Department of Cardiology, Zhongda Hospital, School of Medicine, Southeast University, Nanjing, 210009, People's Republic of China.
Pharmaceutical Department, Shandong Provincial Qianfoshan Hospital, Jinan, 250014, People's Republic of China.
Apoptosis. 2020 Jun;25(5-6):321-340. doi: 10.1007/s10495-020-01592-7.
Arterial media calcification is related to mitochondrial dysfunction. Protective mitophagy delays the progression of vascular calcification. We previously reported that lactate accelerates osteoblastic phenotype transition of VSMC through BNIP3-mediated mitophagy suppression. In this study, we investigated the specific links between lactate, mitochondrial homeostasis, and vascular calcification. Ex vivo, alizarin S red and von Kossa staining in addition to measurement of calcium content, RUNX2, and BMP-2 protein levels revealed that lactate accelerated arterial media calcification. We demonstrated that lactate induced mitochondrial fission and apoptosis in aortas, whereas mitophagy was suppressed. In VSMCs, lactate increased NR4A1 expression, leading to activation of DNA-PKcs and p53. Lactate induced Drp1 migration to the mitochondria and enhanced mitochondrial fission through NR4A1. Western blot analysis of LC3-II and p62 and mRFP-GFP-LC3 adenovirus detection showed that NR4A1 knockdown was involved in enhanced autophagy flux. Furthermore, NR4A1 inhibited BNIP3-related mitophagy, which was confirmed by TOMM20 and BNIP3 protein levels, and LC3-II co-localization with TOMM20. The excessive fission and deficient mitophagy damaged mitochondrial structure and impaired respiratory function, determined by mPTP opening rate, mitochondrial membrane potential, mitochondrial morphology under TEM, ATP production, and OCR, which was reversed by NR4A1 silencing. Mechanistically, lactate enhanced fission but halted mitophagy via activation of the NR4A1/DNA-PKcs/p53 pathway, evoking apoptosis, finally accelerating osteoblastic phenotype transition of VSMC and calcium deposition. This study suggests that the NR4A1/DNA-PKcs/p53 pathway is involved in the mechanism by which lactate accelerates vascular calcification, partly through excessive Drp-mediated mitochondrial fission and BNIP3-related mitophagy deficiency.
动脉中层钙化与线粒体功能障碍有关。保护性自噬可延缓血管钙化的进展。我们之前报道过，乳酸通过 BNIP3 介导的自噬抑制加速血管平滑肌细胞（VSMC）的成骨样表型转变。在这项研究中，我们研究了乳酸、线粒体稳态和血管钙化之间的具体联系。茜素红 S 红色和 von Kossa 染色以及钙含量、RUNX2 和 BMP-2 蛋白水平的测定表明，乳酸加速了动脉中层钙化。我们证明，乳酸诱导主动脉中线粒体分裂和凋亡，同时抑制自噬。在 VSMCs 中，乳酸增加 NR4A1 的表达，导致 DNA-PKcs 和 p53 的激活。乳酸诱导 Drp1 向线粒体迁移，并通过 NR4A1 增强线粒体分裂。LC3-II 和 p62 的 Western blot 分析以及 mRFP-GFP-LC3 腺病毒检测显示，NR4A1 敲低参与了增强的自噬流。此外，NR4A1 抑制与 BNIP3 相关的自噬，这通过 TOMM20 和 BNIP3 蛋白水平以及 LC3-II 与 TOMM20 的共定位得到证实。过多的分裂和不足的自噬破坏了线粒体结构，损害了呼吸功能，通过 mPTP 开放率、线粒体膜电位、TEM 下的线粒体形态、ATP 产生和 OCR 来确定，这通过 NR4A1 沉默得到逆转。在机制上，乳酸通过激活 NR4A1/DNA-PKcs/p53 通路增强分裂但停止自噬，引发凋亡，最终加速 VSMC 的成骨样表型转变和钙沉积。这项研究表明，NR4A1/DNA-PKcs/p53 通路参与了乳酸加速血管钙化的机制，部分通过过度的 Drp 介导的线粒体分裂和 BNIP3 相关的自噬不足。
