Adenosine Inhibits Pro-Inflammatory Thrombin Signaling In Endothelial Cells

Abstract

Adenosine is an important regulatory metabolite which attenuates inflammation when it binds and activates the adenosine A2A receptor subtype on immune cells. However, the effect of adenosine on inflammatory responses in endothelial cells is mostly unknown. Thrombin as a known pro-inflammatory protease is involved in a variety of pathophysiological processes associated with inflammation in stimulated endothelial cells. The present study investigated the effect of adenosine on thrombin-mediated modulation of pro-inflammatory responses in human umbilical vein endothelial cells (HUVECs). Adenosine, in a concentration-dependent manner (1-100µM), inhibited the barrier disruptive effect of thrombin (20nM) on endothelial monolayer. The expression level of adenosine receptors, A1, A2A, A2B and A3 was examined in HUVECs and it was found that A2A and A2B are the highest expressing receptors among the four subtypes (A2B> A2A>A1>A3) in endothelial cells. Further studies revealed that the barrier protective effect of adenosine on thrombin-induced hyperpermeability in HUVECs was abrogated by the A2A receptor specific siRNA or the A2A receptor specific antagonist, ZM-241385, but not by siRNAs targeting the other adenosine receptor subtypes. To further determine the molecular mechanisms of the barrier protective effect of adenosine, its effect on thrombin-induced RhoA activation was assessed. Pretreatment of endothelial cells with adenosine prevented both thrombin-induced RhoA activation (Rho-GTP) and its membrane translocation as evidenced by cell fractionation and Pull-down assays, respectively. Moreover, preincubation of endothelial cells with adenosine down-regulated the expression of cell surface adhesion molecules (VCAM-1 and ICAM-1) and thrombin-mediated activation of nuclear factor-kappaB (NF-kB) pathway. Adenosine also inhibited secretion of the early chemokine, MCP-1, and the late-acting pro-inflammatory cytokine, HMGB-1, by thrombin-stimulated endothelial cells. Taken together, these results suggest that adenosine can inhibit pro-inflammatory thrombin signaling responses in endothelial cells by specifically activating the A2A receptor subtype, thereby protecting endothelium during the activation of coagulation and inflammatory pathways. Disclosures:No relevant conflicts of interest to declare.