Chronic endoplasmic reticulum stress activates unfolded protein response in arterial endothelium in regions of susceptibility to atherosclerosis

Abstract

Rationale: Endothelial function and dysfunction are central to the focal origin and regional development of atherosclerosis; however, an in vivo endothelial phenotypic footprint of susceptibility to atherosclerosis preceding pathological change remains elusive.

Objective: To conduct a comparative multi-site genomics study of arterial endothelial phenotype in atherosusceptible and atheroprotected regions.

Methods and results: Transcript profiles of freshly isolated endothelial cells from 7 discrete arterial regions in normal swine were analyzed to determine the steady state in vivo endothelial phenotypes in regions of varying susceptibilities to atherosclerosis. The most abundant common feature of the endothelium of all atherosusceptible regions was the upregulation of genes associated with endoplasmic reticulum (ER) stress. The unfolded protein response pathway, induced by ER stress, was therefore investigated in detail in endothelium of the atherosusceptible aortic arch and was found to be partially activated. ER transmembrane signal transducers IRE1alpha and ATF6alpha and their downstream effectors, but not PERK, were activated concomitant with a higher transcript expression of protein folding enzymes and chaperones, indicative of ER stress in vivo.

Conclusions: The findings demonstrate the prevalence of chronic endothelial ER stress and activated unfolded protein response in vivo at atherosusceptible arterial sites. We propose that chronic localized biological stress is linked to spatial susceptibility of the endothelium to the initiation of atherosclerosis.

Figure 1

Arterial regions of endothelial isolation. ECs were gently scraped from multiple athero-susceptible and athero-protected regions for transcript and protein analysis. Representative images showing regions of isolation. Scale bar = 1cm.

Figure 2

ER stress and UPR. (A) UPR signaling outline (figure adapted from 30). (B) ER stress marker HSPA5 gene and protein (78 kDa) expression in aortic arch, AA, normalized to descending thoracic aorta, DT, for each paired sample based on their animal origin. Gene (n=6 paired samples) and protein (n=12 paired samples) expression was normalized to GAPDH and β-actin, respectively. Values > 1.0 indicate higher expression in AA. Data represent mean±SEM.*p≤0.05 one-sample, one sided, paired Wilcoxon test.

Figure 3

UPR expression in arterial endothelium. Gene and protein expression of tripartite branches of UPR signaling: (A) ATF6α branch (B) IRE1α branch (C) PERK branch in aortic arch, AA, normalized to descending thoracic aorta, DT, for each paired sample based on their animal origin. Gene (n=6–10 paired samples) and protein (n=10–12 paired samples) expression was normalized to GAPDH and β-actin, respectively. Values > 1.0 indicate higher expression in AA. Data represent mean±SEM. N.S.: non-significant. *p≤0.05 one-sample, one sided, paired Wilcoxon test.

Figure 4

UPR gene expression at renal branch and renal artery. HSPA5, ATF4 and XBP1 transcript in renal branch, RB, normalized to renal artery, RA, for each paired sample based on their animal origin. Gene expression (n= 6 paired samples) was normalized to GAPDH. Values >1.0 indicate higher expression in RB. Data represent mean±SEM. *p≤0.05 one-sample, one sided, paired Wilcoxon test.

Figure 5

Pro-apoptotic and anti-oxidative transcription factor expression in arterial endothelium. (A) Pro-apoptotic transcription factor CHOP gene (n=6 paired) and protein (n=12 paired) expression. (B) Anti-oxidative transcription factor NRF2 gene (n=5 paired) expression in aortic arch, AA, normalized to thoracic aorta, DT. Values > 1.0 indicate higher expression in AA. Data represent mean±SEM. N.S.: non-significant.*p≤0.05 one-sample, one sided, paired Wilcoxon test.