Fluid Flow Induces Upregulation of Synthesis and Release of Tissue Factor Pathway Inhibitor In Vitro

Abstract

Abstract-Fluid flow modulates the synthesis and secretion by endothelial cells (ECs) of several proteins that control the hemostatic properties of the vessel wall. Tissue factor pathway inhibitor (TFPI), also synthesized by ECs, is the main downregulator of tissue factor-dependent procoagulant activity. In the present study, we investigated the effect of physiological shear stress on the expression, distribution, and release of TFPI in cultured ECs. The EA.hy926 cell line was grown in a hollow-fiber perfusion system and exposed for variable times to different shear values: 0.27 dyne/cm 2 (minimal flow), 4.1 dyne/cm 2 (venous flow), and 19 dyne/cm 2 (moderate arterial flow).Step increase of the shear stress from 0.27 to 19 dyne/cm 2 induced a sharp increase of TFPI released into the medium and a parallel decrease and redistribution of cell-associated TFPI, which suggests that an acute release of TFPI occurred from the cellular pools. During 24 hours of high shear stress, cell-associated TFPI antigen and mRNA increased time-dependently. Subjecting ECs to steady shear stress for 72 hours also upregulated the expression and production of TFPI, in direct correlation with the degree of the shear. The secretion of TFPI was enhanced 1.9-fold under venous flow and 2.4-fold under arterial flow compared with minimal flow. Equally, cell-associated TFPI antigen and cell surface TFPI activity increased proportionally with the shear stress. The expression of TFPI mRNA, as determined by Northern blotting, increased up to 2-fold in ECs under venous flow and up to 3-fold under arterial flow. These results suggest that shear forces regulate TFPI by modulating its release and gene expression in ECs in vitro. Key Words: tissue factor pathway inhibitor Ⅲ shear stress Ⅲ flow Ⅲ endothelial cells E ndothelial cells (ECs) serve as a functional barrier between blood and the vessel wall and control the fluidity of the blood by distinct mechanisms. Accordingly, ECs produce antiplatelet aggregants and vasoactive compounds such as prostacyclin (PGI 2 ) and NO; antithrombotic proteins such as thrombomodulin, tissue factor (TF) pathway inhibitor (TFPI), and heparan sulfate proteoglycans; and fibrinolytic proteins such as tissue plasminogen activator (tPA) (for a review, see Reference 1). Because of their strategic location, ECs interface with fluid shear forces, which in turn affect their function and gene expression. Examples include the reorganization of the cytoskeletal components, 2 regulation of gene expression for proteins that play key roles in the maintenance of homeostasis, 3,4 cell migration, 5 and cell growth. 6 Hemodynamic forces also regulate several of the hemostatic proteins produced by ECs. Fluid shear stress induces significant increases in the release of PGI 2 7 and NO, 8,9 as well as enhanced synthesis of regulators that inactivate certain clotting enzymes or cofactors. Recent studies showed that shear stress regulates the generation of thrombomodulin, 10,11 which interacts with protein C and protein S to inactivate factor (F...

Integrated Regional Cardiac Hemodynamic Imaging and RNA Sequencing Reveal Corresponding Heterogeneity of Ventricular Wall Shear Stress and Endocardial Transcriptome

“…These data contrast with reports of arterial EC experiments in vitro that showed no effect of oscillatory flow on TFPI gene expression 34, 35, 36. Nevertheless, a step increase of WSS resulted in a marked increase in TFPI expression35 suggesting that rate of change of WSS and the associated circumferential stretch may be important for TFPI transcriptional regulation.…”

Biology of tissue factor pathway inhibitor

“…66,103,104 Further investigation of human tissue vascular beds is needed to better define cell-surface TFPI isoform expression, as well as the presence and potential physiological function of the intracellular stores of TFPIa identified in cultured endothelial cells.…”

The influence of biomaterials on endothelial cell thrombogenicity

“…In particular fluid shear stress resulting from blood flow over the endothelial surface, sensed via a number of mechanosensor mechanisms [84], produces junction [85], cytoskeleton [86] and integrin [87] re-organization which alters ECM organization [88,89], EC morphology [90], and EC gene expression [91][92][93]. This further affects the expression and secretion of proteins associated with EC function, such as regulation of vascular tone [94][95][96], fibrinolysis [97][98][99], surface adhesion [100] and coagulation proteins such as tissue factor and its inhibitor [101,102], thrombomodulin [103,104], prostacyclin [105,106] and von Willebrand factor [107]. Of interest here, is that KLF-2 [108] (Kruppel-like factor), a flow responsive transcription factor is a primary candidate as a central "switch" between quiescent and activated adult EC.…”