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

Abstract

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.

Fig 1. Direct effects of RANKL±TRAIL on BMP-2 levels in HAECs.

HAECs were treated for 72 hr with RANKL (0–25 ng/mL) in the absence and presence of TRAIL (5 ng/mL) and then analyzed by qPCR for (A) BMP-2 mRNA. Cells and conditioned media were also harvested for ELISA analysis of (B) BMP-2 cellular protein and (C) released BMP-2, respectively. *P≤0.05 versus 0 ng/mL RANKL; ^δP≤0.05 versus corresponding 5 and 25 ng/mL RANKL treatments.

Fig 2. Direct effects of RANKL±TRAIL on OPG levels in HAECs.

HAECs were treated for 72 hr with RANKL (0–25 ng/mL) in the absence and presence of TRAIL (5 ng/mL) and then investigated by qPCR for (A) OPG mRNA. Cells and conditioned media were also harvested for ELISA analysis of (B) OPG cellular protein and (C) released OPG, respectively. *P≤0.05 versus 0 ng/mL RANKL (or control); ^δP≤0.05 versus 25 ng/mL RANKL.

Fig 3. Schematic of the HAEC:HASMC transwell co-culture model.

HAECs within the luminal compartment were treated for 72 hr with RANKL (0–25 ng/mL) in the absence and presence of TRAIL (5 ng/mL). Within the subluminal compartment, HASMCs were then analyzed for key targets (OPG, ALP, Runx2, Sox9, BMP-2.

Fig 4. Effects of RANKL±TRAIL on HASMC OPG levels within a HAEC:HASMC co-culture model.

HAECs within the luminal compartment were treated for 72 hr with RANKL (0–25 ng/mL) in the absence and presence of TRAIL (5 ng/mL). Subluminal HASMCs were then analyzed by qPCR for (A) OPG mRNA. Subluminal HASMCs and conditioned media were also harvested for ELISA analysis of (B) OPG cellular protein and (C) released OPG, respectively. *P≤0.05 versus 0 ng/mL RANKL (or control); ^δP≤0.05 versus corresponding 5 and 25 ng/mL RANKL treatments.

Fig 5. Paracrine effects of RANKL±TRAIL on HASMC ALP levels within a HAEC:HASMC co-culture model.

HAECs within the luminal compartment were treated for 72 hr with RANKL (0–25 ng/mL) in the absence and presence of TRAIL (5 ng/mL). Within the subluminal compartment, HASMCs were then harvested and analyzed for (A) ALP mRNA and (B) ALP enzymatic activity by qPCR and ELISA, respectively. *P≤0.05 versus 0 ng/mL RANKL (or control); ^δP≤0.05 versus 25 ng/mL RANKL.

Fig 6. Paracrine effects of RANKL±TRAIL on HASMC osteoblastic transcription factors within a HAEC:HASMC co-culture model.

HAECs within the subluminal compartment were treated for 72 hr with RANKL (0–25 ng/mL) in the absence and presence of TRAIL (5 ng/mL). Within the subluminal compartment, HASMCs were then analyzed by qPCR for (A) Runx2 and (B) Sox9 mRNA. *P≤0.05 versus 0 ng/mL RANKL (or control); ^δP≤0.05 versus 25 ng/mL RANKL.