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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. [ABSTRACT FROM AUTHOR]

Receptor activator of nuclear factor-κB ligand (RANKL) promotes vascular calcification, while osteoprotegerin (OPG) opposes it by blocking RANKL activity. It remains unclear which vascular cell populations produce and secrete OPG/RANKL. This study characterizes the production of OPG/RANKL from human aortic endothelial and smooth muscle cells (HAECs and HASMCs) under various conditions. HAECs and HASMCs were exposed to inflammatory stimuli (tumor necrosis factor-α or hyperglycemia) or physiological levels of hemodynamic (cyclic) strain. After 72 h, both cells and supernatant media were harvested for assessment of OPG/RANKL production. Based on initial findings, the experiments involving HASMCs were then repeated in the presence of exogenous RANKL. OPG was produced and secreted by HASMCs and (to a considerably lesser degree) HAECs under basal conditions. Inflammatory stimuli upregulated OPG production in both cell populations. Cyclic strain significantly upregulated OPG production in HASMCs. Neither cell population produced RANKL. Exposing HASMCs to exogenous RANKL inhibited basal OPG production and completely abrogated the strain-mediated upregulation of OPG. These data suggest that HASMCs are a significant source of OPG within the vasculature but that RANKL, once present, downregulates this production and appears capable of preventing the “protective” upregulation of OPG seen with HASMCs exposed to physiological levels of cyclic strain. [ABSTRACT FROM AUTHOR]

Background and objectives: Vascular 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 results: 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 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. [ABSTRACT FROM AUTHOR]

Introduction Receptor activator of nuclear factor kappa beta-ligand (RANKL) is thought to promote vascular calcification (VC) by inducing osteoblastic behaviour in vascular smooth muscle cells (VSMC) in an ill-defined process. The present study assessed whether RANKL affects pro-osteoblastic paracrine signalling between human aortic endothelial cells (HAEC) and human aortic smooth muscle cells (HASMC) using both conditioned media transfer and cell co-culture experimental approaches. Methods and results For initial experiments (6-well format), HAEC-conditioned media was harvested following 72 h exposure to RANKL, and transferred to reporter HASMCs with/without noggin, an inhibitor of pro-osteoblastic bone morphogenetic protein (BMP) paracrine signalling. In further experiments, HAECs and HASMCs were co-cultured within the CellMax ® Duo, a perfusing bioreactor unit that mimics the flow-mediated co-interaction of these cells within the arterial wall, and RANKL was added to the perfusing media for 72 h. At the conclusion of each experiment markers of osteoblastic activity were measured in HASMCs, including alkaline phosphatase (ALP) activity, mRNA levels of ALP and Runx2, as well as BMP-2 and BMP-4 concentrations. RANKL increased BMP-2 release from HAECs, while exposure of HASMCs to RANKL-treated HAEC-conditioned media induced osteoblastic behaviour in HASMCs, an effect prevented by noggin. Within the CellMax ® Duo bioreactor, the addition of RANKL to the intraluminal HAECs also produced an increase in BMP-2 and increased osteoblastic behaviour within the co-cultured HASMC population. Conclusions RANKL promotes VC by inducing BMP-2 release from HAECs, which in turn appears to act in a paracrine fashion on the adjacent HASMC population to increase osteoblastic activity. [ABSTRACT FROM AUTHOR]

Vascular calcification (VC), a disorder that causes blood vessel hardening and dysfunction, is a significant risk factor for type-2 diabetes mellitus (T2DM), which invariably manifests associated cardiovascular complications. Although the clinical effects of VC have been well-documented, the precise cellular events underlying the manifestation and progression of VC are only now coming to light. Current research models indicate that VC likely involves signalling pathways traditionally associated with bone remodelling, such as the OPG/RANKL/TRAIL signalling system. In this respect, receptor activator of NF-κB ligand (RANKL) promotes VC whilst osteoprotegerin (OPG) acts as a RANKL decoy receptor to block this effect, events that contrast with the known functional influence of these proteins during bone metabolism. Moreover, evidence suggests that tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), an alternative decoy ligand for OPG, may exert an anti-calcific influence within the vasculature. In the current review, we conduct a timely examination of this complex VC pathology from both mechanistic and therapeutic perspectives. Our objectives are twofold: (i) to critically assess our current understanding of both osteogenic and vascular calcification pathways, with particular focus on the co-interactive roles of OPG, RANKL, and TRAIL. Extensive in vitro , in vivo , and clinical studies will therefore be reviewed and critical findings highlighted; and (ii) to examine a range of therapeutic approaches of potential relevance to VC pathology. In this regard, a clear focus on VC as it applies to T2DM and cardiovascular disease (and particularly atherosclerosis) will be maintained. [ABSTRACT FROM AUTHOR]