Phadwal Kanchan, Koo Eve, Jones Ross A, Forsythe Rachael O, Tang Keyi, Tang Qiyu, Corcoran Brendan M, Caporali Andrea, MacRae Vicky E
The Roslin Institute and R(D)SVS, The University of Edinburgh, Edinburgh, UK.
Edinburgh Medical School, The University of Edinburgh, Edinburgh, UK.
J Cell Physiol. 2022 Nov;237(11):4303-4316. doi: 10.1002/jcp.30887. Epub 2022 Sep 27.
Vascular calcification is associated with aging, type 2 diabetes, and atherosclerosis, and increases the risk of cardiovascular morbidity and mortality. It is an active, highly regulated process that resembles physiological bone formation. It has previously been established that pharmacological doses of metformin alleviate arterial calcification through adenosine monophosphate-activated protein kinase (AMPK)-activated autophagy, however the specific pathway remains elusive. In the present study we hypothesized that metformin protects against arterial calcification through the direct autophagic degradation of runt-related transcription factor 2 (Runx2). Calcification was blunted in vascular smooth muscle cells (VSMCs) by metformin in a dose-dependent manner (0.5-1.5 mM) compared to control cells (p < 0.01). VSMCs cultured under high-phosphate (Pi) conditions in the presence of metformin (1 mM) showed a significant increase in LC3 puncta following bafilomycin-A1 (Baf-A; 5 nM) treatment compared to control cells (p < 0.001). Furthermore, reduced expression of Runx2 was observed in the nuclei of metformin-treated calcifying VSMCs (p < 0.0001). Evaluation of the functional role of autophagy through Atg3 knockdown in VSMCs showed aggravated Pi-induced calcification (p < 0.0001), failure to induce autophagy (punctate LC3) (p < 0.001) and increased nuclear Runx2 expression (p < 0.0001) in VSMCs cultured under high Pi conditions in the presence of metformin (1 mM). Mechanistic studies employing three-way coimmunoprecipitation with Runx2, p62, and LC3 revealed that p62 binds to both LC3 and Runx2 upon metformin treatment in VSMCs. Furthermore, immunoblotting with LC3 revealed that Runx2 specifically binds with p62 and LC3-II in metformin-treated calcified VSMCs. Lastly, we investigated the importance of the autophagy pathway in vascular calcification in a clinical setting. Ex vivo clinical analyses of calcified diabetic lower limb artery tissues highlighted a negative association between Runx2 and LC3 in the vascular calcification process. These studies suggest that exploitation of metformin and its analogues may represent a novel therapeutic strategy for clinical intervention through the induction of AMPK/Autophagy Related 3 (Atg3)-dependent autophagy and the subsequent p62-mediated autophagic degradation of Runx2.
血管钙化与衰老、2型糖尿病和动脉粥样硬化相关，并增加心血管疾病的发病率和死亡率。它是一个活跃的、高度受调控的过程，类似于生理性骨形成。此前已经证实，药理剂量的二甲双胍通过腺苷单磷酸激活的蛋白激酶（AMPK）激活的自噬减轻动脉钙化，然而具体途径仍不清楚。在本研究中，我们假设二甲双胍通过直接自噬降解 runt 相关转录因子 2（Runx2）来预防动脉钙化。与对照细胞相比，二甲双胍以剂量依赖性方式（0.5 - 1.5 mM）抑制血管平滑肌细胞（VSMC）的钙化（p < 0.01）。在高磷（Pi）条件下培养且存在二甲双胍（1 mM）的 VSMC，用巴佛洛霉素 - A1（Baf - A；5 nM）处理后，与对照细胞相比，LC3 斑点显著增加（p < 0.001）。此外，在经二甲双胍处理的钙化 VSMC 的细胞核中观察到 Runx2 表达降低（p < 0.0001）。通过在 VSMC 中敲低 Atg3 来评估自噬的功能作用，结果显示在高 Pi 条件下且存在二甲双胍（1 mM）培养的 VSMC 中，Pi 诱导的钙化加重（p < 0.0001），未能诱导自噬（点状 LC3）（p < 0.001），且细胞核 Runx2 表达增加（p < 0.0001）。采用 Runx2、p62 和 LC3 进行三向共免疫沉淀的机制研究表明，在 VSMC 中用二甲双胍处理后，p62 与 LC3 和 Runx2 均结合。此外，用 LC3 进行免疫印迹显示，在经二甲双胍处理的钙化 VSMC 中，Runx2 与 p62 和 LC3 - II 特异性结合。最后，我们在临床环境中研究了自噬途径在血管钙化中的重要性。对钙化的糖尿病下肢动脉组织进行的体外临床分析突出了 Runx2 和 LC3 在血管钙化过程中的负相关关系。这些研究表明，开发二甲双胍及其类似物可能代表一种新的治疗策略，通过诱导 AMPK/自噬相关 3（Atg3）依赖性自噬以及随后 p62 介导的 Runx2 自噬降解来进行临床干预。
