Shear Stress Modulates Endothelial VCAM‐1 Expression via Endoplasmic Reticulum Stress Response Pathways
Authors: Bailey Keith A , Zakaria Derek L , Simon Scott I , Passerini Anthony G 
Published: November 2023

Introduction

Complications from atherosclerosis remain the leading cause of death and disability in the U.S. This inflammatory disease is characterized by the accumulation of lipid‐rich plaques within the artery wall at predictable locations, which correlate strongly with the local arterial geometry and associated hemodynamic environment, notably low magnitude and reversing fluid shear stress (SS). Mechano‐responsive endothelial cells (EC) moderate vascular inflammation, including cell adhesion molecule (CAM) presentation and subsequent leukocyte recruitment. Endoplasmic reticulum (ER) stress and associated responses have emerged as signaling pathways linking cellular stress to inflammation and cardiovascular risk. Chronic activation of the ER stress response in endothelium has been observed in regions of disturbed flow in the arteries of lesion free swine, and in response to disturbed flow in cultured EC, demonstrating its importance as a possible signature of endothelial dysfunction and atherosusceptibility. The objective of this study was to determine the extent to which ER stress serves as a primary signaling pathway by which SS mediates inflammatory responses in endothelium of functional importance to atherosclerosis.

Methods

To recapitulate flow characteristics of the atherogenic niche in vivo, human aortic EC (HAEC) were exposed to a linear gradient of SS (0 to 16 dynes/cm2) in a microfluidic device and stimulated with the pro‐inflammatory cytokine TNFα in the presence and absence of the ER stress inhibitor 4‐phenylbutyric acid (PBA) for 4 hr. ER morphology was examined by immunofluorescence staining for the ER luminal protein calreticulin. Expression of VCAM‐1, ICAM‐1, and the ER stress sensors eIF2α, XBP1, and ATF6 were examined using automated fluorescence microscopy. HAEC were transfected with shRNA against eIF2α, XBP1, or ATF6 to assess each pathway's contribution to VCAM‐1 expression. To determine if the changes in VCAM‐1 were of functional importance, isolated human monocytes were sheared over HAEC treated as above and adhesion events quantified.

Results

TNFα‐induced VCAM‐1 expression varied significantly with SS magnitude, peaking at ~3‐fold of static expression at 2 dynes/cm2. Peak VCAM‐1 expression under low magnitude SS (LSS) correlated with an increase in the CV for calreticulin pixel intensity, a marker for ER expansion and activity. PBA abrogated the peak in VCAM‐1 expression under LSS, effectively normalizing its expression over the entire gradient, without affecting ICAM‐1 expression. Peak VCAM‐1 expression under LSS was abrogated by knock‐down of eIF2α or XBP1, but not ATF6. At 2 dynes/cm2, there was a 78% decrease in monocytes adhered to the monolayer treated with PBA compared to TNFα alone.

Conclusions

These data support a primary role for ER stress response pathways in the regulation of cytokine‐induced VCAM‐1 expression by SS. This study enhances our understanding of the mechanisms by which SS locally modulates EC inflammatory phenotype. Further elucidation of the pathways involved may have important ramifications for understanding and managing atherosclerosis.

Support or Funding Information

Supported by National Institute of Health grant HL082689 (SIS and AGP)