CLOCK disruption aggravates carotid artery stenosis through endoplasmic reticulum stress-induced endothelial-mesenchymal transition.

Carotid artery stenosis is a leading cause of ischemic stroke, but the underlying mechanism remains unclear. We aimed to determine the molecular mechanisms of carotid plaque progression. We analyzed the molecular and morphometric characteristics of carotid plaque samples obtained from 30 patients who underwent carotid endarterectomy. Additionally, we established a mouse model of carotid atherosclerosis by partially ligating the left common carotid arteries of male ClockΔ19/Δ19 (Clk) and wild-type (WT) C57BL/6J mice fed a high-fat diet. Clk and WT primary mouse aortic endothelial cells (pMAECs) were exposed to disturbed flow (DF) or undisturbed flow (UF) with or without treatment with the IRE-1α inhibitor STF-083010 or the PERK inhibitor GSK2606414. In human carotid artery plaques, CLOCK expression was lower in the lipid-rich necrotic core than in transitional regions, especially in the endothelium. Decreased CLOCK mRNA levels were associated with more extensive stenosis, intraplaque hemorrhage, and complex plaque in human carotid plaques. In mice, the ClockΔ19/Δ19 mutation significantly increased neointima formation and neovascularization but decreased collagen content and lumen area in partially ligated carotid arteries. In addition, ClockΔ19/Δ19 mutants exhibited significantly decreased Cdh5 expression and increased expression of endothelial-mesenchymal transition (EndMT) and endoplasmic reticulum (ER) stress markers in mice with partially ligated carotid arteries and pMAECs exposed to DF. Notably, inhibition of the IRE1α-XBP1 axis abrogated the increased EndMT caused by ClockΔ19/Δ19 mutation and DF in pMAECs. In conclusion, the disruption of CLOCK function aggravates EndMT via the IRE1α-XBP1 axis, contributing to carotid artery stenosis.