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

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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.

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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).

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Authors and Affiliations

  1. School of Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland

    Emma Harper, Keith D. Rochfort & Philip M. Cummins

  2. National Institute for Cellular Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland

    Emma Harper, Keith D. Rochfort & Philip M. Cummins

  3. Department of Academic Endocrinology, Beaumont Hospital, Beaumont Road, Dublin, Ireland

    Diarmuid Smith

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Correspondence to Philip M. Cummins.

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Harper, E., Rochfort, K.D., Smith, D. et al. RANKL treatment of vascular endothelial cells leading to paracrine pro-calcific signaling involves ROS production. Mol Cell Biochem 464, 111–117 (2020). https://doi.org/10.1007/s11010-019-03653-1

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