Adenosine Inhibits Pro-Inflammatory Thrombin Signaling In Endothelial Cells

Adenosine is an important regulatory metabolite which attenuates inflammation when it binds and activates the adenosine A_2A 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, A_2A, A_2B and A₃ was examined in HUVECs and it was found that A_2A and A_2B are the highest expressing receptors among the four subtypes (A_2B> A_2A>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 A_2A receptor specific siRNA or the A_2A 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 A_2A receptor subtype, thereby protecting endothelium during the activation of coagulation and inflammatory pathways.