Biomolecules & Therapeutics

The Korean Society of Applied Pharmacology (한국응용약물학회)
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Niclosamide Inhibits Aortic Valve Interstitial Cell Calcification by Interfering with the GSK-3β/β-Catenin Signaling Pathway

  • Radhika Adhikari (College of Pharmacy and Natural Medicine Research Institute, Mokpo National University) ;
  • Saugat Shiwakoti (College of Pharmacy and Natural Medicine Research Institute, Mokpo National University) ;
  • Eunmin Kim (Division of Cardiology, Department of Internal Medicine, Seoul St. Mary's Hospital, The Catholic University of Korea) ;
  • Ik Jun Choi (Division of Cardiology, Department of Internal Medicine, Seoul St. Mary's Hospital, The Catholic University of Korea) ;
  • Sin-Hee Park (Division of Cardiology, Department of Internal Medicine, Seoul St. Mary's Hospital, The Catholic University of Korea) ;
  • Ju-Young Ko (College of Pharmacy and Natural Medicine Research Institute, Mokpo National University) ;
  • Kiyuk Chang (Division of Cardiology, Department of Internal Medicine, Seoul St. Mary's Hospital, The Catholic University of Korea) ;
  • Min-Ho Oak (College of Pharmacy and Natural Medicine Research Institute, Mokpo National University)
  • Received : 2022.11.11
  • Accepted : 2023.05.23
  • Published : 2023.09.01
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Abstract

The most common heart valve disorder is calcific aortic valve stenosis (CAVS), which is characterized by a narrowing of the aortic valve. Treatment with the drug molecule, in addition to surgical and transcatheter valve replacement, is the primary focus of researchers in this field. The purpose of this study is to determine whether niclosamide can reduce calcification in aortic valve interstitial cells (VICs). To induce calcification, cells were treated with a pro-calcifying medium (PCM). Different concentrations of niclosamide were added to the PCM-treated cells, and the level of calcification, mRNA, and protein expression of calcification markers was measured. Niclosamide inhibited aortic valve calcification as observed from reduced alizarin red s staining in niclosamide treated VICs and also decreased the mRNA and protein expressions of calcification-specific markers: runt-related transcription factor 2 and osteopontin. Niclosamide also reduced the formation of reactive oxygen species, NADPH oxidase activity and the expression of Nox2 and p22phox. Furthermore, in calcified VICs, niclosamide inhibited the expression of β-catenin and phosphorylated glycogen synthase kinase (GSK-3β), as well as the phosphorylation of AKT and ERK. Taken together, our findings suggest that niclosamide may alleviate PCM-induced calcification, at least in part, by targeting oxidative stress mediated GSK-3β/β-catenin signaling pathway via inhibiting activation of AKT and ERK, and may be a potential treatment for CAVS.

Keywords

Acknowledgement

This work was supported in part by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. 2020R1A4A3079570) and Technology Innovation Program (20014873) funded by the Ministry of Trade, Industry & Energy (MOTIE, Korea). We thank to Mr. Tae Joon Hong for providing smooth supply of pig hearts.

References

  1. Adhikari, R., Jung, J., Shiwakoti, S., Park, E.-Y., Kim, H.-J., Ko, J.-Y., You, J., Lee, M. and Oak, M.-H. (2023) Capsaicin inhibits aortic valvular interstitial cell calcification via the redox-sensitive NFκB/AKT/ERK1/2 pathway. Biochem. Pharmacol. 212, 115530.
  2. Adhikari, R., Shiwakoti, S., Ko, J.-Y., Dhakal, B., Park, S.-H., Choi, I. J., Kim, H. J. and Oak, M.-H. (2022) Oxidative stress in calcific aortic valve stenosis: protective role of natural antioxidants. Antioxidants 11, 1169.
  3. Agidigbi, T. S. and Kim, C. (2019) Reactive oxygen species in osteoclast differentiation and possible pharmaceutical targets of ROS-mediated osteoclast diseases. Int. J. Mol. Sci. 20, 3576.
  4. Andrews, P., Thyssen, J. and Lorke, D. (1982) The biology and toxicology of molluscicides, bayluscide. Pharmacol. Ther. 19, 245-295. https://doi.org/10.1016/0163-7258(82)90064-X
  5. Beazley, K. E., Deasey, S., Lima, F. and Nurminskaya, M. V. (2012) Transglutaminase 2-mediated activation of β-catenin signaling has a critical role in warfarin-induced vascular calcification. Arterioscler. Thromb. Vasc. Biol. 32, 123-130. https://doi.org/10.1161/ATVBAHA.111.237834
  6. Benjamin, E. J., Muntner, P., Alonso, A., Bittencourt, M. S., Callaway, C. W., Carson, A. P., Chamberlain, A. M., Chang, A. R., Cheng, S., Das, S. R., Delling, F. N., Djousse, L., Elkind, M. S. V., Ferguson, J. F., Fornage, M., Jordan, L. C., Khan, S. S., Kissela, B. M., Knutson, K. L., Kwan, T. W., Lackland, D. T., Lewis, T. T., Lichtman, J. H., Longenecker, C. T., Loop, M. S., Lutsey, P. L., Martin, S. S., Matsushita, K., Moran, A. E., Mussolino, M. E., O'flaherty, M., Pandey, A., Perak, A. M., Rosamond, W. D., Roth, G. A., Sampson, U. K. A., Satou, G. M., Schroeder, E. B., Shah, S. H., Spartano, N. L., Stokes, A., Tirschwell, D. L., Tsao, C. W., Turakhia, M. P., Vanwagner, L. B., Wilkins, J. T., Wong, S. S. and Virani, S. S.; American Heart Association Council on Epidemiology and Prevention Statistics Committee and Stroke Statistics Subcommittee (2019) Heart disease and stroke statistics-2019 update: a report from the American Heart Association. Circulation 139, e56-e528. https://doi.org/10.1161/CIR.0000000000000659
  7. Bogdanova, M., Kostina, A., Zihlavnikova Enayati, K., Zabirnyk, A., Malashicheva, A., Stenslokken, K.-O., Sullivan, G. J., Kaljusto, M.-L., Kvitting, J.-P. E., Kostareva, A., Vaage, J. and Rutkovskiy, A. (2018) Inflammation and mechanical stress stimulate osteogenic differentiation of human aortic valve interstitial cells. Front. Physiol. 9, 1635.
  8. Bogdanova, M., Zabirnyk, A., Malashicheva, A., Enayati, K. Z., Karlsen, T. A., Kaljusto, M.-L., Kvitting, J.-P. E., Dissen, E., Sullivan, G. J., Kostareva, A., Stenslokken, K.-O., Rutkovskiy, A. and Vaage, J. (2019) Interstitial cells in calcified aortic valves have reduced differentiation potential and stem cell-like properties. Sci. Rep. 9, 12934.
  9. Branchetti, E., Sainger, R., Poggio, P., Grau, J. B., Patterson-Fortin, J., Bavaria, J. E., Chorny, M., Lai, E., Gorman, R. C., Levy, R. J. and Ferrari, G. (2013) Antioxidant enzymes reduce DNA damage and early activation of valvular interstitial cells in aortic valve sclerosis. Arterioscler. Thromb. Vasc. Biol. 33, e66-e74. https://doi.org/10.1161/ATVBAHA.112.300177
  10. Byon, C. H., Javed, A., Dai, Q., Kappes, J. C., Clemens, T. L., Darley-Usmar, V. M., Mcdonald, J. M. and Chen, Y. (2008) Oxidative stress induces vascular calcification through modulation of the osteogenic transcription factor Runx2 by AKT signaling. J. Biol. Chem. 283, 15319-15327. https://doi.org/10.1074/jbc.M800021200
  11. Caira, F. C., Stock, S. R., Gleason, T. G., Mcgee, E. C., Huang, J., Bonow, R. O., Spelsberg, T. C., Mccarthy, P. M., Rahimtoola, S. H. and Rajamannan, N. M. (2006) Human degenerative valve disease is associated with up-regulation of low-density lipoprotein receptor-related protein 5 receptor-mediated bone formation. J. Am. Coll. Cardiol. 47, 1707-1712. https://doi.org/10.1016/j.jacc.2006.02.040
  12. Caraci, F., Gili, E., Calafiore, M., Failla, M., La Rosa, C., Crimi, N., Sortino, M. A., Nicoletti, F., Copani, A. and Vancheri, C. (2008) TGF-β1 targets the GSK-3β/β-catenin pathway via ERK activation in the transition of human lung fibroblasts into myofibroblasts. Pharmacol. Res. 57, 274-282. https://doi.org/10.1016/j.phrs.2008.02.001
  13. Chang, Y.-W., Yeh, T.-K., Lin, K.-T., Chen, W.-C. and Yao, H.-T. (2006) Pharmacokinetics of anti-SARS-CoV agent niclosamide and its analogs in rats. J. Food Drug Anal. 14, 15.
  14. Chaudhary, S. C., Kuzynski, M., Bottini, M., Beniash, E., Dokland, T., Mobley, C. G., Yadav, M. C., Poliard, A., Kellermann, O. and Millan, J. L. (2016) Phosphate induces formation of matrix vesicles during odontoblast-initiated mineralization in vitro. Matrix Biol. 52, 284-300. https://doi.org/10.1016/j.matbio.2016.02.003
  15. Chen, W., Mook, R. A., Jr., Premont, R. T. and Wang, J. (2018) Niclosamide: beyond an antihelminthic drug. Cell. Signal. 41, 89-96. https://doi.org/10.1016/j.cellsig.2017.04.001
  16. Chu, Y., Lund, D. D., Weiss, R. M., Brooks, R. M., Doshi, H., Hajj, G. P., Sigmund, C. D. and Heistad, D. D. (2013) Pioglitazone attenuates valvular calcification induced by hypercholesterolemia. Arterioscler. Thromb. Vasc. Biol. 33, 523-532. https://doi.org/10.1161/ATVBAHA.112.300794
  17. Ding, Q., Xia, W., Liu, J.-C., Yang, J.-Y., Lee, D.-F., Xia, J., Bartholomeusz, G., Li, Y., Pan, Y., Li, Z., Bargou, R. C., Qin, J., Lai, C.-C., Tsai, F.-J., Tsai, C.-H. and Hung, M.-C. (2005) Erk associates with and primes GSK-3β for its inactivation resulting in upregulation of β-catenin. Mol. Cell 19, 159-170. https://doi.org/10.1016/j.molcel.2005.06.009
  18. Dweck, M. R., Boon, N. A. and Newby, D. E. (2012) Calcific aortic stenosis: a disease of the valve and the myocardium. J. Am. Coll. Cardiol. 60, 1854-1863. https://doi.org/10.1016/j.jacc.2012.02.093
  19. Ge, C., Xiao, G., Jiang, D. and Franceschi, R. T. (2007) Critical role of the extracellular signal-regulated kinase-MAPK pathway in osteoblast differentiation and skeletal development. The J. Cell Biol. 176, 709-718. https://doi.org/10.1083/jcb.200610046
  20. Goody, P. R., Hosen, M. R., Christmann, D., Niepmann, S. T., Zietzer, A., Adam, M., Bonner, F., Zimmer, S., Nickenig, G. and Jansen, F. (2020) Aortic valve stenosis. Arterioscler. Thromb. Vasc. Biol. 40, 885-900. https://doi.org/10.1161/ATVBAHA.119.313067
  21. Goto, S., Rogers, M. A., Blaser, M. C., Higashi, H., Lee, L. H., Schlotter, F., Body, S. C., Aikawa, M., Singh, S. A. and Aikawa, E. (2019) Standardization of human calcific aortic valve disease in vitro modeling reveals passage-dependent calcification. Front. Cardiovasc. Med. 6, 49.
  22. Gould, R. A. and Butcher, J. T. (2010) Isolation of valvular endothelial cells. J. Vis. Exp. (46), 2158.
  23. Greenberg, H. Z. E., Zhao, G., Shah, A. M. and Zhang, M. (2022) Role of oxidative stress in calcific aortic valve disease and its therapeutic implications. Cardiovasc. Res. 118, 1433-1451. https://doi.org/10.1093/cvr/cvab142
  24. Gu, X. and Masters, K. S. (2009) Role of the MAPK/ERK pathway in valvular interstitial cell calcification. Am. J. Physiol. Heart Circ. Physiol. 296, H1748-H1757. https://doi.org/10.1152/ajpheart.00099.2009
  25. Hou, Q.-C., Wang, J.-W., Yuan, G., Wang, Y.-P., Xu, K.-Q., Zhang, L., Xu, X.-F., Mao, W.-J. and Liu, Y. (2021) AGEs promote calcification of HASMCs by mediating Pi3k/AKT-GSK3β signaling. Front. Biosci.-Landmark 26, 125-134. https://doi.org/10.52586/4929
  26. Houslay, E. S., Cowell, S. J., Prescott, R. J., Reid, J., Burton, J., Northridge, D. B., Boon, N. A. and Newby, D. E. (2006) Progressive coronary calcification despite intensive lipid-lowering treatment: a randomised controlled trial. Heart 92, 1207-1212. https://doi.org/10.1136/hrt.2005.080929
  27. Huang, H. W., Bow, Y. D., Wang, C. Y., Chen, Y. C., Fu, P. R., Chang, K. F., Wang, T. W., Tseng, C. H., Chen, Y. L. and Chiu, C. C. (2020) DFIQ, a novel quinoline derivative, shows anticancer potential by inducing apoptosis and autophagy in NSCLC cell and in vivo zebrafish xenograft models. Cancers (Basel) 12, 1348.
  28. Hutcheson, J. D., Aikawa, E. and Merryman, W. D. (2014) Potential drug targets for calcific aortic valve disease. Nat. Rev. Cardiol. 11, 218-231. https://doi.org/10.1038/nrcardio.2014.1
  29. Incalza, M. A., D'oria, R., Natalicchio, A., Perrini, S., Laviola, L. and Giorgino, F. (2018) Oxidative stress and reactive oxygen species in endothelial dysfunction associated with cardiovascular and metabolic diseases. Vascul. Pharmacol. 100, 1-19. https://doi.org/10.1016/j.vph.2017.05.005
  30. Jang, W.-G., Kim, E.-J., Kim, D.-K., Ryoo, H.-M., Lee, K.-B., Kim, S.-H., Choi, H.-S. and Koh, J.-T. (2012) BMP2 protein regulates osteocalcin expression via Runx2-mediated Atf6 gene transcription. J. Biol. Chem. 287, 905-915. https://doi.org/10.1074/jbc.M111.253187
  31. Kang, S.-M., Lim, S., Song, H., Chang, W., Lee, S., Bae, S.-M., Chung, J. H., Lee, H., Kim, H.-G., Yoon, D.-H., Kim, T. W., Jang, Y., Sung, J.-M., Chung, N.-S. and Hwang, K.-C. (2006) Allopurinol modulates reactive oxygen species generation and Ca2+ overload in ischemia-reperfused heart and hypoxia-reoxygenated cardiomyocytes. Eur. J. Pharmacol. 535, 212-219. https://doi.org/10.1016/j.ejphar.2006.01.013
  32. Khalid, S., Yamazaki, H., Socorro, M., Monier, D., Beniash, E. and Napierala, D. (2020) Reactive oxygen species (ROS) generation as an underlying mechanism of inorganic phosphate (Pi)-induced mineralization of osteogenic cells. Free Radic. Biol. Med. 153, 103-111. https://doi.org/10.1016/j.freeradbiomed.2020.04.008
  33. Kraler, S., Blaser, M. C., Aikawa, E., Camici, G. G. and Luscher, T. F. (2021) Calcific aortic valve disease: from molecular and cellular mechanisms to medical therapy. Eur. Heart J. 43, 683-697. https://doi.org/10.1093/eurheartj/ehab757
  34. Lassegue, B., Martin, A. S. and Griendling, K. K. (2012) Biochemistry, physiology, and pathophysiology of NADPH oxidases in the cardiovascular system. Circ. Res. 110, 1364-1390. https://doi.org/10.1161/CIRCRESAHA.111.243972
  35. Lerman, D. A., Prasad, S. and Alotti, N. (2015) Calcific aortic valve disease: molecular mechanisms and therapeutic approaches. Eur. Cardiol. 10, 108-112. https://doi.org/10.15420/ecr.2015.10.2.108
  36. Li, Y., Zhu, H., Kuppusamy, P., Roubaud, V., Zweier, J. L. and Trush, M. A. (1998) Validation of lucigenin (bis-N-methylacridinium) as a chemilumigenic probe for detecting superoxide anion radical production by enzymatic and cellular systems. J. Biol. Chem. 273, 2015-2023. https://doi.org/10.1074/jbc.273.4.2015
  37. Liang, L., Huang, M., Xiao, Y., Zen, S., Lao, M., Zou, Y., Shi, M., Yang, X. and Xu, H. (2015) Inhibitory effects of niclosamide on inflammation and migration of fibroblast-like synoviocytes from patients with rheumatoid arthritis. Inflamm. Res. 64, 225-233. https://doi.org/10.1007/s00011-015-0801-5
  38. Liberman, M., Bassi, E., Martinatti, M. K., Lario, F. C., Wosniak, J., Jr., Pomerantzeff, P. M. and Laurindo, F. R. (2008) Oxidant generation predominates around calcifying foci and enhances progression of aortic valve calcification. Arterioscler. Thromb. Vasc. Biol. 28, 463-470. https://doi.org/10.1161/ATVBAHA.107.156745
  39. Liu, F.-L., Chen, C.-L., Lee, C.-C., Wu, C.-C., Hsu, T.-H., Tsai, C.-Y., Huang, H.-S. and Chang, D.-M. (2017) The simultaneous inhibitory effect of niclosamide on RANKL-induced osteoclast formation and osteoblast differentiation. Int. J. Med. Sci. 14, 840-852. https://doi.org/10.7150/ijms.19268
  40. Liu, H., Wang, L., Pan, Y., Wang, X., Ding, Y., Zhou, C., Shah, A. M., Zhao, G. and Zhang, M. (2020) Celastrol alleviates aortic valve calcification via inhibition of NADPH oxidase 2 in valvular interstitial cells. JACC Basic Transl. Sci. 5, 35-49. https://doi.org/10.1016/j.jacbts.2019.10.004
  41. Liu, J., Xiao, Q., Xiao, J., Niu, C., Li, Y., Zhang, X., Zhou, Z., Shu, G. and Yin, G. (2022) Wnt/β-catenin signalling: function, biological mechanisms, and therapeutic opportunities. Signal Transduct. Target. Ther. 7, 3.
  42. Lu, C., Dong, X., Yu, W. P., Ding, J. L., Yang, W., Gong, Y., Liu, J. C., Tang, Y. H., Xu, J. J. and Zhou, J. L. (2020) Inorganic phosphate-osteogenic induction medium promotes osteogenic differentiation of valvular interstitial cells via the BMP-2/Smad1/5/9 and RhoA/ROCK-1 signaling pathways. Am. J. Transl. Res. 12, 3329-3345.
  43. Miller, J. D., Chu, Y., Brooks, R. M., Richenbacher, W. E., Pena-Silva, R. and Heistad, D. D. (2008) Dysregulation of antioxidant mechanisms contributes to increased oxidative stress in calcific aortic valvular stenosis in humans. J. Am. Coll. Cardiol. 52, 843-850. https://doi.org/10.1016/j.jacc.2008.05.043
  44. Munzel, T., Camici, G. G., Maack, C., Bonetti, N. R., Fuster, V. and Kovacic, J. C. (2017) Impact of oxidative stress on the heart and vasculature: part 2 of a 3-part series. J. Am. Coll. Cardiol. 70, 212-229. https://doi.org/10.1016/j.jacc.2017.05.035
  45. Ning, F.-L., Tao, J., Li, D.-D., Tian, L.-L., Wang, M.-L., Reilly, S., Liu, C., Cai, H., Xin, H. and Zhang, X.-M. (2022) Activating BK channels ameliorates vascular smooth muscle calcification through Akt signaling. Acta Pharmacol. Sin. 43, 624-633. https://doi.org/10.1038/s41401-021-00704-6
  46. Okamoto, T., Taguchi, M., Osaki, T., Fukumoto, S. and Fujita, T. (2014) Phosphate enhances reactive oxygen species production and suppresses osteoblastic differentiation. J. Bone Miner. Metab. 32, 393-399. https://doi.org/10.1007/s00774-013-0516-z
  47. Park, J. S., Lee, Y. S., Lee, D. H. and Bae, S. H. (2019) Repositioning of niclosamide ethanolamine (NEN), an anthelmintic drug, for the treatment of lipotoxicity. Free Radic. Biol. Med. 137, 143-157. https://doi.org/10.1016/j.freeradbiomed.2019.04.030
  48. Pasquale, G., Coutsoumbas, G., Zagnoni, S., Filippini, E. and Resciniti, E. (2019) Aortic stenosis in the elderly can be prevented: Old risk factors and a new pathological condition. Eur. Heart J. Suppl. 21, B52-B53. https://doi.org/10.1093/eurheartj/suz017
  49. Pawade, T. A., Newby, D. E. and Dweck, M. R. (2015) Calcification in aortic stenosis: the skeleton key. J. Am. Coll. Cardiol. 66, 561-577. https://doi.org/10.1016/j.jacc.2015.05.066
  50. Qiao, Y. (2022) Reactive oxygen species in cardiovascular calcification: role of medicinal plants. Front. Pharmacol. 13, 858160.
  51. Rajamannan, N. M., Evans, F. J., Aikawa, E., Grande-Allen, K. J., Demer, L. L., Heistad, D. D., Simmons, C. A., Masters, K. S., Mathieu, P., O'brien, K. D., Schoen, F. J., Towler, D. A., Yoganathan, A. P. and Otto, C. M. (2011) Calcific aortic valve disease: not simply a degenerative process. Circulation 124, 1783-1791. https://doi.org/10.1161/CIRCULATIONAHA.110.006767
  52. Richards, J., El-Hamamsy, I., Chen, S., Sarang, Z., Sarathchandra, P., Yacoub, M. H., Chester, A. H. and Butcher, J. T. (2013) Side-specific endothelial-dependent regulation of aortic valve calcification: interplay of hemodynamics and nitric oxide signaling. Am. J. Pathol. 182, 1922-1931. https://doi.org/10.1016/j.ajpath.2013.01.037
  53. Richer, S. C. and Ford, W. C. L. (2001) A critical investigation of NADPH oxidase activity in human spermatozoa. Mol. Hum. Reprod. 7, 237-244. https://doi.org/10.1093/molehr/7.3.237
  54. Rong, S., Zhao, X., Jin, X., Zhang, Z., Chen, L., Zhu, Y. and Yuan, W. (2014) Vascular calcification in chronic kidney disease is induced by bone morphogenetic protein-2 via a mechanism involving the Wnt/β-catenin pathway. Cell. Physiol. Biochem. 34, 2049-2060. https://doi.org/10.1159/000366400
  55. Rutkovskiy, A., Malashicheva, A., Sullivan, G., Bogdanova, M., Kostareva, A., Stenslokken, K. O., Fiane, A. and Vaage, J. (2017) Valve interstitial cells: the key to understanding the pathophysiology of heart valve calcification. J. Am. Heart Assoc. 6, e006339.
  56. Sanchez-Esteban, S., Castro-Pinto, M., Cook-Calvete, A., Reventun, P., Delgado-Marin, M., Benito-Manzanaro, L., Hernandez, I., Lopez-Menendez, J., Zamorano, J. L., Zaragoza, C. and Saura, M. (2022) Integrin-linked kinase expression in human valve endothelial cells plays a protective role in calcific aortic valve disease. Antioxidants 11, 1736.
  57. Serrano, A., Apolloni, S., Rossi, S., Lattante, S., Sabatelli, M., Peric, M., Andjus, P., Michetti, F., Carri, M. T., Cozzolino, M. and D'ambrosi, N. (2019) The S100A4 transcriptional inhibitor niclosamide reduces pro-inflammatory and migratory phenotypes of microglia: implications for amyotrophic lateral sclerosis. Cells 8, 1261.
  58. Shiwakoti, S., Adhikari, D., Lee, J. P., Kang, K. W., Lee, I. S., Kim, H. J. and Oak, M. H. (2020) Prevention of fine dust-induced vascular senescence by humulus lupulus extract and its major bioactive compounds. Antioxidants (Basel) 9, 1243.
  59. Shiwakoti, S., Ko, J.-Y., Gong, D., Dhakal, B., Lee, J.-H., Adhikari, R., Gwak, Y., Park, S.-H., Jun Choi, I., Schini-Kerth, V. B., Kang, K.-W. and Oak, M.-H. (2022) Effects of polystyrene nanoplastics on endothelium senescence and its underlying mechanism. Environ. Int. 164, 107248.
  60. Sugamura, K. and Keaney, J. F., Jr. (2011) Reactive oxygen species in cardiovascular disease. Free Radic. Biol. Med. 51, 978-992. https://doi.org/10.1016/j.freeradbiomed.2011.05.004
  61. Sui, H., Pan, S.-F., Feng, Y., Jin, B.-H., Liu, X., Zhou, L.-H., Hou, F.-G., Wang, W.-H., Fu, X.-L., Han, Z.-F., Ren, J.-L., Shi, X.-L., Zhu, H.-R. and Li, Q. (2014) Zuo Jin Wan reverses P-gp-mediated drug-resistance by inhibiting activation of the PI3K/Akt/NF-κB pathway. BMC Complement. Altern. Med. 14, 279.
  62. Sun, J., Ming, L., Shang, F., Shen, L., Chen, J. and Jin, Y. (2015) Apocynin suppression of NADPH oxidase reverses the aging process in mesenchymal stem cells to promote osteogenesis and increase bone mass. Sci. Rep. 5, 18572.
  63. Tanaka, T., Asano, T., Okui, T., Kuraoka, S., Singh, S. A., Aikawa, M. and Aikawa, E. (2022) Computational screening strategy for drug repurposing identified niclosamide as inhibitor of vascular calcification. Front. Cardiovasc. Med. 8, 826529.
  64. Vasquez-Vivar, J., Kalyanaraman, B., Martasek, P., Hogg, N., Masters, B. S., Karoui, H., Tordo, P. and Pritchard, K. A. Jr. (1998) Superoxide generation by endothelial nitric oxide synthase: the influence of cofactors. Proc. Natl. Acad. Sci. U. S. A. 95, 9220-9225. https://doi.org/10.1073/pnas.95.16.9220
  65. Vossen, L. M., Kroon, A. A., Schurgers, L. J. and De Leeuw, P. W. (2020) Pharmacological and nutritional modulation of vascular calcification. Nutrients 12, 100.
  66. Winter, J. N., Jefferson, L. S. and Kimball, S. R. (2011) ERK and Akt signaling pathways function through parallel mechanisms to promote mTORC1 signaling. Am. J. Physiol. Cell Physiol. 300, C1172-C1180. https://doi.org/10.1152/ajpcell.00504.2010
  67. Yang, X., Meng, X., Su, X., Mauchley, D. C., Ao, L., Cleveland, J. C. and Fullerton, D. A. (2009) Bone morphogenic protein 2 induces Runx2 and osteopontin expression in human aortic valve interstitial cells: role of Smad1 and extracellular signal-regulated kinase 1/2. J. Thorac. Cardiovasc. Surg. 138, 1008-1015.e1001. https://doi.org/10.1016/j.jtcvs.2009.06.024
  68. Yutzey, K. E., Demer, L. L., Body, S. C., Huggins, G. S., Towler, D. A., Giachelli, C. M., Hofmann-Bowman, M. A., Mortlock, D. P., Rogers, M. B., Sadeghi, M. M. and Aikawa, E. (2014) Calcific aortic valve disease: a consensus summary from the Alliance of Investigators on Calcific Aortic Valve Disease. Arterioscler. Thromb. Vasc. Biol. 34, 2387-2393. https://doi.org/10.1161/ATVBAHA.114.302523
  69. Zhang, M., Perino, A., Ghigo, A., Hirsch, E. and Shah, A. M. (2013) NADPH oxidases in heart failure: poachers or gamekeepers? Antioxid. Redox Signal. 18, 1024-1041. https://doi.org/10.1089/ars.2012.4550
  70. Zheng, H., Jia, L., Liu, C.-C., Rong, Z., Zhong, L., Yang, L., Chen, X.-F., Fryer, J. D., Wang, X., Zhang, Y.-W., Xu, H. and Bu, G. (2017) TREM2 promotes microglial survival by activating Wnt/β-catenin pathway. J. Neurosci. 37, 1772-1784. https://doi.org/10.1523/JNEUROSCI.2459-16.2017