ucn-1028-c and Arteriosclerosis

Oxidatively modified low density lipoprotein (LDL) has been implicated in the pathogenesis of atherosclerosis. LDL oxidation may be mediated by several factors, including cellular lipoxygenases. The lipoxygenase product of linoleic acid, 13-hydroperoxyoctadecadienoic acid (13-HPODE), is a significant component of oxidized LDL and has been shown to be present in atherosclerotic lesions. However, the mechanism of action of these oxidized lipids in vascular smooth muscle cells (VSMCs) is not clear. In the present study, we show that 13-HPODE leads to the activation of Ras as well as the mitogen-activated protein kinases, extracellular signal-regulated kinase 1/2, p38, and c-Jun amino-terminal kinase, in porcine VSMCs. 13-HPODE also specifically activated the oxidant stress-responsive transcription factor, nuclear factor-kappaB, but not activator protein-1 or activator protein-2. 13-HPODE-induced nuclear factor-kappaB DNA binding activity was blocked by an antioxidant, N-acetylcysteine, as well as an inhibitor of protein kinase C. 13-HPODE, but not the hydroxy product, 13-(S)-hydroxyoctadecadienoic acid, also dose-dependently increased vascular cell adhesion molecule-1 promoter activation. This was inhibited by an antioxidant as well as by inhibitors of Ras p38 mitogen-activated protein kinase and protein kinase C. Our results suggest that oxidized lipid components of oxidized LDL, such as 13-HPODE, may play a key role in the atherogenic process by inducing the transcriptional regulation of inflammatory genes in VSMCs via the activation of key signaling kinases.

Topics: Active Transport, Cell Nucleus; Animals; Arteriosclerosis; Cell Nucleus; Cells, Cultured; DNA-Binding Proteins; I-kappa B Proteins; Linoleic Acids; Lipid Peroxides; MAP Kinase Signaling System; Muscle, Smooth, Vascular; Naphthalenes; NF-kappa B; NF-KappaB Inhibitor alpha; Oxidative Stress; Promoter Regions, Genetic; Protein Kinase C; Proto-Oncogene Proteins p21(ras); Swine; Transcriptional Activation; Vascular Cell Adhesion Molecule-1

Elevated plasma homocysteine (Hcy) levels have been defined as an increased risk of atherosclerosis. However, the mechanisms that Hcy induces the development of atherosclerosis are not fully understood. Therefore, effect of Hcy on B lymphocyte proliferation and its cellular mechanism were examined in normal and hyperhomocysteinemia ApoE-knockout mice.. Mouse B lymphocytes were incubated with Hcy, related compounds and/or antioxidants and/or inhibitors of PKC, p38 MAPK, NF-kappaB in the presence or absence of lipopolysaccharide. DNA synthesis, production of reactive oxygen species was measured.. Hcy (0.1-3.0 mM) and other compounds with thiol (-SH), such as cysteine and glutathione significantly increased resting and lipopolysaccharide-induced B lymphocyte proliferation. ApoE-knockout mice with hypercysteinemia (plasma Hcy levels were 20.3+/-2.9 vs. 2.6+/-0.6 microM in control, P<0.05) had a significant promotion of B cell proliferation in response to lipopolysaccharide. Hcy also increased intracellular reactive oxygen species production. Radical scavengers reduced Hcy-induced B lymphocyte proliferation. The promotion of Hcy was significantly inhibited by inhibitors of PKC (calphostin C and RO-31-8220), p38 MAPK (SB 202190 and PD 169316) and NF-kappaB (pyrrolidine dithiocarbamate).. The reactive oxygen species generated by thiol (-SH) auto-oxidation of Hcy are essential, and PKC, p38 MAPK and NF-kappaB are involved in the Hcy-induced B lymphocyte proliferation. Hyperhomocysteinemia may increase B lymphocyte susceptibility to inflammatory progression of atherosclerotic lesions.

Topics: Analysis of Variance; Animals; Apolipoproteins E; Arteriosclerosis; B-Lymphocytes; Catalase; Cell Division; Cells, Cultured; Cysteine; Dimethyl Sulfoxide; Enzyme Inhibitors; Free Radical Scavengers; Glutathione; Homocysteine; Imidazoles; Indoles; L-Lactate Dehydrogenase; Lipopolysaccharides; Male; Mice; Mice, Inbred BALB C; Mice, Knockout; Mitogen-Activated Protein Kinases; Naphthalenes; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Protein Kinase C; Pyridines; Pyrrolidines; Reactive Oxygen Species; Signal Transduction; Spleen; Stimulation, Chemical; Sulfhydryl Compounds; Superoxide Dismutase; Thiocarbamates

Vascular smooth muscle cell (VSMC) proliferation is a key event in the development and progression of atherosclerotic lesions. Accumulating evidence suggests that lipoprotein lipase (LPL) produced in the vascular wall may exert proatherogenic effects. The aim of the present study was to examine the effect of LPL on VSMC proliferation. Incubation of growth-arrested human VSMCs with purified endotoxin-free bovine LPL for 48 and 72 hours, in the absence of any added exogenous lipoproteins, resulted in a dose-dependent increase in VSMC growth. Addition of VLDLs to the culture media did not further enhance the LPL effect. Treatment of growth-arrested VSMCs with purified human or murine LPL (1 microg/mL) led to a similar increase in cell proliferation. Neutralization of bovine LPL by the monoclonal 5D2 antibody, irreversible inhibition, or heat inactivation of the lipase suppressed the LPL stimulatory effect on VSMC growth. Moreover, preincubation of VSMCs with the specific protein kinase C inhibitors calphostin C and chelerythrine totally abolished LPL-induced VSMC proliferation. In LPL-treated VSMCs, a significant increase in protein kinase C activity was observed. Treatment of VSMCs with heparinase III (1 U/mL) totally inhibited LPL-induced human VSMC proliferation. Taken together, these data indicate that LPL stimulates VSMC proliferation. LPL enzymatic activity, protein kinase C activation, and LPL binding to heparan sulfate proteoglycans expressed on VSMC surfaces are required for this effect. The stimulatory effect of LPL on VSMC proliferation may represent an additional mechanism through which the enzyme contributes to the progression of atherosclerosis.

Topics: Alkaloids; Animals; Antibodies, Monoclonal; Arteriosclerosis; Benzophenanthridines; Cattle; Cell Division; Cells, Cultured; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Hot Temperature; Humans; Lipoprotein Lipase; Mice; Muscle, Smooth, Vascular; Naphthalenes; Neutralization Tests; Phenanthridines; Polysaccharide-Lyases; Protein Kinase C; Time Factors; Up-Regulation

Hemodynamic forces are important determinants for the formation of atherosclerotic plaques. The recruitment of circulating monocytes into the arterial wall is an important step during atherogenesis. Monocyte chemotactic protein-1 (MCP-1) has been shown to be a key factor for monocyte transmigration. This study examined the effects of cyclic strain on MCP-1 mRNA expression levels of cultured rat aortic smooth muscle cells. The MCP-1 mRNA levels of aortic smooth muscle cells first increased as the duration of cyclic strain increased, reaching the maximum at 6-12 h, maintained at high levels throughout the 48-h strain period. To explore signaling pathways mediating cyclic strain-stimulated MCP-1 mRNA expression, we examined the involvement of tyrosine kinase and protein kinase C (PKC). Tyrosine kinase inhibitors, genistein and tyrphostin 51, at 50 microM blocked cyclic strain-stimulated MCP-1 mRNA expression. Preincubation with a PKC activator, phorbol 12-myristate 13-acetate (PMA), 2 microM, for 24 h to downregulate PKC did not decrease cyclic strain-induced MCP-1 mRNA expression. A 6-h incubation with 0. 1 microM PMA to activate PKC, which stimulated MCP-1 expression when applied alone, abolished the stimulatory effects of cyclic strain. A specific PKC inhibitor, calphostin C (0.1 microM), diminished cyclic strain-stimulated MCP-1 mRNA expression. Angiotensin II at 10 or 1,000 nM induced a moderate upregulation of MCP-1 mRNA, and no synergistic effects were observed between angiotensin II and cyclic strain. These results indicate that cyclic strain stimulates MCP-1 mRNA expression in smooth muscle cells through signaling pathway(s) mediated by tyrosine kinase activation.

Topics: Angiotensin II; Animals; Arteriosclerosis; Cell Movement; Cells, Cultured; Chemokine CCL2; Enzyme Activation; Enzyme Inhibitors; Gene Expression; Monocytes; Muscle, Smooth, Vascular; Naphthalenes; Phosphotyrosine; Protein Kinase C; Protein-Tyrosine Kinases; Rats; RNA, Messenger; Stress, Mechanical

Thrombomodulin (TM), a thrombin receptor protein found on the endothelial cell surface, contains 6 tandem epidermal growth factor (EGF)-like structures. Recombinant human TM peptide containing these 6 EGF-like domains (rTME1-6) exhibits mitogenic activity in Swiss 3T3 cells. We examined the localization of TM in atherosclerotic lesions and the effects of rTME1-6 on the growth of cultured rat vascular smooth muscle cells (SMCs). Immunohistochemical analysis demonstrated that TM antigen was localized on monocytes, macrophages, and vascular SMCs. In cultured vascular SMCs, rTME1-6 accelerated [3H]thymidine uptake into DNA in a dose-dependent manner up to 3.4 times the control level. This mitogenic activity was abolished by addition of polyclonal anti-human TM antibody. The rTME1-6-induced mitogenesis was enhanced by EGF. However, a neutralizing monoclonal antibody against the EGF receptor (monoclonal antibody 225) did not inhibit the mitogenic activity of rTME1-6. Calphostin C, a specific protein kinase C inhibitor, and lavendustin-A, an inhibitor of EGF receptor-specific protein tyrosine kinase, inhibited the mitogenic activities of both rTME1-6 and EGF. Finally, rTME1-6 treatment increased the level of phosphorylated mitogen-activated protein kinase in SMCs. Together, these results suggest that TM expression in atherosclerotic lesions may be associated with promotion of atherosclerosis through its mitogenic activity in vascular SMCs.

Topics: Aged; Animals; Arteriosclerosis; Calcium-Calmodulin-Dependent Protein Kinases; Cell Division; DNA; ErbB Receptors; Female; Humans; Male; Middle Aged; Mitogens; Muscle, Smooth, Vascular; Naphthalenes; Rats; Rats, Wistar; Recombinant Proteins; Thrombomodulin; Thymidine