Thrombin In Endothelial Cells Research Articles

The First EGF Domain of Coagulation Factor IX Increases PAR1 Distributionin Lipid Rafts and Modulates the Response to Thrombin in Endothelial Cells

The first epidermal growth factor 1 (EGF-1) domain of coagulation factor IX (FIX) contains a CXDXXXXYXCXC consensus amino acid sequence, which reportedly induces endocytosis and clustering of lipid rafts in adhesive cells. Endocytosis and lipid rafts regulate signaling via receptors on the plasma membrane. In this study, weinvestigated the effect of FIX EGF-1 domains on intracellular signaling by thrombin (FIIa) in endothelial cells.Recombinant EGF-1 protein of FIX (EGF-FIX) alone did not induce ROCK-dependent phosphorylation ofmyosin light chain (MLC) in human umbilical vein endothelial cells (HUVECs). On the other hand, a high concentration (100 ng/ml) of FIIa increased phosphorylated MLC, and EGF-FIX enhanced it by 1.3-fold. Interestingly, low concentrations (0.3 ng/ml) of FIIa suppressed the phosphorylation of MLC, and the combination oflow-concentration FIIa and EGF-FIX promoted MLC phosphorylation. Methyl-beta-cyclodextrin (MβCD), whichinhibits the formation of lipid rafts, suppressed the phosphorylation of MLC regardless of the FIIa concentration,whereas EGF-FIX attenuated the effects of MβCD. EGF-FIX increased the distribution of the FIIa receptor(PAR1) in the lipid raft fraction. Active FIX could modulate intracellular signaling by other molecules in endothelial cells via its EGF domain.

Effect of low-dose rivaroxaban with low-dose aspirin vs low-dose aspirin on platelet and oxidative biomarkers: a randomized study in diabetes patients with stable peripheral or coronary artery disease

Abstract Background Rivaroxaban (Riva), a direct FXa inhibitor, at 2.5 mg twice-daily (bid) combined with low-dose aspirin (ASA, 100 mg once daily-od) reduced major vascular events vs. ASA alone in subjects with stable coronary artery (CAD) or symptomatic peripheral artery disease (PAD). Whether this benefit is due to the anticoagulant effect or additional FXa-mediated effects through the platelet and endothelial cell thrombin receptors is unknown. Type 2 diabetes mellitus (T2DM) is characterized by high platelet activation and oxidative stress that may contribute to increased cardiovascular risk. Purpose We investigated the effects of Riva (2.5 mg bid) + ASA (100 mg od) vs. ASA (100 mg od) alone on platelet and oxidative biomarkers in subjects with T2DM and stable vascular disease (stable CAD, symptomatic PAD and/or significant carotid stenosis). Methods In this randomized, open-label, cross-over trial, patients were randomized to continue ASA for 4 weeks and then add Riva for 4 weeks, or add Riva in the first 4 weeks and then continue with ASA alone for 4 weeks. Primary endpoints were: in vivo platelet activation and lipid peroxidation, assessed by the urinary excretion of 11-dehydro-TXB2 (TXM) and 8-iso-PGF2alpha (ISOP), respectively. Secondary endpoints included: routine coagulation tests, D-dimer, thrombin generation, serum TXB2, and Riva plasma concentrations. Results Seventy-6 subjects (10 females) were recruited: age 68±7 years (mean±SD); BMI 27.1±3.5 kg/m2; fasting glucose 129±31 mg/dL; HbA1c 6.8±0.9%; serum creatinine 1±0.25 mg/dL; LDL-cholesterol 77±31 mg/dL. Two patients dropped out: one for benign, self-limiting hematuria, one for unwillingness to continue, 8 subjects are completing the study leaving 66 who completed the 8-week randomized treatment and showed no sequence effect. Urinary TXM and ISOP were significantly reduced by Riva+ASA vs. ASA alone: TXM was 260 [195–398] vs. 335 [225–441] pg/mg creatinine and ISOP 722 [601–991] vs. 827 [648–1350] pg/mg creatinine (median [IQR]) on Riva+ASA vs. ASA alone, respectively (p<0.001 for paired samples). Riva plasma concentrations were 48±1.9 ng/ml at peak and 21±1.4 ng/ml at trough. The velocity of thrombin formation significantly decreased with Riva+ASA vs. ASA alone (velocity index, 46±3% vs. 83±3%; peak-height, 66±2% vs. 83±1%, respectively). aPTT levels were slightly but significantly prolonged by Riva vs. ASA alone (44±1 vs. 39±1 sec). Serum TXB2, D-dimer, von Willebrand factor, PT, fibrinogen and endogenous thrombin potential values were similar between treatments. Conclusions In ASA-treated subjects with T2DM and stable vascular disease, the addition of very low-dose Riva restrained incompletely-suppressed lipid peroxidation and platelet activation and modified the kinetics of thrombin formation. These changes may contribute to the beneficial effects of the Riva+ASA combination. Funding Acknowledgement Type of funding sources: Private grant(s) and/or Sponsorship. Main funding source(s): Investigator-initiated study funded by Bayer AG

Occupancy of human EPCR by protein C induces β-arrestin-2 biased PAR1 signaling by both APC and thrombin

AbstractActivation of protease-activated receptor 1 (PAR1) by activated protein C (APC) and thrombin elicits paradoxical cytoprotective and cytotoxic signaling responses in vascular endothelial cells through cleavage of the receptor at Arg-46 and Arg-41 protease recognition sites, respectively. It has been reported that unlike a disruptive G-protein-mediated PAR1 signaling by thrombin, APC induces a protective β-arrestin-2 biased PAR1 signaling by unknown mechanisms. We hypothesize that the occupancy of endothelial protein C receptor (EPCR) by the Gla-domain of protein C/APC is responsible for the β-arrestin-2 biased PAR1 signaling independent of the protease cleavage site. To test this hypothesis, we monitored the signaling specificity of thrombin in endothelial cells in response to lipopolysaccharide (LPS) with or without pretreatment of cells with protein C-S195A. The PAR1-dependent recruitment of β-arrestin-2 in response to LPS by both APC and thrombin was analyzed by functional, gene silencing, and signaling assays. Results indicate that similar to APC, thrombin exerts cytoprotective effects via β-arrestin-2 biased PAR1 signaling. Similar to APC, thrombin triggered β-arrestin-2-dependent recruitment of disheveled 2 (Dvl-2) in PC-S195A pretreated cells. Further studies in HeLa cells transfected with PAR1 constructs revealed that EPCR occupancy initiates β-arrestin-2 biased PAR1 signaling independent of the protease cleavage sites. We demonstrate that EPCR occupancy recruits G-protein coupled receptor kinase 5, thereby inducing β-arrestin-2 biased PAR1 signaling by both APC and thrombin. In support of a physiological relevance for these results, intraperitoneal administration of PC-S195A conferred a cytoprotective effect for thrombin in an in vivo inflammatory model.

Adenosine Regulates the Proinflammatory Signaling Function of Thrombin in Endothelial Cells

The plasma level of the regulatory metabolite adenosine increases during the activation of coagulation and inflammation. Here we investigated the effect of adenosine on modulation of thrombin-mediated proinflammatory responses in HUVECs. We found that adenosine inhibits the barrier-disruptive effect of thrombin in HUVECs by a concentration-dependent manner. Analysis of cell surface expression of adenosine receptors revealed that A2A and A2B are expressed at the highest level among the four receptor subtypes (A2B > A2A > A1 > A3 ) on HUVECs. The barrier-protective effect of adenosine in response to thrombin was recapitulated by the A2A specific agonist, CGS 21680, and abrogated both by the siRNA knockdown of the A2A receptor and by the A2A -specific antagonists, ZM-241385 and SCH-58261. The thrombin-induced RhoA activation and its membrane translocation were both inhibited by adenosine in a cAMP-dependent manner, providing a molecular mechanism through which adenosine exerts a barrier-protective function. Adenosine also inhibited thrombin-mediated activation of NF-κB and decreased adhesion of monocytic THP-1 cells to stimulated HUVECs via down-regulation of expression of cell surface adhesion molecules, VCAM-1, ICAM-1, and E-selectin. Moreover, adenosine inhibited thrombin-induced elevated expression of proinflammatory cytokines, IL-6 and HMGB-1; and chemokines, MCP-1, CXCL-1, and CXCL-3. Taken together, these results suggest that adenosine may inhibit thrombin-mediated proinflammatory signaling responses, thereby protecting the endothelium from injury during activation of coagulation and inflammation.

Transcriptional regulation of platelet-derived growth factor-B chain by thrombin in endothelial cells: involvement of Egr-1 and CREB-binding protein

Thrombin and platelet-derived growth factor-B chain (PDGF-B) are key factors in the stimulation of atherosclerosis. The effect of thrombin on PDGF-B production has been characterized. However, the underlying mechanism is still far clear. Here, we investigate the transcription factors and regulators that are involved in PDGF-B production caused by thrombin in endothelial cells (ECs). Levels of PDGF were analyzed by real-time RT-PCR and ELISA, while levels of early growth response-1 (Egr-1) were analyzed by real-time RT-PCR and western blot. To evaluate the function of CBP and Egr-1 involved in regulation of PDGF-B, small interfering RNA (siRNA) were used to down-regulate their expression in mRNA and protein level. Interaction of Egr-1 and CBP was measured with immunoprecipitation and western blot. Thrombin induced an early and transient up-regulation of transcription factor early Egr-1, which was followed by a delayed increase of PDGF-B. siRNA against Egr-1-inhibited thrombin-induced PDGF-B production. Furthermore, thrombin could enhance the interaction of Egr-1 with its co-activator CREB-binding protein (CBP). CBP knockdown attenuated this interaction, and led to a reduction of PDGF-B expression induced by thrombin. Our results suggest that CBP might be one of the main interaction targets for Egr-1, and the transient activation of Egr-1 and recruitment of CBP are required for thrombin-induced PDGF-B in ECs.

Thrombin induces MCP-1 expression through Rho-kinase and subsequent p38MAPK/NF-κB signaling pathway activation in vascular endothelial cells

Thrombin has been shown to increase expression of chemokines such as monocyte chemoattractant protein 1 (MCP-1) in endothelial cells, leading to the development of atherosclerosis. However, the precise mechanism of this induction remains unknown. In the present study, we investigated whether the small G protein RhoA, and its effector, Rho-kinase are involved in MCP-1 induction by thrombin in endothelial cells. Y-27632, a specific Rho-kinase inhibitor, potently inhibited MCP-1 induction by thrombin. Y-27632 significantly decreased the chemotactic activity of thrombin-stimulated supernatants of endothelial cells on monocytes. Importantly, fasudil, a specific Rho-kinase inhibitor, attenuated MCP-1 gene expression in the aorta of db/db mice. Y-27632 attenuated thrombin-mediated phosphorylation of p38MAPK and p65, indicating that Rho-kinase mediates thrombin-induced MCP-1 expression through p38MAPK and NF-κB activation. Our findings demonstrate that the Rho/Rho-kinase signaling pathway plays a critical role in thrombin-mediated MCP-1 expression and function, and suggest that Rho/Rho-kinase may be an important target in the development of new therapeutic strategies for atherosclerosis.

Hypoxia upregulates PGI-synthase and increases PGI2 release in human vascular cells exposed to inflammatory stimuli

Hypoxia affects vascular function and cell metabolism, survival, growth, and motility; these processes are partially regulated by prostanoids. We analyzed the effect of hypoxia and inflammation on key enzymes involved in prostanoid biosynthesis in human vascular cells. In human vascular smooth muscle cells (VSMC), hypoxia and interleukin (IL)-1β synergistically increased prostaglandin (PG)I₂ but not PGE₂ release, thereby increasing the PGI₂/PGE₂ ratio. Concomitantly, these stimuli upregulated cyclooxygenase-2 (COX-2) expression (mRNA and protein) and COX activity. Interestingly, hypoxia enhanced PGI-synthase (PGIS) expression and activity in VSMC and human endothelial cells. Hypoxia did not significantly modify the inducible microsomal-PGE-synthase (mPGES)-1. Hypoxia-inducible factor (HIF)-1α-silencing abrogated hypoxia-induced PGIS upregulation. PGIS transcriptional activity was enhanced by hypoxia; however, the minimal PGIS promoter responsive to hypoxia (-131 bp) did not contain any putative hypoxia response element (HRE), suggesting that HIF-1 does not directly drive PGIS transcription. Serial deletion and site-directed mutagenesis studies suggested several transcription factors participate cooperatively. Plasma levels of the stable metabolite of PGI₂ and PGIS expression in several tissues were also upregulated in mice exposed to hypoxia. These data suggest that PGIS upregulation is part of the adaptive response of vascular cells to hypoxic stress and could play a role in counteracting the deleterious effect of inflammatory stimuli.

Matrix Metalloproteinase-10 Is Upregulated by Thrombin in Endothelial Cells and Increased in Patients With Enhanced Thrombin Generation

Thrombin is a multifunctional serine protease that promotes vascular proinflammatory responses whose effect on endothelial MMP-10 expression has not previously been evaluated. Thrombin induced endothelial MMP-10 mRNA and protein levels, through a protease-activated receptor-1 (PAR-1)-dependent mechanism, in a dose- and time-dependent manner. This effect was mimicked by a PAR-1 agonist peptide (TRAP-1) and antagonized by an anti-PAR-1 blocking antibody. MMP-10 induction was dependent on extracellular regulated kinase1/2 (ERK1/2) and c-jun N-terminal kinase (JNK) pathways. By serial deletion analysis, site-directed mutagenesis and electrophoretic mobility shift assay an AP-1 site in the proximal region of MMP-10 promoter was found to be critical for thrombin-induced MMP-10 transcriptional activity. Thrombin and TRAP-1 upregulated MMP-10 in murine endothelial cells in culture and in vivo in mouse aorta. This effect of thrombin was not observed in PAR-1-deficient mice. Interestingly, circulating MMP-10 levels (P<0.01) were augmented in patients with endothelial activation associated with high (disseminated intravascular coagulation) and moderate (previous acute myocardial infarction) systemic thrombin generation. Thrombin induces MMP-10 through a PAR-1-dependent mechanism mediated by ERK1/2, JNK, and AP-1 activation. Endothelial MMP-10 upregulation could be regarded as a new proinflammatory effect of thrombin whose pathological consequences in thrombin-related disorders and plaque stability deserve further investigation.

NF-κB regulates thrombin-induced ICAM-1 gene expression in cooperation with NFAT by binding to the intronic NF-κB site in the ICAM-1 gene

Activation of NF-kappaB is essential for protease-activated receptor-1 (PAR-1)-mediated ICAM-1 expression in endothelial cells. Here we show that PAR-1 activation induces binding of both p65/RelA and NFATc1 to the NF-kappaB binding site localized in intron-1 of the ICAM-1 gene to initiate transcription in endothelial cells. We discovered the presence of two NF-kappaB binding sites in intron-1 (+70, NF-kappaB site 1; +611, NF-kappaB site 2) of the human ICAM-1 gene. Chromatin immunoprecipitation results showed that thrombin induced binding of p65/RelA and of NFATc1 specifically to intronic NF-kappaB site 1 of the ICAM-1 gene. Electrophoretic mobility shift and supershift assays confirmed the binding of p65/RelA and NFATc1 to the intronic NF-kappaB site 1 in thrombin-stimulated cells. Thrombin increased the expression of ICAM-1-promoter-intron 1-reporter (-1,385 to +234) construct approximately 25-fold and mutation of intronic NF-kappaB site 1 markedly reduced thrombin-induced reporter expression. Moreover, inhibition of calcineurin, knockdown of either NFATc1 or p65/RelA with siRNA significantly reduced thrombin-induced ICAM-1 expression and polymorphonuclear leukocyte adhesion to endothelial cells. In contrast, NFATc1 knockdown had no effect on TNF-alpha-induced ICAM-1 expression. Thus these results suggest that p65/RelA and NFATc1 bind to the intronic NF-kappaB site 1 sequence to induce optimal transcription of the ICAM-1 gene in response to thrombin in endothelial cells.

Concentration dependent dual effect of thrombin in endothelial cells via Par‐1 and Pi3 Kinase

Disruption of endothelial barrier is a critical pathophysiological factor in inflammation. Thrombin exerts a variety of cellular effects including inflammation and apoptosis through activation of the protease activated receptors (PARs). The activation of PAR-1 by thrombin is known to have a bimodal effect in endothelial cell permeability with a low concentration (pM levels) eliciting a barrier protective and a high concentration (nM levels) eliciting a barrier disruptive response. It is not known whether this PAR-1-dependent activity of thrombin is a unique phenomenon specific for the in vitro assay or it is part of a general anti-inflammatory effect of low concentrations of thrombin that may have a physiological relevance. Here, we report that low concentrations of thrombin or of PAR-1 agonist peptide induced significant anti-inflammatory activities. However, relatively high concentration of thrombin or of PAR-1 agonist peptide showed pro-inflammatory activities. By using function-blocking anti-PAR-1 antibodies and PI3 kinase inhibitor, we show that the direct anti-inflammatory effects of low concentrations of thrombin are dependent on the activation of PAR-1 and PI3 kinase. These results suggest a role for cross communication between PAR-1 activation and PI3 kinase pathway in mediating the cytoprotective effects of low concentrations of thrombin in the cytokine-stimulated endothelial cells. J. Cell. Physiol. 219: 744-751, 2009. (c) 2009 Wiley-Liss, Inc.

TRPC‐1 knockdown abrogates nuclear factor‐kB‐dependent anti‐apoptotic gene expression induced by thrombin in endothelial cells

Thrombin activation of protease‐activated receptor‐1 induces Ca2+ influx through store‐operated Ca2+ entry channels (SOC) in endothelial cells. Previously we have shown that the canonical TRPC isoform TRPC1 is an essential component of SOC in endothelial cells. Further, we showed that thrombin‐induced Ca2+ influx signal activates nuclear factor‐kB (NF‐kB) in endothelial cells. In this study we measured thrombin‐induced expression of NF‐kB regulated genes in human pulmonary artery endothelial cells (ECs) utilizing Gene Microarray. Thrombin stimulation increased at least sixteen genes between 5‐ and 20‐fold over basal in ECs. Thrombin‐induced expression of anti‐apoptotic genes such as A20, IL8, and BCL3 were markedly reduced by pharmacological inhibition of Ca2+ influx in ECs. TRPC1 knockdown with siRNA abolished thrombin‐induced Ca2+ influx and markedly reduced A20, IL8, and BCL3 expression in ECs. Thus our results suggest that Ca2+ influx via SOC activates NF‐kB signaling to induce expression of A20, IL8, and BCL3 to promote cytoprotective effect in endothelial cells.

Glutamic Acid 192 of Activated Protein C Restricts the Specificity of PAR-1 Cleavage in Endothelial Cells.

It is known that residue 192 (chymotrypsin numbering) plays a key role in determining the P3 binding specificity of coagulation proteases. This residue is a Glu in both thrombin and activated protein C. Previous studies have indicated that the substrates containing an acidic residue at the P3 position are not recognized efficiently by either thrombin or APC in the absence of a cofactor. Protease activated receptor 1 (PAR-1), a common substrate for both thrombin and APC contains an acidic Asp at the P3 position. In this study we examined the ability of Glu-192 to Gln (E192Q) substitution mutants of both thrombin and APC to recognize and cleave PAR-1 in human umbilical vein endothelial cells (HUVEC) transfected with a PAR-1 cleavage reporter plasmid in which the exodomain of the receptor has been fused to a cDNA fragment encoding the soluble alkaline phosphatase. Thrombin E192Q cleaved PAR-1 with a catalytic efficiency that was approximately 5-fold higher than that of wild-type thrombin. On the other hand, the activity of APC E192Q toward PAR-1 was improved approximately 100-fold. Thus, unlike an approximately 1000-fold lower activity for APC in cleaving PAR-1 relative to thrombin, the activity of APC E192Q toward PAR-1 was only 10-fold lower than that of thrombin. These results suggest that the inhibitory interaction of Glu-192 of APC with P3-Asp of PAR-1 is responsible for its poor activity toward PAR-1. It is known that the cleavage of PAR-1 by thrombin in endothelial cells elicits a proinflammatory response. However, the cleavage of the same receptor by APC in complex with endothelial protein C receptor (EPCR) invokes a protective antiinflammatory response. The mechanism of the paradoxical effect of PAR-1 signaling by the two proteases is not known. We used these mutants to investigate the possibility that the level of PAR-1 activation by either thrombin or APC dictates the type of the response in endothelial cells. Furthermore, we used these mutants in TNF-a-stimulated endothelial cell permeability and apoptosis assays to understand the mechanism by which EPCR enables APC to activate PAR-1 in endothelial cells. We discovered that the dose of receptor activation is not responsible for the paradoxical effect of PAR-1 signaling by APC and thrombin in endothelial cells. Furthermore, the interaction of APC with EPCR is not associated with an improvement in the catalytic efficiency of the protease toward PAR-1. The possible EPCR and PAR-1 dependent recognition and signaling mechanism of APC and thrombin is discussed.

Rational de novo gene synthesis by rapid polymerase chain assembly (PCA) and expression of endothelial protein-C and thrombin receptor genes

The assembly of synthetic oligonucleotides into genes and genomes is an important methodology. Several methodologies for such synthesis have been developed, but they have two drawbacks: (1) the processes are slow and (2) the error frequencies are high (typically 1–3 errors/kb of DNA). Thermal damage is a major contributor to biosynthetic errors. In this paper, we elucidate the advantages of rapid gene synthesis by polymerase chain assembly (PCA) when used in combination with smart error control strategies. We used a high-speed thermocycler (PCRJet ®) to effectively minimize thermal damage and to perform rapid assembly of synthetic oligonucleotides to construct two different genes: endothelial protein C receptor (EPCR) and endothelial cell thrombin receptor, thrombomodulin (TM). First, the intact EPCR gene (EPCR-1, 612 bp) and a mutant EPCR-2 (576 bp) that lacked 4 N-linked glycosylation sites were constructed from 35 and 33 oligonucleotides, respectively. Next, for direct error comparison, another longer gene, the 1548 bp TM gene was constructed from 87 oligonucleotides by both rapid and conventional PCA. The fidelity and accuracy of the synthetic genes generated in this manner were confirmed by sequencing. The combined steps of PCA and DNA amplification are completed in about 10 and 22 min for EPCR-1, 2 and TM genes, respectively with comparable low errors in the DNA sequence. Furthermore, we subcloned synthetic TM, EPCR-1, EPCR-2 and native EPCR-1 (amplified from cDNA) into a Pichia pastoris expression vector to evaluate the expression ability, and to compare them with the native gene. Here, we illustrate that the synthetic genes, assembled by rapid PCA, successfully directed the expression of functional proteins. And, importantly, the synthetic and the native genes expressed proteins with the same efficiency.

Evidence for Actin Cytoskeleton-dependent and -independent Pathways for RelA/p65 Nuclear Translocation in Endothelial Cells

Activation of the transcription factor NF-kappaB involves its release from the inhibitory protein IkappaBalpha in the cytoplasm and subsequently, its translocation to the nucleus. Whereas the events responsible for its release have been elucidated, mechanisms regulating the nuclear transport of NF-kappaB remain elusive. We now provide evidence for actin cytoskeleton-dependent and -independent mechanisms of RelA/p65 nuclear transport using the proinflammatory mediators, thrombin and tumor necrosis factor alpha, respectively. We demonstrate that thrombin alters the actin cytoskeleton in endothelial cells and interfering with these alterations, whether by stabilizing or destabilizing the actin filaments, prevents thrombin-induced NF-kappaB activation and consequently, expression of its target gene, ICAM-1. The blockade of NF-kappaB activation occurs downstream of IkappaBalpha degradation and is associated with impaired RelA/p65 nuclear translocation. Importantly, thrombin induces association of RelA/p65 with actin and this interaction is sensitive to stabilization/destabilization of the actin filaments. In parallel studies, stabilizing or destabilizing the actin filaments fails to inhibit RelA/p65 nuclear accumulation and ICAM-1 expression by tumor necrosis factor alpha, consistent with its inability to induce actin filament formation comparable with thrombin. Thus, these studies reveal the existence of actin cytoskeleton-dependent and -independent pathways that may be engaged in a stimulus-specific manner to facilitate RelA/p65 nuclear import and thereby ICAM-1 expression in endothelial cells.

Electromagnetic Fields Inhibit Endothelin-1 Production Stimulated by Thrombin in Endothelial Cells

Electromagnetic field (EMF) radiation has been found to induce arteriolar dilatation, but the mechanism of action remains largely unknown. This study investigated the effect of EMF radiation on the production of endothelin-1 (ET-1), a potent vasoconstrictor, by cultured endothelial cells. EMF radiation reduced ET-1 basal levels in human umbilical vein and microvascular endothelial cells, but failed to reduce ET-1 basal levels in bovine and human aortic endothelial cells. EMF radiation significantly inhibited thrombin-stimulated ET-1 production in all four endothelial cell types in a dose-dependent manner. EMF radiation significantly inhibited thrombin-induced endothelin-1 mRNA expression in all four cell types. The inhibitory effect of EMF radiation on ET-1 production was abolished by the nitric oxide synthase inhibitor NG-monomethyl-L-arginine (10(-3) mol/1). These results demonstrate that EMF radiation modulates ET-1 production in cultured vascular endothelial cells and the inhibitory effect of EMF radiation is, at least partly, mediated through a nitric oxide-related pathway.

PAR1 Cleavage and Signaling in Response to Activated Protein C and Thrombin

Activated protein C (APC), a natural anticoagulant protease, can trigger cellular responses via protease-activated receptor-1 (PAR1), a G protein-coupled receptor for thrombin. Whether this phenomenon contributes to the physiological effects of APC is unknown. Toward answering this question, we compared the kinetics of PAR1 cleavage on endothelial cells by APC versus thrombin. APC did cleave PAR1 on the endothelial surface, and antibodies to the endothelial protein C receptor inhibited such cleavage. Importantly, however, APC was approximately 10(4)-fold less potent than thrombin in this setting. APC and thrombin both triggered PAR1-mediated responses in endothelial cells including expression of antiapoptotic (tumor necrosis factor-alpha-induced a20 and iap-1) and chemokine (interleukin-8 (il-8) and cxcl3) genes, but again, APC was approximately 10(4)-fold less potent than thrombin. The addition of zymogen protein C to endothelial cultures did not alter the rate of PAR1 cleavage at low or high concentrations of thrombin, and PAR1 cleavage was substantial at thrombin concentrations too low to trigger detectable conversion of protein C to APC. Thus, locally generated APC did not contribute to PAR1 cleavage beyond that effected by thrombin in this system. Although consistent with reports that sufficiently high concentrations of APC can cleave and activate PAR1 in culture, our data suggest that a significant physiological role for PAR1 activation by APC is unlikely.

Downregulation of TGF-β Signaling by Thrombin in Endothelial Cells.

Thrombin activates protease-activated receptor 1 (PAR1) on endothelial cells (ECs) and regulates many aspects of EC biology. A body of evidence has demonstrated that the thrombin-PAR1 pathway is critical for angiogenesis and vascular development. However, the mechanism underlying the proangiogenic effect of thrombin has not been elucidated yet. Here, we report the discovery of a novel functional link between thrombin-PAR1 and transforming growth factor-β (TGF-β) signaling pathways. Using mass spectrometry, confocal immunofluorescence microscopy, cell surface ELISA and a phospho-specific antibody against the serine-phosphorylated endogin, we demonstrated for the first time that thrombin via PAR1 induced the internalization of endoglin and type-II TGF-β receptor (TβRII) but not the type-I receptors in primary human ECs. This effect was mediated by protein kinase C-ζ (PKC-ζ) that was rapidly activated by thrombin in ECs since specific inhibition of PKC-ζ caused aggregation of endoglin and TβRII on cell surface and blocked their internalization in response to thrombin. Furthermore, acute (30 min) and long-term (24 h) pretreatment of ECs with thrombin or PAR1 peptide agonist suppressed the TGF-β-induced serine phosphorylation of Smad2/3, the critical mediators of TGF-β signaling. Moreover, activation of PAR1 led to a profound clustering formation of Smad2/3 and a more spread cytosolic localization, which sequestered Smad2/3 from nucleus and prevented their nuclear translocation evoked by TGF-β. Since TGF-β and its receptors play a crucial role in the resolution phase of angiogenesis, the downregulation of TGF-β signaling by thrombin-PAR1 pathway may provide a new insight into the mechanism of the proangiogenic effect of thrombin.

Alpha-Tocopherol Inhibits IL-8 synthesis induced by thrombin and high glucose in endothelial cells.

It has been reported that alpha-tocopherol, an antioxidant agent, may play a role in preventing diabetic angiopathy. However, there is little evidence to show the effect of alpha-tocopherol on the production of pro-inflammatory cytokines in endothelial cells. Therefore, we examined the effect of alpha-tocopherol on the regulation of IL-8 synthesis induced by high glucose and/or thrombin in endothelial cells. Thrombin alone markedly increased the IL-8 release. Furthermore, high glucose levels and thrombin combined had additive effects on IL-8 synthesis, and alpha-tocopherol diminished their effect; alpha-tocopherol also inhibited the phosphorylation of IkappaB-alpha induced by high glucose levels and/or thrombin. Our results suggest that the administration of alpha-tocopherol to diabetic patients may have a beneficial effect for the prevention of diabetic vascular complications by the inhibition of IL-8 synthesis from endothelial cells.

Disturbance of endothelial barrier function by bacterial toxins and atherogenic mediators: a role for Rho/Rho kinase.

Disturbance of endothelial barrier function by bacterial toxins and atherogenic mediators: a role for Rho/Rho kinase.

Shifts in the affinity distribution of one class of seven-transmembrane receptors by activation of a separate class of seven-transmembrane receptors

We have demonstrated previously that activation of thrombin receptors causes increased Gα q coupling to thromboxane A 2 receptors and increased thromboxane A 2 receptor ligand affinity. These results led to the hypothesis that thrombin receptor activation stimulates Gα q redistribution to thromboxane A 2 receptors, thereby shifting them to a higher affinity state. The present study investigated three questions regarding this inter-receptor signaling phenomenon: (i) does activation of thrombin receptors cause a redistribution of thromboxane A 2 receptor subpopulations; (ii) does inter-receptor signaling require that participating receptors couple to the same family of G-protein α-subunits; and (iii) does inter-receptor signaling occur in cell types other than platelets? It was found that thrombin receptor activation caused a shift in the thromboxane A 2 receptor binding data from a one-site model to a two-site model ( K i = 0.5 μM vs K i = 10 nM and 1.1 μM for the antagonist 4-[2-[[(4-chlorophenyl)sulfonyl]amino]ethyl]benzeneacetic acid (BM13.505) and K i = 2.5 μM vs K i = 29.5 nM and 2.6 μM for the agonist 9,11-dideoxy-9α,11α-methanoepoxy prostaglandin F 2α (U46619). It also was found that activation of prostaglandin D 2 receptors also caused a shift of prostacyclin receptor binding data from a one-site model ( ic 50 = 10.1 nM) to a two-site model ( ic 50 = 3.3 and 12.5 nM). The physiological manifestation of this inter-receptor signaling between prostacyclin and prostaglandin D 2 receptors was a synergistic inhibition of human platelet aggregation. Finally, the present results established that activation of endothelial cell thrombin receptors shifts thromboxane A 2 receptor affinity from K i = 0.8 μM (control) to K i = 0.2 μM (thrombin receptor-activating peptide), indicating that cells other than platelets have the capability to signal between seven-transmembrane receptors.