Abstract
Atherogenic endothelial activation is driven by both the local arterial microenvironment, marked by altered extracellular matrix (ECM) composition and disturbed blood flow, and soluble proinflammatory cues such as oxidized low-density lipoprotein (oxLDL). Fibronectin, a provisional extracellular matrix protein enriched at atheroprone sites, augments these proinflammatory stimuli. Although endoplasmic reticulum (ER) stress is a hallmark of atheroprone regions, its regulation by extracellular matrix and its precise role in endothelial inflammatory activation are not well defined. Here, we show that oxLDL and disturbed flow induce ER stress selectively in endothelial cells adhered to fibronectin, but not in those adhered to basement membrane proteins. This matrix-specific ER stress response requires activation of the integrin family of ECM receptors, as endothelial cells deficient for integrin activation (talin1 L325R mutation) fail to activate ER stress in response to disturbed flow and oxLDL, while direct stimulation of integrins using CHAMP peptides is sufficient to induce ER stress. Silencing fibronectin-binding integrins (α5, αv) using siRNA blocks ER stress induction in vitro, and endothelial-specific deletion of α5 or αv reduces ER stress at atheroprone regions in vivo. Mechanistically, integrin-dependent ER stress is not associated with increased protein synthesis, unfolded protein accumulation, or superoxide production. Scavenging superoxide with TEMPOL does not alleviate ER stress. However, pharmacological inhibition of ER stress using TUDCA suppresses proinflammatory and metabolic gene expression (bulk RNA-seq), without affecting NF-κB activation. Instead, TUDCA prevents activation of the JNK-c-Jun signaling axis, which we show to be essential for proinflammatory gene induction. Blocking this pathway using a JNK inhibitor (SP600125) or dominant-negative c-Jun (TAM67) abrogates inflammatory gene expression following oxLDL or disturbed flow. Together, these findings identify a novel mechanism by which fibronectin-integrin signaling promotes ER stress in response to mechanical and metabolic stressors, amplifying endothelial inflammation through JNK-c-Jun signaling.