Cold Exposure Attenuates Medial Arterial Calcification via Autophagy

Abstract

Background: Medial arterial calcification (MAC), a systemic vascular disease different from atherosclerosis, is associated with an increased incidence of cardiovascular events. Several studies have demonstrated that ambient temperature is one of the most important factors affecting cardiovascular events. However, there has been limited research on the effect of different ambient temperatures on MAC.

Methods: We first compared the occurrence and development of MAC/senescence in vitamin D (VD)-induced mice treated with room temperature exposure (RT) and cold temperature exposure (CT). Secondly, blood transfusion therapy was conducted to explore whether it affected the process of arterial MAC/senescence in VD-induced mice. Thirdly, we explored whether the exosomes, circulating factor collected from mice under RT and CT exoposure, affect the calcification/aging process of VSMCs. Subsequently, we explored whether autophagy pathway plays a crucial role in calcification/aging of VSMCs. Finally, to sequence miRNAs from exosomes to explore their downstream mechanisms and validate their functions in vivo and in vitro.

Findings: In the present study, we showed that compared with RT or CT in mice decreased VD-induced vascular calcification. To investigate the mechanism involved, we isolated plasma-derived exosomes from mice subjected to CT or RT for 30 days (CT-Exo or RT-Exo, respectively). Compared with RT-Exo, CT-Exo remarkably inhibited the senescence/calcification of vascular smooth muscle cells (VSMCs) and promoted autophagy by activating phosphorylation of AMP-activated protein kinase (p-AMPK) and inhibiting phosphorylation of mammalian target of rapamycin (p-mTOR). At the same time, CT-Exo promoted autophagy in β-glycerophosphate (β-GP)-induced VSMCs. The number of autophagosomes and the expression of autophagy-related proteins ATG5 and LC3B increased, while the expression of p62 decreased. Based on a microRNA chip microarray assay and real-time polymerase chain reaction, miR-320a-3p was highly enriched in CT-Exo as well as thoracic aortic vessels in CT mice. miR-320a-3p downregulation in CT-Exo using AntagomiR-320a-3p inhibited autophagy and blunted its anti-calcification effect on VSMCs. Moreover, we identified that programmed cell death 4 (PDCD4) is a target of miR-320a-3p, and silencing PDCD4 increased autophagy and decreased calcification in VSMCs. Treatment with CT-Exo alleviated MAC and ageing in VD-treated mice, while these effects were partially reversed by GW4869. Furthermore, the anti-arterial calcification effects of CT-Exo were largely abolished by AntagomiR-320a-3p in VD-induced mice.

Interpretation: Our study highlighted that prolonged cold may be a good way to reduce the incidence of MAC. Specifically, miR-320a-3p from CT-Exo could attenuate MAC via the AMPK/mTOR autophagy pathway.

Funding: This study was supported by the National Natural Science Foundation of China (No. 82071593, 81974223, 81770833 and 82100944), Key R&D Plan Hunan Province (2020SK2078) and Natural Science Foundation of Hunan Province (No. 2021JJ30036 and 2021JJ40842).

Declaration of Interest: The authors declare that they have no competing interests.

Ethical Approval: All experiments were reviewed and approved by the Ethics Committee of the Second Xiang-Ya Hospital, Central South University. All the procedures conformed to the Guide for the Care and Use of Laboratory Animals, NIH publication (8th edition, 2011). All the animal protocols were formally approved by the Ethics Committee of the Second Xiang-Ya Hospital, Central South University (2022708).

Keywords: Cold exposure, Arterial calcification, Plasma derived exosomes, Autophagy, Senescence, miR-320a-3p, PDCD4