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


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.


Endothelial cell Smooth muscle cell Calcification RANKL TRAIL Reactive oxygen species 



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.


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© 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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