Pannexin 1‐dependent ATP release from venous endothelium promotes acute vascular inflammation (669.8)

Abstract

Tumor necrosis factor α (TNFα) activates endothelial cells (EC) promoting EC‐leukocyte interactions in the venous circulation which is mediated, in part, by purinergic signaling. However, it is currently not known if ECs release ATP to TNFα. Employing in vitro ATP release assays, we report that TNFα significantly increased ATP release from venous ECs (Human Umbilical and Saphenous) which was not observed in arterial ECs (Human Coronary and Aortic). ATP release was inhibited by WP9QY, a peptide antagonist of the TNF receptor 1 (TNFR1), indicating receptor activation in the initiation of this event. Pharmacological inhibition of Pannexin 1 (Panx1) channels in venous ECs with CBX or 10panx1 and siRNA knockdown of Panx1 significantly inhibited ATP release, whereas inhibition of connexin hemichannels, CALHM1 channels and exocytosis had no effect. This indicates that Panx1 channels are the conduit for ATP liberation. Additionally, we show that Src family kinases mediate Panx1 channel activation, as the Src inhibitor PP2 significantly reduced TNFα‐induced ATP release. Functionally, both Panx1 inhibition and degradation of extracellular ATP attenuated leukocyte‐venous EC interactions to TNFα stimulation. Utilizing ex vivo cannulated mesenteric venules, luminal perfusion of TNFα significantly increased ATP release into the perfusate, supporting a role for TNFα‐mediated ATP release in intact venules. Finally, we developed an EC‐specific, inducible Panx1 knockout mouse (VEcadCreERT2+/Panx1fl/fl) to determine the role of Panx1 in vascular inflammation in vivo. In conclusion, TNFα activates Panx1 channels in venous ECs inducing ATP release to promote leukocyte‐EC interactions.Grant Funding Source: Supported by NIH, AHA