Emma Harper1, Keith D Rochfort2, Hannah Forde3, Colin Davenport4, Diarmuid Smith5, Philip M Cummins6. 1. School of Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland; National Institute for Cellular Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland. Electronic address: emma.harper2@mail.dcu.ie. 2. School of Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland; National Institute for Cellular Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland. 3. School of Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland; Department of Academic Endocrinology, Beaumont Hospital, Beaumont Road, Dublin, Ireland. 4. School of Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland. 5. Department of Academic Endocrinology, Beaumont Hospital, Beaumont Road, Dublin, Ireland. Electronic address: diarmuidsmith@beaumont.ie. 6. School of Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland; National Institute for Cellular Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland. Electronic address: phil.cummins@dcu.ie.
Abstract
BACKGROUND: The intimal endothelium is known to condition the underlying medial smooth muscle cell (SMC) layer of the vessel wall, and is highly responsive to receptor-activator of nuclear factor-κB ligand (RANKL) and tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), pro-calcific and anti-calcific agents, respectively. In this paper, we tested the hypothesis that RANKL-induced activation of endothelial NF-κB signalling is essential for pro-calcific activation of the underlying SMCs. METHODS: For these studies, human aortic endothelial and smooth muscle cell mono-cultures (HAECs, HASMCs) were treated with RANKL (0-25 ng/ml ± 5 ng/ml TRAIL) for 72 h. Non-contact transwell HAEC:HASMC co-cultures were also employed in which the luminal HAECs were treated with RANKL (± 5 ng/ml TRAIL), followed by analysis of pro-calcific markers in the underlying subluminal HASMCs. RESULTS: Treatment of either HAECs or HASMCs with RANKL activated the non-canonical NF-κB/p52 and canonical NF-κB/p65 pathways in both cell types. In RANKL ± TRAIL-treated HAECs, recombinant TRAIL, previously demonstrated by our group to strongly attenuate the pro-calcific signalling effects of RANKL, was shown to specifically block the RANKL-mediated activation of non-canonical NF-κB/p52, clearly pointing to the mechanistic relevance of this specific pathway to RANKL function within endothelial cells. In a final series of HAEC:HASMC transwell co-culture experiments, RANKL treatment of HAECs that had been genetically silenced (via siRNA) for the NF-κB2 gene (the molecular forerunner to NF-κB/p52 generation) exhibited strongly attenuated pro-calcific activation of underlying HASMCs relative to scrambled siRNA controls. SUMMARY: These in vitro observations provide valuable mechanistic insights into how RANKL may potentially act upon endothelial cells through activation of the alternative NF-κB pathway to alter endothelial paracrine signalling and elicit pro-calcific responses within underlying vascular smooth muscle cells.
BACKGROUND: The intimal endothelium is known to condition the underlying medial smooth muscle cell (SMC) layer of the vessel wall, and is highly responsive to receptor-activator of nuclear factor-κB ligand (RANKL) and tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), pro-calcific and anti-calcific agents, respectively. In this paper, we tested the hypothesis that RANKL-induced activation of endothelial NF-κB signalling is essential for pro-calcific activation of the underlying SMCs. METHODS: For these studies, human aortic endothelial and smooth muscle cell mono-cultures (HAECs, HASMCs) were treated with RANKL (0-25 ng/ml ± 5 ng/ml TRAIL) for 72 h. Non-contact transwell HAEC:HASMC co-cultures were also employed in which the luminal HAECs were treated with RANKL (± 5 ng/ml TRAIL), followed by analysis of pro-calcific markers in the underlying subluminal HASMCs. RESULTS: Treatment of either HAECs or HASMCs with RANKL activated the non-canonical NF-κB/p52 and canonical NF-κB/p65 pathways in both cell types. In RANKL ± TRAIL-treated HAECs, recombinant TRAIL, previously demonstrated by our group to strongly attenuate the pro-calcific signalling effects of RANKL, was shown to specifically block the RANKL-mediated activation of non-canonical NF-κB/p52, clearly pointing to the mechanistic relevance of this specific pathway to RANKL function within endothelial cells. In a final series of HAEC:HASMC transwell co-culture experiments, RANKL treatment of HAECs that had been genetically silenced (via siRNA) for the NF-κB2 gene (the molecular forerunner to NF-κB/p52 generation) exhibited strongly attenuated pro-calcific activation of underlying HASMCs relative to scrambled siRNA controls. SUMMARY: These in vitro observations provide valuable mechanistic insights into how RANKL may potentially act upon endothelial cells through activation of the alternative NF-κB pathway to alter endothelial paracrine signalling and elicit pro-calcific responses within underlying vascular smooth muscle cells.
Authors: L Wexler; B Brundage; J Crouse; R Detrano; V Fuster; J Maddahi; J Rumberger; W Stanford; R White; K Taubert Journal: Circulation Date: 1996-09-01 Impact factor: 29.690
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Authors: Emma Harper; Keith D Rochfort; Hannah Forde; Colin Davenport; Diarmuid Smith; Philip M Cummins Journal: PLoS One Date: 2017-11-16 Impact factor: 3.240
Authors: L Hénaut; A B Sanz; D Martin-Sanchez; S Carrasco; R Villa-Bellosta; G Aldamiz-Echevarria; Z A Massy; M D Sanchez-Nino; A Ortiz Journal: Cell Death Dis Date: 2016-07-21 Impact factor: 8.469