scholarly journals Purinergic P2Y2 Receptor Control of Tissue Factor Transcription in Human Coronary Artery Endothelial Cells

2015 ◽  
Vol 291 (4) ◽  
pp. 1553-1563 ◽  
Author(s):  
Yiwei Liu ◽  
Lingxin Zhang ◽  
Chuan Wang ◽  
Shama Roy ◽  
Jianzhong Shen
Keyword(s):  
Endothelial Cells ◽  
Coronary Artery ◽  
Tissue Factor ◽  
Promoter Activity ◽  
Consensus Sequence ◽  
Coagulation Cascade ◽  
Loss Of Function ◽  
P2y2 Receptor ◽  
Human Coronary Artery

We recently reported that the P2Y2 receptor (P2Y2R) is the predominant nucleotide receptor expressed in human coronary artery endothelial cells (HCAEC) and that P2Y2R activation by ATP or UTP induces dramatic up-regulation of tissue factor (TF), a key initiator of the coagulation cascade. However, the molecular mechanism of this P2Y2R-TF axis remains unclear. Here, we report the role of a newly identified AP-1 consensus sequence in the TF gene promoter and its original binding components in P2Y2R regulation of TF transcription. Using bioinformatics tools, we found that a novel AP-1 site at −1363 bp of the human TF promoter region is highly conserved across multiple species. Activation of P2Y2R increased TF promoter activity and mRNA expression in HCAEC. Truncation, deletion, and mutation of this distal AP-1 site all significantly suppressed TF promoter activity in response to P2Y2R activation. EMSA and ChIP assays further confirmed that upon P2Y2R activation, c-Jun, ATF-2, and Fra-1, but not the typical c-Fos, bound to the new AP-1 site. In addition, loss-of-function studies using siRNAs confirmed a positive transactivation role of c-Jun and ATF-2 but unexpectedly revealed a strong negative role of Fra-1 in P2Y2R-induced TF up-regulation. Furthermore, we found that P2Y2R activation promoted ERK1/2 phosphorylation through Src, leading to Fra-1 activation, whereas Rho/JNK mediated P2Y2R-induced activation of c-Jun and ATF-2. These findings reveal the molecular basis for P2Y G protein-coupled receptor control of endothelial TF expression and indicate that targeting the P2Y2R-Fra-1-TF pathway may be an attractive new strategy for controlling vascular inflammation and thrombogenicity associated with endothelial dysfunction.

Abstract 190: Purinergic Control of Tissue Factor Transcription in Human Coronary Artery Endothelial Cells: New AP-1 Site and Negative Regulator

2015 ◽  
Vol 35 (suppl_1) ◽  
Author(s):  
Yiwei Liu ◽  
Lingxin Zhang ◽  
Chuan Wang ◽  
Shama Roy ◽  
Jianzhong Shen
Keyword(s):  
Endothelial Cells ◽  
Coronary Artery ◽  
Tissue Factor ◽  
Promoter Activity ◽  
Purinergic Receptor ◽  
Consensus Sequence ◽  
Negative Regulator ◽  
Coagulation Cascade ◽  
Human Coronary Artery

Previously we reported that the P2Y2 receptor (P2Y2R) is one of the predominant purinergic receptors expressed in human coronary artery endothelial cells (HCAEC), and that P2Y2R activation by ATP or UTP induces dramatic up-regulation of tissue factor (TF), key initiator of the coagulation cascade. However, the molecular mechanism of this P2Y2R-TF axis remains unclear. Here we report a role of a newly identified AP-1 consensus sequence along with its new binding components in P2Y2R regulation of TF transcription. We identified with bioinformatics tools that a novel AP-1 site at -1363 bp of human TF promoter region is highly conserved across multiple species. P2Y2R activation increased TF promoter activity and mRNA expression in HCAEC. Truncation, deletion, and mutation of this new distal AP-1 site all significantly supressed TF promoter activity in response to P2Y2R activation. EMSA and ChIP assays further confirmed that upon P2Y2R activation, c-Jun, ATF-2 and Fra-1, but not the typical c-Fos, bound to the new AP-1 site. In addition, loss-of-function studies using siRNAs confirmed a positive transactivation role of c-Jun and ATF-2, but unexpectedly revealed a strong negative role of Fra-1 in P2Y2R-induced TF up-regulation. Furthermore, we found that P2Y2R activation promoted ERK1/2 phosphorylation, leading to Fra-1 activation while JNK activated c-Jun and ATF-2. These findings reveal the basis for P2Y purinergic receptor regulation of endothelial TF expression and indicate that targeting the P2Y2R-Fra-1-TF pathway may be an attractive new strategy in control of vascular thrombogenicity and/or inflammation associated with endothelial dysfunction.

scholarly journals The Ratio of Factor VIIa:Tissue Factor Content within Microvesicles Determines the Differential Influence on Endothelial Cells

TH Open ◽  
2019 ◽  
Vol 03 (02) ◽  
pp. e132-e145 ◽  
Author(s):  
Yahya Madkhali ◽  
Sophie Featherby ◽  
Mary Collier ◽  
Anthony Maraveyas ◽  
John Greenman ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Endothelial Cells ◽  
Coronary Artery ◽  
Tissue Factor ◽  
Cellular Proliferation ◽  
Molar Ratio ◽  
Activity Levels ◽  
Human Coronary Artery ◽  
Determining Factors ◽  
Mcf 7

AbstractTissue factor (TF)-positive microvesicles from various sources can promote cellular proliferation or alternatively induce apoptosis, but the determining factors are unknown. In this study the hypothesis that the ratio of fVIIa:TF within microvesicles determines this outcome was examined. Microvesicles were isolated from HepG2, BxPC-3, 786-O, MDA-MB-231, and MCF-7 cell lines and microvesicle-associated fVIIa and TF antigen and activity levels were measured. Human coronary artery endothelial cells (HCAECs) were incubated with these purified microvesicles, or with combinations of fVIIa-recombinant TF, and cell proliferation/apoptosis was measured. Additionally, by expressing mCherry-PAR2 on HCAEC surface, PAR2 activation was quantified. Finally, the activation of PAR2 on HCAEC or the activities of TF and fVIIa in microvesicles were blocked prior to addition of microvesicles to cells. The purified microvesicles exhibited a range of fVIIa:TF ratios with HepG2 and 786-O cells having the highest (54:1) and lowest (10:1) ratios, respectively. The reversal from proapoptotic to proliferative was estimated to occur at a fVIIa:TF molar ratio of 15:1, but HCAEC could not be rescued at higher TF concentrations. The purified microvesicles induced HCAEC proliferation or apoptosis according to this ruling. Blocking PAR2 activation on HCAEC, or inhibiting fVIIa or TF-procoagulant function on microvesicles prevented the influence on HCAEC. Finally, incubation of HCAEC with recombinant TF resulted in increased surface exposure of fVII. The induction of cell proliferation or apoptosis by TF-positive microvesicles is dependent on the ratio of fVIIa:TF and involves the activation of PAR2. At lower TF concentrations, fVIIa can counteract the proapoptotic stimulus and induce proliferation.

scholarly journals Interleukin-37: The Effect of Anti-Inflammatory Response in Human Coronary Artery Endothelial Cells

2019 ◽  
Vol 2019 ◽  
pp. 1-12 ◽  
Author(s):  
Xianfeng Yan ◽  
Bin Xie ◽  
Guihai Wu ◽  
Jing Hu ◽  
Di Wang ◽  
...  
Keyword(s):  
Innate Immunity ◽  
Endothelial Cells ◽  
Coronary Artery ◽  
Inflammatory Response ◽  
Atherosclerotic Plaque ◽  
Inflammatory Responses ◽  
Plaque Formation ◽  
Human Coronary Artery ◽  
Atherosclerotic Plaque Formation

Interleukin-37 (IL-37) is unique in the IL-1 family since it broadly suppresses innate immunity and elevates in humans with inflammatory and autoimmune diseases. IL-37 shows definite groups and transcripts for human IL37 gene, but it is still not completely understood the effect and mechanisms of inflammatory response in endothelial cells. It is well accepted that endothelial dysfunction caused by inflammation is a key initiating event in atherosclerotic plaque formation, which leads to the occurrence and development of the cardiovascular adverse events in clinical since the inflammatory responses of endothelial cells could induce and enhance the deposition of extensive lipid and the formation of atherosclerotic plaque in the intima. Thus, it is essential to investigate the role and potential mechanisms in endothelial inflammatory response to prevent the formation and development of many cardiovascular diseases including atherosclerosis. So far, the recent studies have revealed that IL-37 is able to inhibit inflammatory response by suppressing the TLR2-NF-κB-ICAM-1 pathway intracellularly in human coronary artery endothelial cells (HCAECs). Further, the role of IL-37 may be related to the IL-18 pathway extracellularly and involved in the adhesion and transmigration of neutrophils in HCAECs.

scholarly journals Role of Caspases in Ox-LDL–Induced Apoptotic Cascade in Human Coronary Artery Endothelial Cells

2004 ◽  
Vol 94 (3) ◽  
pp. 370-376 ◽  
Author(s):  
Jiawei Chen ◽  
Jawahar L. Mehta ◽  
Nezam Haider ◽  
Xingjian Zhang ◽  
Jagat Narula ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Coronary Artery ◽  
Human Coronary Artery ◽  
Apoptotic Cascade

scholarly journals IL-33 / ST2 Signaling Promotes TF Expression by Regulating NF-κB Activation in Coronary Artery Endothelial Microparticles

2021 ◽  
Author(s):  
Yujuan Yuan ◽  
Hui Cheng ◽  
Jing Tao ◽  
Muyesai Nijiati
Keyword(s):  
Endothelial Cells ◽  
Coronary Artery ◽  
Tissue Factor ◽  
Stable Coronary Artery Disease ◽  
Dimethyl Fumarate ◽  
Human Coronary Artery ◽  
Roc Curve Analysis ◽  
Endothelial Microparticles ◽  
Artery Disease ◽  
St2 Receptor

IntroductionInterleukin (IL)-33 was previously shown to induce angiogenesis and inflammatory activation of endothelial Microparticles(EMPs). Tissue factor (TF) plays a central role in hemostasis and thrombosis.Material and methodsThe study analyzed the coronary blood of level of CD31+EMPs, TF protein and IL-33 protein in Acute Myocardial Infarction (AMI) and stable coronary artery disease (SCAD) patients. Human coronary artery endothelial cells (HCAECs) were treated with IL-33 to obtain EMPs. The TF activity of EMPs was tested by Thermo Fisher by adding the TF antibody. Furthermore, TF and Tissue Factor Pathway Inhibitor (TFPI) protein were tested by ELISA. Finally, NF-κB inhibitor dimethyl fumarate (DMF) and soluble extracellular domain of ST2 coupled to the Fc fragment of human IgG1 (sST2) were added to HCAECs which were treated with IL-33, and the TF protein level was also tested by ELISA.ResultsThe AMI patients had higher level of CD31+EMPs, TF protein and IL-33 protein than the SCAD patients in coronary artery. In AMI patients (N=27), the IL-33 protein positively correlated with CD31+EMPs (r=0.794, p<0.01). According to the ROC curve analysis, the AUC of CD31+EMPs, TF protein and IL-33 protein were 0.888, 0.962 and 0.778 respectively. In the cell culture, the TF activity and TF protein in EMPs increased gradually with time of intervention by the treatment of IL-33. IL-33 binding to the ST2 receptor promoted TF expression by regulating NF-κB activation in EMPs of HCAECs.ConclusionsActivated endothelial cells and EMPs they released simultaneously express TF, which is a risk factor for cardiovascular disease.

Abstract 346: Manipulating P2Y2 Receptor Biased Signaling to Limit Pro-thrombotic Gene Expression in Human Coronary Artery Endothelial Cells

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Lingxin Zhang ◽  
Chuan Wang ◽  
Thamer Alqurashi ◽  
Jianzhong Shen
Keyword(s):  
Endothelial Cells ◽  
Coronary Artery ◽  
Promoter Activity ◽  
Gene Promoter ◽  
Mechanistic Study ◽  
Human Coronary Artery ◽  
Biased Signaling ◽  
Luciferase Activity Assay ◽  
Intracellular Ca ◽  
Tf Gene

Recently we reported that in human coronary artery endothelial cells (HCAEC), the P2Y2 nucleotide receptor (P2Y2R) regulates the pro-thrombotic gene tissue factor (TF) expression through positive and negative signaling mechanisms. Here, we report the identification of a P2Y2R ligand JZS0312 which selectively activates the negative, but not the positive pathways, leading to decreased TF expression. Unlike the endogenous agonist UTP, JZS0312 alone had no effect on TF mRNA transcription, but it unexpectedly inhibited UTP-induced TF expression. Mechanistic study showed that JZS0312 induced intracellular Ca 2+ mobilization in HCAEC which exhibited the same efficacy as UTP and was inhibited by AR-C118925, a P2Y2R-selective antagonist. However, when evaluated in their activation of the MAPK pathways, JZS0312 only activated the ERK1/2, but not JNK and p38, whereas UTP activated all the three, suggesting JZS0312 as a biased ligand for the P2Y2R. This notion was supported by the fact that JZS0312-induced ERK1/2 activation was inhibited by AR-C1189258 and it had no effect on ERK1/2 in P2Y2R-null cells. In addition, JZS0312 selectively activated the TF gene repressor Fra-1, but not the positive AP-1 subunits c-Jun and ATF-2, whereas UTP activated all of them. Furthermore, luciferase activity assay indicates that JZS0312 suppressed TF gene promoter activity via ERK1/2 activation, while UTP increased overall TF promoter activity. These findings reveal that JZS0312 acts as a biased agonist in human endothelial P2Y2R, in which it selectively activates the ERK1/2-Fra1 pathway, leading to suppression of TF gene transcription. Thus, JZS0312 represents the first identified biased ligand in the P2Y receptor family, opening new avenue for potential drug discovery in relevant inflammatory and thrombotic diseases.

Proapoptotic effects of ANG II in human coronary artery endothelial cells: role of AT1receptor and PKC activation

1999 ◽  
Vol 276 (3) ◽  
pp. H786-H792 ◽  
Author(s):  
Dayuan Li ◽  
Baichun Yang ◽  
M. Ian Philips ◽  
Jawahar L. Mehta
Keyword(s):  
Endothelial Cells ◽  
Coronary Artery ◽  
Specific Inhibitor ◽  
Ang Ii ◽  
Human Coronary Artery ◽  
Tnf Α ◽  
Factor Α ◽  
Pkc Activation

Anoxia-reoxygenation, tumor necrosis factor-α (TNF-α), and angiotensin II (ANG II) have been shown to induce apoptosis in myocytes. However, the role of these mediators in causing apoptosis of human coronary artery endothelial cells (HCAEC) is not known. This study was designed to examine the interaction of these mediators in induction of apoptosis in HCAEC. Cultured HCAEC were exposed to anoxia-reoxygenation, TNF-α, and ANG II. TNF-α enhanced apoptosis of HCAEC (determined by DNA nick-end labeling in situ and DNA laddering) caused by anoxia-reoxygenation. ANG II increased apoptosis beyond that caused by anoxia-reoxygenation and TNF-α. Apoptosis caused by ANG II was reduced by losartan, a specific ANG II type 1 receptor (AT1R) blocker, whereas PD-123,177, a specific ANG II type 2 receptor blocker, under identical conditions had minimal effect. The proapoptotic effects of ANG II were associated with the activation of protein kinase C (PKC). The importance of PKC activation as a signal transduction mechanism became evident in experiments wherein treatment of HCAEC with a specific inhibitor of PKC activation decreased ANG II-mediated apoptosis. Thus AT1R activation appears to be responsible for apoptosis caused by ANG II in HCAEC, and AT1R activation-mediated apoptosis involves activation of PKC.

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