Tumour necrosis factor-alpha in uraemic serum promotes osteoblastic transition and calcification of vascular smooth muscle cells via extracellular signal-regulated kinases and activator protein 1/c-FOS-mediated induction of interleukin 6 expression

Background. Vascular calcification is enhanced in uraemic chronic haemodialysis patients, likely due to the accumulation of midsize uraemic toxins, such as interleukin 6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha). Here we have assessed the impact of uraemia on vascular smooth muscle cell (VSMC) calcification and examined the role of IL-6 and TNF-alpha as possible mediators and, most importantly, its underlying signalling pathway in VSMCs. Methods. VSMCs were incubated with samples of uraemic serum obtained from patients treated with haemodialysis for renal failure in the Permeability Enhancement to Reduce Chronic Inflammation-I clinical trial. The VSMCs were assessed for IL-6 gene regulation and promoter activation in response to uraemic serum and TNF-alpha with reporter assays and electrophoretic mobility shift assay and for osteoblastic transition, cellular calcification and cell viability upon osteogenic differentiation. Results. Uraemic serum contained higher levels of TNF-alpha and IL-6 compared with serum from healthy individuals. Exposure of VSMCs to uraemic serum or recombinant TNF-alpha lead to a strong upregulation of IL-6 mRNA expression and protein secretion, which was mediated by activator protein 1 (AP-1)/c-FOS-pathway signalling. Uraemic serum induced osteoblastic transition and calcification of VSMCs could be strongly attenuated by blocking TNF-alpha, IL-6 or AP-1/c-FOS signalling, which was accompanied by improved cell viability. Conclusion. These results demonstrate that uraemic serum contains higher levels of uraemic toxins TNF-alpha and IL-6 and that uraemia promotes vascular calcification through a signalling pathway involving TNF-alpha, IL-6 and the AP-1/c-FOS cytokine-signalling axis. Thus treatment modalities aiming to reduce systemic TNF-alpha and IL-6 levels in chronic haemodialysis patients should be evaluated in future clinical trials.