Cyclic Strain Delays the Expression of Tissue Factor Induced by Thrombin in Human Umbilical Vein Endothelial Cells (original) (raw)
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Journal of Vascular Surgery, 2011
High levels of tissue factor (TF) have been associated with atherosclerotic plaques. The specific pathways linked to TF expression in endothelial cells (ECs) have not been well defined. This study compared TF expression in human umbilical vein ECs (HUVECs) exposed to laminar shear stress (LSS) using a parallel flow chamber and to orbital shear stress (OSS) using an orbital shaker. We also compared the effects of thrombin (TH) stimulation of ECs exposed to different shear forces on the expression of TF and investigated the role that second messengers, p38 and extracellular signal-regulated kinase 1 and 2 (ERK1/2), had in the EC response. Methods: HUVECs were subjected to 2, 4, or 6 hours of LSS or OSS in the presence or absence of 4 U/mL of TH. Western blot analysis of ERK1/2 and p38 activation and polymerase chain reaction analysis of TF in the presence of inhibitors to these second messengers was performed in HUVECs subjected to OSS or LSS in the presence or absence of TH. Results: TF expression was increased and peaked at 2 hours in all HUVECs exposed to LSS or TH. Stimulation of static HUVECs with TH resulted in an increase in TF expression of 5.68 ؎ 1.58-, 3.80 ؎ 1.21-, and 2.54 ؎ 0.38-fold at 2, 4, and 6 hours, respectively (n ؍ 6 experiments). In the absence of TH, HUVECs exposed to LSS demonstrated a 9.51 ؎ 0.62-, 7.31 ؎ 1.43-, and 4.39 ؎ 1.32-fold increase in TF expression at 2, 4, and 6 hours, respectively (n ؍ 6 experiments). TF was increased significantly more when exposed to LSS in the presence of TH (18.85 ؎ 1.43-, 15.05 ؎ 0.95-, and 8.91 ؎ 1.06-fold increases at 2, 4, and 6 hours, respectively [n ؍ 6 experiments], P < .01). Between-group analysis showed a significant difference between groups (P < .001). OSS did not significantly increase TF expression in the presence or absence of TH. ERK1/2 and p38 activation was increased in LSS and LSS ؉ TH but not in OSS or OSS ؉ TH (n ؍ 3 experiments). Conclusion: LSS and TH independently increased TF expression, but OSS did not. LSS ؉ TH stimulation showed a synergistic effect, which suggests that these mechanical and chemical stimuli work through different pathways or that an intracellular interaction between TH and LSS may be present that does not occur in OSS. (J Vasc Surg 2011;54:480-8.) Clinical Relevance: Tissue factor (TF) has been shown to be overexpressed in advanced atherosclerotic lesions where disturbed flow patterns are prevalent. Mechanical stimuli such as shear stress and chemical stimuli such as TH are thought to influence the levels of TF expression and activity. Uncovering how these factors influence TF expression on an intracellular level can lead to a better understanding of atherogenesis and better direct the search for novel therapeutic targets. This study reveals the different effects that TH and distinct hemodynamic stimuli have on TF messenger RNA levels and attempts to elucidate the underlying intracellular signaling pathways leading to these effects.
Varying Effects of Hemodynamic Forces on Tissue Factor RNA Expression in Human Endothelial Cells
Journal of Surgical Research, 2011
Background. Atherosclerotic lesions predominantly localize in areas exposed to distinct hemodynamic conditions. In such lesions, tissue factor (TF) is over-expressed. Therefore, we hypothesized that varying types of mechanical forces may induce different effects on TF expression in endothelial cell, and may also influence the effects of chemical stimuli. Materials and Methods. TF RNA expression in human umbilical vein endothelial cells (HUVEC) exposed to mechanical stress in the presence or absence of chemical stimulation with thrombin (Th) was determined. The forces examined were: steady unidirectional laminar flow (LF), pulsatile unidirectional laminar flow (PF), constant oscillatory flow (OF), pulsatile to-fro flow (TFF), and cyclic strain (CS). Results. Mechanical stimulation of HUVEC with LF for 2 h induced an 8.7 ± 0.7-fold increase in TF RNA expression, while PF induced 4.7 ± 0.9 and TFF induced 8.6 ± 1.7-fold, respectively. These responses were significantly higher than static controls. Exposure to OF or CS did not result in any significant increase, whereas chemical stimulation with Th led to significant TF expression (4.9 ± 0.3-fold). The combination of mechanical-chemical stimuli induced significantly higher TF expression than mechanical stresses alone, and this effect was synergistic. Combination of LFDTh for 2 h induced significantly increased TF expression (16.6 ± 1.7-fold), as did PFDTh (14.8 ± 2.4) and TFFDTh (17.4 ± 1.0). Furthermore, after 6 h exposure, only TFF demonstrated significantly higher TF expression both with and without Th. Conclusions. While uniform laminar flow resulted in transient TF expression, disturbed flow induced sustained amplification of TF expression. Further investigation is needed to elucidate the mechanism of localized atherosclerosis in areas exposed to disturbed flow.
Arteriosclerosis, Thrombosis, and Vascular Biology, 2001
Monolayers of human umbilical vein endothelial cells were activated with 50 U/mL interleukin-1␣ (IL-1␣) for 3 hours and simultaneously conditioned with shear stresses of 0, 0.68, or 13.2 dyne/cm 2 in a parallel-plate flow chamber. In the presence of an inflow buffer containing 100 nmol/L factor X and 10 nmol/L factor VII, production of factor Xa, a measure of functional tissue factor (TF), was determined as the product of outflow concentration of factor Xa (chromogenic assay performed under quasi-static flow conditions after the shear period) and flow rate. Similarly, production of TF pathway inhibitor (TFPI) was estimated as the product of antigenic TFPI (by enzyme-linked immunosorbent assay) in the supernatant and flow rate. In parallel experiments, total RNA was isolated for determination of amplification products of TF mRNA by reverse transcription-polymerase chain reaction. We found that shear stress reduced factor Xa production (meanϮSE; nϭnumber of experiments) from 13.33Ϯ1.14 (nϭ16) fmol/minϫcm 2 at 0 shear stress to 5.70Ϯ2.51 (nϭ5) and 0.54Ϯ0.54 (nϭ4) fmol/minϫcm 2 at shear stresses of 0.68 and 13.2 dyne/cm 2 , respectively. At the same time, immunogold labeling showed that TF antigen on the endothelial surface increased Ͼ5-fold with shear stress, whereas TFPI antigen on the surface increased 2-fold. The secretion of TFPI (appearance of new supernatant TFPI) rose from 7.4Ϯ2.4 (nϭ12) ϫ10 Ϫ3 fmol/minϫcm 2 at 0 shear stress to 23.7Ϯ7.3 (nϭ9) and 50.2Ϯ14.3 (nϭ4) ϫ10 Ϫ3 fmol/minϫcm 2 at 0.68 and 13.2 dyne/cm 2 , respectively. TF mRNA amplification products were not markedly changed by shear stress. We conclude that acute application of shear stress reduces functional, but not antigenic, expression of TF by intact, activated endothelial cell monolayers in a manner associated with shear stress-augmented endothelial cell secretion of TFPI. (Arterioscler Thromb Vasc Biol. 2001;21:157-162.)
PLoS ONE, 2014
Background and Objectives: Thrombomodulin (TM), an integral membrane glycoprotein expressed on the lumenal surface of vascular endothelial cells, promotes anti-coagulant and anti-inflammatory properties. Release of functional TM from the endothelium surface into plasma has also been reported. Much is still unknown however about how endothelial TM is regulated by physiologic hemodynamic forces (and particularly cyclic strain) intrinsic to endothelial-mediated vascular homeostasis. Methods: This study employed human aortic endothelial cells (HAECs) to investigate the effects of equibiaxial cyclic strain (7.5%, 60 cycles/min, 24 hrs), and to a lesser extent, laminar shear stress (10 dynes/cm 2 , 24 hrs), on TM expression and release. Time-, dose-and frequency-dependency studies were performed. Results: Our initial studies demonstrated that cyclic strain strongly downregulated TM expression in a p38-and receptor tyrosine kinase-dependent manner. This was in contrast to the upregulatory effect of shear stress. Moreover, both forces significantly upregulated TM release over a 48 hr period. With continuing focus on the cyclic strain-induced TM release, we noted both dose (0-7.5%) and frequency (0.5-2.0 Hz) dependency, with no attenuation of strain-induced TM release observed following inhibition of MAP kinases (p38, ERK-1/2), receptor tyrosine kinase, or eNOS. The concerted impact of cyclic strain and inflammatory mediators on TM release from HAECs was also investigated. In this respect, both TNFa (100 ng/ml) and ox-LDL (10-50 mg/ml) appeared to potentiate strain-induced TM release. Finally, inhibition of neither MMPs (GM6001) nor rhomboids (3,4-dichloroisocoumarin) had any effect on strain-induced TM release. However, significantly elevated levels (2.1 fold) of TM were observed in isolated microparticle fractions following 7.5% strain for 24 hrs. Conclusions: A preliminary in vitro investigation into the effects of cyclic strain on TM in HAECs is presented. Physiologic cyclic strain was observed to downregulate TM expression, whilst upregulating in a time-, dose-and frequency-dependent manner the release of TM.
Cell Biology International, 2014
Biomechanical environments affect the function of cells. In this study we analysed the effects of five mechanical stimuli on the gene expression of human umbilical vein endothelial cells (HUVECs) in mRNA level using real-time PCR. The following loading regimes were applied on HUVECs for 48 h: intermittent (0-5 dyn/cm 2 , 1 Hz) and uniform (5 dyn/cm 2 ) shear stresses concomitant by 10% intermittent equiaxial stretch (1 Hz), uniform shear stress alone (5 dyn/cm 2 ), and intermittent uniaxial and equiaxial stretches (10%, 1 Hz). A new bioreactor was made to apply uniform/cyclic shear and tensile loadings. Three endothelial suggestive specific genes (vascular endothelial growth factor receptor-2 (VEGFR-2, also known as FLK-1), von Willebrand Factor (vWF) and vascular endothelial-cadherin (VE-cadherin)), and two smooth muscle genes (a-smooth muscle actin (a-SMA) and smooth muscle myosin heavy chain (SMMHC)) were chosen for assessment of alteration in gene expression of endothelial cells and transdifferentiation toward smooth cells following load applications. Shear stress alone enhanced the endothelial gene expression significantly, while stretching alone was identified as a transdifferentiating factor. Cyclic equiaxial stretch contributed less to elevation of smooth muscle genes compared to uniaxial stretch. Cyclic shear stress in comparison to uniform shear stress concurrent with cyclic stretch was more influential on promotion of endothelial genes expression. Influence of different mechanical stimuli on gene expression may open a wider horizon to regulate functions of cell for tissue engineering purposes.
American Journal of Physiology-Cell Physiology, 1999
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 < 0.0...
Cell Biology International, 2015
It has been well established that biomechanical environment can influence functionality of biological cells. There are evidences that show mechanical cyclic stretch can promote smooth muscle cell (SMC) markers in endothelial cells (ECs). The objective of this study was to determine whether mechanical stimuli in the forms of uniaxial and equiaxial cyclic stretches (UNCS & EQCS) can affect endothelial and smooth muscle gene expressions in mRNA level of human umbilical vein endothelial cells (HUVECs). For this purpose, 10% uniaxial UNCS and EQCS (60 cycles/min for 24h) were applied on HUVECs, and using real-time PCR expressions of three EC specific markers, vascular endothelial growth factor receptor-2 (VEGFR-2, also known asFLK-1) , von Willebrand Factor (vWF) and vascular endothelial-cadherin (VE-cadherin) and two SMC specific genes, α-smooth muscle actin (α-SMA) and smooth muscle myosin heavy chain (SMMHC) were quantified. Moreover, alterations in cell height were analyzed by atomic force microscopy (AFM). Results showed that cyclic UNCS for 24h downregulated the expression of all EC markers and upregulated the expression of all SMC markers while low effects on HUVECs height were observed. Cyclic EQCS in the same conditions resulted in minor effect on SMC gene expression in HUVECs, while led to strong reduction in vWF with no significant change in other two endothelial genes. Cyclic EQCS considerably elevated cell height. Results proposed that ECs can transdifferentiate to SMC phenotype under specific microenvironmental conditions.
Induction of Tissue Factor in the Arterial Wall During Recurrent Thrombus Formation
Objective-Tissue factor (TF) is normally expressed at low levels in the media of blood vessels, but it is readily induced after vessel injury. It is not known whether vascular damage per se or thrombus formation is responsible for this phenomenon. Methods and Results-Cyclic flow variations (CFVs), attributable to recurrent thrombus formation, were induced in stenotic rabbit carotid arteries with endothelial injury. CFVs were observed for 30 minutes and 2, 4, and 8 hours in different groups of animals. Another group of rabbits pretreated with hirudin before inducing arterial damage to inhibit thrombus formation was observed for 8 hours. Arterial sections were immunostained for TF. Undamaged arteries served as controls. In additional rabbits, in situ hybridization experiments were performed. No TF expression was observed in the media of control vessels, whereas a progressive increase in TF mRNA and protein expression was observed in carotid arteries as CFVs progressed. No increase in TF expression was observed in animals pretreated with hirudin. In vitro experiments demonstrated that TF mRNA is induced in smooth muscle cells stimulated with activated platelets as well as with some platelet-derived mediators.
Physiological Genomics, 2005
Microarrays were utilized to determine gene expression of vascular endothelial cells (ECs) subjected to mechanical stretch for insight into the role of strain in vascular pathophysiology. Over 4,000 genes were screened for expression changes resulting from cyclic strain (10%, 1 Hz) of human umbilical vein ECs for 6 and 24 h. Comparison of t-statistics and adjusted P values identified genes having significantly different expression between strained and static cells but not between strained and motion control. Relative to static, 6 h of cyclic stretch upregulated two genes and downregulated two genes, whereas 24 h of cyclic stretch upregulated eight genes but downregulated no genes. However, incorporating the motion control revealed that fluid agitation over the cells, rather than strain, is the primary regulator of differential expression. Furthermore, no gene exceeded a threefold change when comparing cyclic strain to either static or motion control. Quantitative real-time polymeras...
International journal of medical sciences, 2016
Inflammation and dysfunction of endothelial cells are thought to be triggers for the secretion of Von Willebrand factor. The aim of this study was to examine the effects of the inflammatory cytokines interleukin-6 (IL-6), interleukin-8 (IL-8) and tumour necrosis factor-alpha (TNF-α) and the coagulation factors, tissue factor and thrombin on the release and cleavage potential of ultra-large von Willebrand factor (ULVWF) and its cleavage protease by cultured human umbilical vein endothelial cells (HUVEC). HUVEC were treated with IL-6, IL-8, and TNF-α, tissue factor (TF) and thrombin, and combinations thereof for 24 hours under static conditions. The cells were then exposed to shear stress after which the VWF-propeptide levels and the VWF cleavage protease, ADAMTS13 content were measured. All treatments and their combinations, excluding IL-6, significantly stimulated the secretion of VWF from HUVEC. The VWF secretion from the HUVEC was stimulated most by the combination of TF with TNF-...