GRP78 Upregulation by Atheroprone Shear Stress Via p38-, α2β1-Dependent Mechanism in Endothelial Cells

Abstract

Objective-The initiation of atherosclerosis is in part dependent on the hemodynamic shear stress environment promoting a proinflammatory phenotype of the endothelium. Previous studies demonstrated increased expression of ER stress protein and unfolded protein response (UPR) regulator, GRP78, within all vascular cells in atherosclerotic lesions and its regulation in the endothelium by several atherosclerotic stressors; however, regulation of GRP78 by shear stress directly has not been established. Method and Results-Using an in vitro model to simulate human arterial shear stress waveforms, atheroprone or atheroprotective flow was applied to human endothelial cells. GRP78 was found to be significantly upregulated (3-fold) in a sustained manner under atheroprone, but not atheroprotective flow up to 24 hours. This response was dependent on both sustained activation of p38, as well integrin ␣2␤1. Increased GRP78 correlated with the activation of the ER stress sensing element (ERSE1) promoter by atheroprone flow as a marker of the UPR. Shear stress regulated GRP78 through increased protein stability when compared to other flow regulated proteins, such as connexin-43 and vascular cell adhesion molecule (VCAM)-1. Increased endothelial expression of GRP78 was also observed in atheroprone versus atheroprotective regions of C57BL6 mice. Conclusions-This study supports a role of the hemodynamic environment in preferentially inducing GRP78 and the UPR in atheroprone regions, before lesion development, and suggests a potential atheroprotective (ie, prosurvival), compensatory effect in response to ER stress within atherosclerotic lesions. Key Words: endothelial Ⅲ GRP78 Ⅲ shear stress Ⅲ atherosclerosis Ⅲ unfolded protein response A therosclerosis is a focal inflammatory disease that develops preferentially in areas of disturbed flow, where variations in shear stress have been shown to alter the phenotypes of endothelial cells toward either an atheroprone or atheroprotective state in vitro and in vivo. 1,2 Therefore, hemodynamic-induced shear stress provides a major mechanical signal, which causes the overlying endothelium to become at risk for the promotion of atherosclerosis.Of many proteins of interest, the chaperone protein, glucose regulated protein 78 (GRP78), a common marker for endoplasmic reticulum (ER) stress, is preferentially expressed in advanced atherosclerotic lesions 3 and on the fibrous cap surface in ApoE-KO mice. 4 Further, cell-surface associated GRP78 has been speculated to serve a protective role in atheroprone environments by inhibiting tissue factor through direct binding to the endothelium overlying the plaque. 5 Hyperhomocysteinemia is associated with increased risk of cardiovascular disease possibly by limiting the antioxidant activity and causing ER stress, leading to the activation of GRP78. 4 ER stress is further linked to oxidative stress through peroxynitrite-induced GRP78 expression. 3 ER stress is present at every stage of atherosclerosis, even preceding free cholesterol accumulation. 6 This su...

A reporter for tracking the UPR in vivo reveals patterns of temporal and cellular stress during atherosclerotic progression

“…3B ). Previous reports have implicated endothelial ER stress as a relatively early event during atherogenesis (21)(22)(23). Quantifi cation of endothelial ERAI fl uorescence showed a prominent increase at 8 weeks ( Fig.…”

Endoplasmic Reticulum Stress As a Therapeutic Target in Cardiovascular Disease

“…79 Indeed, ER stress is markedly increased in endothelial cells subjected to atherosclerosisprone shear stress. 80 Consistently, transcript profiles revealed that the most abundant feature of the endothelium of all atherosusceptible regions is the upregulation of genes associated with ER stress. 81 Dong et al demonstrated that the dysfunction of AMPK significantly increases the level of ER stress and suppresses sarcoplasmic reticulum calciumtransporting ATPase (SERCA) activity in endothelial cells.…”

Molecular Biology of Atherosclerosis

“…Endothelial ER stress was found in areas of disturbed flow in both swine (329) and mice (506). BiP (immunoglobulin heavy chain-binding protein, also known as GRP78), the best studied ER chaperone and mediator of ER stress, could be induced by exposing cultured endothelial cells to disturbed flow with upregulation dependent on MAPK p38 and integrin ␣ 2 ␤ 1 signaling (506).…”