Abstract-Little is known regarding the molecular mechanisms of atherogenicity of triglyceride-rich lipoproteins such as very low-density lipoproteins (VLDLs). We examined the effect of VLDL on proliferation of rat aortic smooth muscle cells, intracellular Ca 2ϩ handling, and activity of cAMP-responsive element binding protein (CREB) and nuclear factor of activated T cells (NFAT) transcription factors. VLDL, isolated from human serum, dose-and time-dependently promoted proliferation. After 4 hours of exposure to VLDL (0.15 g/L proteins), the caffeine-induced Ca 2ϩ release was inhibited and the IP3-sensitive Ca 2ϩ release induced by ATP (10 mol/L) was markedly prolonged. In quiescent cells, CREB was phosphorylated (pCREB) and NFAT was present in the cytosol, whereas in cells exposed to VLDL for 4 to 24 hours, pCREB disappeared and NFAT was translocated to the nucleus. VLDL-induced NFAT translocation and proliferation were blocked by cyclosporin A and LY294002 involving calcineurin and phosphatidylinositol 3-kinase (PI3K) pathways. Indeed, VLDLs rapidly phosphorylate protein kinase B and glycogen synthase kinase-3␤ in a PI3K-dependent way. These results provide the first evidence that VLDLs induce smooth muscle cell proliferation by activating the PI3K pathway and nuclear NFAT translocation. Blockade of the Ca 2ϩ -induced Ca 2ϩ release mechanism and dephosphorylation of pCREB contribute but were not sufficient to induce a proliferating phenotype. (Circ Res. 2003;92:1115-1122.)Key Words: vascular disease Ⅲ smooth muscle Ⅲ calcium signaling Ⅲ transcription factors Ⅲ lipoproteins H ypertriglyceridemia is considered an independent risk factor of atherosclerosis and coronary disease, 1,2 but the mechanisms of atherogenicity are not clearly defined. An increasing body of evidence supports multiple roles for very low-density lipoproteins (VLDLs), the main carriers of triglycerides, in the development of atherosclerosis and cardiovascular diseases. The VLDLs may have an indirect role because of the strong relationship between elevated triglycerides and other atherogenic metabolic changes, including decreased levels of high-density lipoprotein (HDL) and a predominance of atherogenic small and dense low-density lipoprotein (LDL) particles. They may also have a direct influence on atherosclerosis by altering physiological functions of arterial wall cells. 2,3 A potential relationship between hypertriglyceridemia and disease progression has been suggested in studies showing that atherogenic lipoproteins induce smooth muscle cell (SMC) proliferation, a phenomenon of primary importance in atherosclerosis, restenosis after balloon injury, or neointimal occlusion of grafts. How hypertriglyceridemia alters vascular cells toward the atherosclerotic phenotype is still under intense investigation. Previous studies showed that VLDL stimulated SMC DNA replication and growth by themselves 4,5 via activation of the mitogenactivated protein kinase (MAPK) extracellular signal-regulated kinase (ERK1) and ERK2. 5 ␤VLDL, a cholesteryl ester-r...

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Among the fatty acids used, only modification of the membrane composition due to linoleic acid supply disturbed the basal enzymatic activity and Ser1177 phosphorylation of eNOS in a way that limited the role of histamine activation. Linoleic acid might involve the dysfunction of both eNOS basal activity and its phosphorylation status and may then contribute to an impaired vasodilatation in vivo.

Oxidized low density lipoprotein (Ox-LDL) is a well-established risk factor in atherosclerosis and lysophosphatidylcholine (LysoPtdCho) is considered to be one of the major atherogenic component of Ox-LDL. The purpose of this work was to investigate the effects of two membrane n-3 long chain polyunsaturated fatty acids (n-3 PUFAs), EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid) compared to n-6 PUFA, ARA (arachidonic acid), on the activation of endothelial NO synthase (eNOS) by histamine in Ea hy 926 endothelial cells incubated during 24 h in the presence or the absence of LysoPtdCho. DHA (50 muM) produced a ROS induction in cells and aggravated the LysoPtdCho-induced oxidative stress. It did not modify the basal eNOS activity but impaired the stimulation of eNOS induced by histamine and was unable to correct the deleterious effect of LysoPtdCho on histamine-stimulated eNOS activity or phosphorylation of Ser 1177. In contrast, EPA (90 muM) did not modify the ROS level produced in the presence or absence of LysoPtdCho or basal eNOS activity and the stimulating effect of histamine on eNOS. However, it diminished the deleterious effect of LysoPtdCho as well as on the histamine-stimulated eNOS activity on the phosphorylation on Ser 1177 of eNOS. The beneficial effect of EPA but not DHA on endothelial eNOS activity in Ea hy 926 could be also partially due to a slight decrease in membrane DHA content in EPA-treated cells. Consequently, the equilibrium between NO generated by eNOS and ROS due to oxidative stress could explain, in part, the beneficial effect of EPA on the development of cardiovascular diseases. By contrast ARA an n-6 PUFA was devoid of any effect on ROS generation or eNOS activity in the basal state or after histamine-induced stimulation. In vivo experiments should be undertaken to confirm these results.