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.
Highlights
Vascular calcification (VC) afflicts multiple patient populations, where it manifests as elevated levels of mineral deposition within the intimal and medial regions of blood vessels
Consistent with this, numerous studies have demonstrated that RANKL may promote trans-differentiation of vascular smooth muscle cells (VSMCs) to an osteoblastic/chondroblastic phenotype either through direct interaction with the VSMC RANK receptor [10,11], or by inducing the release of endothelial-derived proosteoblastic paracrine signals such as bone morphogenetic protein-2/4 (BMP-2/4), which in turn may act upon the underlying VSMCs [12,13]
It should be noted that receptors for RANKL (RANK) and tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) (DCR1, DCR2, DR4, and DR5) were all expressed at the mRNA level in both cell types
Summary
Vascular calcification (VC) afflicts multiple patient populations, where it manifests as elevated levels of mineral deposition within the intimal and medial regions of blood vessels. This has adverse consequences for vessel wall homeostasis, and constitutes a significant risk factor for elevated rates of cardiovascular (CV) morbidity and mortality [1,2]. OPG, produced in substantial quantities by VSMCs (and non-vascular sources), acts as a soluble decoy receptor for RANKL to neutralize its biological actions within the vasculature [14] This anti-calcific action of OPG within the vessel wall is supported by research demonstrating that OPG-/- mice display severe VC burden [15], whilst atherogenic mice treated with recombinant OPG exhibit reduced levels of plaque calcification and aortic osteocalcin [16]. It is noteworthy that the roles for RANKL and OPG within the vascular wall are, paradoxically, opposite to those observed for these same ligands within bone morphogenesis [8]
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