scholarly journals Coagulation Factor Xa Induces Proinflammatory Responses in Cardiac Fibroblasts via Activation of Protease-Activated Receptor-1

Cells ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 2958
Author(s):  
Elisa D’Alessandro ◽  
Billy Scaf ◽  
Chantal Munts ◽  
Arne van Hunnik ◽  
Christopher J. Trevelyan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cardiac Fibroblasts ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Conditioned Media ◽  
Cell Type ◽  
Protease Activated Receptors ◽  
Protein Levels ◽  
Proinflammatory Responses ◽  
Protease Activated Receptor 1

Coagulation factor (F) Xa induces proinflammatory responses through activation of protease-activated receptors (PARs). However, the effect of FXa on cardiac fibroblasts (CFs) and the contribution of PARs in FXa-induced cellular signalling in CF has not been fully characterised. To answer these questions, human and rat CFs were incubated with FXa (or TRAP-14, PAR-1 agonist). Gene expression of pro-fibrotic and proinflammatory markers was determined by qRT-PCR after 4 and 24 h. Gene silencing of F2R (PAR-1) and F2RL1 (PAR-2) was achieved using siRNA. MCP-1 protein levels were measured by ELISA of FXa-conditioned media at 24 h. Cell proliferation was assessed after 24 h of incubation with FXa ± SCH79797 (PAR-1 antagonist). In rat CFs, FXa induced upregulation of Ccl2 (MCP-1; >30-fold at 4 h in atrial and ventricular CF) and Il6 (IL-6; ±7-fold at 4 h in ventricular CF). Increased MCP-1 protein levels were detected in FXa-conditioned media at 24 h. In human CF, FXa upregulated the gene expression of CCL2 (>3-fold) and IL6 (>4-fold) at 4 h. Silencing of F2R (PAR-1 gene), but not F2RL1 (PAR-2 gene), downregulated this effect. Selective activation of PAR-1 by TRAP-14 increased CCL2 and IL6 gene expression; this was prevented by F2R (PAR-1 gene) knockdown. Moreover, SCH79797 decreased FXa-induced proliferation after 24 h. In conclusion, our study shows that FXa induces overexpression of proinflammatory genes in human CFs via PAR-1, which was found to be the most abundant PARs isoform in this cell type.

A Novel Mechanism of Plasmin-Induced Mitogenesis in Fibroblasts.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2628-2628 ◽  
Author(s):  
Samir K. Mandal ◽  
L. Vijaya Mohan Rao ◽  
Thuy Tien T. Tran ◽  
Usha Pendurthi
Keyword(s):  
Dna Synthesis ◽  
Tissue Factor ◽  
Protease Activity ◽  
Neutralizing Antibodies ◽  
Factor Viia ◽  
Early Response ◽  
Conditioned Media ◽  
Induced Response ◽  
Protease Activated Receptors ◽  
Protein Levels

Abstract The plasminogen activator/plasmin system plays an important role, in addition to fibrinolysis, in many pathophysiological processes. Our recent studies showed that plasmin, similar to factor VIIa/tissue factor and thrombin, upregulates Cyr61, a growth factor like immediate early response gene, in fibroblasts via protease-activated receptor-mediated signaling. In preliminary studies we found that when fibroblasts were treated with VIIa (100 nM), thrombin (10 nM) or plasmin (50 nM), in concentrations that were shown to induce Cyr61 expression maximally, VIIa and thrombin increased the DNA synthesis by only about 20–30% whereas plasmin increased the DNA synthesis by 200 to 500%. The present study was carried out to investigate potential mechanism(s) by which plasmin promotes the DNA synthesis in fibroblasts and the role of protease-activated receptors and Cyr61 in plasmin-induced DNA synthesis. The plasmin-induced DNA synthesis in fibroblasts was dependent on its protease activity as the addition of active site-inhibited plasmin had no effect; and tissue factor pathway inhibitor-2 (TFPI-2) completely abrogated the plasmin-induced response. Neutralizing antibodies against PAR-1 and not PAR-2 attenuated the plasmin-induced DNA synthesis. Cyr61 antibodies completely blocked the plasmin-induced DNA synthesis suggesting that Cyr61 acts as a down-stream mediator. Surprisingly, we found no detectable increase in Cyr61 protein in plasmin-treated cell lysates whereas Cyr61 protein levels were clearly increased in thrombin-treated cells. These data suggest that plasmin might be cleaving newly synthesized Cyr61, which accumulates in ECM. Studies performed with purified recombinant Cyr61 and tumor cells that constitutively express abundant Cyr61 provided support for this notion. To test the hypothesis that plasmin release of Cyr61 from ECM plays a role in promoting DNA synthesis, we investigated the effect of conditioned media from control, plasmin-, and thrombin-treated fibroblasts in promoting DNA synthesis. The data revealed that the conditioned media of plasmin-treated cells (the protease activity in the media was inactivated) and not others significantly increased 3H-thymidine incorporation in fibroblasts. Addition of Cyr61 antibodies to the conditioned media of plasmin-treated cells attenuated the enhancing effect of the conditioned media. In summary, the data presented herein provide a novel mechanism by which plasmin promotes cell proliferation. In this mechanism, plasmin first induces the expression of Cyr61 via activation of PAR-1 mediated signaling. Next, plasmin releases/cleaves newly synthesized Cyr61 from the ECM, making it accessible to cells. The ability of plasmin to induce mitogenesis, in addition to its pericellular proteolysis, may provide a coordinated spatiotemporal effect that is required for would healing and tissue remodeling.

Gene induction by coagulation factor Xa is mediated by activation of protease-activated receptor 1

Blood ◽  
2001 ◽  
Vol 97 (10) ◽  
pp. 3109-3116 ◽  
Author(s):  
Matthias Riewald ◽  
Vladimir V. Kravchenko ◽  
Ramona J. Petrovan ◽  
Peter J. O'Brien ◽  
Lawrence F. Brass ◽  
...  
Keyword(s):  
Cell Surface ◽  
Cellular Response ◽  
Expression Patterns ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Gene Induction ◽  
Tissue Growth ◽  
Thrombin Inhibitor ◽  
Local Concentration ◽  
Protease Activated Receptor 1

Abstract Cell signaling by coagulation factor Xa (Xa) contributes to pro-inflammatory responses in vivo. This study characterizes the signaling mechanism of Xa in a HeLa cell line that expresses protease-activated receptor 1 (PAR-1) but not PAR-2, -3, or -4. Xa induced NF-κB in HeLa cells efficiently but with delayed kinetics compared to thrombin. This delay caused no difference in gene expression patterns, as determined by high-density microarray analysis. Both proteases prominently induced the angiogenesis-promoting geneCyr61 and connective tissue growth factor. Inhibition of PAR-1 cleavage abolished MAP kinase phosphorylation and gene induction by Xa, demonstrating that Xa signals through PAR-1 and not through a novel member of the PAR family. Activation of cell surface prothrombin with the snake venom enzyme Ecarin also produced PAR-1–dependent signaling. However, though the response to Ecarin was completely blocked by the thrombin inhibitor hirudin, the response to Xa was not. This suggests that the Xa response is not mediated by locally generated thrombin. The concentration dependence of Xa for PAR-1 activation is consistent with previously characterized Xa-mediated PAR-2 signaling, suggesting that local concentration of Xa on the cell surface, rather than sequence-specific recognition of the PAR scissile bond, determines receptor cleavage. This study demonstrates that PAR-1 cleavage by Xa can elicit the same cellular response as thrombin, but mechanistic differences in receptor recognition may be crucial for specific roles for Xa in signaling during spatial or temporal separation from thrombin generation.

Abstract 487: Dihyrosphingosine 1 Phosphate Mediates Collagen Synthesis in Cardiac Fibroblasts Throught Pi3k/akt- Mtor Signalling Pathway

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Ruth R Magaye ◽  
Feby Sevira ◽  
Xin Xiong ◽  
Bernard Flynn ◽  
Bing Wang
Keyword(s):  
Gene Expression ◽  
Collagen Synthesis ◽  
Cardiac Fibrosis ◽  
Cardiac Fibroblasts ◽  
Mtor Pathway ◽  
Neonatal Rat ◽  
Protein Levels ◽  
Proline Incorporation ◽  
Exogenous Addition ◽  
First Time

Background: Cardiac fibrosis is one of the hallmarks of cardiac remodelling in cardiomyopathies such as heart Failure (HF). Dyslipidemia plays a role in the progression of HF. The sphingolipid, dihydrosphingosine 1 phosphate (dhS1P) has been shown to bind to high density lipids in plasma. Unlike its analog, spingosine 1 phosphate (S1P), the role of dhS1P in cardiac fibrosis is not known. The aim of this study is to determine the role dhS1P plays in cardiac fibrosis through the PI3K/Akt- mTOR pathway. Method: Neonatal rat cardiac fibroblasts (NCF) were isolated from 1-2 day old pups with enzymic digestion. After pre-treating with the PI3K inhibitor, Wortmannin (W, 0.1 - 10.0μM), cells were stimulated with dhS1P for 48 hours. NCF collagen synthesis was determined by 3H-proline incorporation. NCF were also treated for protein and gene expression analysis. Results: Exogenous addition of 3 μM dhS1P stimulated significant increase in collagen synthesis (p<0.005) which was dose dependently inhibited by W (p < 0.0001, Fig. 1A). Western blot analysis showed that W reduced Akt, mTOR, and S6 activation in the presence of dhS1P. dhS1P also increased protein levels of TGFβ, Coll 1 and TIMP1. W reduced dhS1P elevated TIMP1, and SK1, but not TGFβ1 gene expression (Fig. 1B). Conclusion: Our study demonstrates for the first time that dhS1P can cause cardiac cellular fibrosis via PI3K/Akt- mTOR pathway. Its inhibition may represent a novel therapeutic strategy for cardiac fibrosis.

scholarly journals Single cell RNA sequencing identifies IGFBP5 and QKI as ciliated epithelial cell genes associated with severe COPD

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Xiuying Li ◽  
Guillaume Noell ◽  
Tracy Tabib ◽  
Alyssa D. Gregory ◽  
Humberto E. Trejo Bittar ◽  
...  
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Single Cell ◽  
Rna Sequencing ◽  
Cell Types ◽  
Cell Type ◽  
Protein Levels ◽  
Single Cell Rna Sequencing ◽  
Ciliated Epithelial Cells ◽  
Severe Copd

Abstract Background Whole lung tissue transcriptomic profiling studies in chronic obstructive pulmonary disease (COPD) have led to the identification of several genes associated with the severity of airflow limitation and/or the presence of emphysema, however, the cell types driving these gene expression signatures remain unidentified. Methods To determine cell specific transcriptomic changes in severe COPD, we conducted single-cell RNA sequencing (scRNA seq) on n = 29,961 cells from the peripheral lung parenchymal tissue of nonsmoking subjects without underlying lung disease (n = 3) and patients with severe COPD (n = 3). The cell type composition and cell specific gene expression signature was assessed. Gene set enrichment analysis (GSEA) was used to identify the specific cell types contributing to the previously reported transcriptomic signatures. Results T-distributed stochastic neighbor embedding and clustering of scRNA seq data revealed a total of 17 distinct populations. Among them, the populations with more differentially expressed genes in cases vs. controls (log fold change >|0.4| and FDR = 0.05) were: monocytes (n = 1499); macrophages (n = 868) and ciliated epithelial cells (n = 590), respectively. Using GSEA, we found that only ciliated and cytotoxic T cells manifested a trend towards enrichment of the previously reported 127 regional emphysema gene signatures (normalized enrichment score [NES] = 1.28 and = 1.33, FDR = 0.085 and = 0.092 respectively). Among the significantly altered genes present in ciliated epithelial cells of the COPD lungs, QKI and IGFBP5 protein levels were also found to be altered in the COPD lungs. Conclusions scRNA seq is useful for identifying transcriptional changes and possibly individual protein levels that may contribute to the development of emphysema in a cell-type specific manner.

scholarly journals Spatiotemporal dynamics of SETD5-containing NCoR–HDAC3 complex determines enhancer activation for adipogenesis

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yoshihiro Matsumura ◽  
Ryo Ito ◽  
Ayumu Yajima ◽  
Rei Yamaguchi ◽  
Toshiya Tanaka ◽  
...  
Keyword(s):  
Gene Expression ◽  
Histone Acetylation ◽  
Ubiquitin Ligase ◽  
Cellular Differentiation ◽  
Specific Gene ◽  
Cell Type ◽  
Set Domain ◽  
Protein Levels ◽  
Primed State ◽  
Cell Type Specific

AbstractEnhancer activation is essential for cell-type specific gene expression during cellular differentiation, however, how enhancers transition from a hypoacetylated “primed” state to a hyperacetylated-active state is incompletely understood. Here, we show SET domain-containing 5 (SETD5) forms a complex with NCoR-HDAC3 co-repressor that prevents histone acetylation of enhancers for two master adipogenic regulatory genes Cebpa and Pparg early during adipogenesis. The loss of SETD5 from the complex is followed by enhancer hyperacetylation. SETD5 protein levels were transiently increased and rapidly degraded prior to enhancer activation providing a mechanism for the loss of SETD5 during the transition. We show that induction of the CDC20 co-activator of the ubiquitin ligase leads to APC/C mediated degradation of SETD5 during the transition and this operates as a molecular switch that facilitates adipogenesis.

scholarly journals Cell type-specific roles of PAR1 in Coxsackievirus B3 infection

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Michael F. Bode ◽  
Clare M. Schmedes ◽  
Grant J. Egnatz ◽  
Vanthana Bharathi ◽  
Yohei M. Hisada ◽  
...  
Keyword(s):  
Cardiac Myocytes ◽  
Cardiac Fibroblasts ◽  
Coxsackievirus B3 ◽  
Poly I:C ◽  
Cell Type ◽  
Embryonic Fibroblasts ◽  
Cell Type Specific ◽  
Protease Activated Receptor 1 ◽  
Inflammatory Infiltrates ◽  
Specific Deletion

AbstractProtease-activated receptor 1 (PAR1) is widely expressed in humans and mice, and is activated by a variety of proteases, including thrombin. Recently, we showed that PAR1 contributes to the innate immune response to viral infection. Mice with a global deficiency of PAR1 expressed lower levels of CXCL10 and had increased Coxsackievirus B3 (CVB3)-induced myocarditis compared with control mice. In this study, we determined the effect of cell type-specific deletion of PAR1 in cardiac myocytes (CMs) and cardiac fibroblasts (CFs) on CVB3-induced myocarditis. Mice lacking PAR1 in either CMs or CFs exhibited increased CVB3 genomes, inflammatory infiltrates, macrophages and inflammatory mediators in the heart and increased CVB3-induced myocarditis compared with wild-type controls. Interestingly, PAR1 enhanced poly I:C induction of CXCL10 in rat CFs but not in rat neonatal CMs. Importantly, activation of PAR1 reduced CVB3 replication in murine embryonic fibroblasts and murine embryonic cardiac myocytes. In addition, we showed that PAR1 reduced autophagy in murine embryonic fibroblasts and rat H9c2 cells, which may explain how PAR1 reduces CVB3 replication. These data suggest that PAR1 on CFs protects against CVB3-induced myocarditis by enhancing the anti-viral response whereas PAR1 on both CMs and fibroblasts inhibits viral replication.

Coagulation proteases and human cancer

2002 ◽  
Vol 30 (2) ◽  
pp. 201-207 ◽  
Author(s):  
M. T. Sampson ◽  
A. K. Kakkar
Keyword(s):  
Gene Expression ◽  
Tissue Factor ◽  
Serine Proteases ◽  
Factor Viia ◽  
Human Cancer ◽  
Factor Xa ◽  
Protease Activated Receptors ◽  
Clotting Cascade ◽  
Cancer Procoagulant ◽  
Tumour Cell Growth

Tumours are capable of activating blood coagulation through the expression of procoagulant molecules such as tissue factor, cancer procoagulant and hepsin. Initiation of the clotting cascade results in the generation of the activated serine proteases factor VIIa, factor Xa and thrombin. These proteases act via protease-activated receptors and tissue factor to alter gene expression, thereby modulating tumour cell growth, invasion, metastasis and angiogenesis.

scholarly journals Single Cell RNA Sequencing Identifies IGFBP5 And QKI In Ciliated Epithelial Cell Genes Associated With Severe COPD

2020 ◽  
Author(s):  
Xiuying Li ◽  
Guillaume Noell ◽  
Tracy Tabib ◽  
Alyssa D Gregory ◽  
Humberto E Trejo Bittar ◽  
...  
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Single Cell ◽  
Rna Sequencing ◽  
Cell Types ◽  
Cell Type ◽  
Protein Levels ◽  
Single Cell Rna Sequencing ◽  
Ciliated Epithelial Cells ◽  
Severe Copd

Abstract Background: Whole lung tissue transcriptomic profiling studies in chronic obstructive pulmonary disease (COPD) have led to the identification of several genes associated with the severity of airflow limitation and/or the presence of emphysema, however, the cell types driving these gene expression signatures remain unidentified.Methods: To determine cell specific transcriptomic changes in severe COPD, we conducted single-cell RNA sequencing (scRNA seq) on n= 29,961 cells from the peripheral lung parenchymal tissue of nonsmoking subjects without underlying lung disease (n=3) and patients with severe COPD (n=3). The cell type composition and cell specific gene expression signature was assessed. Gene set enrichment analysis (GSEA) was used to identify the specific cell types contributing to the previously reported transcriptomic signatures.Results: T-distributed stochastic neighbor embedding and clustering of scRNA seq data revealed a total of 17 distinct populations. Among them, the populations with more differentially expressed genes in cases vs. controls (log fold change >|0.4| and FDR=0.05) were: monocytes (n=1499); macrophages (n=868) and ciliated epithelial cells (n= 590), respectively. Using GSEA, we found that only ciliated and cytotoxic T cells manifested a trend towards enrichment of the previously reported 127 regional emphysema gene signatures (normalized enrichment score [NES] = 1.28 and =1.33, FDR= 0.085 and =0.092 respectively). Among the significantly altered genes present in ciliated epithelial cells of the COPD lungs, QKI and IGFBP5 protein levels were also found to be altered in the COPD lungs. Conclusions: scRNA seq is useful to identify transcriptional changes and possibly individual protein levels that may contribute to the development of emphysema in a cell-type specific manner.

Spatiotemporal dynamics of SETD5-containing NCoR-HDAC3 complex determines enhancer activation for adipogenesis

2021 ◽  
Author(s):  
Yoshihiro Matsumura ◽  
Ryo Ito ◽  
Ayumu Yajima ◽  
Rei Yamaguchi ◽  
Kenta Magoori ◽  
...  
Keyword(s):  
Gene Expression ◽  
Histone Acetylation ◽  
Ubiquitin Ligase ◽  
Cellular Differentiation ◽  
Specific Gene ◽  
Cell Type ◽  
Set Domain ◽  
Protein Levels ◽  
Primed State ◽  
Cell Type Specific

Abstract Enhancer activation is essential for cell-type specific gene expression during cellular differentiation, however, how enhancers transition from a hypoacetylated “primed” state to a hyperacetylated-active state is incompletely understood. Here, we show SET domain-containing 5 (SETD5) forms a complex with NCoR-HDAC3 co-repressor that prevents histone acetylation of enhancers for two master adipogenic regulatory genes Cebpa and Pparg early during adipogenesis. The loss of SETD5 from the complex is followed by enhancer hyperacetylation. SETD5 protein levels were transiently increased and rapidly degraded prior to enhancer activation providing a mechanism for the loss of SETD5 during the transition. We show that induction of the CDC20 co-activator of the ubiquitin ligase leads to APC/C mediated degradation of SETD5 during the transition and this operates as a molecular switch that facilitates adipogenesis.

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