RANKL enhances macrophage paracrine pro-calcific activity in high phosphate-treated smooth muscle cells: dependence on IL-6 and TNF-α.

BACKGROUND

Vascular calcification is highly correlated with cardiovascular disease (CVD) morbidity and mortality, and it is associated with inflammation. Receptor activator of NF-ĸB ligand (RANKL) inhibition in vivo has been shown to reduce vascular calcification in a mouse model of atherosclerosis. Therefore, we tested the hypothesis that RANKL regulates smooth muscle cell (SMC) calcification by modulating macrophage production of pro-calcific cytokines.

METHODS

We used a bone marrow-derived macrophage (BMDM)/SMC co-culture system and examined the effects of RANKL on BMDM activation and SMC matrix calcification.

RESULTS

Treatment with RANKL alone did not stimulate SMC calcification induced by elevated phosphate. BMDMs differentiated with macrophage colony-stimulating factor and placed in co-culture with SMCs increased phosphate-induced SMC calcification. RANKL added to the BMDM/SMC co-cultures further enhanced SMC calcification. Treatment of BMDMs with RANKL resulted in increased expression of IL-6 and TNF-α. Thus, increased expression of these pro-calcific cytokines in macrophages may mediate RANKL-induced SMC calcification in a paracrine fashion. Addition of neutralizing IL-6 and TNF-α antibodies together with RANKL treatment significantly reduced the RANKL induction of SMC calcification.

CONCLUSION

RANKL activation of pro-inflammatory and pro-calcific pathways in macrophages may contribute to vascular calcification and inflammation.