We have previously demonstrated that toxic doses of mildly oxidized LDL evokes in cultured cells a delayed and sustained rise of cytosolic [Ca2+], eliciting in turn irreversible cell damage and leading finally to cell death. HDL and delipidated apolipoprotein (apo). A prevented effectively the toxic effect of oxidized LDL to bovine aortic endothelial cells, in a time- and dose-dependent manner. The major part of the protective effect was mimicked by purified apoA-I, whereas purified apoA-II exhibited only very low protective activity. The protective effect was independent of the paraoxonase-linked HDL activity. The protective effect of HDL is independent of the contact of HDL with oxidized LDL, as shown by preincubation of oxidized LDL with HDL or apoA. In contrast, the protective effect was dependent on the integrity of apoA and on the contact of HDL with cells, thus suggesting that HDL acts directly on cells by enhancing their resistance against oxidized LDL. Preincubation experiments show that the protective effect is dependent on the duration of the contact of cells with HDL (maximal effect observed after 12 to 16 hours' preincubation), is also dependent on protein synthesis, and is persistent for at least 48 hours after the end of the contact of HDL with cells. Finally, effective concentrations of HDL inhibit the Ca2+ peak, which is directly involved in the cytotoxic effect of oxidized LDL, as shown by the inhibitory effect of Ca2+ chelators. All together, these results suggest that HDL, mainly apoA-I, increases the resistance of endothelial cells against oxidized LDL and prevents its toxic (apoptotic) effect by blocking the pathogenic intracellular signaling (culminating in sustained Ca2+ rise) involved in cell death.

Oxidized low density lipoproteins (oxLDL) are thought to play a major role in atherosclerosis. OxLDL exhibit a wide variety of biological effects resulting from their ability to interfere with intracellular signaling. The cellular targets and primary signaling events of oxLDL are unknown. We report that oxLDL elicit, in intact cells, tyrosine phosphorylation of the epithelial growth factor receptor (EGFR) and activation of its signaling pathway. This activation triggered by oxLDL was associated with derivatization of reactive amino groups of EGFR and was mimicked by 4-hydroxynonenal (4-HNE, a major lipid peroxidation product of oxLDL). Immunopurified EGFR was derivatized and activated in vitro by oxLDL lipid extracts and 4-HNE, thus indicating that 1) EGFR may be a primary target of oxidized lipids and 2) EGFR derivatization may be associated with activation. The reported data suggest that EGFR acts as a sensor for oxidized lipids. We therefore propose a novel concept of the mechanism by which oxidized lipids (contained in oxLDL or more generally produced during oxidative stress) are able to activate receptor tyrosine kinase and subsequent signaling pathways, resulting finally in a gain of function.

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-

Abstract-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 ϩ 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. (Circ Res. 1999;85:892-899.)