Exosomal ALKBH5 Alleviates Vascular Calcification by Suppressing Cell Apoptosis via m6A-Modified GSDME

IF 3.5 4区医学 Q2 CHEMISTRY, MEDICINAL

Drug Development Research Pub Date : 2025-02-14 DOI:10.1002/ddr.70065

Guian Xu, Qingman Li, Lijie Zhu, Tingjie Yang, Yapan Yang, Honghui Yang

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Abstract

This study aimed to explore the function and regulatory mechanism of ALKBH5 in the progression of coronary artery calcification. Human aortic vascular smooth muscle cells (HA-VSMCs) were treated with inorganic phosphate (Pi) and exosomes derived from bone marrow mesenchymal stem cell (BMSC) carrying ALKBH5, a GSDME overexpression vector or si-GSDME. The morphology and size of the exosomes were assessed using nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Calcium deposition was measured using Alizarin red staining and cell pyroptosis was evaluated using Hoechst 33342/PI staining. The association between ALKBH5 and m6A modifications was confirmed by methylated-RNA immunoprecipitation assay (MeRIP) and dot blot assays. The expression levels of ALKBH5 and GSDME were quantified by quantitative real-time polymerase chain reaction (qRT-PCR), and protein levels were quantified by western blot. BMSCs-derived exosomes reduced calcium deposition and cell pyroptosis in Pi-treated HA-VSMCs. Exosomes containing ALKBH5 overexpression inhibited high mobility group box 1 (HMGB1) and cell apoptosis, thereby promoting vascular calcification, whereas ALKBH5 knockdown in exosomes exerted the opposite effect on calcification development. Additionally, ALKBH5 was found to regulate the m6A modification of GSDME. Overexpression of GSDME reversed the effects of ALKBH5 in exosomes on HMGB1 expression and cell apoptosis. Exosomal ALKBH5 mitigated HMGB1 expression and cell pyroptosis by modulating the m6A modification of GSDME, thus influencing the progression of coronary artery calcification.

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外泌体ALKBH5通过m6a修饰的GSDME抑制细胞凋亡减轻血管钙化

本研究旨在探讨ALKBH5在冠状动脉钙化进程中的功能及调控机制。用携带ALKBH5 （GSDME过表达载体）或si-GSDME的骨髓间充质干细胞（BMSC）外泌体和无机磷酸盐（Pi）处理人主动脉血管平滑肌细胞（HA-VSMCs）。利用纳米颗粒跟踪分析（NTA）和透射电子显微镜（TEM）评估外泌体的形态和大小。茜素红染色检测细胞钙沉积，Hoechst 33342/PI染色检测细胞焦亡。通过甲基化rna免疫沉淀法（MeRIP）和斑点印迹法证实了ALKBH5和m6A修饰之间的关联。采用实时荧光定量pcr （quantitative real-time polymerase chain reaction, qRT-PCR）检测ALKBH5和GSDME的表达水平，western blot检测蛋白表达水平。骨髓间充质干细胞衍生的外泌体减少了pi处理的HA-VSMCs的钙沉积和细胞焦亡。高表达ALKBH5的外泌体抑制高迁移率组盒1 （HMGB1）和细胞凋亡，从而促进血管钙化，而低表达ALKBH5的外泌体则对钙化的发展起到相反的作用。此外，ALKBH5被发现调节GSDME的m6A修饰。GSDME过表达逆转了外泌体中ALKBH5对HMGB1表达和细胞凋亡的影响。外泌体ALKBH5通过调节GSDME的m6A修饰减轻HMGB1的表达和细胞焦亡，从而影响冠状动脉钙化的进展。

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来源期刊

Drug Development Research 医学-药学

CiteScore

6.40

自引率

2.60%

发文量

104

审稿时长

6-12 weeks

期刊介绍： Drug Development Research focuses on research topics related to the discovery and development of new therapeutic entities. The journal publishes original research articles on medicinal chemistry, pharmacology, biotechnology and biopharmaceuticals, toxicology, and drug delivery, formulation, and pharmacokinetics. The journal welcomes manuscripts on new compounds and technologies in all areas focused on human therapeutics, as well as global management, health care policy, and regulatory issues involving the drug discovery and development process. In addition to full-length articles, Drug Development Research publishes Brief Reports on important and timely new research findings, as well as in-depth review articles. The journal also features periodic special thematic issues devoted to specific compound classes, new technologies, and broad aspects of drug discovery and development.