P2X4 receptors mediate atheroprotective gene expression in an in vitro model of steady flow in vascular endothelial cells (696.6)

Abstract

Vascular endothelial cells are in direct contact with blood flow and are exposed to fluid shear stress. The role of P2X4 receptors in shear stress‐mediated mechanotransduction is well documented. Studies have shown endothelial cells to release ATP in response to steady flow that in turn modulates cellular functions via P2 receptors in the vasculature. However, the molecular mechanisms governing ATP‐mediated P2 receptor signaling downstream of steady flow in endothelial cells remains largely unknown. Although steady flow‐induced KLF2 expression implicated in atheroprotection is well studied, whether ATP regulates KLF2 remains unanswered and is the aim of this study.Human umbilical vein endothelial cells (HUVECs) were either exposed to steady flow at 12 dynes/cm2 or to exogenous administration of a non‐hydrolysable analog of ATP (ATPγS) in the presence of the P2X4 antagonist (PSB‐12253) and P2X4 siRNA. We validated the in vitro orbital shaker model for steady flow showing cell alignment in the direction of flow and upregulation of KLF2 and NOS3. Further, addition of ATPγS to static cultures led to an increase in KLF2 mRNA as early as 3h. Exposing HUVECs to apyrase and PSB‐12253 reduced KLF2 and NOS3 expression under steady flow. Furthermore, the knockdown of P2X4 confirmed its role in regulating KLF2. These results suggest that ATP is involved in regulating KLF2 in part via the P2X4 receptor.