Role of Sphingosine-1-Phosphate in Transplant Vasculopathy Evoked by Anti-HLA Antibody.

Transplant vasculopathy (TV) represents the main cause of late graft failure and limits the long-term success of organ transplantation. Cellular and humoral immune responses contribute to the pathogenesis of the concentric and diffuse intimal hyperplasia of arteries of the grafted organ. We recently reported that the mitogenic signaling, evoked in human vascular smooth muscle cells (hmSMC) by the anti-HLA class I monoclonal antibody W6/32, implicates neutral sphingomyelinase-2, suggesting a role for sphingolipids in intimal hyperplasia of TV. Here, we investigated whether the mitogenic sphingolipid, sphingosine-1-phosphate (S1P), is involved in intimal hyperplasia elicited by W6/32. Studies were done on cultured hmSMC and on an in vivo model of TV, consisting of human mesenteric arteries grafted into SCID/beige mice, injected weekly with W6/32. hmSMC migration and DNA synthesis elicited by W6/32 were inhibited by the sphingosine kinase-1 (SK1) inhibitor dimethylsphingosine, the anti-S1P antibody Sphingomab and the S1PR1/R3 inhibitor VPC23019. W6/32 stimulated SK1 activity, while siRNA silencing SK1, S1PR1 and S1PR3 inhibited hmSMC migration. In vivo, Sphingomab significantly reduced the intimal thickening induced by W6/32. These data emphasize the role of S1P in intimal hyperplasia elicited by the humoral immune response, and open perspectives for preventing TV with S1P inhibitors.

HLA class I antibodies provoke graft arteriosclerosis in human arteries transplanted into SCID/beige mice.

Antibodies toward HLA class I and/or MICA are commonly observed in transplanted patients suffering from allograft arteriosclerosis, also called chronic vascular rejection (CVR). The relative importance of cellular versus humoral alloreactivity for CVR is still disputed. We demonstrate that antibodies toward HLA class I provoke lesions typical for CVR in human arteries in vivo in the absence of cellular immunity. To show this, we grafted segments of human mesenteric arteries from 8 deceased organ donors into 36 immunodeficient SCID/beige mice in the infrarenal aortic position. Three mice died postoperatively. The remaining 33 mice received weekly i.v. injections of either a monoclonal antibody toward HLA class I, toward MICA or an irrelevant monoclonal antibody. At sacrifice after 6 weeks, mice receiving the HLA antibody showed a significant neointimal thickening in the grafted artery due to smooth muscle cell (SMC) proliferation while control mice receiving anti-MICA or irrelevant antibody showed little or no thickening. Whereas antibodies toward HLA class I were mitogenic to SMC in vitro, those directed toward MICA did not have any effect. Humoral alloreactivity toward HLA may thus play a causal role for the development of CVR and this opens new possibilities for the treatment of CVR.

FTY720 Inhibits Tumor Necrosis Factor-alpha-Induced Proliferation and Extracellular Signal-Regulated Kinase Phosphorylation of Human Smooth Muscle Cells.

We investigated the effects of the sphingolipid FTY720 on tumor necrosis factor-alpha (TNF-alpha)-induced proliferation and signal transduction in human smooth muscle cells (SMC). We showed that clinically relevant concentrations of FTY720 inhibited TNF-alpha-induced SMC proliferation and extracellular signal-regulated kinase (ERK) phosphorylation. We concluded that FTY720 may be a useful drug to inhibit chronic vascular rejection.

Effect of FTY720 on apoptosis of smooth muscle cells.

BACKGROUND: Hyperproliferation of smooth muscle cells (SMCs) plays a key role in allograft arteriosclerosis. This prompted us to investigate the effect of the novel immune modulator and synthetic sphingolipid FTY720 on apoptosis of SMCs. METHODS: Rabbit SMC cultures were treated with FTY720 and apoptosis and necrosis were detected by fluorescence microscopy. RESULTS: We investigated dose- and time-dependent effects of FTY720 and found that clinically relevant low doses of FTY720 (<1 micromol/L) did not induce apoptosis, whereas 10 micromol/L FTY720 induced apoptosis after 48 hours incubation. CONCLUSION: At doses of FTY720 used in clinics for treatment of renal allografts and multiple sclerosis. FTY720 did not induce SMC apoptosis.

Sphingolipid mediators in cardiovascular cell biology and pathology.

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.

Sphingomyelin metabolites in vascular cell signaling and atherogenesis.

The atherosclerotic lesion most probably develops through a number of cellular events which implicate all vascular cell types and include synthesis of extracellular proteins, cell proliferation, differentiation and death. Sphingolipids and sphingolipid metabolizing enzymes may play important roles in atherogenesis, not only because of lipoprotein alterations but also by mediating a number of cellular events which are believed to be crucial in the development of the vascular lesions such as proliferation or cell death. Exogenous sphingolipids may mediate various biological effects such as apoptosis, mitogenesis or differentiation depending on the cell type. Moreover, several molecules present in the atherogenic lesion, such as oxidized LDL, growth factors or cytokines, which activate intracellular signaling pathways leading to vascular cell modifications, can stimulate sphingomyelin hydrolysis and generation of ceramide (and other metabolites as sphingosine-1-phosphate). Here we review the potential implication of the sphingomyelin/ceramide cycle in vascular cell signaling related to atherosclerosis, and more generally the role of sphingolipids in the events observed during the atherosclerotic process as cell differentiation, migration, adhesion, retraction, proliferation and death.

Stress-induced apoptosis is not mediated by endolysosomal ceramide.

A major lipid-signaling pathway in mammalian cells implicates the generation of ceramide from the ubiquitous sphingolipid sphingomyelin (SM). Hydrolysis of SM by a sphingomyelinase present in acidic compartments has been reported to mediate, via the production of ceramide, the apoptotic cell death triggered by stress-inducing agents. In the present study, we investigated whether the ceramide formed within or accumulated in lysosomes indeed triggers apoptosis. A series of observations strongly suggests that ceramide involved in stress-induced apoptosis is not endolysosomal: 1) Although short-chain ceramides induced apoptosis, loading cells with natural ceramide through receptor-mediated endocytosis did not result in cell death. 2) Neither TNF-alpha nor anti-CD95 induced the degradation to ceramide of a natural SM that had been first introduced selectively into acidic compartments. 3) Stimulation of SV40-transformed fibroblasts by TNF-alpha or CD40 ligand resulted in apoptosis equally well in cells derived from control individuals and from patients affected with Farber disease, having a genetic defect of acid ceramidase activity leading to lysosomal accumulation of ceramide. Also, induction of apoptosis using anti-CD95 (Fas) or anti-CD40 antibodies, TNF-alpha, daunorubicin, and ionizing radiation was similar in control and Farber disease lymphoid cells. In all cases, apoptosis was preceded by a comparable increase of intracellular ceramide levels. 4) Retroviral-mediated gene transfer and overexpression of acid ceramidase in Farber fibroblasts, which led to complete metabolic correction of the ceramide catabolic defect, did not affect the cell response to TNF-alpha and CD40 ligand.

Activation of epithelial growth factor receptor pathway by unsaturated fatty acids.

Nonesterified fatty acids (NEFAs) are acutely liberated during lipolysis and are chronically elevated in pathological conditions, such as insulin resistance, hypertension, and obesity, which are known risk factors for atherosclerosis. The purpose of this study was to investigate the effect and mechanism of action of NEFAs on the epithelial growth factor (EGF) receptor (EGFR). In the ECV-304 endothelial cell line, unsaturated fatty acids triggered a time- and dose-dependent tyrosine phosphorylation of EGFR (polyunsaturated fatty acids [PUFAs] were the most active), whereas saturated FAs were inactive. Although less potent than PUFAs, oleic acid (OA) was used because it is prominent in the South European diet and is only slightly oxidizable (thus excluding oxidation derivatives). EGFR is activated by OA independent of any autocrine secretion of EGF or other related mediators. OA-induced EGFR autophosphorylation triggered EGFR signaling pathway activation (as assessed through coimmunoprecipitation of SH2 proteins such as SHC, GRB2, and SHP-2) and subsequent p42/p44 mitogen-activated protein kinase (as shown by the use of EGFR- deficient B82L and EGFR- transduced B82LK(+) cell lines). OA induced in vitro both autophosphorylation and activation of intrinsic tyrosine kinase of immunopurified EGFR, thus suggesting that EGFR is a primary target of OA. EGFR was also activated by mild surfactants, Tween-20 and Triton X-100, both in vitro (on immunopurified EGFR) and in intact living cells, thus indicating that EGFR is sensitive to amphiphilic molecules. These data suggest that EGFR is activated by OA and PUFAs, acts as a sensor for unsaturated fatty acids (and amphiphilic molecules), and is a potential transducer by which diet composition may influence vascular wall biology.

Role of sphingosine 1-phosphate in the mitogenesis induced by oxidized low density lipoprotein in smooth muscle cells via activation of sphingomyelinase, ceramidase, and sphingosine kinase.

Oxidized LDL (oxLDL) have been implicated in diverse biological events leading to the development of atherosclerotic lesions. We previously demonstrated that the proliferation of cultured vascular smooth muscle cells (SMC) induced by oxLDL is preceded by an increase in neutral sphingomyelinase activity, sphingomyelin turnover to ceramide, and stimulation of mitogen-activated protein kinases (Augé, N., Escargueil-Blanc, I., Lajoie-Mazenc, I., Suc, I., Andrieu-Abadie, N., Pieraggi, M. T., Chatelut, M., Thiers, J. C., Jaffrézou, J. P., Laurent, G., Levade, T., Nègre-Salvayre, A., and Salvayre, R. (1998) J. Biol. Chem. 273, 12893-12900). Since ceramide can be converted to other bioactive metabolites, such as the well established mitogen sphingosine 1-phosphate (S1P), we investigated whether additional ceramide metabolites are involved in the oxLDL-induced SMC proliferation. We report here that incubation of SMC with oxLDL increased the activities of both acidic and alkaline ceramidases as well as sphingosine kinase, and elevated cellular sphingosine and S1P. Furthermore, the mitogenic effect of oxLDL was inhibited by D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol and N,N-dimethylsphingosine which are inhibitors of ceramidase and sphingosine kinase, respectively. These findings suggest that S1P is a key mediator of the mitogenic effect of oxLDL. In agreement with this conclusion, exogenous addition of sphingosine stimulated the proliferation of cultured SMC, and this effect was abrogated by dimethylsphingosine but not by fumonisin B1, an inhibitor of the acylation of sphingosine to ceramide. Exogenous S1P also promoted SMC proliferation. Altogether, these results strongly suggest that the mitogenic effect of oxLDL in SMC involves the combined activation of sphingomyelinase(s), ceramidase(s), and sphingosine kinase, resulting in the turnover of sphingomyelin to a number of sphingolipid metabolites, of which at least S1P is critical for mitogenesis.

Retrovirus-mediated correction of the metabolic defect in cultured Farber disease cells.

Farber disease is a rare severe lysosomal storage disorder due to a deficient activity of the enzyme acid ceramidase (AC). Patients have granulomas along with lipid-laden macrophages that accumulate in a number of tissues, leading to multiple diverse clinical symptoms. There is no therapy for the disorder and most patients succumb to the disease in early childhood. The severity of the disease progression seems to correlate with the amount of the accumulated ceramide substrate. Since the cDNA for human AC has been elucidated we sought to establish if genetic transfer of this sequence would lead to enzymatic and, especially, functional correction of the catabolic defect in Farber patient cells. To do this, a novel amphotropic recombinant retrovirus was constructed that engineers transfer of the human AC cDNA. On infection of patient fibroblasts, AC enzyme activity in cell extracts was completely restored. Further, substrate-loading assays of intact living cells showed a fully normalized catabolism of lysosomal ceramide. Lastly, as reported for some other corrected enzymatic defects of lysosomes, metabolic cooperativity was seen, in that functionally corrected patient fibroblasts secreted AC that was taken up through the mannose 6-phosphate receptor and used by uncorrected fibroblasts as well as recipient Farber lymphoblastoid cells. This overall transduction and uptake scenario for Farber disease allows future treatment of this severe disorder to be envisioned using gene transfer approaches.

Uptake of 13-hydroperoxylinoleic acid by cultured cells.

Oxidized free fatty acids have profound effects on cultured cells. However, little is known about whether these effects depend on their uptake and metabolism by cells or primarily involve their interaction with cell-surface components. We determined the uptake and metabolism of unoxidized (linoleic or oleic acid) and oxidized linoleic acid (13-hydroperoxyoctadecadienoic acid, 13-HPODE) by endothelial cells, smooth muscle cells, and macrophages. We show that 13-HPODE is poorly taken up by cells. The levels of uptake were dependent on the cell type but were independent of the expression of CD36. 13-HPODE was also poorly used by microsomal lysophosphatidylcholine acyltransferase that is involved in the formation of phosphatidylcholine. Based on these results, we suggest that most of the biological effects of 13-HPODE and other oxidized free fatty acids on cells might involve a direct interaction with cell-surface components. Alternatively, very small amounts of oxidized free fatty acids that enter the cell may have effects, analogous to those of hormones or prostanoids.

Sphingomyelin-degrading pathways in human cells role in cell signalling.

The ubiquitous sphingophospholipid sphingomyelin (SM) can be hydrolysed in human cells to ceramide by different sphingomyelinases (SMases). These enzymes exert a dual role, enabling not only the turnover of membrane SM and the degradation of exogenous (lipoprotein) SM, but also the signal-induced generation of the lipid second messenger ceramide. This review focuses on the function(s) of the different SMases in living cells. While both lysosomal and non-lysosomal pathways that ensure SM hydrolysis in intact cells can be distinguished, the precise contribution of each of these SM-cleaving enzymes to the production of ceramide as a signalling molecule remains to be clarified.

Oxidized low-density lipoprotein, a two-faced Janus in coronary artery disease?

The word antioxidant has become a household term, and every day we are bombarded with claims of antioxidant protection against a host of diseases. Atherosclerosis, cancer, gastric ulcers, memory loss, rheumatoid arthritis, endometriosis, pregnancy complications, hypertension, stroke, and a host of other diseases have been suggested to be induced by oxidative stress, and antioxidants have been suggested to be beneficial in the prevention and treatment of these disorders. While some of these may be exuberant claims, atherosclerosis is one disease in which the oxidation hypothesis has taken firm roots. The oxidation of low-density lipoprotein (LDL) has been suggested to be a key step in the initiation of the early atherosclerotic lesion. A number of proatherogenic effects have been described for both the protein and lipid components of oxidized low-density lipoprotein. In this commentary, a brief description of the involvement of oxidation and the potential for antioxidant treatment for cardiovascular disease will be provided. However, there are innumerable questions plaguing the hypothesis; this commentary, therefore, will also serve as a devil's advocate and propose that some form of oxidation might actually be beneficial.

Implications of lag time concept in the oxidation of LDL.

Oxidation of low density lipoprotein (LDL) has been implicated in the pathogenesis of atherosclerosis. The most common technique for measuring the oxidation of lipoproteins is the continuous measurement of the formation of conjugated diene at OD 234 nm. The concept of "lag time", derived from such measurements, has been used to test the efficacy of various antioxidants for their ability to inhibit the oxidation of LDL. This review will elaborate on some of the factors that might affect the lag time.

Potential role for ceramide in mitogen-activated protein kinase activation and proliferation of vascular smooth muscle cells induced by oxidized low density lipoprotein.

Proliferation of vascular smooth muscle cells (SMC) is a hallmark in the pathogenesis of atherosclerotic lesions. Mildly oxidized low density lipoproteins (UV-oxLDL), which are mitogenic to cultured AG-08133A SMC, activate the sphingomyelin (SM)-ceramide pathway. We report here the following. (i) UV-oxLDL elicited a biphasic and sustained activation of MBP kinase activity, phosphorylation and nuclear translocation of p44/42 mitogen-activated protein kinase (MAPK), and [3H]thymidine incorporation, which were inhibited by PD-098059, a MAPK kinase inhibitor. (ii) The use of preconditioned media (from SMC pre-activated by UV-oxLDL) transferred to native SMC and blocking antibodies against growth factors suggest that UV-oxLDL-induced activation of MAPK and [3H]thymidine incorporation seem to be independent of any autocrine secretion of growth factors. (iii) UV-oxLDL-induced activation of a neutral sphingomyelinase, SM hydrolysis, ceramide production, and [3H]thymidine incorporation were inhibited by two serine-protease inhibitors (serpins), suggesting that a serpin-sensitive proteolytic pathway is involved in the activation of the SM-ceramide signaling pathway. (iv) UV-oxLDL-induced MAPK activation and [3H]thymidine incorporation were mimicked by ceramide generated in the plasma membrane by bacterial sphingomyelinase treatment or by addition of the permeant C2-ceramide. Serpins did not inhibit the MAPK activation and [3H]thymidine incorporation induced by C2-ceramide, indicating that activation of the MAPK and [3H]thymidine incorporation is subsequent to the stimulation of the SM-ceramide pathway. Taken together, these data suggest that mitogenic concentrations of UV-oxLDL are able to stimulate the SM-ceramide pathway through a protease-dependent mechanism and activate p44/42 MAPK, leading to proliferation of vascular SMC.

Mildly oxidized LDL evokes a sustained Ca(2+)-dependent retraction of vascular smooth muscle cells.

Oxidized low density lipoprotein (LDL) is thought to play a major role in atherogenesis. Atherosclerotic arteries exhibit structural changes associated with profound alterations in vascular tone that are potentially involved in arterial spasm and ischemic heart disease. We report here the role of oxidized LDL in the retraction of vascular smooth muscle cells. Mildly oxidized LDL elicited a broad and sustained peak in cytosolic calcium concentration ([Ca2+]i) in cultured arterial smooth muscle cells. Concomitant with the [Ca2+]i rise, oxidized LDL evoked a sustained and intense retraction of smooth muscle cells, as shown by the changes in cross-sectional area of single cells. Cell retraction was dependent on time, the concentration of oxidized LDL, and the level of LDL oxidation (native LDL induced neither a significant [Ca2+]i rise nor cell retraction). Oxidized LDL but not native LDL also elicited a delayed (12 +/- 2 hours) and sustained (14 +/- 2 hours) increase in isometric tension in deendothelialized arterial rings only, thus suggesting a protective role of intact endothelium. When triggered by nontoxic doses of oxidized LDL, retraction of cultured cells and the contractile response of aortic rings was reversible, whereas with higher (toxic) doses (> or = 200 micrograms apoB/mL), cell retraction was irreversible and led progressively to detachment and cell death. Cell retraction can be prevented in three ways: (1) by inhibiting LDL oxidation with supplements of antioxidants (indirect inhibition); (2) by blocking the pathogenic intracellular signaling elicited by oxidized LDL (direct inhibition), eg, by inhibiting calcium influx with EGTA or the calcium channel blocker nisoldipine or by blocking intracellular signaling (at a still-unknown step) by the lipophilic antioxidant alpha-tocopherol; and (3) by directly inhibiting myosin light chain kinase by 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1, 4-diazepine. In conclusion, oxidized LDL evoked a sustained and intense calcium-dependent retraction of cultured smooth muscle cell, which can be prevented by inhibiting LDL oxidation or by blocking the intracellular signaling induced by oxidized LDL.

The sphingomyelin-ceramide signaling pathway is involved in oxidized low density lipoprotein-induced cell proliferation.

Development of atherosclerosis is believed to involve proliferation of smooth muscle cells (SMC). Our laboratory previously demonstrated that the growth of bovine aortic SMC was stimulated by mildly oxidized low density lipoproteins (oxLDL) and that the mitogenic effect of oxLDL was greater than that induced by native LDL (Augé, N., Pieraggi, M. T., Thiers, J. C., Nègre-Salvayre, A., and Salvayre R.(1995) Biochem. J. 309, 1015-1020). Since the lipid mediator ceramide has been described to be proliferative, the present work aimed at studying the potential involvement of the so-called sphingomyelin-ceramide pathway in the signal transduction cascade induced by oxLDL. Incubation of SMC with UV-oxidized LDL induced sphingomyelin hydrolysis (32%), which peaked at 60 min and was accompanied by a concomitant increase of intracellular ceramide level. The effect of oxidized LDL on sphingomyelin turnover exhibited the same LDL dose dependence as their mitogenic effect. Exogenous bacterial sphingomyelinase induced sphingomyelin hydrolysis and ceramide generation and also stimulated cell growth, in contrast to exogenous phospholipases A2, C, or D. This mitogenic effect was reproduced by incubating the cells with the cell-permeant ceramides, N-acetyl- and N-hexanoylsphingosines. Altogether, these data strongly suggest for the first time that activation of the sphingomyelin-ceramide pathway may play a pivotal role in the oxLDL-induced SMC proliferation and atherogenesis.

Proliferative and cytotoxic effects of mildly oxidized low-density lipoproteins on vascular smooth-muscle cells.

We have investigated the role of low-density lipoprotein (LDL) oxidation in the proliferative effect of LDLs on cultured bovine aortic smooth-muscle cells and compared it with their effect on bovine aortic endothelial cells. The following conclusions were reached. (1) Non-toxic doses of mildly oxidized LDLs elicit a proliferative effect on smooth-muscle cells significantly higher than that of native LDLs or lipoprotein-depleted serum. The proliferative effect is dependent on time (relatively slow), dose (high doses are cytotoxic) and the level of LDL oxidation. (2) The proliferative effect on smooth-muscle cells is counterbalanced at high concentrations of mildly oxidized LDLs (or at high oxidation levels) by their cytotoxic effect. (3) The same dose of mildly oxidized LDLs exhibits no proliferative effect on endothelial cells but rather a cytotoxic one. Endothelial cells may therefore be intrinsically more susceptible to the cytotoxic effect of mildly oxidized LDLs than are smooth-muscle cells. (4) The proliferative effect of native LDLs on smooth-muscle cells results (at least in part) from cell-induced LDL oxidation during cell culture as suggested by (i) the progressive LDL oxidation over the 3 days of contact between LDLs and smooth-muscle cells and (ii) the concomitant inhibition of LDL oxidation and proliferative effect by butylated hydroxytoluene. The hypothetical mechanisms and potential involvement in atherogenesis are discussed.