Induction Of Tissue Factor In Endothelial Cells Research Articles

Tissue factor induction by protease-activated receptor 1 requires intact caveolin-enriched membrane microdomains in human endothelial cells.

Protease-activated receptors (PARs) comprise a family of G-protein-coupled receptors with a unique mechanism of proteolytic activation. PARs regulate a broad range of cellular functions and are active in the pathogenesis of disorders characterized by chronic inflammation or activation of the coagulation cascade. Signaling through PAR1 and PAR2 shifts the endothelium towards a prothrombotic phenotype, thereby exacerbating the initial pathophysiologic condition. This study aimed to analyze the localization of PARs in the cell membrane and how their compartmentalization affects tissue factor (TF) in human endothelial cells. TF expression was determined by quantitative real-time polymerase chain reaction analysis and by activity assays. The interaction of PARs with caveolin was investigated through: (i) caveolin-1 gene knockdown performed by transfection with specific small interfering RNA (siRNA); (ii) caveolin-enriched membrane microdomain disruption; and (iii) coimmunoprecipitation assay. We have shown that PAR1, but not PAR2, is present in endothelial caveolin-enriched membrane microdomains, where it is bound to caveolin-1, and that these structures must be intact if PAR1-induced signaling is to increase TF activity. Cholesterol depletion of endothelial cells by cholesterol-sequestering agents caused the PAR1 to relocate to high-density membranes, and impaired the induction of TF (P < 0.01) without affecting the PAR2-mediated procoagulant effect. In addition, siRNA directed against caveolin-1 inhibited TF activation by PAR1 (P < 0.01 and P < 0.01, respectively). PAR1 localization in the caveolin-enriched membrane microdomain, bound to caveolin-1, represents a crucial requirement for TF induction in endothelial cells.

Suppression of human monocyte tissue factor induction by red wine phenolics and synthetic derivatives of resveratrol

Prevention of cardiovascular disease through nutritional supplements is growing in popularity throughout the world. Multiple epidemiologic studies found that moderate consumption of alcohol, particularly red wine, lowers mortality rates from coronary heart diseases (CHD). Chronic inflammation and atherosclerosis associated with CHD culminate in aberrant intravascular expression of tissue factor (TF), which triggers blood coagulation leading to thrombosis, a major cause for heart attack. We showed earlier that two red wine phenolics, resveratrol and quercetin, suppressed TF induction in endothelial cells. In the present study, we investigated efficacy of seven resveratrol derivatives, which were shown to be effective in regulating cancer cell growth in vitro at much lower concentrations than the parent compound resveratrol, in inhibiting TF induction in peripheral blood mononuclear cells (PBMCs). We also tested possible synergistic effects of resveratrol and quercetin with the other major red wine phenolics in suppression of lipopolysaccharide-induced TF expression in human PBMCs. We found that several resveratrol derivatives were 2- to 10-fold more efficient than resveratrol in inhibiting TF induction. Our study found no evidence for synergism among red wine polyphenolics. These data suggest that structural alterations of resveratrol can be effective in producing potent antithrombotic agents that will have therapeutic potential in the improvement of cardiovascular health and prevention of CHD. Among major red wine phenolics, quercetin appears to be the predominant suppressor of TF induction.

Effect of Wine Phenolics and Their Analogs on Suppression of Tissue Factor Induction in Mononuclear Cells.

Several epidemiologic studies suggest that moderate consumption of alcohol, particularly red wine, lowers mortality rates from coronary heart diseases. Aberrant expression of tissue factor (TF), a cellular receptor of coagulation factor VIIa, is thought to contribute to the pathogenesis of coronary heart diseases (CHD) and acute coronary events associated with atherosclerosis. Therefore we hypothesized earlier that consumption of red wine provides protection against CHD by suppressing the aberrant expression of TF in vascular cells, and showed two red wine phenolics, resveratrol and quercetin, suppressed TF induction in endothelial cells and mononuclear cells. However these studies revealed that relatively high concentrations of these compounds (10 to 100 μM), which were unlikely to be achieved in vivo after moderate consumption of red wine, were needed to suppress TF induction. The present study was undertaken to investigate whether other red wine phenolics potentiate the inhibitory effects of resveratrol or quercetin and evaluate the effect of various resveratrol analogs, which were shown to be 10 to 1000 times more effective than resveratrol in modulating other cellular effects, on TF induction in mononuclear cells. Mononuclear cells isolated from healthy human blood donors were treated with various wine phenolics alone or in a combination for 1 h and the cells were stimulated with LPS (10 ng/ml for 6 h) to induce TF expression. TF activity in cell lysates was measured as its ability to support factor VIIa catalyzed activation of factor X. Studies of treating mononuclear cells with varying concentrations of resveratrol and quercetin alone or in a combination revealed that their inhibitory effects were additive and not synergistic. Treatment of cells with a cocktail of red wine phenolics (in concentrations that present in red wine) completely suppressed LPS-induced TF activity. Exclusion resveratrol, epicatechin, catechin or rutin from the cocktail did not diminish the inhibitory effect. In contrast, exclusion of quercetin from the cocktail fully attenuated the inhibitory effect of the cocktail. Further experiments showed that quercetin (55 μM) alone fully suppressed TF induction. Among various resveratrol analogs tested, the cis-form methylated derivative of resveratrol (3,5,4′-trimethoxystilbene, TMS) was 10-times more effective than resveratrol in inhibiting TF induction. A 2 μM of TMS inhibited the induction of TF by more than 75%. None of the wine phenolics or resveratrol analogs had a significant effect on the cell viability. Overall these data suggest that quercetin is the principal inhibitor of TF induction among red wine phenolics and no synergism exists among various wine phenolics in inhibiting TF induction. Comparison of our data on the effect of resveratrol analogs on TF induction with previously published studies on other cellular functions suggests that resveratrol analogs exhibit varying potencies in modulating different cellular activities.