inhibitor of NADPH oxidase. In contrast, inhibitors of NADH oxidase (KCN) had no effects on this reaction. Similarly, allopurinol, an inhibitor of xanthine oxidase, and an NO synthase inhibitor, N-methylarginine, did not abrogate LacCer-induced superoxide generation and cell proliferation. Glutathione, on the other hand, decreased the LacCer-induced phenomena above. LacCer-induced O₂^- generation or cell proliferation was not dependent on intact protein kinase C (PKC),⁵⁸ since inhibitors of PKC, staurosporine and phorbol myristoyl acetate, did not prevent those effects of LacCer. Interestingly, neither the inhibitors mentioned above nor Cu²⁺ altered the activity of GalT-2 in ASMCs. On the other hand, preincubation of cells with LacCer and buthionine sulfoximine, an inducer of superoxide generation that by itself stimulates cell proliferation, caused further induction of superoxide generation, and Ras GTP-loading and cell proliferation were observed. In contrast, inhibitors of NADPH oxidase, such as DPI and PDTC, inhibited the Ras GTP loading, p⁴⁴MAPK phosphorylation, and the proliferation of ASMCs.⁵⁸ These findings are in agreement with a recent report that probucol can markedly decrease the proliferation of ASMCs in patients who have undergone balloon angioplasty.⁶¹

In summary, Ox-LDL stimulates the activity of GalT-2; this in turn produces LacCer. Our recent finding that cells have significant GalT-2 activity associated with the plasma membrane and the rapid kinetics of GalT-2 activation by Ox-LDL suggest that such reactions may occur on or at the cell surface. LacCer activates NADPH oxidase, resulting in rapid generation of superoxide (Figure 3). This phenomenon may be due to LacCer-mediated stimulation of the translocation of cytosolic proteins p47^phox and a GTP binding protein to the cell surface. Once superoxides are generated, they activate the entire signaling process involving Ras GTP loading, activation of the kinase cascade, proto-oncogene activation, PCNA expression, and cell proliferation (Figure 4). Thus, free radicals are intimately involved in LacCer-induced cell proliferation of ASMCs. Moreover, since structural homologues of LacCer such as GlcCer, GbOse₃Cer, GM₃, and ceramide do not induce O₂^- generation in ASMCs, the focus is on GalT-2 as the potential target for the action of Ox-LDL in this signaling pathway.

In Vivo Oxidation of LDL and Its Consequences on ASMC Proliferation

Because LDLs are in close proximity to erythrocytes in the circulation, further studies were pursued to determine the effect of oxidative modification of LDL on reaction with red blood cells and hemoglobin under hypoxic conditions on ASMC proliferation. The increased oxidation of hemoglobin under reduced oxygen pressure is due to a high population of double E and triple E liganded states of hemoglobin, which oxidize more rapidly than does fully liganded oxyhemoglobin. Hemoglobin is also released during bleeding episodes. Moreover, immediately after myocardial infarction, there is an increase in myoglobulin in the circulating blood from ruptured cardiomyocytes.^{62 63} Increased local generation of H₂O₂ has also been found from superoxides at the site of injury, which may interact with myoglobulin and hemoglobin. In fact, the activities of superoxide radical and H₂O₂ are amplified by the availability of transition metal ions, such as iron derived from hemoglobin or myoglobin, or copper. Using oxygen levels of 60% to 70% as are found in the venous circulation, we discovered that LDL is more rapidly modified by red cells and hemoglobin under hypoxic conditions compared with Cu²⁺-mediated LDL oxidation. When such minimally modified LDL was added to cultured ASMCs, it markedly increased cell proliferation that was considerably greater than that seen with LDL modified with Cu²⁺.⁶³ Since LDL may have been modified by the hemoglobin, hypoxic hemoglobin, or normoxic hemoglobin, we pursued further studies in which we investigated several of the LDLs modified with different hemoglobin-based preparations, including fully oxygenated normoxic hemoglobin, partially oxygenated hypoxic hemoglobin, and oxidized hemoglobin, in addition to Cu²⁺-modified LDL.⁶³ These studies revealed that minimally modified LDL as derived above markedly stimulated the phosphorylation of p⁴⁴MAPK and increased ASMC proliferation. Incubation of ASMCs with 10 µg/mL of normoxic hemoglobin–modified LDL, hypoxic hemoglobin–modified LDL, and Cu²⁺-oxidized LDL all stimulated the activity of p⁴⁴MAPK on the order of 2.5-fold.⁶³ Unmodified LDL did not affect MAPK activity in these cells. This phenomenon was accompanied by a concentration-dependent stimulation in [³H]thymidine incorporation (an index of cell proliferation) of ASMCs.⁶³ At the present time, the significance of LDL modification by the above mediators is not clearly understood. Nevertheless, it can be assumed that in situations such as restenosis or inflammation when erythrocyte lysis occurs, LDL modification by interactions with hemoglobin or methemoglobin may contribute to additional superoxide generation and ASMC proliferation.

Identification of a Biologically Active Compound in MM-LDL That Mediates Cell Proliferation Via GalT-2, LacCer, and p⁴⁴MAPK

Recently, the molecular structures of molecules present in minimally modified LDL, which induce the interaction of monocytes to endothelial cells, have been reported.⁶⁴ These are 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phospho-choline (POPVC; m/z, 594.3), 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphatidylcholine (PGPC; m/z, 610.2), and 1-palmitoyl-2-isopentanoyl-sn-glycero-3-phosphocholine (PIPPC; m/z, 828.6). These molecules were found in fatty streak lesions from cholesterol-fed rabbits. In a collaborative study, it was found that among the molecules above, only POPVC exerted a time- and concentration-dependent stimulation of GalT-2, activation/phosphorylation of p⁴⁴MAPK, and proliferation of ASMCs (S.C., unpublished observations, 1998). Interestingly, in ASMCs, D-PDMP abrogated the signal transduction cascade initiated by POPVC. It remains to be determined whether POPVC binds to a cell surface receptor, is internalized, and then exerts its effect on the Golgi GalT-2, or whether it reacts directly with the cell surface GalT-2. Clearly, further work is required to elaborate the biochemical mechanisms in POPVC-induced GalT-2 activation and ASMC proliferation. Nevertheless, these studies define the biologically active compound in Ox-LDL and minimally modified LDL in ASMC proliferation involving LacCer as a lipid second messenger. Our following observations support the tenet that POPVC may react with cell surface GalT-2. First, our time kinetics data reveal that in ASMCs, Ox-LDL or oxidized phospholipids maximally stimulate GalT-2 activity within 1 to 2 minutes of incubation.⁶⁰ Second, we have detected significant GalT-2 activity associated with the plasma membrane. Third, dipalmitoylphosphatidylcholine specifically stimulated GalT-2 activity in a time- and concentration-dependent fashion.³⁷

Role of Sphingoglycolipids in Cell Death (Apoptosis)

Apoptosis is a genetically defined biological phenomenon that predetermines a cell to die. Several GSLs have now been shown to cause apoptosis (Table 2). During our studies on the effects of Ox-LDL in ASMCs, we have uncovered a novel signaling mechanism of apoptosis in these cells.

Previously, our laboratory has shown that high concentrations of Ox-LDL (100 µg/mL) can induce the death of ASMCs.⁴³ A novel aspect of Ox-LDL–mediated signal transduction to explain the phenomenon above was subsequently uncovered. We found that in ASMCs, Ox-LDL stimulates the activity of N-SMase, an enzyme that cleaves sphingomyelin to ceramide and phosphocholine.²⁰ The activity of N-SMase increased 5-fold within 5 minutes of incubation of cells with Ox-LDL, but not LDL, and then returned to baseline values after 30 minutes. This was accompanied by marked apoptosis, as evidenced by (1) a DNA ladder; (2) [³H]thymidine release; and (3) fluorescence-assisted cell sorting analysis after dual staining of cells with annexin-V (specific stain for phosphatidylserine exposed on the outer membrane leaflet in apoptotic cells), and propidium iodide (specific for viable cells) in cells incubated with Ox-LDL.

Preincubation of cells with antibody against N-SMase, but not preimmune serum IgG (1:200 dilution), abrogated the Ox-LDL–induced apoptosis in these cells. In contrast, transient transfection of ASMCs with cDNA for N-SMase resulted in a 5-fold increase in the activity of N-SMase and sensitivity to Ox-LDL induced apoptosis. Increased activity of N-SMase was also correlated with elevated levels of ceramide and apoptosis in plaque and calcified plaques in patients who died of atherosclerosis at the Johns Hopkins Hospital.

In summary, Ox-LDL–mediated induction of apoptosis in ASMCs involves the activation of N-SMase, ceramide generation, and apoptosis, and this may contribute to plaque instability. Apoptosis may be compromised by the use of antibody against N-SMase.

Functional Role of Sphingolipids in Vascular Cell Biology and in Other Systems

A comprehensive review of the functional role of sphingoglycolipids in cell-cell recognition and signaling has appeared recently^{3 4 5 6 7 65 66 67 68} and is summarized in Table 2 with appropriate references.^{68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90} In general, the physiological reactions imparted by glycosphingolipid and ceramide-mediated signaling include modulation of cells, adhesion, transport of ions, regulation of cell growth, differentiation, and programmed cell death. Several sphingolipids serve as adhesion molecules and can regulate cell proliferation via signal transduction pathways involving NADPH oxidase–mediated superoxide generation, as well as growth factor receptor phosphorylation/dephosphorylation mechanisms.^{78 85 86} While several of the signaling systems above initiated by tyrosine kinase–linked receptors such as PKC, MEK, Raf-1, MAPK, and other kinases in other systems, other kinases and/or alteration of cytosolic Ca²⁺ levels led to changes in cells, differentiation, proliferation, and apoptosis. In other studies, macrophages⁹⁶ and platelets treated with thrombin⁹⁷ were shown to secrete a hitherto-uncharacterized SMase. Moreover, the macrophage-derived SMase was shown to stimulate the aggregation of VLDL and LDL. The supplementation of apoE in VLDL and LDL derived from apoE knockout mice did not alter the in vitro aggregation of mouse lipoproteins; however, sphingomyelin enrichment did.⁹⁸ The finding that sphingosine-1-phosphate may be an inhibitor of platelet-derived growth factor–induced migration of ASMCs is of considerable significance.^{74 75} In addition, the protective role of trimethylsphingosine of the myocardium and endothelium in ischemia/reperfusion injury has been suggested.⁷¹ Several mechanisms have been implicated in the initiation of early atherogenesis. Some of these processes are LDL oxidation,⁹¹ lipoprotein retention and aggregation,^{92 93} endothelial cell dysfunction, the expression of adhesion molecules,⁹⁴ monocyte/macrophage adhesion to the endothelial cells, foam cell formation, and smooth muscle cell phenotypic changes involving proliferation as well as migration.⁹⁵ Recent studies have implicated several sphingolipids to be involved in the phenomenon above. For example, sphingosine-1-phosphate was shown to serve as a ligand for the G protein–coupled receptors for the endothelial differentiation gene–coupled orphan receptor EDG-1. Overexpression of EDG-1 in human kidney cells induced cell-cell aggregation as well as the expression of adhesins.⁹⁵

Perspectives

Recently, there has been a surge of interest in glycosphingolipids and sphingomyelin in biomedical research, as these compounds themselves and various products of metabolism of such compounds, are now know to serve second-messenger functions in a variety of cellular signaling pathways. These lipids play an active role in transducing signals received from the external environment to the cells' interiors. Alternatively, exogenously added GSLs and ceramide have been shown to reconstitute the functions of various cytokines and growth factors. Moreover, since the plaque intima is enriched with sphingomyelin^{99 100} and N-SMase,¹⁴ this may contribute to an increase in the aggregation of the atherogenic, cholesterol-rich lipoproteins, VLDL and LDL, to the vascular wall.⁹⁸ On the other hand, in human liver cells, N-SMase action may mediate TNF-–induced maturation of sterol regulatory-element-binding protein (SREBP-1) and consequently, LDL receptor upregulation. Since TNF-, N-SMase, and a cell-permeable ceramide (C₂ ceramide) could activate the phenomenon above in the presence of cholesterol and 25- hydroxycholesterol in the medium, this indicates that sphingolipids may be involved in the regulation of LDL receptors and sterol metabolism via sterol-independent pathways. Clearly, further work is required to elaborate such pathways, as they may provide novel avenues for therapy.

The localized attachment of circulating leukocytes and monocytes to the endothelium in inflamed vessels is an important event in the initiation and progression of atherosclerotic plaque formation.⁶¹ These adherent monocytes subsequently migrate through the endothelium into the subendothelial space, where they differentiate into macrophages and internalize Ox-LDL, leading to foam cell proliferation. The latter events contribute to the progression of atherosclerotic plaque formation and consequent vessel stenosis. As a response to injury, the endothelium secretes inflammatory cytokines such as TNF-, interleukin-8, platelet-derived activation factor, and monocyte chemotactic factor. These, in turn, induce the expression of intracellular cell adhesion molecules (ICAMs) in endothelial cells, whereas others have been implicated in the expression of CD11/CD8 on the surface of leukocytes and macrophages. In fact, in the endothelial cells, ICAM-1 serves as a receptor for CD11/CD8, and this recognition (binding) of ICAM-1 by leukocyte/monocyte CD11/CD8 precipitates the adhesion of the endothelium to leukocytes/macrophages. Our studies indicate that LacCer can induce superoxide generation in human neutrophils and human endothelial cells.^{89 90} In turn, these cells express elevated levels of CD11/CD8 and ICAM-1 expression, respectively, resulting in the adhesion of neutrophils/monocytes to endothelial cells. The superoxides generated by endothelial cells, neutrophils, and Ox-LDL collectively and/or the superoxides generated by LacCer in ASMCs independently could conceivably stimulate the proliferation of ASMCs. Thus, at least in vitro in cultured human cells, LacCer appears to play a significant role in the 2 major events in atherosclerosis, ie, the initiation of neutrophil adhesion to endothelial cells and ASMC proliferation. I am tempted to predict that LacCer-induced O₂^- generation may well play an important role in the expression of other adhesion molecules in vascular cells and in other systems, such as the adhesion of eosinophils to the airway epithelium. Clearly, these provocative studies needs to be substantiated by in vivo experiments to further elucidate the role of LacCer and superoxides in the phenomena above that lead to atherosclerosis and restenosis in patients who have undergone balloon angioplasty.

Acknowledgments

This work was supported by National Institutes of Health grant RO-1-DK31722, Specialized Center of Research I Atherosclerosis PO-1 HL47212, and awards from the American Heart Association. This work was pursued through the collaborative efforts of many colleagues over the past 2 decades. I wish to particularly acknowledge Drs Peter O. Kwiterovich, Nupur Ghosh, and Anil Bhunia. I would also like to thank Dr Peter O. Kwiterovich, Jr, for critically reviewing this manuscript and Tammy DeMoss for the skillful preparation of this article

Received October 22, 1997; accepted April 9, 1998.

References

1. Thudichum JLW. Reports of the Medical Officer of Privy Council and Local Government Board. 1876;N serial No. VIII, 117; 1874;N serial No. III, 113.

2. Desnick R, Ioannou YA, Eng CM. -Galactodase deficiency in Fabry's disease. In: Scriver CR, Beaudet Al, Sly WS, Valle D, eds. The Metabolic and Molecular Basis of Inherited Diseases. New York, NY: McGraw-Hill Inc; 1995;II:2484.

3. Miyake M, Hakomori S. A specific surface glycoconjugate controlling cell motility: evidence by functional monoclonal antibodies that inhibit cell motility and tumor metastasis. Biochemistry. 1991;30:3328–3334.[Medline] [Order article via Infotrieve]

4. Hannun YA. The sphingomyelin cycle and the second messenger function of ceramide. J Biol Chem. 1994;269:3125–3128.[Free Full Text]

5. Spiegel S, Merrill A. Sphingolipid metabolism and cell growth regulation. FASEB J. 1996;10:1388–1397.[Abstract]

6. Pena LA, Fuks Z, Kolesnick R. Stress-induced apoptosis and the sphingomyelin pathway. Biochem Pharmacol. 1997;53:615–621.[Medline] [Order article via Infotrieve]

7. Shayman JA. Sphingolipids: their role in intracellular signaling and renal growth. J Am Soc Nephrol. 1996;7:171–182.[Abstract]

8. Merril AH Jr, Jones DD. An update of the enzymology and regulation of sphingomyelin metabolism. Biochim Biophys Acta. 1990;1044:1–12.[Medline] [Order article via Infotrieve]

9. Radin NS. Biosynthesis of the sphingoid bases: a provation. J Lipid Res. 1984;25:1536–1540.[Medline] [Order article via Infotrieve]

10. Obeid L, Linardic CM, Kerolac LA, Hannun Y. Programmed cell death induced by ceramide. Science. 1992;259:1769–1771.

11. Basu S, Basu M. Biosynthesis and regulation of glycosphingolipids. In: Pinto BM, Pastgheits S, Harves JW, eds. Comprehensive Natural Products Chemistry—Carbohydrate. New York, NY: Pergamon Press; 1997.

12. Chatterjee S, Sekerke CS, Kwiterovich PO Jr. Increased urinary excretion of glycosphingolipids in familial hypercholesterolemia. J Lipid Res. 1982;23:513–522.[Abstract]

13. Chatterjee S, Dey S, Shi W-Y, Thomas K, Hutchins GM. Accumulation of glycosphingolipids in human atherosclerotic plaque and unaffected aorta tissue. Glycobiology. 1997;7:57–65.[Abstract/Free Full Text]

14. Mukhin D, Chao F-F, Kruth HS. Glycosphingolipid accumulation in the aortic wall is another feature of human atherosclerosis. Arterioscler Thromb Vasc Biol. 1995;15:1607–1615.[Abstract/Free Full Text]

15. Beutler E, Grabowski G. Gaucher's disease. In: Scriver CR, Beaudet Al, Sly WS, Valle D, eds. The Metabolic and Molecular Basis of Inherited Diseases.New York, NY: McGraw-Hill Inc; 1995;II:2601–2624.

16. Moser HW. Ceramide deficiency: Farber's disease lipogranulomatosis in the metabolic and molecular basis of inherited disease. In: Scriver CR, Beaudet Al, Sly WS, Valle D, eds. The Metabolic and Molecular Basis of Inherited Diseases. New York, NY: McGraw-Hill Inc; 1994;II:2589–2599.

17. Desnick RJ. Niemann-Pick disease types A and B: acid sphingomyelin deficiency. In: Scriver CR, Beaudet Al, Sly WS, Valle D, eds. The Metabolic and Molecular Basis of Inherited Diseases. New York, NY: McGraw-Hill Inc; 1995;II:2601–2624.

18. Ito M, Yamagata T. A novel glycosphingolipid-degrading enzyme cleaves the linkage between the oligosaccharide and ceramide of neutral and acidic glycosphingolipids. J Biol Chem. 1986;261:14278–14282.[Abstract/Free Full Text]

19. Zhou B, Li SC, Laine R, Huang TC, Li YT. Isolation and characterization of ceramide glycanase from the leech, Macrobdella decora. J Biol Chem. 1989;264:12272–12277.[Abstract/Free Full Text]

20. Chatterjee S. Neutral sphingomyelinase. Adv Lipid Res. 1993;26:25–48.[Medline] [Order article via Infotrieve]

21. Spence M. Sphingomyelinases. Adv Lipid Res. 1993;26:1–23.

22. Rock P, Allietta M, Young WW Jr, Thompson TE, Tillack TW. Ganglioside GM₁ and asialo-GM₁ at low concentrations are preferentially incorporated into the gel phase in two-component, two-phase phosphatidylcholine bilayers. Biochemistry. 1991;30:19–25.[Medline] [Order article via Infotrieve]

23. Chatterjee S, Kwiterovich PO Jr, Gupta P, Erozan Y, Alving CA, Richard R. Localization of urinary lactosylceramide in cytoplasmic vesicles of renal tubular cells in homozygous familial hypercholesterolemia. Proc Natl Acad Sci U S A. 1983;80:1313–1318.[Abstract/Free Full Text]

24. Symington FW, Murray WA, Bearman SI, Hakomori S. Intracellular localization of lactosylceramide, the major human neutrophil glycosphingolipid. J Biol Chem. 1987;262:11356–11363.[Abstract/Free Full Text]

25. Steck TL, Kezdy FJ, Lange Y. An activation-collision mechanism for cholesterol transfer between membranes. J Biol Chem. 1988;263:13023–13031.[Abstract/Free Full Text]

26. Svennerholm E, Svennerholm L. The separation of neutral blood-serum glycolipids by thin-layer chromatography. Biochim Biophys Acta. 1963;70:432–441.[Medline] [Order article via Infotrieve]

27. Chatterjee S, Kwiterovich PO Jr. Glycosphingolipids of lipoproteins in normal and hyperlipoproteinemic states. Lipids. 1976;11:462–466.[Medline] [Order article via Infotrieve]

28. Dawson G, Kruski AW, Scanu AM. Distribution of glycosphingolipids in the serum lipoproteins of normal human subjects and patients with hypo- and hyperlipidemias. J Lipid Res. 1976;17:125–131.[Abstract]

29. Chatterjee S, Bell W, Kwiterovich PO Jr. Distribution of antithrombin III and glucosyl-ceramide in human plasma lipoproteins and lipoprotein deficient plasma. Lipids. 1984;19:363–366.[Medline] [Order article via Infotrieve]

30. Chatterjee S, Clarke KS, Kwiterovich PO Jr. Regulation of synthesis of lactosylceramide and long chain bases in normal and familial hypercholesterolemic cultured proximal tubular cells. J Biol Chem. 1986;261:13474–13479.[Abstract/Free Full Text]

31. Chatterjee S, Castiglione E. Characterization of UDP-galactose:glucosylceramide ß14:galactosyltransferase activity in cultured human proximal tubular cells. Biochim Biophys Acta. 1987;923:136–142.[Medline] [Order article via Infotrieve]

32. Deleted in proof.

33. Chatterjee S, Clarke KS, Kwiterovich PO Jr. Uptake and metabolism of lactosylceramide in cultured proximal tubular cells from normal and familial hypercholesterolemic homozygotes. J Biol Chem. 1986;261:13480–13486.[Abstract/Free Full Text]

34. Chatterjee S, Ghosh N, Castiglione E, Kwiterovich PO Jr. Regulation of glycosphingolipids glycosyltransferases by low density lipoproteins in cultured human proximal tubular cells. J Biol Chem. 1988;263:13017–13022.[Abstract/Free Full Text]

35. Goldstein JL, Brown MS. Familial hypercholesterolemia. In: Scriver CR, Beaudet Al, Sly WS, Valle D, eds. The Metabolic and Molecular Basis of Inherited Diseases. New York, NY: McGraw-Hill Inc; 1995; 1981–2030.

36. Chatterjee S. Role of low density lipoprotein receptor on the regulation of synthesis of lactosylceramide in cultured normal human proximal tubular cells. Ind J Biochem Biophys. 1988;25:85–89.[Medline] [Order article via Infotrieve]

37. Chatterjee S, Ghosh N. Phosphatidylcholine stimulates the activity of glucosylceramideB1–4galactosyltransferase (GalT-2) in cultured human proximal tubular (PT) cells. Ind J Biochem Biophys. 1990;27:375–378.[Medline] [Order article via Infotrieve]

38. Chatterjee S. Regulation of synthesis of lactosylceramide in cultured normal and tumor proximal tubular cells. Biochim Biophys Acta. 1993;1167:339–344.[Medline] [Order article via Infotrieve]

39. Morton RE, West GA, Hoff HF. A low density lipoprotein-sized particle isolated from human atherosclerotic lesions is internalized by macrophages via a non-scavenger-receptor mechanism. J Lipid Res. 1986;27:1124–1134.[Abstract]

40. Carew TE, Schwenke DC, Steinberg D. Antiatherogenic effect of probucol unrelated to its hypocholesterolemic effect: evidence that antioxidants in vivo can selectively inhibit low density lipoprotein degradation in macrophage-rich fatty streaks and slow the progression of atherosclerosis in the Watanabe heritable hyperlipidemic rabbit. Proc Natl Acad Sci U S A. 1987;84:7725–7729.[Abstract/Free Full Text]

41. Steinbrecher UP, Parthasarathy S, Leake DS, Witztum JL, Steinberg D. Modification of low density lipoprotein by endothelial cells involves lipid peroxidation and degradation of low density lipoprotein phospholipids. Proc Natl Acad Sci U S A. 1984;81:3883–3887.[Abstract/Free Full Text]

42. Hessler JR, Robertson AL, Chisholm GM. Low density lipoprotein cytotoxicity induced by free radical peroxidation of lipids. J Lipid Res. 1983;24:1070–1076.[Abstract]

43. Chatterjee S. Role of oxidized low density lipoproteins in atherosclerosis: effects on smooth muscle cell proliferation. J Mol Cell Biochem. 1992;111:143–147.

44. Radin NS, Shayman JA, Inokuchi J-I. Metabolic effects of inhibiting glucosylceramide synthesis with PDMP and other substances. Adv Lipid Res. 1993;26:183–214.[Medline] [Order article via Infotrieve]

45. Chatterjee S, Ghosh N. Oxidized low density lipoprotein stimulates aortic smooth muscle cell proliferation. Glycobiology. 1996;6:303–311.[Abstract/Free Full Text]

46. Chatterjee S. Lactosylceramide stimulates aortic smooth muscle cell proliferation. Biochem Biophys Res Commun. 1991;181:554–561.[Medline] [Order article via Infotrieve]

47. Chatterjee S, Bhunia AK, Han H, Snowden A. Oxidized low density lipoproteins stimulate galactosyltransferase activity, ras activation, p⁴⁴ mitogen activated protein kinase, and c-fos expression in human aortic smooth muscle cells. Glycobiology. 1997;7:703–710.[Abstract/Free Full Text]

48. Lo YC, Cruz TF. Involvement of reactive oxygen species in cytokine and growth factor induction of c-fos expression in chondrocytes. J Biol Chem. 1995;270:11727–11730.[Abstract/Free Full Text]

49. Halliwell B, Gutteridge JMC. Role of free radicals and catalytic metal ions in human disease: an overview. Methods Enzymol. 1990;186:1–85.[Medline] [Order article via Infotrieve]

50. Guyton KZ, Kensler TW. Oxidative mechanisms of carcinogens. Br Med Bull. 1993;49:523–544.[Abstract/Free Full Text]

51. Kehrer JP. Free radicals as mediators of tissue injury and disease. Crit Rev Toxicol. 1993;23:21–48.[Medline] [Order article via Infotrieve]

52. Schreck R, Rieber P, Baeuerle PA. Reactive oxygen intermediates as apparently widely used messengers in the activation of NF-B transcription factor and HIV-1. Eur Mol Biol Organ J. 1991;10:2247–2258.[Medline] [Order article via Infotrieve]

53. Larsson R, Cerutti P. Oxidants induce phosphorylation of ribosome protein S6. J Biol Chem. 1988;263:17452–17458.[Abstract/Free Full Text]

54. Staal FJT, Anderson MT, Staal GE, Herzenberg LA, Gitler C, Hergenberg LA. Intracellular thiols regulate activation of nuclear factor B and transcription of human immunodeficiency virus. Proc Natl Acad Sci U S A. 1994;91:3619–3622.[Abstract/Free Full Text]

55. Sundaresan M, Yu Z, Ferrans VJ, Irani K, Finkel T. Requirement for generation of H₂O₂ for platelet-derived growth factor signal transduction. Science. 1995;270:296–299.[Abstract/Free Full Text]

56. Puri PL, Avantaggiati ML, Burgio VL, Chirillo P, Collepardo D, Natoli G, Balsano C, Levero M. Reactive oxygen intermediates mediate angiotensin II-induced c-jun C-fos heterodimer DNA binding activity and proliferation of hypertrophic responses in myogenic cells. J Biol Chem. 1995;270:22129–22134.[Abstract/Free Full Text]

57. Pinkus R, Weiner LM, Daniel V. Role of oxidants and antioxidants in the induction of Ap-1, NF-B and glutathione-S-transferase gene expression. J Biol Chem. 1996;270:13422–13429.[Abstract/Free Full Text]

58. Bhunia A, Han, H, Snowden A, Chatterjee S. Lactosylceramide stimulates Ras GTP loading, kinases (MEK, Raf), p⁴⁴ mitogen activated protein kinase and c-fos expression in human aortic smooth muscle cells. J Biol Chem. 1996;271:10660–10666.[Abstract/Free Full Text]

59. Bhunia AK, Han H, Snowden A, Chatterjee S. Redox regulated signaling by lactosylceramide in the proliferation of aortic smooth muscle cells. J Biol Chem. 1997;272:15642–15649.[Abstract/Free Full Text]

60. Chatterjee S. Oxidized low density lipoprotein and lactosylceramide both stimulate the expression of proliferating nuclear antigen and the proliferation of aortic smooth muscle cells. Ind J Biochem Biophys. 1997;34:56–61.[Medline] [Order article via Infotrieve]

61. Epstein FH. Mechanisms of disease: antioxidants and atherosclerotic heart disease. N Engl J Med. 1997;6:408–416.

62. Balagopalakrishna C, Nirmala R, Rifkind JM, Chatterjee S. Modification of low density lipoproteins by erythrocytes and hemoglobin under hypoxic conditions: oxygen transport to tissues. Adv Exp Biol Med. 1997;18:337–345.

63. Balagopalakrishna C, Bhunia AK, Snowden A, Rifkind JM, Chatterjee S. Minimally modified low density lipoproteins induce aortic smooth muscle cell proliferation via the activation of mitogen activated protein kinase. Mol Cell Biochem. 1997;170:85–89.[Medline] [Order article via Infotrieve]

64. Watson AD, Leitinger N, Navab M, Faull KF, Horkko S, Witztum JL, Palinski W, Schwenke D, Salomon RG, Sha W, Subbanagounder G, Fogelman AM, Berliner JA. Structural identification by mass spectrometry of oxidized phospholipids in minimally oxidized low density lipoprotein that induce monocyte/endothelial interactions and evidence for their presence in vivo. J Biol Chem. 1997;272:13597–13607.[Abstract/Free Full Text]

65. Chatterjee S, Ghosh N. Regulation of lactosylceramide biosynthesis: a challenge and an opportunity for glycobiologists. Trends Glycosci Glycotech. 1994;6:187–198.

66. Ghosh S, Strum JC, Bell RM. Lipid biochemistry: function of glycolipids and sphingolipids in cellular signaling. FASEB J. 1997;11:45–50.[Abstract]

67. Testi R. Sphingomyelin breakdown and cell fate. Trends Biochem Sci. 1996;21:468–471.[Medline] [Order article via Infotrieve]

68. Hakomori S-I, Igarashi Y. Functional role of glycosphingolipid in cell recognition and signaling. J Biochem. 1995;118:1091–1103.[Abstract/Free Full Text]

69. Ghosh TK, Bian T, Gill DL. Intracellular calcium release mediated by sphingosine derivative generated via cells. Science. 1990;248:1643–1656.[Abstract/Free Full Text]

70. Okazaki T, Bell RM, Hannun Y. Sphingomyelin turnover induced by vitamin D3 in HL-60 cells: role in cell differentiation. J Biol Chem. 1989;264:19076–19080.[Abstract/Free Full Text]

71. Murohara T, Buerke M, Margiotta J, Ruan F, Igarashi Y, Hakomori S, Lefer AM. Myocardial and endothelial protection by TMS in ischemia-reperfusion injury. Am J Physiol. 1995;269:H504–H514.[Abstract/Free Full Text]

72. Ohta H, Yatomi Y, Sweeney EA, Hakomori S, Igarashi Y. A possible role of sphingosine in induction of apoptosis by tumor necrosis factor- in human neutrophils. FEBS Lett. 1994;355:267–270.[Medline] [Order article via Infotrieve]

73. Ohta H, Sweeney EA, Masamune A, Yatomi Y, Hakomori S, Igarashi Y. Induction of apoptosis from sphingosine in human leukemic HL-60 cells: a possible exogenous modulator of apoptotic DNA fragmentation occurs during phorbol ester-induced differentiation. Cancer Res. 1995;55:691–697.[Abstract/Free Full Text]

74. Bornfeldt KE, Graves LM, Raines EW, Igarashi Y, Wayman G, Yamamura S, Yatomi Y, Sidhu JS, Krebs EG, Hakomori S, Ross R. Sphingosine-1-phosphate inhibits PDGF-induced chemotaxis of human arteria smooth muscle cells: spatial and temporal modulation of PDGF chemotactic signal transduction. J Cell Biol. 1995;130:193–206.[Abstract/Free Full Text]

75. Sadarhira Y, Ruan F, Hakomori S, Igarashi Y. Sphingosine-1-phosphate, a specific endogenous signaling molecule controlling cell motility and tumor cell invasiveness. Proc Natl Acad Sci U S A. 1992;89:9686–9690.[Abstract/Free Full Text]

76. Igarashi Y, Hakomori S, Toyokuni T, Dean B, Fujita S, Sugimoto M, Ogawa T, El-Ghendy K, Racker E. Effect of chemically well-defined sphingosine and its N-methyl derivatives on protein kinase C and src kinase activities. Biochemistry. 1989;28:6796–6800.[Medline] [Order article via Infotrieve]

77. Desai NN, Zhang H, Olivera A, Mattie ME, Spiegel S. Sphingosine-1-phosphate, a metabolite of sphingosine, increases phosphatidic acid levels by phospholipase D activation. J Biol Chem. 1992;267:23122–23128.[Abstract/Free Full Text]

78. Olivera A, Spiegel S. Sphingosine-1-phosphate, second messenger in cell proliferation and induced by PDGF and mitogens. Nature. 1993;365:557–560.[Medline] [Order article via Infotrieve]

79. Dyer CA, Benjamins JA. Glycolipids and transmembrane signaling: antibodies to galactocerebroside cause an influx of calcium in oligodendrocytes. J Cell Biol. 1990;111:625–633.[Abstract/Free Full Text]

80. Shayman JA, Makdiyoum S, Dedmuich G, Radin NSA. Glycosphingolipid dependence of hormone stimulated inositol triphosphate formation. J Biol Chem. 1990;265:12135–12138.[Abstract/Free Full Text]

81. Marsh NL, Elias PM, Holleran WM. Glucosylceramides stimulate murine epidermal hyperproliferation. J Clin Invest. 1995;95:2903–2909.

82. Chatterjee S, Shi W Y, Wilson P, Mazumdar A. Role of lactosylceramide and map kinase in the proliferation of proximal tubular cells in human polycystic kidney disease. J Lipid Res. 1996;37:1334–1344.[Abstract]

83. Karlsson KA. Animal glycosphingolipids as membrane attachment sites for bacteria. Annu Rev Biochem. 1989;58:309–350.[Medline] [Order article via Infotrieve]

84. Mangeney M, Lingwood C, Taga S, Caillou B, Tursz T, Wiels J. Apoptosis induced in Burkitt's lymphoma cells via Gb3/GD77, a glycolipid antigen. Cancer Res. 1993;53:5314–5319.[Abstract/Free Full Text]

85. Bremer EG, Schlessinger J, Hakomori S. Ganglioside-mediated modulation of cell growth: specific effects of GM₃ on tyrosine phosphorylation of the epidermal growth factor receptor. J Biol Chem. 1986;261:2434–2440.[Abstract/Free Full Text]

86. Zhou Q, Hakomori S, Kitamura K, Igarashi Y. GM₃ directly inhibits tyrosine phosphorylation and de-N-acetyl-GM₃ directly enhances serine phosphorylation of epidermal growth factor receptor, independently of receptor-receptor interaction. J Biol Chem. 1994;269:1959–1965.[Abstract/Free Full Text]

87. De Maria R, Lenti L, Malisan F, d'Agostino F, Tomassini B, Zeuner A, Rippo MR, Testi R. Requirement for GD3 ganglioside in CD95-and ceramide-induced apoptosis. Science. 1997;277:1652–1655.[Abstract/Free Full Text]

88. Hannun Y, Bell RM. Lysosphingolipids inhibit protein kinase C: implications for the sphingolipodoses. Science. 1987;235:670–674.[Abstract/Free Full Text]

89. Arai, T, Bhunia, AK, Chatterjee, S, Bulkley, G. Lactosylceramide stimulates the expression of Mac-1 receptor in human neutrophils and superoxide production. Circ Res. 1998;82:540–547.[Abstract/Free Full Text]

90. Bhunia AK, Arai T, Bulkley G, Chatterjee S. Lactosylceramide-mediated redox signaling stimulates intercellular adhesion molecule-1 (ICAM-1) expression and neutrophil adhesion in human endothelial cells. In press.

91. Steinberg D. Low density lipoprotein and its biological significance. J Biol Chem. 1997;272:20963–20966.[Free Full Text]

92. Nievelstein PFEM, Fogelman AM, Mottino G, Frank JS. Lipid accumulation in rabbit aortic intima 2 hours after bolus infusion of low density lipoprotein. Arterioscler Thromb. 1991;11:1795–1805.[Abstract/Free Full Text]

93. Williams KJ, Tabas I. The response-to-retention hypothesis of early atherosclerosis. Arterioscler Thromb Vasc Biol. 1995;15:551–561.[Free Full Text]

94. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993;362:801–809.[Medline] [Order article via Infotrieve]

95. Lee M-J, Van Brocklyn JR, Thangada S, Liu CH, Hand AR, Menzeleev R, Spiegel S, Hla T. Sphingosine-1-phosphate as a ligand for the G protein-coupled receptor EGD-1. Science. 1998;279:1552–1555.[Abstract/Free Full Text]

96. Schissel SL, Jiang X-C, Tweedie-Hardman J, Jeong T-S, Camejo EH, Najib J, Rapp JH, Williams KJ, Tabas I. Secretory sphingomyelinase, a product of the acid sphingomyelinase gene, can hydrolyze atherogenic lipoproteins at neutral pH. J Biol Chem. 1998;273:2738–2746.[Abstract/Free Full Text]

97. Simon CG, Chatterjee S, Gear ARL. Sphingomyelinase activity in human platelets. 1998;90:155–161.

98. Jeong T-S, Schissel SL, Tabas I, Pownall HJ, Tall AR, Jiang X-C. Increased sphingomyelin content of plasma lipoproteins in apolipoprotein E knockout mice reflects combined production and catabolic defects and enhances reactivity with mammalian sphingomyelinase. J Clin Invest. 1998;101:905–912.[Medline] [Order article via Infotrieve]

99. McCandless EL, Zilversmit DB. The effect of cholesterol on the turnover of lecithin, cephalin and sphingomyelin in rabbits. Arch Biochem Biophys. 1956;62:402–410.[Medline] [Order article via Infotrieve]

100. Chatterjee S, Gakenheimer K, Han H, Dey S, Hutchins G, Dobromilskaya, Snowden A. Oxidized low density lipoprotein stimulates apoptosis via activation of neutral sphingomyelinase in human aortic smooth muscle cells. In press.

101. Lawler JF Jr, Yin M, Diehl AM, Roberts E, Chatterjee S. Tumor necrosis factor- stimulates the maturation of sterol regulatory element binding protein-1 in human hepatocytes through the action of neutral sphingomyelinase. J Biol Chem. 1998;273:5053–5059.[Abstract/Free Full Text]

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				H. Mu, X. Wang, H. Wang, P. Lin, Q. Yao, and C. Chen Lactosylceramide promotes cell migration and proliferation through activation of ERK1/2 in human aortic smooth muscle cells Am J Physiol Heart Circ Physiol, July 1, 2009; 297(1): H400 - H408. [Abstract] [Full Text] [PDF]

				J. M. Haus, S. R. Kashyap, T. Kasumov, R. Zhang, K. R. Kelly, R. A. DeFronzo, and J. P. Kirwan Plasma Ceramides Are Elevated in Obese Subjects With Type 2 Diabetes and Correlate With the Severity of Insulin Resistance Diabetes, February 1, 2009; 58(2): 337 - 343. [Abstract] [Full Text] [PDF]

				E. Bonzon-Kulichenko, D. Schwudke, N. Gallardo, E. Molto, T. Fernandez-Agullo, A. Shevchenko, and A. Andres Central Leptin Regulates Total Ceramide Content and Sterol Regulatory Element Binding Protein-1C Proteolytic Maturation in Rat White Adipose Tissue Endocrinology, January 1, 2009; 150(1): 169 - 178. [Abstract] [Full Text] [PDF]

				Y.-T. Yen, H.-C. Chen, Y.-D. Lin, C.-C. Shieh, and B. A. Wu-Hsieh Enhancement by Tumor Necrosis Factor Alpha of Dengue Virus-Induced Endothelial Cell Production of Reactive Nitrogen and Oxygen Species Is Key to Hemorrhage Development J. Virol., December 15, 2008; 82(24): 12312 - 12324. [Abstract] [Full Text] [PDF]

				E. O. Apostolov, A. G. Basnakian, X. Yin, E. Ok, and S. V. Shah Modified LDLs induce proliferation-mediated death of human vascular endothelial cells through MAPK pathway Am J Physiol Heart Circ Physiol, April 1, 2007; 292(4): H1836 - H1846. [Abstract] [Full Text] [PDF]

				F. Samad, K. D. Hester, G. Yang, Y. A. Hannun, and J. Bielawski Altered Adipose and Plasma Sphingolipid Metabolism in Obesity: A Potential Mechanism for Cardiovascular and Metabolic Risk Diabetes, September 1, 2006; 55(9): 2579 - 2587. [Abstract] [Full Text] [PDF]

				A. C.M. Mulders, M. C. Hendriks-Balk, M.-J. Mathy, M. C. Michel, A. E. Alewijnse, and S. L.M. Peters Sphingosine Kinase-Dependent Activation of Endothelial Nitric Oxide Synthase by Angiotensin II Arterioscler. Thromb. Vasc. Biol., September 1, 2006; 26(9): 2043 - 2048. [Abstract] [Full Text] [PDF]

				S.-K. Moon, S.-K. Kang, and C.-H. Kim Reactive oxygen species mediates disialoganglioside GD3-induced inhibition of ERK1/2 and matrix metalloproteinase-9 expression in vascular smooth muscle cells FASEB J, July 1, 2006; 20(9): 1387 - 1395. [Abstract] [Full Text] [PDF]

				E. Gulbins and P. L. Li Physiological and pathophysiological aspects of ceramide Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2006; 290(1): R11 - R26. [Abstract] [Full Text] [PDF]

				D. K. Sharma, J. C. Brown, Z. Cheng, E. L. Holicky, D. L. Marks, and R. E. Pagano The Glycosphingolipid, Lactosylceramide, Regulates {beta}1-Integrin Clustering and Endocytosis Cancer Res., September 15, 2005; 65(18): 8233 - 8241. [Abstract] [Full Text] [PDF]

				E. N. Glaros, W. S. Kim, C. M. Quinn, J. Wong, I. Gelissen, W. Jessup, and B. Garner Glycosphingolipid Accumulation Inhibits Cholesterol Efflux via the ABCA1/Apolipoprotein A-I Pathway: 1-PHENYL-2-DECANOYLAMINO-3-MORPHOLINO-1-PROPANOL IS A NOVEL CHOLESTEROL EFFLUX ACCELERATOR J. Biol. Chem., July 1, 2005; 280(26): 24515 - 24523. [Abstract] [Full Text] [PDF]

				S. A. Summers and D. H. Nelson A Role for Sphingolipids in Producing the Common Features of Type 2 Diabetes, Metabolic Syndrome X, and Cushing's Syndrome Diabetes, March 1, 2005; 54(3): 591 - 602. [Abstract] [Full Text] [PDF]

				S.-J. Lin, S.-K. Shyue, Y.-Y. Hung, Y.-H. Chen, H.-H. Ku, J.-W. Chen, K.-B. Tam, and Y.-L. Chen Superoxide Dismutase Inhibits the Expression of Vascular Cell Adhesion Molecule-1 and Intracellular Cell Adhesion Molecule-1 Induced by Tumor Necrosis Factor-{alpha} in Human Endothelial Cells Through the JNK/p38 Pathways Arterioscler. Thromb. Vasc. Biol., February 1, 2005; 25(2): 334 - 340. [Abstract] [Full Text] [PDF]

				S. P. Didion and F. M. Faraci Ceramide-Induced Impairment of Endothelial Function Is Prevented by CuZn Superoxide Dismutase Overexpression Arterioscler. Thromb. Vasc. Biol., January 1, 2005; 25(1): 90 - 95. [Abstract] [Full Text] [PDF]

				M. Yeh, A. L. Cole, J. Choi, Y. Liu, D. Tulchinsky, J.-H. Qiao, M. C. Fishbein, A. N. Dooley, T. Hovnanian, K. Mouilleseaux, et al. Role for Sterol Regulatory Element-Binding Protein in Activation of Endothelial Cells by Phospholipid Oxidation Products Circ. Res., October 15, 2004; 95(8): 780 - 788. [Abstract] [Full Text] [PDF]

				S.-K. Moon, H.-M. Kim, Y.-C. Lee, and C.-H. Kim Disialoganglioside (GD3) Synthase Gene Expression Suppresses Vascular Smooth Muscle Cell Responses via the Inhibition of ERK1/2 Phosphorylation, Cell Cycle Progression, and Matrix Metalloproteinase-9 Expression J. Biol. Chem., August 6, 2004; 279(32): 33063 - 33070. [Abstract] [Full Text] [PDF]

				R. Bhatia, K. Matsushita, M. Yamakuchi, C. N. Morrell, W. Cao, and C. J. Lowenstein Ceramide Triggers Weibel-Palade Body Exocytosis Circ. Res., August 6, 2004; 95(3): 319 - 324. [Abstract] [Full Text] [PDF]

				T. Matsunaga, S. Kotamraju, S. V. Kalivendi, A. Dhanasekaran, J. Joseph, and B. Kalyanaraman Ceramide-induced Intracellular Oxidant Formation, Iron Signaling, and Apoptosis in Endothelial Cells: PROTECTIVE ROLE OF ENDOGENOUS NITRIC OXIDE J. Biol. Chem., July 2, 2004; 279(27): 28614 - 28624. [Abstract] [Full Text] [PDF]

				D. T. U. Abeytunga, J. J. Glick, N. J. Gibson, L. A. Oland, A. Somogyi, V. H. Wysocki, and R. Polt Presence of unsaturated sphingomyelins and changes in their composition during the life cycle of the moth Manduca sexta J. Lipid Res., July 1, 2004; 45(7): 1221 - 1231. [Abstract] [Full Text] [PDF]

				R. Pannu, J.-S. Won, M. Khan, A. K. Singh, and I. Singh A Novel Role of Lactosylceramide in the Regulation of Lipopolysaccharide/Interferon-{gamma}-Mediated Inducible Nitric Oxide Synthase Gene Expression: Implications for Neuroinflammatory Diseases J. Neurosci., June 30, 2004; 24(26): 5942 - 5954. [Abstract] [Full Text] [PDF]

				J. W. Helge, A. Dobrzyn, B. Saltin, and J. Gorski Exercise and training effects on ceramide metabolism in human skeletal muscle Exp Physiol, January 1, 2004; 89(1): 119 - 127. [Abstract] [Full Text] [PDF]

				K. A. Walton, A. L. Cole, M. Yeh, G. Subbanagounder, S. R. Krutzik, R. L. Modlin, R. M. Lucas, J. Nakai, E. J. Smart, D. K. Vora, et al. Specific Phospholipid Oxidation Products Inhibit Ligand Activation of Toll-Like Receptors 4 and 2 Arterioscler. Thromb. Vasc. Biol., July 1, 2003; 23(7): 1197 - 1203. [Abstract] [Full Text] [PDF]

				D. X. Zhang, A.-P. Zou, and P.-L. Li Ceramide-induced activation of NADPH oxidase and endothelial dysfunction in small coronary arteries Am J Physiol Heart Circ Physiol, February 1, 2003; 284(2): H605 - H612. [Abstract] [Full Text] [PDF]

				D. Y. Hui and P. N. Howles Carboxyl ester lipase: structure-function relationship and physiological role in lipoprotein metabolism and atherosclerosis J. Lipid Res., December 1, 2002; 43(12): 2017 - 2030. [Abstract] [Full Text] [PDF]

				D. X. Zhang, F.-X. Yi, A.-P. Zou, and P.-L. Li Role of ceramide in TNF-alpha -induced impairment of endothelium-dependent vasorelaxation in coronary arteries Am J Physiol Heart Circ Physiol, November 1, 2002; 283(5): H1785 - H1794. [Abstract] [Full Text] [PDF]

				H. Li, P. Junk, A. Huwiler, C. Burkhardt, T. Wallerath, J. Pfeilschifter, and U. Forstermann Dual Effect of Ceramide on Human Endothelial Cells: Induction of Oxidative Stress and Transcriptional Upregulation of Endothelial Nitric Oxide Synthase Circulation, October 22, 2002; 106(17): 2250 - 2256. [Abstract] [Full Text] [PDF]

				I. GOUNI-BERTHOLD and A. SACHINIDIS Does the coronary risk factor low density lipoprotein alter growth and signaling in vascular smooth muscle cells? FASEB J, October 1, 2002; 16(12): 1477 - 1487. [Abstract] [Full Text] [PDF]

				Y.-J. Geng and P. Libby Progression of Atheroma: A Struggle Between Death and Procreation Arterioscler. Thromb. Vasc. Biol., September 1, 2002; 22(9): 1370 - 1380. [Abstract] [Full Text] [PDF]

				L Van Heerebeek, C Meischl, W Stooker, C J L M Meijer, H W M Niessen, and D Roos NADPH oxidase(s): new source(s) of reactive oxygen species in the vascular system? J. Clin. Pathol., August 1, 2002; 55(8): 561 - 568. [Abstract] [Full Text] [PDF]

				A. K. Bhunia, G. Schwarzmann, and S. Chatterjee GD3 Recruits Reactive Oxygen Species to Induce Cell Proliferation and Apoptosis in Human Aortic Smooth Muscle Cells J. Biol. Chem., May 3, 2002; 277(19): 16396 - 16402. [Abstract] [Full Text] [PDF]

				A. Loidl, R. Claus, H. P. Deigner, and A. Hermetter High-precision fluorescence assay for sphingomyelinase activity of isolated enzymes and cell lysates J. Lipid Res., May 1, 2002; 43(5): 815 - 823. [Abstract] [Full Text] [PDF]

				H. Deguchi, J. A. Fernandez, and J. H. Griffin Neutral Glycosphingolipid-dependent Inactivation of Coagulation Factor Va by Activated Protein C and Protein S J. Biol. Chem., March 8, 2002; 277(11): 8861 - 8865. [Abstract] [Full Text] [PDF]

				Y. Ryu, N. Takuwa, N. Sugimoto, S. Sakurada, S. Usui, H. Okamoto, O. Matsui, and Y. Takuwa Sphingosine-1-Phosphate, a Platelet-Derived Lysophospholipid Mediator, Negatively Regulates Cellular Rac Activity and Cell Migration in Vascular Smooth Muscle Cells Circ. Res., February 22, 2002; 90(3): 325 - 332. [Abstract] [Full Text] [PDF]

				B. Garner, D. A. Priestman, R. Stocker, D. J. Harvey, T. D. Butters, and F. M. Platt Increased glycosphingolipid levels in serum and aortae of apolipoprotein E gene knockout mice J. Lipid Res., February 1, 2002; 43(2): 205 - 214. [Abstract] [Full Text] [PDF]

				T. Levade, N. Auge, R. J. Veldman, O. Cuvillier, A. Negre-Salvayre, and R. Salvayre Sphingolipid Mediators in Cardiovascular Cell Biology and Pathology Circ. Res., November 23, 2001; 89(11): 957 - 968. [Abstract] [Full Text] [PDF]

				I. Gouni-Berthold, C. Seul, Y. Ko, J. Hescheler, and A. Sachinidis Gangliosides GM1 and GM2 Induce Vascular Smooth Muscle Cell Proliferation via Extracellular Signal-Regulated Kinase 1/2 Pathway Hypertension, November 1, 2001; 38(5): 1030 - 1037. [Abstract] [Full Text] [PDF]

				P. Boutouyrie, S. Laurent, B. Laloux, O. Lidove, J.-P. Grunfeld, and D. P Germain Non-invasive evaluation of arterial involvement in patients affected with Fabry disease J. Med. Genet., September 1, 2001; 38(9): 629 - 631. [Full Text] [PDF]

				H. Deguchi, J. A. Fernandez, I. Pabinger, J. A. Heit, and J. H. Griffin Plasma glucosylceramide deficiency as potential risk factor for venous thrombosis and modulator of anticoagulant protein C pathway Blood, April 1, 2001; 97(7): 1907 - 1914. [Abstract] [Full Text] [PDF]

				F. D. Kolodgie, A. Farb, and R. Virmani Local Delivery of Ceramide for Restenosis : Is There a Future for Lipid Therapy? Circ. Res., August 18, 2000; 87(4): 264 - 267. [Full Text] [PDF]

				R. Charles, L. Sandirasegarane, J. Yun, N. Bourbon, R. Wilson, R. P. Rothstein, S. W. Levison, and M. Kester Ceramide-Coated Balloon Catheters Limit Neointimal Hyperplasia After Stretch Injury in Carotid Arteries Circ. Res., August 18, 2000; 87(4): 282 - 288. [Abstract] [Full Text] [PDF]

				A. Iwakura, M. Fujita, K. Hasegawa, T. Sawamura, R. Nohara, S. Sasayama, and M. Komeda Pericardial fluid from patients with unstable angina induces vascular endothelial cell apoptosis J. Am. Coll. Cardiol., June 1, 2000; 35(7): 1785 - 1790. [Abstract] [Full Text] [PDF]

				T. Zheng, W. Li, J. Wang, B. T. Altura, and B. M. Altura Sphingomyelinase and ceramide analogs induce contraction and rises in [Ca2+]i in canine cerebral vascular muscle Am J Physiol Heart Circ Physiol, May 1, 2000; 278(5): H1421 - H1428. [Abstract] [Full Text] [PDF]

				H. Vesper, E.-M. Schmelz, M. N. Nikolova-Karakashian, D. L. Dillehay, D. V. Lynch, and A. H. Merrill Jr. Sphingolipids in Food and the Emerging Importance of Sphingolipids to Nutrition J. Nutr., July 1, 1999; 129(7): 1239 - 1250. [Abstract] [Full Text]

				Y. Ryu, N. Takuwa, N. Sugimoto, S. Sakurada, S. Usui, H. Okamoto, O. Matsui, and Y. Takuwa Sphingosine-1-Phosphate, a Platelet-Derived Lysophospholipid Mediator, Negatively Regulates Cellular Rac Activity and Cell Migration in Vascular Smooth Muscle Cells Circ. Res., February 22, 2002; 90(3): 325 - 332. [Abstract] [Full Text] [PDF]

				D. X. Zhang, A.-P. Zou, and P.-L. Li Ceramide Reduces Endothelium-Dependent Vasodilation by Increasing Superoxide Production in Small Bovine Coronary Arteries Circ. Res., April 27, 2001; 88(8): 824 - 831. [Abstract] [Full Text] [PDF]

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