Myoendothelial gap junctions mediate regulation of angiopoietin-2-induced vascular hyporeactivity after hypoxia through connexin 43-gated cAMP transfer

Abstract

Angiopoietin-2 (Ang-2) contributes to vascular hyporeactivity after hemorrhagic shock and hypoxia through upregulation of inducible nitric oxide synthase (iNOS) in a vascular endothelial cell (VEC)-specific and Ang-2/Tie2 receptor-dependent manner. While iNOS is primarily expressed in vascular smooth muscle cells (VSMCs), the mechanisms of signal transfer from VECs to VSMCs are unknown. A double-sided coculture model with VECs and VSMCs from Sprague-Dawley rats was used to investigate the role of myoendothelial gap junctions (MEGJs), the connexin (Cx) isoforms involved, and other relevant mechanisms. After hypoxia, VSMCs treated with exogenous Ang-2 showed increased iNOS expression and hyporeactivity, as well as MEGJ formation and communication. These Ang-2 effects were suppressed by the MEGJ inhibitor 18_-glycyrrhetic acid (18-GA), Tie2 siRNA, or Cx43 siRNA. Reagents antagonizing cAMP or protein kinase A (PKA) in VECs inhibited Cx43 expression in MEGJs, decreasing MEGJ formation and associated communication, after hypoxia following Ang-2 treatment. The increased cAMP levels in VSMCs and transfer of Alexa Fluor 488-labeled cAMP from VECs to VSMCs, after hypoxia following Ang-2 treatment, was antagonized by Cx43 siRNA. A cAMP antagonist added to VECs or VSMCs inhibited both increased iNOS expression and hyporeactivity in VSMCs subjected to hypoxia following Ang-2 treatment. Based on these findings, we propose that Cx43 was the Cx isoform involved in MEGJ-mediated VEC-dependent regulation of Ang-2, which induces iNOS protein expression and vascular hyporeactivity after hypoxia. Cx43 was upregulated by cAMP and PKA, permitting cAMP transfer between cells.