Abstract-Oxidized low-density lipoproteins (oxLDLs) trigger various biological responses potentially involved in atherogenesis. Disturbing endoplasmic reticulum (ER) function results in ER stress and unfolded protein response, which tends to restore ER homeostasis but switches to apoptosis when ER stress is prolonged. We aimed to investigate whether ER stress is induced by oxLDLs and can be prevented by the ER-associated chaperone ORP150 (150-kDa oxygen-regulated protein). oxLDLs and the lipid oxidation products 7-ketocholesterol and 4-hydroxynonenal induce ER stress in human endothelial cells (HMEC-1), characterized by the activation of ER stress sensors (phosphorylation of Ire1␣ and PERK, nuclear translocation of ATF6) and of their subsequent pathways (eukaryotic initiation factor 2␣ phosphorylation, expression of XBP1/spliced XBP1, CHOP, and KDEL chaperones GRP78, GRP94, ORP150). ER stress was inhibited by the antioxidant N-acetylcysteine. In advanced atherosclerotic lesions, phospho-Ire1␣, KDEL, and ORP150 staining were localized in lipid-rich areas with 4-hydroxynonenal adducts and CD68-positive macrophagic cells. By comparison, staining for 4-hydroxynonenal, phospho-Ire1␣, KDEL, and ORP were faint and more diffuse in intimal hyperplasia. ER stress takes part in the apoptotic effect of oxLDLs, through the Ire1␣/c-Jun N-terminal kinase pathway, as assessed by the protective effect of specific small interfering RNAs and c-Jun N-terminal kinase inhibitor. Key Words: ER stress Ⅲ apoptosis Ⅲ ORP150 Ⅲ oxidized LDL Ⅲ atherosclerosis L ow oxidized low-density lipoprotein (oxLDL) concentration exhibit proinflammatory and mitogenic effects, whereas higher concentrations elicit a progressive inhibition of survival mechanisms and the induction of proapoptotic signaling pathways. 1-5 oxLDLs may alter the fragile balance between survival and death of vascular cells, thereby leading to stable-to-vulnerable plaque transition and finally to atherothrombotic events. 6 -8 The stress of the endoplasmic reticulum (ER) occurs in atherosclerotic lesions of hypercholesterolemic (apolipoprotein [apo]E Ϫ/Ϫ ) mice, 9 and could be associated with acute coronary syndrome. 10 ER stress emerges as a new adaptive system determining the fate of cells to survive or die 11 and may thereby be implicated in the erosion or rupture of atherosclerotic plaques.Altered protein folding and ER stress occurs in various pathological conditions, including ischemia, hypoxia, heat shock, proteasome inhibition, glycosylation inhibition, oxidative stress, and calcium depletion of ER stores. 12,13 The accumulation of unfolded protein aggregates leads to the activation of transmembrane sensors/transducers inositolrequiring enzyme (Ire)1␣, PERK (RNA-dependent protein kinase-like endoplasmic reticulum kinase), and activating transcription factor (ATF)6 that regulate several signaling pathways, gene expression, and protein synthesis. 11 The presence of ER-resident chaperones is crucial for facilitating and maintaining the protein folding, thereby preventing ER...

Background-The cytokines interleukin 1 (IL-1) and tumor necrosis factor (TNF) are secreted by the different cell populations of the vascular wall and have been suggested to promote atherosclerosis. Methods and Results-Their respective roles in fatty-streak formation in apolipoprotein E-deficient mice were investigated by use of IL-1 receptor antagonist and TNF binding protein. Estradiol-17␤ was used as a positive control. Blocking TNF seemed to be active in female animals but not in males. IL-1 receptor antagonist was as effective as or more effective than estradiol in both sexes. Conclusions-IL-1 plays a crucial role in the initial step of the atherosclerotic process in this animal model, and blocking the activity of this cytokine should be considered as a therapeutic possibility. (Circulation. 1998;97:242-244.)Key Words: atherosclerosis Ⅲ apolipoproteins Ⅲ interleukins Ⅲ tumor necrosis factor I t is known that IL-1␣ and -1␤ and TNF-␣ and -␤ are secreted in the vascular wall by endothelial and smooth muscle cells as well as by monocytes/macrophages. 1,2 These cytokines have been shown to increase permeability of the endothelial cell barrier, 3 induce the expression of surface leukocyte adhesion molecules, 1,4,5 and enhance the production of other cytokines and growth factors, such as IL-6 6 and macrophage colony-stimulating factor, 7-9 all such activities being considered to promote atherosclerosis. The objective of the present study was therefore to clarify the role of IL-1 and TNF in the initial steps of the atherosclerotic process, ie, fatty-streak formation, using apo E KO mice as an animal model of atherosclerosis 10,11 and human IL-1ra and TNFbp as the specific cytokine antagonists. IL-1ra is a recombinant 17-kD protein, which binds to IL-1 receptors and competes with both IL-1␣ and IL-1␤ without detectable IL-1 agonistic effects. 12,13 TNFbp is a specific TNF inhibitor consisting of two molecules of the extracellular domain of the human type 1 TNF receptor added to both ends of a molecule of polyethylene glycol. TNFbp binds with equal affinity to TNF-␣ and TNF-␤.14 -16 E 2 treatment was used as a positive control, because we and others have shown that this hormone prevents fatty-streak formation in the apo E KO mouse animal model. 17,18 The data obtained showed that TNFbp was active in female animals but not in males. Like E 2 , IL-1ra was active in both sexes, suggesting that IL-1 plays a crucial role in the initial step of the atherosclerotic process in this animal model. Methods Study ProtocolApo E KO mice, originally obtained from the Jackson Laboratory, Bar Harbor, Me (sixth generation of backcross from 129/B6 F1 heterozygous to C57BL/6), were housed as previously described 18 and fed normal laboratory mouse chow containing 4.3% fat and 0.02% cholesterol. Four-week-old animals were gonadectomized under general anesthesia. At 2 months of age, these animals were given 0.2 mg 60-day time-release E 2 pellets (Innovative Research of America), a dose that was found to exert a maximal effect on fatty-strea...

Background-Oxidized LDLs (oxLDLs) and matrix metalloproteinases (MMPs) are present in atherosclerotic lesions.OxLDLs activate various signaling pathways potentially involved in atherogenesis. OxLDLs induce smooth muscle cell (SMC) proliferation mediated by the activation of the sphingomyelin/ceramide pathway and tyrosine kinase receptors. MMPs are also able to induce SMC migration and proliferation in addition to extracellular matrix degradation. The present study was designed to investigate whether MMPs play a role in the mitogenic effect of oxLDLs. Methods and Results-OxLDLs induce the release of activated MMP-2 in SMC culture medium. MMP-2 was identified by its 65-kDa gelatinase activity on zymography and by using specific blocking antibodies and MMP-2 Ϫ/Ϫ cells. MMP inhibitors (batimastat and Ro28-2653) and the blocking antibodies anti-MMP-2 and anti-membrane type 1-MMP inhibited the oxLDL-induced sphingomyelin/ceramide pathway activation and subsequent activation of ERK1/2 and DNA synthesis but did not inhibit the oxLDL-induced epidermal growth factor receptor and platelet-derived growth factor receptor activation. Exogenously added activated MMP-2 or membrane type 1-MMP-1 triggered the activation of both sphingomyelin/ceramide and ERK1/2 pathways and DNA synthesis. Conversely, suppression of MMP-2 expression in MMP-2 Ϫ/Ϫ cells or in SMCs treated by small-interference RNA also blocked both sphingomyelin/ceramide signaling and DNA synthesis. Conclusions-Together, these data demonstrate that MMP-2 plays a pivotal role in oxLDL-induced activation of the sphingomyelin/ceramide signaling pathway and subsequent SMC proliferation. These pathways may constitute a potential therapeutic target for modulating the oxLDL-induced proliferation of SMCs in atherosclerosis or restenosis.

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, ...

Development of atherosclerosis is believed to involve proliferation of smooth muscle cells (SMC).

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-

No abstract

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.