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Hepatocyte growth factor (HGF) is a potent regeneration factor for endothelial and epithelial cells, and has also been shown to modulate extracellular matrix synthesis and matrix metalloproteinase activity in renal epithelial cells and tumor cells. Controversial results have been published concerning the possible role of HGF in the pathogenesis of coronary atherosclerosis. In this study, we have investigated the effect of oxidized low density lipoproteins (LDL) and elevated glucose concentrations on HGF synthesis in cultured human coronary artery smooth muscle cells. In addition, we have studied whether HGF modulates the release of extracellular matrix, extracellular matrix metalloproteinase inducer (EMMPRIN) and matrix metalloproteinases (MMP) by coronary artery smooth muscle cells. Oxidized LDL (1-10 microg/ml) induced a significant dose-dependent decrease of HGF release and a concomitant decrease of HGF mRNA expression, whereas native LDL and elevated glucose concentrations induced no significant changes of HGF synthesis. Incubation of cultured human coronary smooth muscle cells with human HGF (1-100 ng/ml) did not significantly alter cell migration and collagen I, fibronectin, EMMPRIN, MMP-1, MMP-2 and MMP-9 release. In summary, our results provide evidence that HGF does not promote coronary plaque growth or plaque destabilization. Regarding the fact that HGF is a potent endothelial cell regeneration factor, the observed downregulation of HGF synthesis by oxidized LDL supports the concept that HGF might be a protective factor in coronary atherosclerosis and that a decrease rather than an increase of HGF synthesis might promote coronary atherosclerosis.

Background: The presence of accelerated atherosclerosis in patients with kidney disease cannot be entirely explained by traditional cardiovascular risk factors. Exposure to urea, which is normally present in human blood plasma and elevated in patients with kidney disease, leads to the carbamylation of proteins. We postulated that low-density lipoprotein (LDL) carbamylated by urea has biologic effects relevant to atherosclerosis., Methods: To produce carbamylated LDL (cLDL), human native LDL (nLDL) was chemically modified in vitro by exposure to potassium cyanate. Human coronary artery endothelial cells (HCAECs) and human coronary artery smooth muscle cells (CASMCs) were treated in vitro with cLDL or nLDL. Irreversible cell death was measured using the lactate dehydrogenase (LDH) assay, apoptosis was assessed by annexin V binding, and proliferation was determined using bromodeoxyuridine (BrdU) incorporation. Total plasma protein carbamylation and plasma cLDL were measured in hemodialysis patients using the homocitrulline assay and enzyme-linked immunosorbent assay (ELISA)., Results: Our studies demonstrated that cLDL but not nLDL induced dose-dependent vascular cell injuries relevant to atherosclerosis, which included the proliferation of vascular smooth muscle cells and endothelial cell death. Under light microscopy, endothelial cells treated with cLDL showed signs of morphologic alterations. The injury to endothelial cells measured by LDH release was time-dependent and correlated with the degree of LDL carbamylation. At least a part of the endothelial cell population treated with cLDL died by apoptosis. In patients with advanced renal disease on hemodialysis, total plasma protein carbamylation and plasma cLDL were several times higher than in control healthy individuals., Conclusion: Collectively these data suggest the potential role of carbamylated LDL in accelerated atherosclerosis in patients with chronic renal disease and, possibly, in healthy individuals.

Native low density lipoprotein (n-LDL) is a major risk factor for cardiovascular diseases by inducing inflammatory processes and vascular smooth muscle cell proliferation in vessel cells. It has previously been reported that LDL enhances inflammatory reactions by the up-regulation of interleukin (IL)-8 via the activation of p38 kinase and activator protein (AP)-1 in human aortic smooth muscle cells (hAoSMCs). The findings of this study show, for the first time, that the peroxisome proliferator-activated receptor (PPARalpha) agonist, fenofibrate, completely abolishes the LDL-induced IL-8 up-regulation at the transcriptional level. Pretreatment of hAoSMCs with fenofibrate abolishes the effects of LDL on AP-1 activation without affecting nuclear factor (NF)-kappaB. In contrast, fenofibrate failed to modulate the activation state of p38 and JNK kinases or the levels of c-fos and phospho-Jun. These data suggest that AP-1 is likely to be located at the crossroads between LDL signaling and the regulation of IL-8 modulation by PPARalpha.

Objectives: A healthy, intact coronary artery endothelium is important because most common coronary artery diseases result from loss of endothelial integrity. In this study, we explored the biological significance of the angiopoietin-Tie2 system in porcine coronary artery., Methods: Cultured porcine coronary artery endothelial cells and explanted coronary arteries were used., Results: Immunohistochemical analyses indicated that Ang1 is selectively expressed in vascular muscular cells, whereas angiopoietin-2 (Ang2) and Tie2 are selectively expressed in endothelial cells. Accordingly, Ang1 mRNA is mainly expressed in cultured porcine coronary artery vascular smooth muscle cells, whereas Ang2 and Tie2 mRNAs are mainly expressed in cultured porcine coronary artery endothelial cells (PCAECs). Ang1 (200 ng/ml) induced Tie2 phosphorylation, while Ang2 (200 ng/ml) did not produce Tie2 phosphorylation. Ang1 increased the survival of cultured PCAECs during apoptosis induced by oxidized low-density lipoprotein (OxLDL). This survival effect was does-dependent and PI. Furthermore, Ang1 also protected endothelial cells of explanted coronary artery against OxLDL-induced apoptosis artery., Conclusion: These results suggest that adult coronary artery contains Ang1-Tie2 components that enhance endothelial cell survival to help maintain the normal integrity of the coronary artery endothelium.

Statins competitively inhibit 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity reducing mevalonate synthesis. In this study, antiproliferative and antimigratory effects of the new compound cerivastatin were analyzed and compared with classic statins of the first and second generation using mono- and cocultures of human arterial smooth muscle (haSMC) and endothelial (haEC) cells. Effects on the mitotic index and mitochondrial activity of haEC and haSMC monocultures were tested using BrdU enzyme-linked immunosorbent assay (ELISA) and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) tests, respectively. In lactate dehydrogenase (LDH) assays, cytotoxicity of statins was studied. Transfilter cocultures were performed for 14 days to evaluate haSMC growth under the stimulatory effect of proliferating haEC, which release growth factors [e.g., platelet-derived growth factor (PDGF)]. The hydrophobic statins simvastatin, lovastatin, and atorvastatin significantly inhibited haSMC and haEC growth in monocultures at 0.5-50 microM. However, most potent effects were exerted by cerivastatin in 10- to 30-fold lower doses without any significant cytotoxicity. More important, cerivastatin showed also significant effects on haSMC proliferation and migration in transfilter cocultures at extremely low doses (IC50, 0.04-0.06 microM), even when applied exclusively to the endothelial side and in the presence of low-density lipoprotein (LDL). Addition of mevalonate abolished the effects of cerivastatin completely. Even in the presence of growth-stimulating haEC and LDL, cerivastatin was found to be the most potent inhibitor of haSMC proliferation and migration in doses that also can be reached in human serum after oral drug administration. The results support the concept that statins seems to influence additional cellular mechanisms beyond cholesterol reduction, which might also have a relevance for the prevention of restenosis.

Background: Tissue factor, which is required for the initiation of the extrinsic coagulation cascade, is known to be upregulated in cells within atherosclerotic lesions, including smooth muscle cells. Tissue factor expression on the smooth muscle cell surface could be of pathological significance as a contributor to plaque growth, thrombus formation, and the acute coronary syndrome after plaque rupture., Methods and Results: In this study, we show that LDL increased tissue factor mRNA and cell surface protein in smooth muscle cells without a marked increase in surface tissue factor activity. Hydrogen peroxide activated tissue factor on the cell surface but did not increase tissue factor mRNA or cell surface protein. Sequentially added LDL and hydrogen peroxide increased mRNA, cell surface protein, and activity; surface activity was greater than that observed with hydrogen peroxide alone. The action of hydrogen peroxide did not involve a regulatory mechanism associated with the cytoplasmic tail of tissue factor because a truncated tissue factor lacking the cytoplasmic tail was activated by hydrogen peroxide., Conclusions: These results suggest a novel 2-step pathway for increased tissue factor activity on smooth muscle cell surfaces in which lipoproteins regulate synthesis of a latent tissue factor and oxidants activate the protein complex.

In the present study, we describe possible mechanisms by which hypercholesterolemia may contribute to the development of cardiovascular diseases. Treatment of rat aortic smooth muscle cells for 20 hours with cholesterol-rich liposomes (500 micrograms/mL cholesterol, 100 micrograms/mL low-density lipoprotein) resulted in a 76 +/- 12% increase in total cholesterol content. The effects of cholesterol enrichment were examined by determination of changes in cell membrane fluidity. Fluidity of the cholesterol-enriched cell membranes was decreased at all temperatures between 15 degrees C and 40 degrees C. Changes in membrane fluidity in whole cell membranes represented changes in fluidity of microsomal membranes isolated by Percoll gradient ultracentrifugation. The basal [Ca2+]i and the maximal platelet-derived growth factor (PDGF)-BB-induced [Ca2+]i was elevated by 30% and 90% in cholesterol-enriched cells, respectively. In contrast, the resting pH, and the PDGF-BB-induced stimulation of the Na+/H+ exchange were not affected in cholesterol-enriched cells. The effect of PDGF-BB on [3H]thymidine incorporation in cholesterol-enriched cells was elevated by 40% in comparison with untreated cells. Our findings show that cellular cholesterol may be involved in the development of vascular diseases via modulation of the PDGF-induced increase in [Ca2+]i and DNA synthesis in vascular smooth muscle cells.

The effects of fluvastatin, a new fully synthetic inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, on growth and cell-cycle kinetics of porcine and human vascular smooth muscle cells (SMC) were studied by growth curves and flow cytometric determination of cell-cycle distribution. Growth curves were obtained from counting after 2, 4, 7, 9, 11, and 14 days of incubation in Dulbecco's minimum essential medium and 10% fetal calf serum (FCS). For cell-cycle phase determination, cells were synchronized in G0 by 48 hours of incubation in serum-free medium, then stimulated by incubation in 10% FCS, with or without fluvastatin. There was a concentration-dependent decrease in the proliferation of human and porcine SMC when cells were incubated in the presence of fluvastatin. The reduction in the number of cells was significant with 10(-5) M and 10(-4) M fluvastatin. The G/S-phase transition of human and porcine vascular SMC was reduced to 50% of controls by 10(-4) M fluvastatin, as revealed by cell-cycle analysis. The effects of fluvastatin on growth kinetics and cell-cycle distribution could be completely reversed by the addition of 1 mM mevalonolactone, indicating that the fluvastatin effects are due to specific inhibition of HMG-CoA reductase. The addition of low density lipoprotein as a source of cholesterol failed to support SMC growth and phase transition. Addition of squalene or cholesterol to the culture medium also failed to normalize cell growth. It is concluded that nonsterol products synthesized from mevalonate are necessary for the growth of SMC.(ABSTRACT TRUNCATED AT 250 WORDS)

Hyperlipidemia and hypertension are risk factors for cardiovascular diseases. To obtain insight into intracellular mechanisms underlying these phenomenon, the influence of low density lipoprotein (LDL) on the intracellular free calcium concentration, the intracellular pH, the calcium influx as well as proliferation rate of cultured vascular smooth muscle cells was studied. 1-15 micrograms/ml of LDL increased dose-dependently the concentration of intracellular free calcium and led to biphasic pH-shifts. 3-7 micrograms/ml of LDL augmented thymidine incorporation into cell DNA by 70% and enhanced calcium influx by 80%. LDL (1-15 micrograms/ml) clearly increased contractile response of aortic rings. The findings indicate that low concentrations of LDL may contribute to the pathogenesis of cardiovascular diseases by enhancing cell proliferation and vasoconstriction via changing intracellular calcium and intracellular pH.

A biochemical correlation between vascular cholesterol metabolism and eicosanoid biosynthesis has not been fully elucidated. To assess the effects of cholesteryl ester (CE) accretion on eicosanoid synthesis, we studied eicosanoid metabolism in cultured rabbit aortic smooth muscle cells (SMC) following lipid-enrichment by incubation with cationized LDL (cLDL). SMC exposed to cLDL synthesized 50% less immunoreactive 6-keto-PGF1 alpha than untreated cells when exposed to the calcium ionophore, A-23187. In addition, cLDL-treatment reduced arachidonate acid (AA)-induced prostacyclin (PGI2) production sevenfold. Components of cLDL decreased eicosanoid biosynthesis in the following rank-order: linoleate greater than cholesterol greater than apo-cLDL. Lipid-enriched cells incorporated amounts of [1-14C]AA into phosphatidylcholine and phosphatidylethanolamine equal to control cells, but subsequent exposure to ionophore released significantly less radioactivity as free arachidonate (AA), with proportionally less conversion to eicosanoids. Ionophore released equivalent amounts of AA from all phospholipids, suggesting specificity for uptake, but not release of AA by cellular phospholipases. Cells enriched in CE had an eightfold decrease in percentage of phospholipid-derived AA relative to linoleate as compared to controls. Taken together, our data demonstrate that SMC metabolism of cLDL leads to cholesterol and CE accretion concomitant with diminished production of eicosanoids. Potential mechanisms for this effect include competitive inhibition of eicosanoid production by linoleate derived from LDL, direct inhibition of phospholipase A2 activity by cholesterol, and decrease in cyclooxygenase activity. These findings may have pathophysiological significance in that a reduction in PGI2 synthetic capacity of arterial SMC may exacerbate CE deposition since PGI2 promotes intracellular CE hydrolysis.