Proliferation of vascular smooth muscle cells (SMC) isAtherosclerosis, and its complications, namely myocardial infarction, stroke, and peripheral vascular diseases, is one of the most prevalent cause of morbidity and mortality in Western countries. During atherogenesis, focal lesions spread out progressively and lead to the formation of fibro-atheroma plaques, in which smooth muscle cell (SMC) 1 proliferation plays a critical role (1, 2). Among the risk factors identified, low density lipoprotein (LDL) cholesterol level is strongly predictive of coronary heart disease. LDL are believed to have an important role in atherogenesis (3), following oxidative modifications (4 -6), because oxidized LDL are present in atherosclerotic lesions (7) and possess a wide range of biological properties potentially occurring during atherogenesis in vivo (8). Oxidized LDL have recently been shown to be mitogenic to vascular SMC (9 -11). These studies suggest that oxidized LDL may be considered as an additional mitogenic factor, alongside the classical growth factors implicated in SMC proliferation during atherogenesis (6). To date, the mechanism of the oxidized LDL proliferative effect is poorly elucidated and may result from the triggering of a mitogenic intracellular signal either directly by oxidized LDL or indirectly through an autocrine effect involving growth factor secretion and/or growth factor receptor over-expression.Recently, sphingolipids have emerged as key signaling molecules involved in the regulation of cell growth and differentiation (for reviews, see . In particular, the sphingomyelin (SM; ceramide phosphocholine)-ceramide pathway appears as a prototypic sphingolipid signaling pathway implicated in the positive or negative regulation of cell growth. Activation of this pathway leads to SM hydrolysis and subsequent generation of ceramide, the backbone of all sphingolipids, which serves as an intracellular second messenger. To date, several agents have been described to stimulate the SMceramide pathway (reviewed in Refs. 12 and 14 -17), including cytokines such as TNF␣, interleukin-1␤, interferon ␥, nerve growth factor, anti-CD28, anti-Fas antibodies, anticancer drugs, and ionizing radiations (18 -21). Cell-permeant ceram-

Since the generation upon cell stimulation of the second messenger ceramide has been reported to occur in an endosomal/lysosomal compartment, we investigated whether ceramide formed in the lysosomes can escape this compartment. The metabolic fate of radiolabelled ceramide produced by intralysosomal hydrolysis of LDL-associated [ceramide-Q H]sphingomyelin or [stearoyl-1-14 C]sulfatide was examined in fibroblasts from control individuals and a patient with inborn lysosomal ceramidase deficiency (Farber disease). The behavior of this radioactive ceramide was compared to that of a fluorescent (lissamine-rhodaminyl) ceramide analogue deriving from sulfatide degradation. While in Farber cells the natural, radiolabelled ceramide remained completely undegraded and accumulated in the lysosomes, the fluorescent derivative was rapidly converted to sphingomyelin. These findings strongly suggest that, in contrast to fluorescent derivatives, endogenous long-chain ceramide is unable to exit from lysosomes, therefore making the lysosomal ceramide unlikely to be a biomodulatory molecule.z 1998 Federation of European Biochemical Societies.

In loading tests using galactosylceramide which had been labelled with tritium in the ceramide moiety, living skin fibroblast lines derived from the original prosaposin-deficient patients had a markedly reduced capacity to degrade galactosylceramide. The hydrolysis of galactosylceramide could be partially restored in these cells, up to about half the normal rate, by adding pure saposin A, pure saposin C, or a mixture of these saposins to the culture medium. By contrast, saposins B and D had little effect on galactosylceramide hydrolysis in the prosaposin-deficient cells. Cells from P-galactocerebrosidasedeficient (Krabbe) patients had a relatively high residual galactosylceramide degradation, which was similar to the rate observed for prosaposin-deficient cells in the presence of saposin A or C. An SV40-transformed fibroblast line from the original saposin C-deficient patient, where saposin A is not affected, showed normal degradation of galactosylceramide. The findings support the hypothesis, which was deduced originally from in vitro experiments, that saposins A and C are the in vivo activators of galactosylceramide degradation. Although the results with saposin C-deficient fibroblasts suggest that the presence of only saposin A allows galactosylceramide breakdown to proceed at a normal rate in fibroblasts, it remains to be determined whether saposins A and C can substitute for each other with respect to their effects on galactosylceramide metabolism in the whole organism. © 1997 Federation of European Biochemical Societies.

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