Tissue factor activity is increased in human endothelial cells cultured under elevated static pressure

Abstract

We tested the hypothesis that elevated blood pressure, a known stimulus for vascular remodeling and an independent risk factor for the development of atherosclerotic disease, can modulate basal and cytokine-induced tissue factor (TF; CD 142) expression in cultured human endothelial cells (EC). Using a chromogenic enzymatic assay, we measured basal and tumor necrosis factor-α (TNF-α; 10 ng/ml, 5 h)-induced TF activities in human aortic EC (HAEC) and vena cava EC (HVCEC) cultured at atmospheric pressure and at 170 mmHg imposed pressure for up to 48 h. Basal TF activities were 22 ± 10 U/mg protein for HAEC and 14 ± 9 U/mg protein for HVCEC and were upregulated in both cell types >10-fold by TNF-α. Exposure to pressure for 5 h induced additional elevation of basal TF activity by 47 ± 16% ( P < 0.05, n = 6) for HAEC and 17 ± 5% ( P < 0.05, n = 3) for HVCEC. Pressurization also enhanced TF activity in TNF-α-treated cells from 240 ± 28 to 319 ± 32 U/mg protein in HAEC ( P < 0.05, n = 4) and from 148 ± 25 to 179 ± 0.8 U/mg protein ( P < 0.05, n = 3) in HVCEC. Cytokine stimulation caused an ∼100-fold increase in steady-state TF mRNA levels in HAEC, whereas pressurization did not alter either TF mRNA or cell surface antigen expression, as determined by quantitative RT-PCR methodology and ELISA. Elevated pressure, however, modulated the EC plasma membrane organization and/or permeability as inferred from the increased cellular uptake of the fluorescent amphipathic dye merocyanine 540 (33 ± 7%, P < 0.05). Our data suggest that elevated static pressure modulates the hemostatic potential of vascular cells by modifying the molecular organization of the plasma membrane.

Tissue factor expression and activity are not increased in peripheral monocytes isolated from uncomplicated hypertensive patients

“…Moreover, circulating monocytes derived from patients with elevated levels of blood pressure were found to express significantly higher levels of the inflammatory cytokines, TNF-a, a TF inducer, and IL-1b, than normal donors, suggesting an activation of these cells in systemic hypertension [3,4]. In the past few years a number of in-vitro studies have demonstrated that the exposure of human endothelial cells to haemodynamic forces may modify the expression of target genes involved in regulating vascular homeostasis, including the TF gene [22][23][24][25]. The human TF gene promoter region contains binding sites for the transcription factor activator protein 1, nuclear factor kappa B/Rel, Egr-1 and Sp-1 [28,29].…”

“…Circulating monocytes isolated from hypertensive individuals have been seen to produce more inflammatory cytokines and have a greater power to adhere to cultivated endothelial cells compared with monocytes obtained from normotensive controls [3,4]. A number of experimental studies have indicated an increase in TF expression and activity in human endothelial cells cultured under altered flow conditions [22][23][24][25], such as shear stress, cyclic strain and elevated static pressure.…”

Relationships between Brachial Artery Flow Mediated Dilation and Carotid Artery Intima-Media Thickness in Patients with Suspected Coronary Artery Disease.

“…TNF-α stimulates TF and PAI-1 production in endothelium. 21,22) IL-6 is induced by TNF-α 23) and has proinflammatory and procoagulant properties. 24) TNF-α and IL-6 are elevated in patients with unstable angina and acute myocardial infarction.…”

Fine-tuning of a three-dimensional microcarrier-based angiogenesis assay for the analysis of endothelial-mesenchymal cell co-cultures in fibrin and collagen gels

“…Both the HAECs and BAECs used for the experiments described here were pooled from several independent isolations following purification of the primary isolate (to > 99%) by manual weeding and/or flow cytometric sorting [21,22]. Subsequently, in order to minimize the differences between the various isolates, several batches of 2nd-passage cells from three individual preparations each were pooled.…”