Impaired UPR^ER is associated with Chronic Endoplasmic Reticulum Stress in Aging Endothelial Cells

Abstract

Purpose : Age-related endothelial dysfunction is associated with multiple eye diseases. One potential mechanism is the disturbance of the endoplasmic reticulum environment (ER stress). In healthy cells, ER stress is constantly sensed and mitigated by a sophisticated adaptive mechanism namely the unfolded protein response of the ER (UPR^ER). In this study, we characterize the status of ER homeostasis and the UPR^ER in aging endothelial cells and elucidate a potential mechanism underlying vascular aging.

Methods : Brain microvascular endothelial cells (BMECs) were isolated from mice at ages of 2-24 months. To induce ER stress, BMECs were treated with either 0.5 µM of thapsigargin (TG) or 0.5 µg/ml of tunicamycin (TM) for 6 hours. Expression of UPR^ER genes was assessed by quantitative RT-PCR (qRT-PCR).

Results : Cell morphology and cell growth appeared to be similar among BMECs from different-age groups. In unstimulated cells, the basal levels of UPR^ER, indicative by the expression of X-box binding protein 1 (XBP1), activating transcription factor 4 (ATF4), activating transcription factor 6 (ATF6), binding-immunoglobulin protein (BiP, also known as GRP78), and protein disulfide isomerase (PDI), were significantly higher in aging BMECs (20-24 months) compared to the young group (2-4 months). In stress conditions, TG and TM treatment induced a robust increase of the UPR^ER in young BMECs, and this response was significantly decreased with age. Specifically, aging significantly reduced the expression of XBP1, ATF4, ATF6, and C/EBP-homologous protein (CHOP). Likewise, the expression levels of ER chaperons and foldases, such as BiP, DNAJC3/p58IPK, and PDI were markedly upregulated by TM and TG treatment in young BMECs, but to a significantly less extent in aging cells. Interestingly, TM, but not TG treatment, significantly upregulated NADPH oxidase 4 (NOX4) expression in young BMECs and the response was blunted in the aging group.

Conclusions : Our results suggest that there is a sustained ER stress in aging endothelial cells that may contribute to the increased endothelial dysfunction, inflammation, and apoptosis in aging vasculature. Moreover, the ability of activation of UPR^ER is drastically impaired in endothelial cells with age. The dysfunction of the adaptive response to stress conditions may lead to the accumulation of unfolded/misfolded proteins resulting in chronic ER stress in aging endothelial cells.

This is a 2021 ARVO Annual Meeting abstract.