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RANKL treatment of vascular endothelial cells leading to paracrine pro-calcific signaling involves ROS production

  • Emma Harper
  • Keith D. Rochfort
  • Diarmuid Smith
  • Philip M. CumminsEmail author
Article

Abstract

Numerous studies have highlighted the causal link between over-production of reactive oxygen species (ROS) and cardiovascular complications such as vascular calcification (VC). Receptor-activator of nuclear factor-κB ligand (RANKL) has previously been shown to act on endothelial cells, eliciting the production/release of paracrine pro-calcific signals that act, in-turn, upon underlying vascular smooth muscle cells (VSMCs) to induce osteoblastic differentiation and VC. A role for endothelial ROS signaling in this process has not been established however. In the current paper, we investigate the possibility that RANKL leads to ROS signaling within the endothelial layer as part of the RANKL-driven VC signaling cascade. Human aortic endothelial cells (HAECs) were treated with RANKL (25 ng/ml, 72 h) and monitored for ROS production, in parallel with various pro-calcific signaling indices. Antioxidant co-treatments included TRAIL (5 ng/ml), apocynin (10 mM) and N-acetylcysteine (5 mM). Treatment of HAECs with RANKL-induced robust ROS production. This surge could be partially attenuated by TRAIL and strongly attenuated by apocynin and N-acetylcysteine. RANKL also elicited a range of signaling events in HAECs that we have previously demonstrated are coupled to osteoblastic differentiation in underlying VSMCs. These include non-canonical NF-κB/p52 activation, elevated BMP-2 release and increased alkaline phosphatase (ALP) enzyme activity (cellular and extracellular). Importantly, these RANKL-induced signaling events could be completely prevented by co-treatment of HAECs with antioxidants. In summary, RANKL elicits ROS generation in HAECs with direct consequences for generation of paracrine pro-calcific signals known to effect calcification in underlying VSMCs.

Keywords

Endothelial cell Smooth muscle cell Calcification RANKL TRAIL Reactive oxygen species 

Notes

Acknowledgements

The authors wish to acknowledge the generous financial support (to EH) from the DCU O’Hare Scholarship and the Government of Ireland/Irish Research Council Postgraduate Scholarship scheme (Grant reference GOIPG/2015/3758). Support (to PMC) was also provided through the Science Foundation Ireland US-Ireland R&D Partnership Programme (Grant reference 14/US/B3116).

Compliance with ethical standards

Conflict of interest

The authors declare that there are no conflict of interest regarding the publication of this paper.

References

  1. 1.
    Demer LL, Tintut Y (2008) Vascular calcification: pathobiology of a multifaceted disease. Circulation 117:2938–2948.  https://doi.org/10.1161/CIRCULATIONAHA.107.743161 CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Harper E, Forde H, Davenport C, Rochfort KD, Smith D, Cummins PM (2016) Vascular calcification in type-2 diabetes and cardiovascular disease: integrative roles for OPG, RANKL and TRAIL. Vascul Pharmacol 82:30–40.  https://doi.org/10.1016/j.vph.2016.02.003 CrossRefPubMedGoogle Scholar
  3. 3.
    Harper E, Rochfort KD, Forde H, Davenport C, Smith D, Cummins PM (2017) TRAIL attenuates RANKL-mediated osteoblastic signalling in vascular cell mono-culture and co-culture models. PLoS ONE 12:e0188192.  https://doi.org/10.1371/journal.pone.0188192 CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Panizo S, Cardus A, Encinas M, Parisi E, Valcheva P, López-Ongil S, Coll B, Fernandez E, Valdivielso JM (2009) RANKL increases vascular smooth muscle cell calcification through a RANK-BMP4-dependent pathway. Circ Res 104:1041–1048.  https://doi.org/10.1161/CIRCRESAHA.108.189001 CrossRefPubMedGoogle Scholar
  5. 5.
    Davenport C, Harper E, Forde H, Rochfort KD, Murphy RP, Smith D, Cummins PM (2016) RANKL promotes osteoblastic activity in vascular smooth muscle cells by upregulating endothelial BMP-2 release. Int J Biochem Cell Biol 77:171–180.  https://doi.org/10.1016/j.biocel.2016.06.009 CrossRefPubMedGoogle Scholar
  6. 6.
    Osako MK, Nakagami H, Koibuchi N, Shimizu H, Nakagami F, Koriyama H, Shimamura M, Miyake T, Rakugi H, Morishita R (2010) Estrogen inhibits vascular calcification via vascular RANKL system: common mechanism of osteoporosis and vascular calcification. Circ Res 107:466–475.  https://doi.org/10.1161/CIRCRESAHA.110.216846 CrossRefPubMedGoogle Scholar
  7. 7.
    Harper E, Rochfort KD, Forde H, Davenport C, Smith D, Cummins PM (2018) Activation of the non-canonical NF-κB/p52 pathway in vascular endothelial cells by RANKL elicits pro-calcific signalling in co-cultured smooth muscle cells. Cell Signal 47:142–150.  https://doi.org/10.1016/j.cellsig.2018.04.004 CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Forde H, Harper E, Davenport C, Rochfort KD, Wallace R, Murphy RP, Smith D, Cummins PM (2016) The beneficial pleiotropic effects of tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) within the vasculature: a review of the evidence. Atherosclerosis 247:87–96.  https://doi.org/10.1016/j.atherosclerosis.2016.02.002 CrossRefPubMedGoogle Scholar
  9. 9.
    Jha JC, Ho F, Dan C, Jandeleit-Dahm K (2018) A causal link between oxidative stress and inflammation in cardiovascular and renal complications of diabetes. Clin Sci (Lond) 132:1811–1836.  https://doi.org/10.1042/CS20171459 CrossRefGoogle Scholar
  10. 10.
    Pitocco D, Tesauro M, Alessandro R, Ghirlanda G, Cardillo C (2013) Oxidative stress in diabetes: implications for vascular and other complications. Int J Mol Sci 14:21525–21550.  https://doi.org/10.3390/ijms141121525 CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Choi SY, Ryu HM, Oh EJ, Choi JY, Cho JH, Kim CD, Kim YL, Park SH (2017) Dipeptidyl peptidase-4 inhibitor gemigliptin protects against vascular calcification in an experimental chronic kidney disease and vascular smooth muscle cells. PLoS ONE 12:e0180393.  https://doi.org/10.1371/journal.pone.0180393 CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Luong TTD, Schelski N, Boehme B, Makridakis M, Vlahou A, Lang F, Pieske B, Alesutan I, Voelkl J (2018) Fibulin-3 attenuates phosphate-induced vascular smooth muscle cell calcification by inhibition of oxidative stress. Cell Physiol Biochem 46:1305–1316.  https://doi.org/10.1159/000489144 CrossRefPubMedGoogle Scholar
  13. 13.
    Liberman M, Johnson RC, Handy DE, Loscalzo J, Leopold JA (2011) Bone morphogenetic protein-2 activates NADPH oxidase to increase endoplasmic reticulum stress and human coronary artery smooth muscle cell calcification. Biochem Biophys Res Commun 413:436–441.  https://doi.org/10.1016/j.bbrc.2011.08.114 CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Farrar EJ, Huntley GD, Butcher J (2015) Endothelial-derived oxidative stress drives myofibroblastic activation and calcification of the aortic valve. PLoS ONE 10:e0123257.  https://doi.org/10.1371/journal.pone.0123257 CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Kim HJ, Park C, Kim GY, Park EK, Jeon YJ, Kim S, Hwang HJ, Choi YH (2018) Sargassum serratifolium attenuates RANKL-induced osteoclast differentiation and oxidative stress through inhibition of NF-κB and activation of the Nrf2/HO-1 signalling pathway. Biosci Trends 12:257–265.  https://doi.org/10.5582/bst.2018.01107 CrossRefPubMedGoogle Scholar
  16. 16.
    Thummuri D, Naidu VGM, Chaudhari P (2017) Carnosic acid attenuates RANKL-induced oxidative stress and osteoclastogenesis via induction of Nrf2 and suppression of NF-κB and MAPK signalling. J Mol Med (Berl) 95:1065–1076.  https://doi.org/10.1007/s00109-017-1553-1 CrossRefGoogle Scholar
  17. 17.
    Davenport C, Mahmood WA, Forde H, Ashley DT, Agha A, McDermott J, Sreenan S, Thompson CJ, McGrath F, McAdam B, Cummins PM, Smith D (2015) The effects of insulin and liraglutide on osteoprotegerin and vascular calcification in vitro and in patients with type 2 diabetes. Eur J Endocrinol 173:53–61.  https://doi.org/10.1530/EJE-14-1137 CrossRefPubMedGoogle Scholar
  18. 18.
    Rochfort KD, Collins LE, Murphy RP, Cummins PM (2014) Downregulation of blood-brain barrier phenotype by proinflammatory cytokines involves NADPH oxidase-dependent ROS generation: consequences for interendothelial adherens and tight junctions. PLoS ONE 9:e101815.  https://doi.org/10.1371/journal.pone.0101815 CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Manuneedhi CP, Cartland SP, Dang L, Rayner BS, Patel S, Thomas SR, Kavurma MM (2018) TRAIL protects against endothelial dysfunction in vivo and inhibits angiotensin-II-induced oxidative stress in vascular endothelial cells in vitro. Free Radic Biol Med 126:341–349.  https://doi.org/10.1016/j.freeradbiomed.2018.08.031 CrossRefGoogle Scholar
  20. 20.
    Frey RS, Ushio-Fukai M, Malik AB (2009) NADPH oxidase-dependent signalling in endothelial cells: role in physiology and pathophysiology. Antioxid Redox Signal 11:791–810.  https://doi.org/10.1089/ARS.2008.2220 CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Brodeur MR, Bouvet C, Barrette M, Moreau P (2013) Palmitic acid increases medial calcification by inducing oxidative stress. J Vasc Res 50:430–441.  https://doi.org/10.1159/000354235 CrossRefPubMedGoogle Scholar
  22. 22.
    Brodeur MR, Bouvet C, Bouchard S, Moreau S, Leblond J, Deblois D, Moreau P (2014) Reduction of advanced-glycation end products levels and inhibition of RAGE signaling decreases rat vascular calcification induced by diabetes. PLoS ONE 9:e85922.  https://doi.org/10.1371/journal.pone.0085922 CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Feng W, Zhang K, Liu Y, Chen J, Cai Q, Zhang Y, Wang M, Wang J, Huang H (2016) Apocynin attenuates angiotensin II-induced vascular smooth muscle cells osteogenic switching via suppressing extracellular signal-regulated kinase 1/2. Oncotarget 7:83588–83600.  https://doi.org/10.18632/oncotarget.13193 CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Tada Y, Yano S, Yamaguchi T, Okazaki K, Ogawa N, Morita M, Sugimoto T (2013) Advanced glycation end products-induced vascular calcification is mediated by oxidative stress: functional roles of NAD(P)H-oxidase. Horm Metab Res 45:267–272.  https://doi.org/10.1055/s-0032-1329965 CrossRefPubMedGoogle Scholar
  25. 25.
    Kang IS, Kim C (2016) NADPH oxidase gp91phox contributes to RANKL-induced osteoclast differentiation by upregulating NFATc1. Sci Rep 6:38014.  https://doi.org/10.1038/srep38014 CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Liu X, Gao X, Liu Y, Liang D, Fu T, Song Y, Zhao C, Dong B, Han W (2018) Daphnetin inhibits RANKL-induced osteoclastogenesis in vitro. J Cell Biochem.  https://doi.org/10.1002/jcb.27555 CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Taye AH, Saad AH, Kumar H (2010) Morawietz. Effect of apocynin on NADPH oxidase-mediated oxidative stress-LOX-1-eNOS pathway in human endothelial cells exposed to high glucose. Eur J Pharmacol 627:42–48.  https://doi.org/10.1016/j.ejphar.2009.10.045 CrossRefPubMedGoogle Scholar
  28. 28.
    Chrissobolis S, Banfi B, Sobey CG, Faraci FM (2012) Role of Nox isoforms in angiotensin II-induced oxidative stress and endothelial dysfunction in brain. J Appl Physiol 113:184–191.  https://doi.org/10.1152/japplphysiol.00455.2012 CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Beloqui O, Moreno MU, San José G, Pejenaute Á, Cortés A, Landecho MF, Díez J, Fortuño A, Zalba G (2017) Increased phagocytic NADPH oxidase activity associates with coronary artery calcification in asymptomatic men. Free Radic Res 51:389–396.  https://doi.org/10.1080/10715762.2017.1321745 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.School of BiotechnologyDublin City UniversityDublin 9Ireland
  2. 2.National Institute for Cellular Biotechnology, Dublin City UniversityDublin 9Ireland
  3. 3.Department of Academic EndocrinologyBeaumont HospitalDublinIreland

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