Mechanosensitive Membrane Domains Regulate Calcium Entry in Arterial Endothelial Cells to Protect Against Inflammation

Endothelial cells (ECs) in the descending aorta are exposed to high laminar shear stress, which supports an anti-inflammatory phenotype. High laminar shear stress also supports flow-aligned cell elongation and front-rear polarity, but whether these are required for the anti-inflammatory phenotype is unclear. Here, we show that Caveolin-1-rich microdomains polarize to the downstream end of ECs exposed to continuous high laminar flow. These microdomains are characterized by high membrane rigidity, filamentous actin (F-actin), and raft-associated lipids. Transient receptor potential vanilloid-type 4 (Trpv4) ion channels are ubiquitously expressed on the plasma membrane but mediate localized Ca2+ entry only at these microdomains where they physically interact with clustered Caveolin-1. The resultant focal Ca2+ bursts activate endothelial nitric oxide synthase (eNOS) within the confines of these domains. Importantly, we find that signaling at these domains requires both cell body elongation and sustained flow. Finally, Trpv4 signaling at these domains is necessary and sufficient to suppress inflammatory gene expression, and ectopic activation of Trpv4 channels ameliorates the inflammatory response to stimuli both in vitro and in vivo. Our work reveals a novel polarized mechanosensitive signaling hub that dampens inflammatory gene expression in arterial ECs. To investigate the effect of Trpv4 channel inhibition on endothelial cell phenotype, primary human aortic endothelial cells (HAECs) were seeded on 6-well plates and subjected to uni-directional laminar flow. After 48 h, the flow-conditioned HAEC monolayers were treated with either DMSO or Trpv4-specific antagonist GSK205 (20 uM) for 2 h under flow. We then performed gene expression profiling analysis using data obtained from RNA sequencing of three replicates per treatment condition. Comparative gene expression profiling analysis of RNA-seq data for DMSO-control HAECs and Trpv4-antagonist GSK205-treated HAECs in the presence of laminar flow (20 dynes/cm2).