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Circulation Research. 2001;89:957-968
doi: 10.1161/hh2301.100350
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(Circulation Research. 2001;89:957.)
© 2001 American Heart Association, Inc.

Reviews

Sphingolipid Mediators in Cardiovascular Cell Biology and Pathology

Thierry Levade, Nathalie Augé, Robert Jan Veldman, Olivier Cuvillier, Anne Nègre-Salvayre, Robert Salvayre

From INSERM U466, CHU Rangueil, Toulouse, France. The present address for R.J.V. is The Netherlands Cancer Institute, Division of Cellular Biochemistry, Amsterdam, The Netherlands.

Correspondence to Thierry Levade, MD, PhD, INSERM U466, CHU Rangueil, 1 Avenue Jean Poulhès, 31403 Toulouse Cedex 4, France. E-mail levade{at}toulouse.inserm.fr

Abstract

Sphingolipids have emerged as a new class of lipid mediators. In response to various extracellular stimuli, sphingolipid turnover can be stimulated in vascular cells and cardiac myocytes. Subsequent generation of sphingolipid molecules such as ceramide, sphingosine, and sphingosine-1-phosphate, is followed by regulation of ion fluxes and activation of various signaling pathways leading to smooth muscle cell proliferation, endothelial cell differentiation or apoptotic cell death, cell contraction, retraction, or migration. The importance of sphingolipids in cardiovascular signaling is illustrated by recent observations implicating them in physiological processes such as vasculogenesis as well as in frequent pathological conditions, including atherosclerosis and its complications.


Key Words: sphingomyelin • ceramide • sphingosine-1-phosphate • signal transduction • vascular cells

Sphingolipids (SLs), which are found in many living organisms (from yeast, plants, to humans) and in virtually all cell types, comprise more than 300 species. At least in mammalian cells, they have been regarded mostly as structural elements of membranes for almost one century. It is only recently that the first evidence was provided in favor of a biomodulatory role of some SLs, in particular the sphingomyelin (SM) metabolites.1,2 A wealth of reports has now accumulated, on a wide variety of cell types of different animal species, supporting an unquestionable role of SLs in regulating numerous cell functions and possibly mediating the effects of extracellular stimuli.

We will review the actions of SLs on the different cardiovascular system cell types. The potential implications of SLs in physiological and pathological processes affecting cardiac and vascular cells will also be discussed. Because the particular role of glycosphingolipids, and notably lactosylceramide (LacCer), in vascular signaling has recently been reviewed,3 attention will be mainly paid here to the sphingophospholipids and the other SM-derived molecules. In addition, only the potential second-messenger functions of SLs in mammalian cells will be considered. Thus, the role of SL-metabolizing enzymes such as the secreted sphingomyelinase in the conversion of circulating lipoproteins to atherogenic particles will not be detailed (the reader is referred to recent reviews4,5).

Sphingolipid Structure and Metabolism

Understanding the possible cell regulatory functions of SLs needs a precise knowledge of how they are generated, degraded, or interconverted, and where they are located or metabolized. We will briefly review these biochemical aspects (see other references for details6–8).

Structure and Subcellular Distribution
SLs are defined by the presence of a long-chain sphingoid backbone, generally sphingosine (Figure 1). Acylation of the sphingoid base, ie, addition of a C16 to C24 fatty acid, on the amino group yields ceramide, which serves as a building block for most of the SLs. Different substitutions at the 1-hydroxyl position of ceramide define the various SL classes: addition of a phosphate or phosphocholine group results in the formation of sphingophospholipids (ceramide-1-phosphate and SM [ceramide-phosphocholine], respectively) whereas incorporation of carbohydrates leads to the complex group of glycosphingolipids. Lysosphingolipids represent SL variants consisting of N-deacylated derivatives. They include sphingosine-1-phosphate (S1P) and sphingosylphosphocholine (SPC), which are also sphingophospholipids (Figure 1).



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  		Figure 1. Structure and metabolism of signaling-involved sphingolipids. Details on the enzymes and their subcellular localization are provided in Table 1 according to the indicated reaction numbers. FA indicates fatty acid.


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  		Table 1. Sphingolipid-Metabolizing Enzymes Potentially Implicated in Signaling

Because of their amphiphilic structure, most SLs are located in membrane bilayers. Most subcellular membranes contain SLs, although the plasma membrane and functionally related organelles are enriched in SLs8,9 (Figure 2). Within the plasma membrane, they are predominantly found in the outer leaflet. Importantly, the lateral distribution of SLs at the cell surface is not homogeneous. Rather, SLs are concentrated in microdomains, either rafts or caveolae, now believed to serve as signal-initiating platforms since a variety of proteins involved in signal transduction are located (or are translocated) there.10 As components of these structures, SM and glycosphingolipids could be implicated in initiating signaling events.11 Finally, SLs (in particular SM) are also found in each of the lipoprotein classes.9



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  		Figure 2. Main intracellular pathways for sphingolipid metabolism. The indicated numbers refer to the enzyme reactions given in Figure 1 and Table 1. DH-Sph indicates dihydrosphingosine; Sph, sphingosine; and GSL, glycosphingolipids.

Sphingolipid Turnover
The following section gives an overview of the main routes of the (glyco)sphingolipid metabolism, together with the involved enzymes and their subcellular localization (Figure 2 and Table 1), concentrating on the SLs that are believed to be involved in signal transduction.

De novo biosynthesis of ceramide takes place in the endoplasmic reticulum (ER) and starts with the formation by serine palmitoyl-transferase of 3-keto-sphinganine, which is then reduced to sphinganine (dihydrosphingosine) by 3-keto-sphinganine reductase.12 Ceramide synthase subsequently acylates sphinganine with an unbranched acyl chain, yielding dihydroceramide. Immediately, introduction of the C4–5 trans double bond by dihydroceramide desaturase occurs at the cytosolic face of the ER.13,14

Ceramide is primarily used for the synthesis of the phospholipid SM (Figure 2). In this reaction, a phosphocholine headgroup is transferred from phosphatidylcholine to ceramide.6,9 The SM synthase responsible for this reaction mainly resides in the cis and medial Golgi stacks,15,16 but contributions by the plasma membrane and a site in the endocytic recycling pathway have also been reported.17,18 The exact mechanism of ceramide transport from the ER to the Golgi and its translocation across the Golgi membrane (a prerequisite for the luminal event of SM biosynthesis) remain unknown.

Glucose and/or galactose can also be added to ceramide, yielding glucosylceramide (GlcCer) and galactosylceramide, respectively. Thereafter, GlcCer can be further glycosylated by the addition of galactose, resulting in the formation of LacCer, which in turn can give rise to more complex glycosphingolipids.7 These glycosylation steps are carried out by transferases restricted to the luminal face of the Golgi. An exception is the cytosol-oriented GlcCer synthase.19 After synthesis, SLs are transported to the plasma membrane, mostly by vesicular bulk flow.8

Ceramide can also be subjected to a phosphorylation/dephosphorylation cycle, which is mediated by a specific ceramide kinase20 and a ceramide-1-phosphate phosphatase,21 respectively.

Constitutive turnover of plasma membrane SLs mainly occurs after endocytosis (Figure 2). While a part of the internalized lipids is then recycled to the Golgi, or back to the plasma membrane, the remainder is degraded in the lysosomes through stepwise hydrolytic cleavage.22 Sequential removal of the terminal hydrophilic portions of the glycosphingolipids by a series of glycosidases is eased by the so-called SL activator proteins and eventually results in ceramide release. SM is cleaved in acidic compartments by the lysosomal sphingomyelinase (SMase),23 releasing phosphocholine and ceramide. After removal of the amide-linked fatty acid by a ceramidase,24 the remaining sphingosine presumably translocates across the lysosomal membrane.

Along with the well-characterized lysosomal pathways, nonlysosomal degradation also exists implicating membrane-bound hydrolases with neutral/alkaline optimum pH (Table 1). These ill-defined enzymes, which include SMases and ceramidases,25–30 may play a significant role in signal transduction (see Enzymes Involved in Sphingolipid Signaling). Moreover, no details are known concerning the subsequent trafficking of sphingosine from the plasma membrane and/or the lysosomes to the cytosolic site of the ER, where it can be reutilized for ceramide synthesis. Sphingosine can also be phosphorylated by a cytosolic sphingosine kinase to form S1P,31 which can be dephosphorylated by an S1P phosphatase32,33 or degraded by an ER-associated S1P lyase.34

Sphingolipids as Mediators: Generation and Mode of Action

Enzymes Involved in Sphingolipid Signaling
Despite that various SLs are now regarded as biomodulators, it is still unclear which enzymes are implicated in the generation of these signaling lipids. Since the same SL can be synthesized or degraded in distinct compartments by different enzyme entities (see Figure 2 and Table 1), a given SL may have different sources. Indeed, various pathways exhibiting different subcellular locations have been proposed for the generation of SLs.35 For instance, production of ceramide has most often been reported to arise from SM turnover through increased SMase activity but also from enhanced de novo synthesis. Different enzymes have been described to be activated upon cell stimulation and to be responsible for SM hydrolysis, implicating different sites such as the plasma membrane (inner leaflet, outer leaflet, and microdomain pools) or the acidic compartments. The nature of the signaling SMase is still intensely debated.35 The same applies for the production of S1P, the level of which is controlled by the concerted activities of sphingosine kinase, S1P phosphatase, and S1P lyase.36 These fundamental questions well illustrate the current debate on SL functions in cell signaling, obviously awaiting the availability of specific enzyme inhibitors, further characterization of the enzymes, and molecular tools.

Another important aspect for the production of SL mediators is the apparent tight regulation of SL-generating enzymes. For instance, the extracellular stimuli-responsive SMase activity is activated by lipids, such as arachidonic acid and ceramide itself, and serine proteases; on the other hand, neutral SMase stimulation can be negatively manipulated by protein kinase C (PKC) isoforms and glutathione levels.35

Sphingolipid Targets
Although few proteins have been reported to interact in vitro with some SLs, most of the direct molecular targets of SLs remain unknown. Because of the pleiotropic effects ascribed to ceramide or S1P, it was soon suspected that these SLs have different targets. The ceramide targets include kinases (a serine-threonine and proline-directed ceramide-activated protein kinase),37 members of the mitogen-activated protein kinases (MAPKs),38–40 classic and atypical PKC,41–43 a cytosolic ceramide-activated protein phosphatase,44 and transcription factors45,46 (Table 2). The activating role of ceramide on nuclear factor-{kappa}B (NF-{kappa}B) has, however, been challenged.47–49 The subcellular location of ceramide generation is likely to play an important role in dictating its downstream targets and thereby the biological response of the cell to this mediator, also accounting for the diversity of ceramide actions.


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  		Table 2. Intracellular Sphingolipid Targets

With regard to S1P, which is relatively water soluble, its effects depend on its source (ie, extracellular versus intracellular).50 Support for this dual mode of action has been brought by microinjection of S1P, the use of sphingosine kinase inhibitors, inhibition of S1P receptors by pertussis toxin, or more recently overexpression of sphingosine kinase. Agonist-stimulated sphingosine kinase leads to activation of MAPK, calcium mobilization, and inhibition of apoptogenic proteins.36 When released from cells (eg, platelets51,52), extracellular S1P acts as a ligand for the endothelial differentiation gene (EDG) family of G protein–coupled receptors.53 EDG-1 was identified as an immediate-early gene product in endothelial cells54 and was suggested to play a role in angiogenesis. The S1P-sensitive EDG family now includes EDG-1, -3, -5, -6, and -8.55 Upon binding of S1P to EDG-1, downstream events include the Gi-mediated activation of mitogenic pathways such as Ras and MAPK.56 In addition, EDG-1–coupled Gi protein induces a Fyn-mediated tyrosine phosphorylation of the cytoskeletal docking protein Cas (Crk-associated substrate), suggesting a role for EDG-1 in cytoskeletal remodeling and motility of (endothelial) cells.57 EDG-3 and EDG-5 are both coupled to G12/13 and to Gq.58 These routes converge on the small GTPase Rho that in turn activates p125FAK (focal adhesion kinase) and cadherin, again implicating EDG receptors in endothelial cell migration and differentiation.36 Little is known concerning the downstream signaling mechanisms of EDG-6 and EDG-8. The eventual cellular response to exogenous S1P will depend on the types of EDG receptors expressed and the extent of crosstalk between the involved signaling pathways.59

Biological Effects of Sphingolipids in Cardiovascular Cells

Exogenous SLs are able to induce in vitro various biological effects depending on the cell type. Moreover, several nonsphingolipidic molecules such as oxidized lipoproteins, cytokines, and growth factors, which are crucial players in vascular wall development and vascular pathology, stimulate various signaling pathways involving SLs. We have reviewed the main actions of SLs on cardiac myocytes and cells of the vascular wall (Table 3) (see also online Tables 1 and 2 available in the data supplement at http://www.circresaha.org).


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  		Table 3. Pathophysiological Conditions Resulting in the Generation of Sphingolipids in Cardiovascular Cells

Sphingolipids in Modulation of Cell Proliferation
Several observations underline the role of SLs in the regulation of cell growth responses (see reviews3,5). Intracellularly produced ceramide, S1P, SPC, and LacCer can stimulate DNA synthesis in endothelial and smooth muscle cells (SMCs).60–62 They also potentiated the mitogenesis induced by various growth factors including platelet-derived growth factor (PDGF). Addition of these SLs to the cells or treatment with bacterial SMase was accompanied by an increased activity of MAPK,62 a key factor in cell proliferation.

The SMC proliferation induced by mitogens such as PDGF, fibroblast growth factor (FGF), epidermal growth factor (EGF), or insulin-like growth factor (IGF) but not endothelin-1 or the inflammatory cytokines interleukin-1 or tumor necrosis factor (TNF) (in contrast to other observations63) has been linked to the production of sphingosine through stimulation of a ceramidase.64 In fact, a decrease in ceramide content via an increased activity of ceramidase, SM synthase, or GlcCer synthase often correlates with a proliferative response. Activation of sphingosine kinase and generation of S1P is also stimulated by PDGF.65 Our group and others have reported that SMC proliferation could be due to a direct mitogenic effect of mildly or extensively oxidized LDL.60–62 Indeed, oxidized LDL–induced proliferation appeared to be partly dependent on SL generation,5 in particular through a cascade from SM to S1P,66 resulting in MAPK activation.62

In contrast, treatment with TNF (which produced ceramide) or with exogenous ceramide led to an inhibition of cell growth (online Table 1). On endothelial cells, this effect was related to a decrease in the retinoblastoma protein content and increased p53 level.67 Recently, the role of ceramide in the inhibition of SMC proliferation has been evaluated in a model of hypertensive rats: whereas in vascular SMCs from hypertensive rats a mitogenic response to TNF was found that was associated with inhibition of ceramide generation and SMase mRNA expression, none of these events were observed in SMCs from control animals.68

Sphingolipids in Apoptotic Cell Death
Exogenously added short-chain ceramides, but also sphingosine and its N-methyl derivative, as well as intracellular SMase overexpression, can induce apoptosis of various vascular wall cell types, cardiomyocytes, and macrophages (online Table 1). The particular issue of ceramide as a proapoptotic signaling molecule has recently been challenged.69,70 This debate was mostly based on the use of short-chain ceramide analogues, which, because of their physicochemical properties, might not fully mimic the natural SL. However, a number of environmental stresses result in intracellular ceramide production that precedes hallmarks of apoptosis2 (online Table 1). Apoptosis of endothelial cells during septic shock, due to lipopolysaccharide, has also been linked to ceramide generation.71

The signaling pathways involved in SL-induced endothelial cell apoptosis may include inhibition of survival pathways such as the PKB/Akt and/or activation of the classical caspase cascade.72 As for oxidized LDL–induced apoptosis, the proapoptotic role of ceramide production in endothelial cells is still uncertain.72–74 Interestingly, endothelial cell apoptosis triggered by exogenous ceramide could be prevented by overexpression of a Bcl-2 family member,75 addition of vascular endothelial growth factor (VEGF76), or generation of S1P,63 two factors promoting survival in this cell type. Of note, as reported in other cell types,77 a ceramide/S1P biostat seems to operate in vascular cells that modulate cell death or survival depending on the fine regulation of key SL-metabolizing enzymes. Hence, S1P can protect endothelial cells from apoptosis induced by growth factor withdrawal or ceramide treatment,51,78,79 possibly through action on EDG-1.78

Sphingolipids in Cell Migration
S1P and possibly also SPC mediate endothelial cell chemotaxis, suggesting a role as proangiogenic factors (see Sphingolipids in Angiogenesis and Vasculogenesis).59,80,81 S1P-induced cell migration involves activation of a Gi-coupled receptor phospholipase C calcium signaling, leading to tyrosine phosphorylation of p125FAK.52,82 Activation by S1P of Rho, Rac, and Cas (which are also critical for cell migration responses) through EDG-1 and EDG-3 receptors57,78,80,83–85 has also been reported. EDG-1 expression has recently been shown to be required for PDGF-directed chemotaxis of human aortic SMCs.84 Because PDGF stimulates sphingosine kinase,65 a crosstalk between PDGF and EDG-1 signaling probably exists.

Sphingolipids and Cell Adhesion
Several agents seem to induce expression of adhesion molecules via SL generation. For example, N-octanoyl-sphingosine, bacterial SMase, or TNF-induced ceramide production elicits E-selectin–dependent adhesion of quiescent neutrophils to human endothelial cells.86 Other investigators, however, have found a dissociation between TNF and ceramide in induction of gene expression.49 Conversely, TNF can induce sphingosine kinase activation and S1P generation resulting in increased E-selectin and vascular cell adhesion molecule (VCAM)-1 expression via NF-{kappa}B and MAPK activation.87,88 Moreover, VEGF induction of intercellular adhesion molecule (ICAM)-1, VCAM-1, and E-selectin has also been associated with sphingosine kinase, NF-{kappa}B, and PKC activation.89 On the other hand, TNF-induced expression of ICAM-1 in endothelial cells has been linked to the production of LacCer90 but not ceramide,91 which could act through NADPH oxidase–mediated superoxide generation.

Sphingolipids and Ion Fluxes
Ten years ago, Ghosh et al92 discovered that sphingosine mediated rapid and profound release of calcium from intracellular stores and proposed that this effect might be mediated by S1P. Later, it was observed that both sphingosine and S1P were potent calcium-mobilizing agonists (online Table 2). More recently, several studies have indicated that S1P-induced calcium mobilization may also rely on EDG receptors.93,94 Ample evidence also indicates that SPC could mobilize calcium in SMCs or endothelial cells (online Table 2), although it is possible that it acts independently of EDG but through its high-affinity receptor.95 In myocytes, whereas S1P and SPC cause calcium mobilization by the ryanodine receptor,94,96 ceramide and sphingosine substantially inhibit the L-type calcium channel current.97–99 Similarly, sphingosine along with S1P (in a pertussis toxin–independent manner) leads to a pronounced inhibitory effect on inward sodium current in myocytes.99,100 In atrial myocytes, S1P activated Gi protein–regulated inwardly rectifying potassium channels93 whereas both S1P and SPC could activate muscarinic potassium current via a G-coupled receptor,101 such as EDG-3.102

Sphingolipids and Vascular Tone Regulation
Although a clear picture on the role of SLs in contractile functions within the blood vessel wall has not yet emerged, SLs modulate the vascular tone (online Table 1). Ceramide is capable of attenuating the contractile response to phenylephrine,103,104 an effect correlated with decreased intracellular calcium mobilization. Ceramide has also been suggested to account for the endothelium-independent, phospholipase A2–mediated vasodilation response to TNF.105 On the other hand, ceramide added to untreated arterial rings provoked a sustained vasoconstriction that was endothelium-independent.106 This contractile effect of ceramide on SMCs has been linked to a calcium-dependent activation of an Src kinase.107 Sphingosine could also impair endothelium-dependent relaxation.108 Finally, the sphingophospholipids S1P and SPC have been reported to induce contraction of mesenteric and intrarenal microvessels109 as well as coronary arterial strips.110 On precontracted coronary arteries, however, SPC induced an endothelium-dependent relaxation.111

These apparently divergent effects of SLs might be related to the ability of ceramide or lysosphingolipids to activate NO synthase (Table 2). Although S1P- or SPC-induced vasoconstriction was not affected by NO synthase inhibition109 and implicated Rho-kinase activation,110 the relaxing effect of SPC was associated with NO production and was inhibited by NO synthase blockers.111 In a different context, ceramide could attenuate the vasodilator response to bradykinin by decreasing NO in vascular endothelial cells.112 Further complexity in the vessel wall response is due to the fact that whereas SLs can induce NO production, NO can activate ceramide generation.113

Sphingolipids in Cardiovascular Biology and Pathology

Many observations indicate that SLs play an important role in normal and pathological situations, although their participation in the pathophysiology or progression of cardiovascular diseases is still speculative.

Sphingolipids in Angiogenesis and Vasculogenesis
Angiogenesis is the formation of new capillary networks from preexisting vasculature by sprouting and/or splitting of capillaries. This fundamental process is involved in physiological processes such as wound healing and uterine development. In addition, angiogenesis has been implicated in cancer, rheumatoid arthritis, diabetic retinopathy, and other pathological conditions.114 Angiogenesis involves proliferation, migration, adhesion, and differentiation of endothelial cells, which, in a later stage, are then lined up by SMCs. This intricate process is under the control of both pro- and antiangiogenic factors. In addition to VEGF and FGF, certain lipids, including gangliosides, prostaglandins, and lysophosphatidic acid, are also implicated. Recent data have underlined a major regulatory role for the lysosphingolipid S1P in angiogenesis.

The involvement of S1P in angiogenesis was first established in an in vitro model system.52,78,115 Not only is S1P able to increase proliferation and chemotaxis of endothelial cells, but it also stimulates tube formation of cells grown on collagen gels.80,115 The S1P released by platelets during clotting would account for most of the chemotactic activity of serum.81 Both EDG-1 and EDG-3 receptors were reported to regulate adherens junction assembly and formation of capillary-like networks.78 Furthermore, the observation that homozygous EDG-1–deficient mice die in utero as a result of massive embryonic hemorrhage demonstrated in vivo that S1P receptors are involved in blood vessel formation and maturation.116 Moreover, although S1P alone was unable to promote vessel formation in the avascular mouse cornea, it markedly enhanced the response to basic fibroblast growth factor (bFGF).81

In addition, studies on the zebrafish embryo have demonstrated that an S1P receptor plays a pivotal role in the regulation of cellular movements during heart morphogenesis.117 Indeed, the underlying cause of the myocardial cell migration defect found in the zebrafish mil phenotype is a mutation in a G protein–coupled receptor for S1P that alters its downstream signaling events including calcium mobilization and MAPK activation.

Sphingolipids in Atherosclerosis and Its Complications
Atherosclerotic lesions accumulate glycosphingolipids3 as well as ceramide, which could be generated by the secretory SMase, abundant in atheromata and able to aggregate LDL.4 Besides these observations, several lines of evidence involve SLs as potential mediators of the atherogenic process. First, some SLs may participate in the proliferation of vascular wall cells, favoring thickening and plaque stabilization. This is the case for S1P either released from activated platelets52,81 or produced intracellularly on oxidized LDL entry.66 Second, through an inflammatory response initiated by cytokines or oxidized LDL, lipids such as ceramide, LacCer, or S1P can upregulate adhesion molecule expression and induce adhesion and migration of monocytes, which are crucial events in initiation and progression of atherogenesis. In this respect, one of the antiatherogenic effects of HDL could be the inhibition of TNF-stimulated sphingosine kinase in endothelial cells.88 Third, SLs, eg, ceramide or sphingosine derivatives, may promote cell death (mostly by apoptosis) in the vascular wall, a process implicated in plaque erosion and associated thrombosis.118 SL-mediated apoptosis of cardiac myocytes could probably contribute to progressive heart failure and arrhythmia. Cardiac dysfunction in obesity has also been linked to increased myocardial levels of ceramide.119 Fourth, by modulating platelet activation and aggregation, glycolipids and sphingosine derivatives may favor thrombosis, as ceramide could do by affecting tissue factor91 or plasminogen activator inhibitor (PAI)-1120 release. A low plasma level of GlcCer has also been proposed as a risk factor for venous thrombosis.121 Finally, because of their effects on aortic or renal vasculature, SLs might be involved in hypertension.

Sphingolipids in Ischemia/Reperfusion
Ischemia and reperfusion lead to distinctive effects on tissues that are related to the metabolic changes induced by both hypoxia and reoxygenation. The reduction or arrest of blood supply in heart is followed by apoptosis, necrosis, and infarction.122 The reoxygenation process, although efficient in reducing ischemic damage, initiates additional cell injury including apoptosis,123 this effect being partly due to the generation of reactive oxygen species. The SM-ceramide signaling pathway is one of the pathways123 activated in response to myocardial ischemia/reperfusion.124 It is also involved in ischemic acute renal failure125 and postischemic neuronal cell death.126–128 Hypoxia/reoxygenation activates a neutral SMase and ceramide accumulation, which implicated the production of free radicals.129 This oxidative stress–sensitive ceramide production could activate in turn the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) pathway, and antioxidants prevented both ceramide release and JNK activation. Further indication that ceramide may mediate some of the ischemic brain response was provided by the finding that ceramide increased expression of c-Jun and death-inducing ligands, ceramide induced both necrosis and apoptosis, and that the immunosuppressant FK506 inhibited the release of ceramide and apoptosis in a stroke model.126 Another recent study showed that the defect or inhibition of acid SMase activity led to reduced ischemic neuronal injury.128

Disagreeing with the above data, ceramide, either directly or as a downstream messenger of TNF, can induce tolerance to hypoxic/ischemic insults and partially reduces infarct size.130 These observations suggest that ceramide could in some cases exhibit a protective role against ischemic injury, as previously reported in heart or liver for sphingosine derivatives131,132 or glycolipids.133

Sphingolipids in Chemotherapy-Induced Cardiotoxicity and Irradiation-Induced Cytotoxicity
Some anticancer treatments are known to induce irreversible myocardial injury and/or endothelial cell death. This is the case for the anthracycline doxorubicin that causes cardiac toxicity134 and for ionizing radiation leading to endothelial cell apoptosis. Ceramide has been implicated in anthracycline-induced apoptosis of cardiac myocytes, not only because this SL was found to accumulate as a result of SMase action but also because its generation was blocked by L-carnitine, a compound known to prevent the drug-induced cardiac damage and which reduced apoptosis.135 Pretreatment of myocytes with fumonisin B1 did not protect from anthracycline-induced apoptosis, indicating that de novo ceramide formation is not required for apoptosis induction.136 Interestingly, SL involvement has also been reported in cardiomyocyte apoptosis triggered by TNF, another cytotoxic effector137; in this case, sphingosine was reported to act as mediator.

Microvascular damage seems to represent a critical factor in ionizing radiation injury of normal and tumor tissues, including the lung and central nervous sytem.138 Apoptosis of the endothelium participates in this acute radiation toxicity and implicates the rapid production of ceramide from membrane SM breakdown.139 Studies on the murine model of Niemann-Pick disease, which lacks acid SMase, have led to the conclusion that ceramide is required for radiation-induced apoptosis of the lung, spinal cord, or small intestine endothelium.140–142 Indeed, reduction of the apoptotic response was observed in the microvasculature of the above tissues, but not in thymus, of acid SMase–deficient mice. Moreover, ceramide-mediated apoptosis of endothelial cells in response to irradiation was antagonized by activation of PKC by phorbol esters or bFGF.141

Future Prospects: From Bench to Clinic?

Evidence has accumulated that SLs play crucial roles in modulating cell functions. Not only are SL-metabolizing enzymes or SL receptors present in yeast, nematodes, fishes, and mammals, suggesting an evolutionary conservation of these pathways, but also similar SL-mediated responses exist in human and yeast, insect, or amphibian cells. Although recent work has shown that some SLs appear to transduce signals leading to a series of events associated with the physiology and pathology of the cardiovascular system, further investigations are needed to clarify the role of SLs. Future studies should lead toward the characterization of the whole spectrum of SL-metabolizing enzymes, the structure-function relationships of SL (EDG) receptors, and the molecular targets of SLs. In particular, a clearer understanding of the role of SLs should be provided by studies on animals harboring disrupted genes of SL metabolism.

In addition, solving these issues is expected to result in the development of new strategies to prevent or limit cardiovascular diseases. Potential targets of therapeutic intervention are already identified, including pharmacological manipulation of SL metabolism and genetic approaches. Pharmacological inhibitors include various mycotoxins, which at micromolar concentrations are able to inhibit enzymes of ceramide synthesis143 or SMase,144 and various synthetic drugs such as PDMP and N-butyl-deoxynojirimycin, which block ceramide glucosylation. Although the fungal toxins cannot yet readily be used in humans because of their potent adverse effects, the chemical compounds have already been successfully used to lower the amount of trihexosylceramide in a murine model of Fabry disease, a lysosomal disorder mainly affecting endothelial cells.145 Future enzymatic studies will also probably result in the development of specific inhibitors of S1P kinase, phosphatase, and lyase, allowing manipulation of the level of this bioactive lipid. Another potential therapeutic strategy will be the use of S1P receptor antagonists (or agonists). Elucidation of the specific responses to S1P elicited by the different EDG receptors should yield compounds selective for a given receptor.

Of particular interest is the possibility of a local rather than systemic delivery of enzyme inhibitors or even SL analogues. For instance, balloon catheters coated with short-chain ceramide have recently been shown to reduce neointimal hyperplasia after stretch injury of rabbit carotid arteries,146 thus offering a novel pharmacotherapy to restenosis. A similar approach could be envisaged for antisense oligonucleotides directed against genes of SL-metabolizing enzymes. Thus, it is reasonably hoped that deciphering the signaling pathways triggered by SLs in cardiac and vascular cells will offer novel potential targets and promising approaches for therapeutic intervention in atherosclerosis and its complications, ischemic injury, neovascularization, wound healing, or other pathological states.

Acknowledgments

Financial support from INSERM and Université Paul Sabatier is gratefully acknowledged. The authors thank their collaborators for fruitful discussion.

Received June 8, 2001; revision received October 8, 2001; accepted October 8, 2001.

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