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Lactate accelerates vascular calcification through NR4A1-regulated mitochondrial fission and BNIP3-related mitophagy

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Abstract

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.

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Abbreviations

VSMC:

Vascular smooth muscle cell

BNIP3:

BCL2/adenovirus E1B 19 kDa protein-interacting protein 3

RUNX2:

Runt-related transcription factor 2

BMP-2:

Bone morphogenetic protein 2

NR4A1:

Nuclear receptor subfamily 4 group A member 1

DNA-PKcs:

DNA-dependent protein kinase, catalytic subunit

Drp1:

Dynamin-related protein 1

LC3-II:

Microtubule-associated protein 1 light chain 3B

p62:

SQSTM1

TOMM20:

Translocase of outer mitochondrial membrane 20

mPTP:

Mitochondrial permeability transition pore

TEM:

Transmission electron microscope

ATP:

Adenosine triphosphate

OCR:

Oxygen consumption rate

References

  1. Dunlay SM, Givertz MM, Aguilar D et al (2019) Type 2 diabetes mellitus and heart failure, a scientific statement from the American Heart Association and Heart Failure Society of America. J Cardiac Fail 50(19):1914–1931

    Google Scholar 

  2. Weng L, Zhang F, Wang R, Ma W, Song Y (2019) A review on protective role of genistein against oxidative stress in diabetes and related complications. Chemico-Biol Interact. https://doi.org/10.1016/j.cbi.2019.05.031

    Article  Google Scholar 

  3. Tolle M, Reshetnik A, Schuchardt M, Hohne M, van der Giet M (2015) Arteriosclerosis and vascular calcification: causes, clinical assessment and therapy. Eur J Clin Invest 45:976–985

    Article  PubMed  Google Scholar 

  4. Viegas C, Araujo N, Marreiros C, Simes D (2019) The interplay between mineral metabolism, vascular calcification and inflammation in chronic kidney disease (CKD): challenging old concepts with new facts. Aging 11:4274–4299

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Zhu Y, Ma WQ, Han XQ, Wang Y, Wang X, Liu NF (2018) Advanced glycation end products accelerate calcification in VSMCs through HIF-1alpha/PDK4 activation and suppress glucose metabolism. Sci Rep 8:13730

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  6. Koike S, Yano S, Tanaka S, Sheikh AM, Nagai A, Sugimoto T (2016) Advanced glycation end-products induce apoptosis of vascular smooth muscle cells: a mechanism for vascular calcification. Int J Mol Sci 17:1567

    Article  PubMed Central  CAS  Google Scholar 

  7. Wang Y, Ma WQ, Zhu Y, Han XQ, Liu N (2018) Exosomes derived from mesenchymal stromal cells pretreated with advanced glycation end product-bovine serum albumin inhibit calcification of vascular smooth muscle cells. Front Endocrinol 9:524

    Article  Google Scholar 

  8. Lee KM, Lee EO, Lee YR et al (2017) APE1/ref-1 inhibits phosphate-induced calcification and osteoblastic phenotype changes in vascular smooth muscle cells. Int J Mol Sci 18(10):2053

    Article  PubMed Central  CAS  Google Scholar 

  9. Li X, Fang F, Gao Y et al (2019) ROS induced by killerred targeting mitochondria (mtkr) enhances apoptosis caused by radiation via cyt c/caspase-3 pathway. Oxid Med Cell Longev 2019:4528616

    PubMed  PubMed Central  Google Scholar 

  10. Zhang G, Yang W, Jiang F et al (2019) PERK regulates Nrf2/ARE antioxidant pathway against dibutyl phthalate-induced mitochondrial damage and apoptosis dependent of reactive oxygen species in mouse spermatocyte-derived cells. Toxicol Lett 308:24–33

    Article  CAS  PubMed  Google Scholar 

  11. Rovira-Llopis S, Banuls C, Diaz-Morales N, Hernandez-Mijares A, Rocha M, Victor VM (2017) Mitochondrial dynamics in type 2 diabetes: pathophysiological implications. Redox Biol 11:637–645

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Huang Q, Zhan L, Cao H et al (2016) Increased mitochondrial fission promotes autophagy and hepatocellular carcinoma cell survival through the ROS-modulated coordinated regulation of the NFKB and TP53 pathways. Autophagy 12:999–1014

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. **an H, Liou YC (2019) Loss of MIEF1/MiD51 confers susceptibility to BAX-mediated cell death and PINK1-PRKN-dependent mitophagy. Autophagy 20:1–19

    CAS  Google Scholar 

  14. Zhou H, Hu S, ** Q et al (2017) Mff-dependent mitochondrial fission contributes to the pathogenesis of cardiac microvasculature ischemia/reperfusion injury via induction of mROS-mediated cardiolipin oxidation and HK2/VDAC1 disassociation-involved mPTP opening. J Am Heart Assoc 6:e005328

    PubMed  PubMed Central  Google Scholar 

  15. Zhou H, Shi C, Hu S, Zhu H, Ren J, Chen Y (2018) BI1 is associated with microvascular protection in cardiac ischemia reperfusion injury via repressing Syk-Nox2-Drp1-mitochondrial fission pathways. Angiogenesis 21:599–615

    Article  CAS  PubMed  Google Scholar 

  16. Rogers MA, Maldonado N, Hutcheson JD et al (2017) Dynamin-related protein 1 inhibition attenuates cardiovascular calcification in the presence of oxidative stress. Circ Res 121:220–233

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Chan CM, Huang DY, Sekar P, Hsu SH, Lin WW (2019) Reactive oxygen species-dependent mitochondrial dynamics and autophagy confer protective effects in retinal pigment epithelial cells against sodium iodate-induced cell death. J Biomed Sci 26:40

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  18. Ma WQ, Sun XJ, Wang Y, Zhu Y, Han XQ, Liu NF (2019) Restoring mitochondrial biogenesis with metformin attenuates beta-GP-induced phenotypic transformation of VSMCs into an osteogenic phenotype via inhibition of PDK4/oxidative stress-mediated apoptosis. Mol Cell Endocrinol 479:39–53

    Article  CAS  PubMed  Google Scholar 

  19. Qi J, Wang F, Yang P et al (2018) Mitochondrial fission is required for angiotensin ii-induced cardiomyocyte apoptosis mediated by a Sirt1-p53 signaling pathway. Front Pharmacol 9:176

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  20. Li H, Feng J, Zhang Y et al (2019) Mst1 deletion attenuates renal ischaemia-reperfusion injury: the role of microtubule cytoskeleton dynamics, mitochondrial fission and the GSK3beta-p53 signalling pathway. Redox Biol 20:261–274

    Article  CAS  PubMed  Google Scholar 

  21. Wang EY, Gang H, Aviv Y, Dhingra R, Margulets V, Kirshenbaum LA (2013) p53 mediates autophagy and cell death by a mechanism contingent on Bnip3. Hypertension 62:70–77

    Article  CAS  PubMed  Google Scholar 

  22. Zhou H, Du W, Li Y et al (2018) Effects of melatonin on fatty liver disease: the role of NR4A1/DNA-PKcs/p53 pathway, mitochondrial fission, and mitophagy. J Pineal Res 64:e12450

    Article  CAS  Google Scholar 

  23. **ng M, Oksenych V (2019) Genetic interaction between DNA repair factors PAXX, XLF, XRCC4 and DNA-PKcs in human cells. FEBS Open Bio 8:426–434

    Google Scholar 

  24. Yan Q, Zhu H, Lan L, Yi J, Yang J (2017) Cleavage of Ku80 by caspase-2 promotes non-homologous end joining-mediated DNA repair. DNA Repair 60:18–28

    Article  CAS  PubMed  Google Scholar 

  25. Koike M, Yutoku Y, Koike A (2011) Accumulation of p21 proteins at DNA damage sites independent of p53 and core NHEJ factors following irradiation. Biochem Biophys Res Commun 412:39–43

    Article  CAS  PubMed  Google Scholar 

  26. Li P, Bai Y, Zhao X et al (2018) NR4A1 contributes to high-fat associated endothelial dysfunction by promoting CaMKII-Parkin-mitophagy pathways. Cell Stress Chaperones 23:749–761

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Hanna RN, Shaked I, Hubbeling HG et al (2012) NR4A1 (Nur77) deletion polarizes macrophages toward an inflammatory phenotype and increases atherosclerosis. Circ Res 110:416–427

    Article  CAS  PubMed  Google Scholar 

  28. Zhao BX, Chen HZ, Du XD et al (2011) Orphan receptor TR3 enhances p53 transactivation and represses DNA double-strand break repair in hepatoma cells under ionizing radiation. Mol Endocrinol 25:1337–1350

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Yu Y, Cai Z, Cui M et al (2015) The orphan nuclear receptor Nur77 inhibits low shear stress-induced carotid artery remodeling in mice. Int J Mol Med 36:1547–1555

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Zhou H, Wang J, Zhu P et al (2018) NR4A1 aggravates the cardiac microvascular ischemia reperfusion injury through suppressing FUNDC1-mediated mitophagy and promoting Mff-required mitochondrial fission by CK2alpha. Basic Res Cardiol 113:23

    Article  PubMed  CAS  Google Scholar 

  31. Vallee A, Vallee JN, Lecarpentier Y (2019) Metabolic reprogramming in atherosclerosis: Opposed interplay between the canonical WNT/beta-catenin pathway and PPARgamma. J Mol Cell Cardiol 133:36–46

    Article  CAS  PubMed  Google Scholar 

  32. Yang Q, Xu J, Ma Q et al (2018) PRKAA1/AMPKalpha1-driven glycolysis in endothelial cells exposed to disturbed flow protects against atherosclerosis. Nat Commun 9:4667

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  33. Crawford SO, Hoogeveen RC, Brancati FL et al (2010) Association of blood lactate with type 2 diabetes: the atherosclerosis risk in communities carotid MRI study. Int J Epidemiol 39:1647–1655

    Article  PubMed  PubMed Central  Google Scholar 

  34. Shantha GP, Wasserman B, Astor BC et al (2013) Association of blood lactate with carotid atherosclerosis: the atherosclerosis risk in communities (ARIC) carotid MRI study. Atherosclerosis 228:249–255

    Article  CAS  PubMed  Google Scholar 

  35. Juraschek SP, Bower JK, Selvin E et al (2015) Plasma lactate and incident hypertension in the atherosclerosis risk in communities study. Am J Hypertens 28:216–224

    Article  CAS  PubMed  Google Scholar 

  36. Petsophonsakul P, Furmanik M, Forsythe R et al (2019) Role of vascular smooth muscle cell phenotypic switching and calcification in aortic aneurysm formation. Arterioscler Thromb Vasc Biol 39:1351–1368

    Article  CAS  PubMed  Google Scholar 

  37. Yang L, Gao L, Nickel T et al (2017) Lactate promotes synthetic phenotype in vascular smooth muscle cells. Circ Res 121:1251–1262

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Zhu Y, Ji JJ, Yang R et al (2019) Lactate accelerates calcification in VSMCs through suppression of BNIP3-mediated mitophagy. Cell Signal 58:53–64

    Article  CAS  PubMed  Google Scholar 

  39. Akiyoshi T, Ota H, Iijima K et al (2016) A novel organ culture model of aorta for vascular calcification. Atherosclerosis 244:51–58

    Article  CAS  PubMed  Google Scholar 

  40. Makela J, Tselykh TV, Kukkonen JP, Eriksson O, Korhonen LT, Lindholm D (2016) Peroxisome proliferator-activated receptor-gamma (PPARgamma) agonist is neuroprotective and stimulates PGC-1alpha expression and CREB phosphorylation in human dopaminergic neurons. Neuropharmacology 102:266–275

    Article  CAS  PubMed  Google Scholar 

  41. Andrade MC, Carmo LS, Farias-Silva E, Liberman M (2017) Msx2 is required for vascular smooth muscle cells osteoblastic differentiation but not calcification in insulin-resistant ob/ob mice. Atherosclerosis 265:14–21

    Article  CAS  PubMed  Google Scholar 

  42. Lewis TL Jr, Kwon SK, Lee A, Shaw R, Polleux F (2018) MFF-dependent mitochondrial fission regulates presynaptic release and axon branching by limiting axonal mitochondria size. Nat Commun 9:5008

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  43. Macchioni L, Petricciuolo M, Davidescu M et al (2018) Palmitate lipotoxicity in enteric glial cells: Lipid remodeling and mitochondrial ROS are responsible for cyt c release outside mitochondria. Biochim Biophys Acta 1863:895–908

    Article  CAS  Google Scholar 

  44. Bessueille L, Magne D (2015) Inflammation: a culprit for vascular calcification in atherosclerosis and diabetes. Cell Mol Life Sci CMLS 72:2475–2489

    Article  CAS  PubMed  Google Scholar 

  45. Ong KL, McClelland RL, Rye KA et al (2014) The relationship between insulin resistance and vascular calcification in coronary arteries, and the thoracic and abdominal aorta: the multi-ethnic study of atherosclerosis. Atherosclerosis 236:257–262

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Beloqui O, Moreno MU, San Jose G et al (2017) Increased phagocytic NADPH oxidase activity associates with coronary artery calcification in asymptomatic men. Free Radical Res 51:389–396

    Article  CAS  Google Scholar 

  47. **a W, Li Y, Wu M et al (2019) Inhibition of mitochondrial activity ameliorates atherosclerosis in ApoE(-/-) mice via suppressing vascular smooth cell activation and macrophage foam cell formation. J Cell Biochem 120:17767–17778

    Article  CAS  PubMed  Google Scholar 

  48. Durham AL, Speer MY, Scatena M, Giachelli CM, Shanahan CM (2018) Role of smooth muscle cells in vascular calcification: implications in atherosclerosis and arterial stiffness. Cardiovasc Res 114:590–600

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Feng H, Wang JY, Yu B et al (2019) Peroxisome proliferator-activated receptor-gamma coactivator-1alpha inhibits vascular calcification through sirtuin 3-mediated reduction of mitochondrial oxidative stress. Antioxid Redox Signal 31:75–91

    Article  CAS  PubMed  Google Scholar 

  50. Shi Y, Wang S, Peng H et al (2019) Fibroblast growth factor 21 attenuates vascular calcification by alleviating endoplasmic reticulum stress mediated apoptosis in rats. Int J Biol Sci 15:138–147

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Li MY, Ding JQ, Tang Q et al (2019) SIRT1 activation by SRT1720 attenuates bone cancer pain via preventing Drp1-mediated mitochondrial fission. Biochim Biophys Acta 1865:587–598

    Article  CAS  Google Scholar 

  52. Zhang T, Wu P, Budbazar E et al (2019) Mitophagy reduces oxidative stress via Keap1 (Kelch-like epichlorohydrin-associated protein 1)/Nrf2 (nuclear factor-E2-related Factor 2)/PHB2 (prohibitin 2) pathway after subarachnoid hemorrhage in rats. Stroke 50:978–988

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Santulli G (2015) microRNAs distinctively regulate vascular smooth muscle and endothelial cells: functional implications in angiogenesis, atherosclerosis, and in-stent restenosis. Adv Exp Med Biol 887:53–77

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Kim DY, Jung SY, Kim YJ et al (2018) Hypoxia-dependent mitochondrial fission regulates endothelial progenitor cell migration, invasion, and tube formation. Korean J Physiol Pharmacol 22:203–213

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. ** H, Zhu Y, Li Y et al (2019) BDNF-mediated mitophagy alleviates high-glucose-induced brain microvascular endothelial cell injury. Apoptosis 24:511–528

    Article  CAS  PubMed  Google Scholar 

  56. Meyer JN, Leuthner TC, Luz AL (2017) Mitochondrial fusion, fission, and mitochondrial toxicity. Toxicology 391:42–53

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Li N, Wang H, Jiang C, Zhang M (2018) Renal ischemia/reperfusion-induced mitophagy protects against renal dysfunction via Drp1-dependent-pathway. Exp Cell Res 369:27–33

    Article  CAS  PubMed  Google Scholar 

  58. Cho HM, Ryu JR, Jo Y et al (2019) Drp1-Zip1 interaction regulates mitochondrial quality surveillance system. Mol Cell 73:364–376.e368

    Article  CAS  PubMed  Google Scholar 

  59. Yau WW, Singh BK, Lesmana R et al (2019) Thyroid hormone (T3) stimulates brown adipose tissue activation via mitochondrial biogenesis and MTOR-mediated mitophagy. Autophagy 15:131–150

    Article  CAS  PubMed  Google Scholar 

  60. O'Sullivan TE, Johnson LR, Kang HH, Sun JC (2015) BNIP3- and BNIP3L-mediated mitophagy promotes the generation of natural killer cell memory. Immunity 43:331–342

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Han X, Zhu J, Zhang X et al (2018) Plin4-dependent lipid droplets hamper neuronal mitophagy in the MPTP/p-induced mouse model of Parkinson's disease. Front Neurosci 12:397

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Liu N, Luo J, Kuang D et al (2019) Lactate inhibits ATP6V0d2 expression in tumor-associated macrophages to promote HIF-2alpha-mediated tumor progression. J Clin Investig 129:631–646

    Article  PubMed  Google Scholar 

  63. Woods DS, Sears CR, Turchi JJ (2015) Recognition of DNA termini by the C-terminal region of the Ku80 and the DNA-dependent protein kinase catalytic subunit. PLoS ONE 10:e0127321

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  64. Koenis DS, Medzikovic L, van Loenen PB et al (2018) Nuclear receptor Nur77 limits the macrophage inflammatory response through transcriptional reprogramming of mitochondrial metabolism. Cell Rep 24:2127–2140.e2127

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Kim JH, Bae KH, Byun JK et al (2017) Lactate dehydrogenase-A is indispensable for vascular smooth muscle cell proliferation and migration. Biochem Biophys Res Commun 492:41–47

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This work was supported by the National Nature Science Foundation of China (No. 81770451), the Fundamental Research Funds for the Central Universities and Postgraduate Research & Practice Innovation Program of Jiangsu Province (No. KYCX19_0117), and the Jiangsu Provincial Health and Wellness Committee Research Project (No. H2018001).

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Authors and Affiliations

  1. Department of Cardiology, Zhongda Hospital, School of Medicine, Southeast University, Nan**g, 210009, People’s Republic of China

    Yi Zhu, **-Qiong Han, Xue-Jiao Sun, Wen-Qi Ma & Nai-Feng Liu

  2. Pharmaceutical Department, Shandong Provincial Qianfoshan Hospital, **an, 250014, People’s Republic of China

    Rui Yang

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  1. Yi Zhu
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  2. **-Qiong Han
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  3. Xue-Jiao Sun
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YZ was involved in the experimental design, biochemistry detection, data analysis, and writing of the manuscript. WQM, XQH, SXJ, and YR contributed to the animal model construction and cell culture. NFL was the supervisor and provided expertise in experimental design.

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Correspondence to Nai-Feng Liu.

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Zhu, Y., Han, XQ., Sun, XJ. et al. Lactate accelerates vascular calcification through NR4A1-regulated mitochondrial fission and BNIP3-related mitophagy. Apoptosis 25, 321–340 (2020). https://doi.org/10.1007/s10495-020-01592-7

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