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.

中文翻译：

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乳酸通过NR4A1调节的线粒体裂变和BNIP3相关的线粒体细胞加速血管钙化。

动脉介质钙化与线粒体功能障碍有关。保护性自噬延迟了血管钙化的进程。我们先前曾报道，乳酸通过BNIP3介导的线粒体抑制作用来加速VSMC的成骨细胞表型转变。在这项研究中，我们调查了乳酸，线粒体稳态与血管钙化之间的具体联系。在体外，茜素红和von Kossa染色除了测量钙含量，RUNX2和BMP-2蛋白水平外，还显示乳酸促进了动脉介质钙化。我们证明，乳酸可引起主动脉线粒体的分裂和凋亡，而线粒体则受到抑制。在VSMC中，乳酸会增加NR4A1表达，从而激活DNA-PKcs和p53。乳酸诱导Drp1迁移至线粒体，并通过NR4A1增强线粒体裂变。对LC3-II和p62的Western印迹分析以及mRFP-GFP-LC3腺病毒检测表明NR4A1敲低与自噬通量的增加有关。此外，NR4A1抑制了BNIP3相关的细胞吞噬作用，这一点已通过TOMM20和BNIP3蛋白水平以及LC3-II与TOMM20的共定位得到了证实。裂变的过度和线粒体的缺乏破坏了线粒体的结构并损害了呼吸功能，这取决于mPTP的打开率，线粒体膜电位，TEM下的线粒体形态，ATP的产生和OCR，而NR4A1沉默则可以逆转。从机制上讲，乳酸可通过激活NR4A1 / DNA-PKcs / p53途径增强裂变，但阻止线粒体吞噬，引起凋亡，最终加速了VSMC的成骨细胞表型转变和钙沉积。这项研究表明，NR4A1 / DNA-PKcs / p53途径参与了乳酸加速血管钙化的机制，部分原因是过度的Drp介导的线粒体裂变和BNIP3相关的线粒体缺乏。