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

Elisa D’Alessandro,Frans A Van Nieuwenhoven,Neil A Turner,Ulrich Schotten,Arne Van Hunnik,Billy Scaf,Hugo Ten Cate,Christopher J Trevelyan,Henri M H Spronk,Sander Verheule,Chantal Munts

doi:10.3390/cells10112958

Abstract

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.

Highlights

The strong induction of Ccl2 expression mediated by FXa was reflected by the 20.6- and 36.0-fold upregulation of monocyte chemoattractant protein (MCP)-1 protein levels in the conditioned media of cardiac fibroblasts (CFs) exposed to FXa for 24 h
FXa pro-inflammatory was mainly mediated by protease-activated receptors (PARs)-1 activation, which ap- 264 the FXa pro-inflammatory effect was mainly mediated by PAR-1 activation, which appears pears to be the most abundant isoform expressed in CFs
In line with what was previously described by Guo et al in neonatal rat CFs, we showed that FXa enhanced the proliferation of human CFs and that this effect seemed to be attenuated by PAR-1 antagonist [21]

Summary

Introduction

In the acute phase after myocardial infarction (MI), CFs respond to proinflammatory stimuli (e.g., reactive oxygen species (ROS), interleukin (IL)-1, and tumour necrosis factor alpha (TNF-α)) in the ischemic microenvironment by altering their gene expression profile and acting as inflammatory supporter cells [6]. In this phase, CFs produce large amounts of cytokines, such as IL-6, IL-1, and chemokines, such as IL-8 and monocyte chemoattractant protein (MCP)-1, supporting and modulating the recruitment of leukocytes that clear the tissue of dead cells and matrix debris [5,6]

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