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.

Atherosclerosis and its complications, namely heart attack, stroke, and peripheral vascular disease, are the most prevalent causes of human death in Western countries. During atherogenesis, focal lesions spread out progressively and lead to the formation of fibroatheroma plaques in which accumulation of macrophagic foam cells and proliferation of smooth muscle cells (SMC) play a crucial role (24,25). Oxidatively modified low density lipoproteins (LDL) are present in atherosclerotic areas (26,27) and exhibit a variety of biological properties potentially involved in atherogenesis (reviewed in Ref. 28). LDL oxidation is a progressive process leading at first to the formation of mildly oxidized LDL (oxLDL), which are defined by a low content of lipid peroxidation derivatives and slight apolipoprotein B modifications, and later to extensively oxidized LDL which contain high levels of lipid peroxidation products and severe apolipoprotein B alterations (29).
In atherosclerotic areas, SMC proliferation may be mediated through a complex network of growth factors (30). We recently demonstrated that UV-treated LDL, that is mildly oxidized LDL, are mitogenic to cultured bovine aortic SMC (31), an effect also observed with copper-oxidized LDL (32). We report here that the mitogenic effect of oxLDL is transduced through the SPM-ceramide pathway in cultured SMC as shown by the induction of SPM hydrolysis and as mimicked by exogenous sphingomyelinase or short-chain synthetic C 2 -and C 6 France). N-Acetyl-and N-hexanoylsphingosines (C 2 -and C 6ceramides), B. cereus sphingomyelinase, Naja naja phospholipase A2, cabbage phospholipase D, and BHT were obtained from Sigma. All solvents and other reagents obtained from Merck (Darmstadt, Germany) were of analytical grade.
Lipoprotein Isolation and Oxidation by UV-C or Human Endothelial Cells-LDL (d 1.019 -1.063) and lipoprotein-depleted serum were isolated from human pooled fresh sera by sequential ultracentrifugation (33), dialyzed, sterilized by filtration (0.2-m Millipore membrane), and stored at 4°C under nitrogen until use (up to 2 weeks), as described previously (31). The electrophoretic mobility was monitored by electrophoresis on Hydragel ® . Cholesterol and apolipoprotein B concentrations were determined using automated analyzers.
OxLDL were obtained by UV-oxidation. LDL in 0.15 M NaCl containing 0.3 mM EDTA were irradiated by UV-C (0.5 milliwatts/cm 2 for 2 h) under the previously used conditions (31). Alternatively, oxLDL were obtained by cell-mediated oxidation. Endothelial cells (100,000 cells/ml) were seeded in RPMI containing 10% FCS, depleted for 48 h in RPMI medium containing 1% FCS, and further incubated with native LDL (50, 100, or 200 g of apoB/ml) for 16 h in medium containing 1% FCS. Then, the culture medium was collected, filtered, and added to SMC. These procedures were set up to obtain the formation of mildly oxidized LDL, i.e. LDL characterized by a moderate amount of lipid peroxidation products (34) (2.5 Ϯ 0.3 and 3.6 Ϯ 0.4 nmol of TBARS/mg of apolipoprotein B, for cell-and UV-mediated oxLDL, respectively), by a minor loss of trinitrobenzene sulfonic acid-reactive amino groups (35), and by a cellular uptake at a rate similar to that of nonoxidized LDL (31).
Cell Proliferation and Cytotoxicity Measurements-After trypsinization, bovine aortic SMC were seeded at a density of 50,000 cells/ml in 6-multiwell Nunc culture plaques, in medium containing 10% FCS for 24 h. Then the medium was removed, and cells were washed once with RPMI and grown for 24 h in RPMI medium containing 1% FCS (without any loss of cell viability). Cells were then incubated with the indicated concentration of mitogenic agent (native LDL, oxLDL, sphingomyelinase, short-chain ceramides, or phospholipases) for 24, 48, or 72 h and labeled for the last 12 h of the experiment with [ 3 H]thymidine (0.5 Ci/ml). Cells were washed 3 times with PBS, harvested, precipitated by 3% perchloric acid, and centrifuged at 1000 ϫ g for 10 min. The precipitate was dissolved in 1 M NaOH and 1% SDS overnight and mixed with Aquasafe-300 (Zinsser, BAI) for liquid scintillation counting (Packard Tri-Carb-4530). At each time, the [ 3 H]thymidine radioactivity was expressed as percent of that measured in control cells grown in RPMI containing only 1% FCS.
Alternatively, cells were labeled for the last 16 h with 10 M BrdUrd in the presence of 10 M 5-fluorodeoxyuridine. Cells that have incorporated BrdUrd were immunologically detected (36), using a ternary system anti-BrdUrd monoclonal antibody, antimouse biotinylated sheep Ig, and fluorescein isothiocyanate-streptavidin.
The cytotoxicity was evaluated by lactate dehydrogenase (LDH) activity released into the culture medium (Roche assay kit, MA kit 10 ® ) or using the MTT test (37).
Metabolic Labeling of Cellular Choline Phospholipids-For SPM determination, SMC were metabolically labeled to equilibrium with [methyl-3 H]choline (0.5 Ci/ml) in RPMI medium containing 1% FCS. After 48 h of incubation, cells were washed once with PBS and chased for 2 h in fresh RPMI containing 1% FCS. Then, the medium was replaced by fresh 1% FCS-containing medium with or without UV-oxLDL (100 g of apoB/ml) or bacterial sphingomyelinase (100 milliunits/ml). At the indicated times, cells were washed with ice-cold PBS, harvested using a rubber policeman, and sedimented by centrifugation (300 ϫ g for 5 min). Cell pellets were immediately frozen at Ϫ20°C.
Metabolic Labeling with [9, H]Palmitic Acid-For the estimation of SPM and ceramide, cells were incubated for 48 h with [9, H]palmitic acid in 1% FCS-containing medium (1 Ci/ml). After 2 h of chase, cells were treated with LDL or sphingomyelinase exactly as described for the experiments with radioactive choline.
Lipid Extraction and Analyses-Cell pellets were suspended in 0.6 ml of distilled water and homogenized by sonication (2 ϫ 10 s, using a MSE probe sonicator). An aliquot was saved for protein determination (38). Lipids from 0.5 ml of the cell lysate were extracted by 2.5 ml of chloroform/methanol (39). The lipid phase was evaporated under nitro-gen. [ 3 H]Choline-labeled SPM was quantified as described previously (40). The [9,10-3 H]palmitic acid-labeled lipids were separated by TLC on Silica Gel G 60 analytical plates, using 4 successive runs, 1 run with chloroform/methanol/water (100:42:6, by volume) up to 14 cm, and 3 successive runs with hexane/diethyl ether/formic acid (55:45:1, by volume) up to 19 cm. Radioactive lipids were localized using a Berthold radiochromatoscan, SPM and ceramide spots were scraped off and counted by liquid scintillation.

RESULTS AND DISCUSSION
Exposure of LDL to cultured endothelial cells or to UV-C irradiation results in mild lipoprotein oxidation (29,34,41,42). In contrast to extensively oxidized LDL, mildly oxidized LDL used here are taken up by cultured cells (42), including SMC (data not shown), through the apoB/E receptor pathway. As illustrated in Fig. 1, both endothelial cell-and UV-oxidized LDL (50 and 100 g of apoB/ml) were mitogenic to vascular SMC. Incubation of SMC with higher concentrations (200 g of apoB/ml) of oxLDL did not increase DNA synthesis but were rather cytostatic, in agreement with previous observations (31). Native LDL exhibited a lower mitogenic effect than that of oxLDL, and their effect was prevented by the addition of BHT (10 mol/liter). However, the mitogenic effect of UV-oxidized LDL was not inhibited by the same dose of BHT (see Fig. 1B,  inset). This result suggests that (i) BHT is not antiproliferative per se, (ii) BHT blocks the mitogenic effect of native LDL by inhibiting their oxidation in the culture medium (data not shown), and (iii) the major part of the mitogenic effect of LDL is subsequent to their oxidation. In the next experiments, UVoxidized LDL were employed because the culture medium containing cell-oxidized LDL may contain growth factors secreted by endothelial cells which would interfere with the mitogenic effect of oxidized LDL.
In order to investigate whether oxLDL activate the SPM- ceramide signaling pathway, SMC phospholipids were metabolically labeled to equilibrium either with [methyl-3 H]choline or [9,10-3 H]palmitic acid. As shown in Fig. 2, A and B, effective mitogenic doses of UV-oxidized LDL (100 g of apoB/ml) induced a time-dependent degradation of SPM. Maximal hydrolysis of [ 3 H]choline-labeled SPM (32 Ϯ 9%) was observed within 50 -70 min after addition of oxLDL. Then, SPM levels returned progressively toward baseline ( Fig. 2A). Similar results of SPM hydrolysis were obtained when using cells labeled with [ 3 H]palmitic acid (Fig. 2B). Activation of SPM hydrolysis was accompanied by a concomitant increase of cellular ceramide levels (Fig. 2B), supporting the conclusion that oxLDL stimulate a sphingomyelinase activity. Both SPM and ceramide levels recovered to baseline within 2 h, similarly to responses seen with various activators of the SPM cycle, such as vitamin D3 (9), TNF␣ (10, 43), interferon-␥ (10), nerve growth factor (17), or anti-Fas (20).
To examine further the connection between oxLDL-induced cell proliferation and activation of the SPM pathway, the dose dependence of the effects of oxLDL on SPM degradation was investigated. While 100 g of apoB/ml of oxLDL consistently promoted ϳ30% SPM hydrolysis, a concentration of 50 g/ml caused only a 15% reduction (which was also shorter in duration), and, quite unexpectedly, no significant SPM degradation was detected after incubation (for 15 to 120 min) with 200 g/ml oxLDL (Fig. 2C). Furthermore, as observed with thymidine incorporation assays (see Fig. 1B, inset), addition of BHT did not significantly inhibit the oxLDL-induced activation of SPM breakdown (Fig. 2C). Thus, the effects of oxLDL on SPM turnover closely paralleled their effects on mitogenesis (see Fig.  1, A and B).
Since native LDL were also found to stimulate SMC growth ( Fig. 1 and Ref. 31), the effects on increasing concentrations of native LDL on SPM turnover were examined. Interestingly, only elevated concentrations of native LDL promoted SPM hydrolysis (Fig. 2D). It is noteworthy that the extent of SPM degradation induced by 100 g of apoB/ml of native LDL approximated that induced by 50 g of apoB/ml of oxLDL, thereby correlating with the effects on cell growth (see Fig. 1, A and B). Finally, the effects of native LDL on SPM degradation were severely inhibited by BHT, again in accordance with the inhibition by BHT of the mitogenic effect of native LDL. Based on these observations, it is tempting to speculate that native LDL may stimulate SPM hydrolysis and subsequent cell proliferation because they get oxidized when added to the cell culture medium.
As the SPM-ceramide pathway has been reported to be involved in cell growth regulation (5,8,44), the above data suggested that SPM hydrolysis might be involved in the intracellular signaling triggered by oxLDL in vascular SMC. To further define the potential role of SPM degradation and subsequent ceramide generation, we investigated the ability of membrane SPM hydrolysis and of cell-permeant ceramide analogs to mimic the mitogenic effect of oxLDL. Treatment of SMC by bacterial sphingomyelinase, under nontoxic conditions, induced an intense incorporation of [ 3 H]thymidine (205 Ϯ 20% of the control after a 48-h incubation; Fig. 3A), that was associated with an extensive hydrolysis of radiolabeled SPM (72 Ϯ 9% degradation of cellular radiolabeled SPM within 15 min) and a concomitant production of ceramide (Fig. 3B). In contrast, under nontoxic conditions, addition of exogenous phospholipases A2, C, and D led to the hydrolysis of 35-40% of cellular radiolabeled phospholipids, in particular phosphatidylcholine, but induced no significant [ 3 H]thymidine incorporation (Fig. 3, C and D). This strongly suggests that, under the used conditions, hydrolysis of the SPM present in the plasma membrane triggers a mitogenic signal in vascular SMC, whereas, under similar conditions, phosphatidylcholine hydrolysis by various phospholipases is unable to produce any mitogenic effect.
Among the various metabolites derived from SPM hydrolysis, ceramide has been shown to be mitogenic per se in Swiss 3T3 fibroblasts (45,46). This led us to test whether addition of cell-permeable, short-chain ceramide analogs, such as C 2 -ceramide (N-acetylsphingosine) and C 6 -ceramide (N-hexanoylsphingosine) were mitogenic to the vascular SMC used here. Fig. 4 shows that treatment of SMC with 1 M C 2 -ceramide or 5 M C 6 -ceramide (i.e. under nontoxic conditions), resulted in a significant [ 3 H]thymidine incorporation, thereby mimicking the effect of endogenously produced ceramide.
Taken together, the present findings demonstrate that mildly oxidized LDL activate the SPM-ceramide pathway in SMC with the same dose dependence as their effect on cell growth. In addition, the mitogenic effect of oxLDL can be recapitulated by production or addition of ceramides. Such a proliferative effect has previously been documented on Swiss 3T3 cells directly treated with synthetic ceramides or exogenous sphingomyelinase (45,46). On the same cells, ceramide has also been shown to potentiate the proliferation induced by platelet-derived growth factor (47). While in those studies the effect of exogenously added ceramides or of ceramides produced by bacterial sphingomyelinase was investigated, the question still remained whether the mitogenic response could be elicited by the natural ceramide generated in situ through the action of an extracellular agent. The present study strongly supports this idea, corroborating previous reports which indicated that triggering of the SPM-ceramide pathway is involved in cell proliferation. For instance, the SPM-ceramide pathway has been shown to be activated in the TNF␣ or interleukin-1␤induced proliferation of human skin fibroblasts (40), as well as in the T cell proliferation triggered by anti-CD28 (19).
To our knowledge, the present study is the first report on the initiation of the SPM-ceramide turnover in SMC and on the overlap of SPM and oxLDL signaling pathways. Since ceramide can induce the phosphorylation and activation of mitogen-activated protein kinases (MAPK) (47,48), which are classically associated with cell proliferation signaling, it is tempting to speculate that the mitogenic effect of oxLDL is mediated by the stimulation of ceramide-activated MAPK. Indeed, preliminary results from our laboratory show that oxLDL stimulate MAPK. 2 The proliferative effect of ceramide might also be due to another metabolite of SPM hydrolysis (5). However, it has been demonstrated that exogenous ceramides most probably exert their effect without any metabolic conversion (13,45), suggesting that ceramide actually represents the effective mediator.
Oxidized LDL exhibit a variety of biological properties, being able to modulate gene expression of growth factors, adhesion molecules, and tissue factor, altering the motility of monocytes/ macrophages and the vasomotor properties of arteries, being cytotoxic (reviewed in Ref. 28) and mitogenic to vascular SMC (31,32). The signaling pathways involved in the cellular effects triggered by oxidized LDL are only poorly known, and no coherent picture has emerged to account for all of the data. Although the participation of other signaling pathways cannot be excluded (49), our data provide strong evidence for the involvement of the SPM-ceramide signal transduction pathway in the mitogenic effect of mildly oxidized LDL on vascular SMC. FIG. 4. Mitogenic effect of synthetic short-chain ceramides,  N-acetylsphingosine (A) or N-hexanoylsphingosine (B). Cell-permeant ceramides (squares, 1 M; circles, 5 M; triangles, 10 M) were introduced into the culture medium at time 0, and [ 3 H]thymidine incorporation was determined at the indicated times, exactly under the conditions described in Fig. 1. Inset, cytotoxicity evaluated by LDH activity (IU/liter) released into the culture medium (means Ϯ S.E. of 3 separate experiments).