Lipid oxidation products and oxidized low-density lipoproteins impair platelet-derived growth factor receptor activity in smooth muscle cells: implication in atherosclerosis.

The platelet-derived growth factor receptor-beta (PDGFRbeta) signaling pathway regulates smooth muscle cell (SMC) migration and proliferation in the vascular wall. Oxidized low-density lipoproteins (oxLDLs) and 4-hydroxynonenal (4-HNE) induce a dual effect on PDGFRbeta signaling. Short-term incubation of SMCs with oxLDLs and 4-HNE induced PDGFRbeta activation. Long-term incubation triggered a desensitization of PDGFR to its own agonist, with a progressive inhibition of PDGFRbeta phosphorylation, associated with increased formation of HNE-PDGFR adducts in SMC and in vivo, in the aortae of apoE-deficient mice. Hydralazine used as carbonyl scavenger prevented PDGFRbeta inhibition in vitro and in vivo In conclusion, PDGFRbeta is a target for 4-HNE, acrolein and oxidative stress and its progressive inhibition may contribute to defective SMC proliferation and decrease the stability of a vulnerable plaque.

Desensitization of platelet-derived growth factor receptor-beta by oxidized lipids in vascular cells and atherosclerotic lesions: prevention by aldehyde scavengers.

The platelet-derived growth factor receptor-beta (PDGFRbeta) 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 PDGFRbeta pathway, but the long-term effects of oxLDL on PDGFRbeta function are not well understood. We found that oxLDL induced a dual effect on PDGFRbeta signaling. Initial activation of the PDGFR was followed by desensitization of the receptor. PDGFRbeta desensitization was not attributable to PDGFRbeta degradation or changes in localization to the caveolae but instead resulted from decreased PDGF binding and inhibition of PDGFRbeta tyrosine kinase activity. This inhibition was associated with formation of (4HNE)- and acrolein-PDGFRbeta adducts and was mimicked by preincubation of cells with 4HNE. These PDGFRbeta 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 PDGFRbeta 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.

Two distinct calcium-dependent mitochondrial pathways are involved in oxidized LDL-induced apoptosis.

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.

Oxidized LDL and 4-hydroxynonenal modulate tyrosine kinase receptor activity.

Among the diverse risk factors involved in atherosclerosis, LDL are thought to become atherogenic after undergoing oxidative modifications, characterized by oxidized lipid formation and structural alterations of apoB. Oxidized LDL alter various signaling pathways and exhibit a broad range of biological responses including inflammation, gene expression, cell proliferation or apoptosis. The biological effects of oxidized LDL are related to the presence of peroxidation products such as hydroperoxides, lysophosphatidylcholines, oxysterols and aldehydes.4-Hydroxynonenal (HNE) is one of the most abundant aldehydes formed during the oxidation of polyunsaturated fatty acids in LDL and in membranes. It is able to react with thiols and free amino group residues of proteins. HNE is involved in apoB modifications that alter LDL metabolism and cell protein-adduct formation which may mediate in part the biological effects of oxidized LDL. We report here that HNE delivered to cells by oxidized LDL reacts with cellular proteins, for instance with tyrosine kinase receptors (RTK) such as EGFR and PDGFR. HNE induces in vitro derivatization and tyrosine phosphorylation of RTK (the fine molecular mechanism and conformational changes remain to be elucidated). In intact living cells, oxidized LDL (and pure HNE) trigger HNE-adduct formation and activation of PDGFR and EGFR, through an antioxidant-insensitive and reactive oxygen species independent mechanism. The presence of HNE-PDGFR adducts in atherosclerotic areas lead one to hypothesize that oxidized lipids may also react in vivo with membrane RTK, thereby disturbing their cellular functions.

Mildly oxidized LDL particle subspecies are distinct in their capacity to induce apoptosis in endothelial cells: role of lipid hydroperoxides.

The risk of atherosclerosis is intimately related to the heterogeneity of low-density lipoprotein (LDL) particles. The potential relationship between oxidative modification of distinct LDL subspecies and induction of apoptosis in arterial wall cells is indeterminate. The capacity of light LDL3 versus dense LDL5 to induce cytotoxicity in endothelial cells as a function of the degree of copper-mediated oxidation was compared. Mildly oxidized LDL3 (oxLDL3) exerted potent cytotoxicity, which was intimately related to both the degree of oxidation and the oxLDL3 concentration based on either cholesterol content or particle number. In contrast, dense LDL5 particles exerted a minor effect on cell viability. Cells incubated with oxLDL3 exhibited apoptotic features, with cytoplasmic condensation, cell or nuclear fragmentation, and accumulation of DNA fragments. OxLDL3-induced apoptosis involved cytoplasmic release of cytochrome c, with a concomitant increase in caspase-3-like protease activity. OxLDL3 particles were uniquely distinct from oxLDL5 particles in their elevated content of lipid hydroperoxides. Hydroperoxide removal by NaBH4 markedly reduced oxLDL3-induced cytotoxicity, leading to an increase in cell viability. Lipid hydroperoxide content of oxidatively modified LDL subclasses is therefore a major determinant of the induction of apoptosis in endothelial cells. These data are highly relevant to atherogenic hypercholesterolemia, in which the LDL phenotype is dominated by elevated concentrations of light LDL3.