Calcification of Vascular Smooth Muscle Cells is Induced by Secondary Calciprotein Particles and Enhanced by Tumor Necrosis Factor-α

Background And Aims: Vascular calcification is prevalent in clinical states characterized by low-grade chronic inflammation, such as chronic kidney disease (CKD). Calciprotein particles (CPP) are calcium phosphate-containing nano-aggregates, which have been found in the blood of CKD patients and appear pro-inflammatory in vitro. The interplay of CPPs and inflammatory cytokines with regard to the calcification of vascular smooth muscle cells (VSMC), in vitro, has not been investigated yet.

Methods: Primary or secondary CPP were generated using phosphate-enriched culture medium (DMEM/10% FBS) incubated at 37 °C. Human VSMC were cultured with these media and mineralization was measured. Expression of TNF-α was detected by qPCR, ELISA and Western blot in calcified VSMC. To further characterize the significance of TNF-α and its receptors for the calcification of VSMC, RNA interference experiments using siTNF-α, siTNFR1 and siTNFR2 were performed.

Results: The addition of phosphate to cell culture medium containing DMEM/10% FBS led to the rapid formation of primary CPP, which underwent spontaneous transformation to secondary CPP. Exposure of VSMC towards secondary CPP led to pronounced and concentration-dependent calcification, whereas exposure towards primary CPP did not. Importantly, secondary CPP induced oxidative stress, and led to the up-regulation and release of TNF-α. Addition of TNF-α to the cell culture medium enhanced, whereas the suppression of endogenous TNF-α or TNF receptor type 1 (TNFR1) expression by siRNA, ameliorated calcification.

Conclusions: Secondary, but not primary CPP, induce VSMC calcification. Secondary CPP induce the expression and release of TNF-α, which enhances calcification via its receptor TNFR1.