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We examined the mechanisms underlying the activation of group IVA cytosolic phospholipase A2 (cPLA2α) contributing to the supply of fatty acids required for the formation of cholesteryl ester in oxidized low-density lipoprotein (oxLDL)-stimulated macrophages. The possible involvement of oxidized lipids was also examined. In [3H]arachidonic acid-labeled mouse peritoneal macrophages, oxLDL stimulated the release of arachidonic acid, which was suppressed by methyl arachidonyl fluorophosphonate (MAFP), a cPLA2α inhibitor. oxLDL induced an increase in PLA2α levels in the membrane fraction without affecting those in whole cells or the activity in the lysate. Among 13-hydroxyoctadecadienoic acid (13-HODE), 7-ketocholesterol, and 25-hydroxycholesterol, oxidized lipids present in oxLDL particles, only 13-HODE induced the release of arachidonic acid, which was also sensitive to MAFP. Under conditions where addition of Ca2+ to the cell lysate induced an increase in cPLA2α protein in the membrane fraction, preincubation with 13-HODE facilitated the Ca2+-dependent translocation of cPLA2α. Furthermore, 13-HODE increased cholesteryl ester formation in the presence of [3H]cholesterol. These results suggest that 13-HODE mediates the oxLDL-induced activation of cPLA2α through an increase in cPLA2α protein in the membranes, thus contributing, in part, to the supply of fatty acids required for the esterification of cholesterol in macrophages. [Copyright &y& Elsevier]

To determine if lysophosphatidylcholine (lysoPC) is able to induce proinflammatory changes in monocytes, its ability to stimulate arachidonic acid (AA) release, a product of phospholipase A2 (PLA2) activity, has been analyzed. LysoPC increased AA release in THP-1 and Mono Mac6 cells in a time- and concentration-dependent manner. The monocytes expressed both secretory and cytosolic PLA2 enzymes and AA release was strongly reduced by cellular pretreatment with different PLA2 inhibitors and by pertussis toxin, an inhibitor of Gi-protein activation. This indicates that both cytosolic and secretory PLA2 enzymes regulate specific lysoPC receptor-induced AA release, suggesting lysoPC participation in monocyte proinflammatory activation. [Copyright &y& Elsevier]

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. [Copyright &y& Elsevier]

Monitoring of differential gene expression is an important step towards understanding of gene function. We describe a comparison of the representational difference analysis (RDA) subtraction process with corresponding microarray analysis. The subtraction steps are followed in a quantitative manner using a shotgun cloning and sequencing procedure that includes over 1900 gene sequences. In parallel, the enriched transcripts are spotted onto microarrays facilitating large scale hybridization analysis of the representations and the difference products. We show by the shotgun procedure that there is a high diversity of gene fragments represented in the iterative RDA products (92–67% singletons) with a low number of shared sequences (<9%) between subsequent subtraction cycles. A non redundant set of 1141 RDA clones were immobilized on glass slides and the majority of these clones (97%) gave repeated good fluorescent signals in a subsequent hybridization of the labelled and amplified original cDNA. We observed only a low number of false positives (<2%) and a more than twofold differential expression for 32% (363) of the immobilized RDA clones. In conclusion, we show that by random sequencing of the difference products we obtained an accurate transcript profile of the individual steps and that large-scale confirmation of the obtained transcripts can be achieved by microarray analysis. [Copyright &y& Elsevier]

Interaction of advanced glycation end products (AGE) with AGE receptors induces several cellular phenomena potentially relating to diabetic complications. We here show that AGE-modified bovine serum albumin (BSA) is endocytosed by adipocytes via CD36. Upon differentiation, 3T3-L1 and human subcutaneous adipose cells showed marked increases in endocytic uptake and subsequent degradation of [125I]AGE-BSA, which were inhibited effectively by the anti-CD36 antibody. Ligand specificity of CD36 for modified BSAs was compared with that of LOX-1 and scavenger receptor class A. Effect of fucoidan on [125I]AGE-BSA binding showed a sharp contrast to that on [125I]-oxidized low density lipoprotein. These results implicate that CD36-mediated interaction of AGE-modified proteins with adipocytes might play a pathological role in obesity or insulin-resistance. [Copyright &y& Elsevier]

Electronegative LDL (LDL(−)) constitutes a plasma subfraction of LDL with proinflammatory properties. Its proportion is increased in familial hypercholesterolemia (FH); however, the characteristics of LDL(−) isolated from FH subjects have not been previously studied. In this work, the composition, oxidative status, and inflammatory capacity on endothelial cells of LDL(−) from FH and normolipemic (NL) subjects were evaluated. LDL(−) from FH was relatively enriched in esterified and free cholesterol and triglyceride, and had lower apoB and phospholipid content compared with the non-electronegative fraction (LDL(+)). LDL(−) also contained increased amounts of apoE, apoC-III, sialic acid, and non-esterified fatty acids (NEFAs). The same was observed in NL subjects, except that esterified cholesterol and phospholipid were similar in LDL(−) and LDL(+). No difference was observed between the two fractions concerning malondialdehyde, fatty acid hydroxides, and antioxidants, thereby indicating the absence of increased oxidation of LDL(−) compared with LDL(+). When LDL(−) (100 mg/l) from NL and FH subjects was incubated for 24 h with human umbilical vein endothelial cells (HUVECs), interleukin 8 (IL-8) and monocyte chemotactic protein 1 (MCP-1) increased twofold in the culture medium compared with LDL(+). Vascular cell adhesion molecule 1 (VCAM-1) expression was not increased by LDL(−). Our data indicate that LDL(−) from FH or NL subjects shows no evidence of increased oxidative modification compared to LDL(+); however, LDL(−) induces twofold the release of chemokines by endothelial cells. This effect, which may contribute to leukocyte recruitment and promote atherogenesis, may be greater in FH subjects in which LDL(−) can be up to eightfold higher than in NL subjects. [Copyright &y& Elsevier]