The RANKL/RANK/OPG signaling pathway mediates medial arterial calcification in diabetic Charcot neuroarthropathy

OBJECTIVE

The receptor activator of nuclear factor-κB (RANK), RANK ligand (RANKL), and osteoprotegerin (OPG) signaling pathway (RANKL/RANK/OPG signaling) is implicated in the osteolysis associated with diabetic Charcot neuroarthropathy (CN); however, the links with medial arterial calcification (MAC) seen in people with CN are unclear. This study aimed to investigate the role of RANKL/OPG in MAC in patients with CN.

RESEARCH DESIGN AND METHODS

Enzyme-linked immunosorbent assay and Bio-plex multiarray technology were used to quantify a range of cytokines, including RANKL and OPG in sera from 10 patients with diabetes, 12 patients with CN, and 5 healthy volunteers. Human tibial artery segments were immunohistochemically stained with Alizarin red and human RANKL antibody. Human vascular smooth muscle cells (VSMCs) were also explanted from arterial segments for in vitro studies.

RESULTS

We demonstrate colocalization and upregulation of RANKL expression in areas displaying MAC. Systemic levels of RANKL, OPG, and inflammatory cytokines (interleukin-8, granulocyte colony–stimulating factor) were elevated in those with CN compared with diabetic patients and healthy control subjects. Human VSMCs cultured in CN serum showed accelerated osteoblastic differentiation (alkaline phosphatase activity) and mineralization (alizarin red staining) compared with cells treated with diabetic or control serum (P < 0.05). Coincubation with OPG, the decoy receptor for RANKL, attenuated osteogenic differentiation of VSMCs and was independent of a high calcium-phosphate milieu. The accelerated mineralization induced by RANKL and CN serum correlated with nuclear translocation of nuclear factor-κB, a process abrogated by OPG.

CONCLUSIONS

Our data provide direct evidence that RANKL/RANK/OPG signaling is modulated in patients with CN and plays a role in vascular calcification. This study highlights this pathway as a potential target for intervention.Vascular calcification is a strong independent predictor of cardiovascular mortality (1). In people with diabetes, medial arterial calcification (MAC) has emerged as a strong predictor of lower limb amputation and cardiovascular mortality (2,3). This may be to the result of an increase in arterial stiffness, pulse wave velocity, and systolic blood pressure, ultimately leading to reduced coronary perfusion and ventricular hypertrophy (4). MAC in people with diabetes is more common in those with peripheral neuropathy, who also display increased bone resorption (osteolysis) (5–7), typically seen in Charcot neuroarthropathy (CN). The signaling pathway of the receptor activator of nuclear factor-κB (RANK), RANK ligand (RANKL), and its decoy receptor osteoprotegerin (OPG) has been suggested as the link between vascular and bone metabolism (8,9). In fact, RANKL has been shown to mediate osteolysis in CN by stimulating osteoclastic differentiation of monocytes/macrophages, an effect that is attenuated by OPG, the decoy receptor (10). This has led to nascent theories implicating RANKL/OPG signaling as the potential pathogenetic basis for CN.RANKL exists in two biologically active soluble forms secreted by T cells, endothelial cells, or osteoblasts or proteolytically cleaved from cell surfaces. RANKL binds to its target receptor RANK on cell surfaces (including vascular smooth muscle cells [VSMCs]) to generate multiple intracellular signals that regulate cell differentiation, function, and survival (8,11,12). In the vasculature, RANKL is expressed and upregulated in calcifying vascular cells (13) and enhances the recruitment and infiltration of cells that have been shown to stimulate VSMC mineralization (14).Most of the evidence for a direct role of RANKL/OPG signaling in vascular calcification is derived from animal studies with limited human data. For instance, with the use of VSMCs from rat aorta, RANKL has been shown to increase VSMC calcification via activation of the alternate nuclear factor-κB (NF-κB) pathway (15). However, to enhance translational applications, we extend these data to human VSMCs and use of patient serum.In diabetic CN, there is osteolysis and simultaneous vascular calcification, potentially leading to amputation (16,17). Therefore, the aim of this study was to determine the role of RANKL/OPG signaling in MAC in diabetic CN by using an in vitro model of vascular calcification.