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 LDLs are thought to play a central role in atherogenesis. Among their wide variety of biological properties, oxidized LDLs exhibit a cytotoxic effect on cultured vascular cells. Toxic doses of mildly oxidized LDLs elicited massive apoptosis in both primary and immortalized cultures of endothelial cells as shown by characteristic morphological and biochemical changes. Cytoplasmic and nucleic modifications (eg, chromatin condensation and nucleus fragmentation) were visualized by using electron and fluorescence microscopy of intact cells labeled by the fluorescent DNA probe SYTO-11. DNA fragmentation was quantified by ultracentrifugation of chromatin fragments, evaluated in situ by using the TUNEL (Terminal transferase-mediated dUTP-biotin nick end labeling) procedure, and visualized by electrophoresis of radiolabeled DNA fragments showing the characteristic apoptotic ladder. Apoptotic cells became rapidly detached and underwent postapoptotic necrosis that led to cell disintegration. Apoptosis was subsequent to a sustained and delayed peak of cytosolic calcium. Both the calcium peak and apoptosis were blocked by chelating the extracellular calcium with EGTA or by inhibiting the calcium influx by the calcium-channel blockers nifedipine and nisoldipine, thus suggesting that the apoptotic process induced by oxidized LDLs is clearly calcium dependent. Aurintricarboxylic acid, an inhibitor of endonucleases, also blocked the apoptotic process without blocking the calcium peak. These results suggest that toxic doses of mildly oxidized LDLs induce massive apoptosis of endothelial cells through a calcium-dependent mechanism and that this apoptotic process can be prevented by inhibiting the rise of cytosolic calcium or by inhibiting cellular endonucleases by aurintricarboxylic acid.

Oxidized low density lipoproteins (LDL) elicit in cultured lymphoblastoid cell lines a delayed and sustained calcium rise followed by a progressive of DNA fragmentation, endogenous proteolysis, and morphological features of necrosis and apoptosis. All these events were blocked by chelating the calcium of the culture medium by EGTA, thus suggesting that the two types of cell death induced by oxidized LDL (necrosis and apoptosis) were subsequent to the rise in calcium. The protease inhibitors leupeptin and antipain were able to block (at least in part) the endogenous proteolysis and the necrotic process, but exhibited no effect on apoptosis and DNA fragmentation. At the opposite, aurintricarboxylic acid and spermine (inhibitors of DNA degradation by endonucleases) inhibit DNA fragmentation and morphological apoptosis, but not endogenous proteolysis and necrosis. These data suggest that cell death induced by oxidized LDL occurs through two calcium-dependent processes triggering 1) on one hand, proteolysis and subsequently necrosis characterized by the loss of cell membrane integrity; and 2) on the other hand, internucleosomal DNA cleavage and subsequently morphological apoptosis. The RNA or protein synthesis inhibitors, actinomycin D and cycloheximide, were completely ineffective in preventing endogenous proteolysis, DNA fragmentation, necrosis, and apoptosis induced by oxidized LDL. The subclassification of the type of apoptosis elicited by oxidized LDL is discussed.

Objective-Oxidized low-density lipoprotein (oxLDL)-induced apoptosis of vascular endothelial cells may contribute to plaque erosion and rupture. We aimed to clarify the relationship between the oxLDL-induced calcium signal and induction of apoptotic pathways. Methods and Results-Apoptosis was evaluated by biochemical methods, including studies of enzyme activities, protein processing, release of proapoptotic factors, chromatin cleavage, and especially by morphological methods that evaluate apoptosis/necrosis by SYTO-13/propidium iodide fluorescent labeling. The oxLDL-induced sustained calcium rise activated 2 distinct calcium-dependent mitochondrial apoptotic pathways in human microvascular endothelial cells. OxLDLs induced calpain activation and subsequent Bid cleavage and cytochrome C release, which were blocked by calpeptin. Cyclosporin-A inhibited cytochrome C release, possibly by inhibiting the opening of the mitochondrial permeability transition pore (mPTP). Calcineurin, another cyclosporin-sensitive step, was not implicated, because oxLDLs inhibited calcineurin and FK-506 treatment was ineffective. Cytochrome C release in turn induced caspase-3 activation. In addition, oxLDLs triggered release and nuclear translocation of mitochondrial apoptosis-inducing factor through a mechanism dependent on calcium but independent of calpains, mPTP, and caspases. Conclusions-OxLDL-induced apoptosis involves 2 distinct calcium-dependent pathways, the first mediated by calpain/ mPTP/cytochrome C/caspase-3 and the second mediated by apoptosis-inducing factor, which is cyclosporin-insensitive and caspase-independent. Key Words: calpain Ⅲ caspase Ⅲ mitochondria Ⅲ apoptosis-inducing factor Ⅲ oxidized low-density lipoprotein Ⅲ atherosclerosis A therogenesis is characterized by lipid deposition, a chronic inflammatory response, and chronic wound healing processes. 1,2 Apoptosis may play a role in endothelial cell lining defects, necrotic core formation, or plaque erosion and rupture. [3][4][5] Among the variety of proapoptotic factors present in atherosclerotic plaques, oxidized low-density lipoproteins (oxLDLs) are thought to play a crucial role by concomitantly inducing lipid storage, local inflammation, and toxic events. 4,[5][6][7][8] OxLDLs trigger apoptosis or necrosis of cultured vascular cells 7-9 and may therefore participate in vascular wall injury, plaque erosion/rupture, and subsequent athero-thrombotic events. 4,5 The proapoptotic effects of oxLDLs are mediated through a complex sequence of signaling events that lead to activation of several caspase-dependent or -independent apoptotic pathways. 8,9 Two separate caspase-dependent apoptotic pathways have been implicated in oxLDL-induced apoptosis. 7-9 The extrinsic apoptotic pathway, mediated by death receptors, Fas, and/or tumor necrosis factor receptor (TNFR) and downstream by caspase-8/caspase-3, is involved in oxLDLinduced apoptosis in endothelial cells. 8,10,11 However, a recent report contests this hypothesis. 12 The intrinsic mitochondrial apoptotic pathway, ...

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-

Oxidized low-density lipoproteins (oxLDL) play a role in the genesis of atherosclerosis. OxLDL are able to induce apoptosis of vascular cells, which is potentially involved in the formation of the necrotic center of atherosclerotic lesions, plaque rupture, and subsequent thrombotic events. Because oxLDL may induce structural modifications of cell protein and altered proteins may impair cell viability, the present work aimed to evaluate the extent of protein alterations, the degradation of modified proteins through the ubiquitin-proteasome system (a major degradative pathway for altered and oxidatively modified proteins) and their role during apoptosis induced by oxLDL. This paper reports the following: 1) oxLDL induce derivatization of cell proteins by 4-hydroxynonenal (4-HNE) and ubiquitination. 2) Toxic concentrations of oxLDL elicit a biphasic effect on proteasome activity. An early and transient activation of endogenous proteolysis is followed rapidly by a subsequent decay (resulting probably from the 26S proteasome inhibition) and followed later by the inhibition of the 20S proteasome (as assessed by inhibition of sLLVY-MCA hydrolysis). 3) Specific inhibitors of proteasome (lactacystin and proteasome inhibitor I) potentiated considerably the toxicity of oxLDL (nontoxic doses of oxLDL became severely toxic). The defect of the ubiquitination pathway (in temperature-sensitive mutants) also potentiated the toxicity of oxLDL. This suggests that the ubiquitin-proteasome pathway plays a role in the cellular defenses against oxLDL-induced toxicity. 4) Dinitrophenylhydrazine (DNPH), an aldehyde reagent, prevented both the oxLDL-induced derivatization of cell proteins and subsequent cytotoxicity. Altogether, the reported data suggest that both derivatization of cell proteins (by 4-HNE and other oxidized lipids) and inhibition of the proteasome pathway are involved in the mechanism of oxLDL-induced apoptosis.

Background-Mildly oxidized LDL (moxLDL) is thought to play a role in atherogenesis. MoxLDL induces derivatization of cell proteins and triggers a variety of intracellular signaling. We aimed to investigate whether moxLDL-induced protein derivatization may influence the activity of platelet-derived growth factor receptor ␤ (PDGFR␤), a tyrosine kinase receptor of major importance in vascular biology and atherogenesis. Methods and Results-In cultured rabbit arterial smooth muscle cells, moxLDL induces activation of the PDGFR␤ signaling pathway, as shown by PDGFR␤ tyrosine phosphorylation on Western blot and coimmunoprecipitation of SH2-containing proteins. The cellular events involved in the moxLDL-induced PDGFR␤ activation can be summarized as follows. Oxidized lipids from moxLDL trigger two phases of PDGFR␤ activation involving two separate mechanisms, as shown by experiments on cultured cells (in situ) and on immunopurified PDGFR␤ (in vitro): (1) the first phase may be mediated by 4-hydroxynonenal, which induces PDGFR␤ adduct formation and subsequent PDGFR␤ activation (antioxidant-insensitive step); (2) the second phase involves ceramide-mediated generation of H 2 O 2 (these steps being inhibited by tosylphenylalanylchloromethylketone, an inhibitor of ceramide formation, and by antioxidant BHT, exogenous catalase, or overexpressed human catalase). Because 4-hydroxynonenal-PDGFR␤ adducts are also detected in atherosclerotic aortas, it is suggested that this novel mechanism of moxLDL-induced PDGFR␤ activation may occur during atherogenesis. Conclusions-MoxLDL acts as a local autoparacrine mediator in the vascular wall, and PDGFR␤ acts as a sensor for both oxidized lipids and oxidative stress. This constitutes a novel mechanism of PDGFR␤ activation in atherosclerotic areas.

Abstract-The platelet-derived growth factor receptor-␤ (PDGFR␤) signaling pathway regulates smooth muscle cell (SMC) migration and proliferation and plays a role in the vascular wall response to injury. Oxidized low-density lipoprotein (oxLDL) in atherosclerotic lesions can activate the PDGFR␤ pathway, but the long-term effects of oxLDL on PDGFR␤ function are not well understood. We found that oxLDL induced a dual effect on PDGFR␤ signaling. Initial activation of the PDGFR was followed by desensitization of the receptor. PDGFR␤ desensitization was not attributable to PDGFR␤ degradation or changes in localization to the caveolae but instead resulted from decreased PDGF binding and inhibition of PDGFR␤ tyrosine kinase activity. This inhibition was associated with formation of (4HNE)-and acrolein-PDGFR␤ adducts and was mimicked by preincubation of cells with 4HNE. These PDGFR␤ adducts were also detected in aortae of apolipoprotein-deficient mice and hypercholesterolemic rabbits and in human carotid plaques. The aldehyde scavengers DNPH and Hydralazine prevented both oxLDL-and 4HNE-induced structural modification and PDGFR␤ signaling dysfunction in cells and in vivo. OxLDL inhibition of PDGF signaling may contribute to defective SMC proliferation and decrease the stability of a vulnerable plaque. Key Words: atherosclerosis Ⅲ oxidized LDL Ⅲ PDGF receptor Ⅲ cell proliferation Ⅲ signaling D uring atherogenesis, the formation of fibroatheroma plaques involves a complex sequence of events, including endothelial activation, transendothelial migration of mononuclear cells, lipoprotein oxidation, lipid accumulation in macrophagic cells, smooth muscle cell (SMC) migration and proliferation, and local inflammatory response. 1,2 Low-density lipoproteins (LDLs) are atherogenic 3 after undergoing oxidative modifications. 4 Oxidized LDLs (oxLDLs), present in atherosclerotic areas, exert various biological effects potentially involved in atherogenesis, including lipid accumulation in macrophages (foam cells), changes in gene expression of adhesion molecules, cytokines, growth factors and coagulation proteins, cell migration, cell proliferation, apoptosis, and local inflammatory/immune response. 4,5 SMC migration and proliferation play a critical role in the formation of fibrous cap of atherosclerotic plaques, whereas toxic events are potentially involved in endothelial cell injury, necrotic core formation, and plaque rupture. 1,2,6 Excessive SMC proliferation and extracellular matrix biosynthesis may lead to occlusive lesions. 1 In contrast, the "thin cap fibroatheroma" containing inflammatory cells and a lipid-rich core is generally a vulnerable plaque prone to atherothrombotic events. [7][8][9] Proliferation of SMC is regulated by numerous growth factors and cytokines 8,10 and by oxLDL. 11,12 Platelet-derived growth factor (PDGF) plays a major role in the fibroproliferative response during atherogenesis and restenosis. 1,13 PDGF-induced signaling is mediated by PDGF receptor-␣ (PDGFR␣) and PDGFR␤, which belong to the large fa...