TRAIL attenuates RANKL-mediated osteoblastic signalling in vascular cell mono-culture and co-culture models

Abstract

Background and objectivesVascular calcification (VC) is a major risk factor for elevated cardiovascular morbidity/mortality. Underlying this process is osteoblastic signalling within the vessel wall involving complex and interlinked roles for receptor-activator of nuclear factor-κB ligand (RANKL), osteoprotegerin (OPG), and tumour necrosis factor-related apoptosis-inducing ligand (TRAIL). RANKL promotes vascular cell osteoblastic differentiation, whilst OPG acts as a neutralizing decoy receptor for RANKL (and TRAIL). With respect to TRAIL, much recent evidence points to a vasoprotective role for this ligand, albeit via unknown mechanisms. In order to shed more light on TRAILs vasoprotective role therefore, we employed in vitro cell models to test the hypothesis that TRAIL can counteract the RANKL-mediated signalling that occurs between the vascular cells that comprise the vessel wall.Methods and resultsHuman aortic endothelial and smooth muscle cell mono-cultures (HAECs, HASMCs) were treated with RANKL (0–25 ng/mL ± 5 ng/mL TRAIL) for 72 hr. Furthermore, to better recapitulate the paracrine signalling that exists between endothelial and smooth muscle cells within the vessel wall, non-contact transwell HAEC:HASMC co-cultures were also employed and involved RANKL treatment of HAECs (±TRAIL), subsequently followed by analysis of pro-calcific markers in the underlying subluminal HASMCs. RANKL elicited robust osteoblastic signalling across both mono- and co-culture models (e.g. increased BMP-2, alkaline phosphatase/ALP, Runx2, and Sox9, in conjunction with decreased OPG). Importantly, several RANKL actions (e.g. increased BMP-2 release from mono-cultured HAECs or increased ALP/Sox9 levels in co-cultured HASMCs) could be strongly blocked by co-incubation with TRAIL. In summary, this paper clearly demonstrates that RANKL can elicit pro-osteoblastic signalling in HAECs and HASMCs both directly and across paracrine signalling axes. Moreover, within these contexts we present clear evidence that TRAIL can block several key signalling actions of RANKL in vascular cells, providing further evidence of its vasoprotective potential.

Targeting IL-17 attenuates hypoxia-induced pulmonary hypertension through downregulation of β-catenin

“…A non-contact transwell coculture model9 was used to evaluate the role of PAECs dysfunction on proliferation and migration of PASMCs. The details are in supplements.…”

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 culture experiments were subsequently conducted in two formats: (i) Mono-culture experiments - HAECs and HASMCs were grown to confluency in 6-well dishes and separately treated for 72 h with either RANKL (0–25 ng/ml), TRAIL (0–5 ng/ml), or a combination of both (RANKL: 5 & 25 ng/ml + TRAIL: 5 ng/ml). This treatment period and dose range were previously established by Davenport et al [8,16] and Harper et al [7] and yielded robust responses. Post-treatment, cell lysates were harvested as previously described [17] in order to monitor activation of NF- κ B/ p52 (normalized to NF- κ B/ p100) and NF-κB/Phospho-p65 (normalized to NF- κ B/ p65) by Western blotting.…”

“…Post-treatment, cell lysates were harvested as previously described [17] in order to monitor activation of NF- κ B/ p52 (normalized to NF- κ B/ p100) and NF-κB/Phospho-p65 (normalized to NF- κ B/ p65) by Western blotting. All samples were stored at — 80 °C and assayed within three months; (ii) Co-culture experiments - The effect of HAEC paracrine signalling upon HASMC osteoblastic activity was also investigated using a non-contact transwell co-culture model as previously described [7]. Subluminal compartment; HASMCs were seeded at a density of 1.5 × 10 5 cells per well into standard 6-well culture dishes and grown to confluency.…”

“…DuoSet ™ ELISA Kits (R&D Systems), used in conjunction with F96 Maxisorp ™ Nunc-Immuno ™ 96-well plates (Bio-Sciences Ltd., Dun Laoghaire, IRL), were employed to accurately measure absolute levels of OPG, BMP-2 and IL-6 within cell lysates and subluminal conditioned media according to the method of Harper et al [7]. For normalization purposes, OPG and BMP-2 levels in protein lysates were routinely presented as pg/mg of total cellular protein, whilst conditioned media levels were presented as pg/10 5 cells.…”

“…Indeed, our own group has recently demonstrated that RANKL can elicit pro-calcific signalling in vascular cells (e.g. increased bone morphogenetic protein-2/BMP-2, alkaline phosphatase/ALP, Runx2, and Sox9 levels, in conjunction with decreased osteoprotegerin/OPG levels) using both mono-culture (direct SMC stimulation) and co-culture (EC:VSMC paracrine stimulation) models [7]. Interestingly, we recently demonstrated that several of the observed pro-calcific actions of RANKL in either model were strongly prevented by RANKL co-incubation with tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), reinforcing the widely held view that TRAIL has vasoprotective potential (for review see [10]).…”