Calcification of vascular smooth muscle cells is induced by secondary calciprotein particles and enhanced by tumor necrosis factor-α

Atherosclerosis. 2016 Aug;251:404-414. doi: 10.1016/j.atherosclerosis.2016.05.044. Epub 2016 May 27.


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.

Keywords: CPP; Calciprotein particles; Inflammation; Mineralization; TNF-α; Vascular smooth muscle cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Apoptosis
  • Calcium / blood
  • Cell Survival
  • Cells, Cultured
  • Culture Media
  • Cytokines / metabolism
  • Gene Expression Profiling
  • Gene Expression Regulation
  • Gene Silencing
  • Humans
  • Inflammation
  • Muscle, Smooth, Vascular / cytology*
  • Myocytes, Smooth Muscle / cytology*
  • Myocytes, Smooth Muscle / pathology
  • Phosphates / blood
  • Phosphates / chemistry
  • Phosphorylation
  • Receptors, Tumor Necrosis Factor, Type I / metabolism
  • Tumor Necrosis Factor-alpha / metabolism*
  • Vascular Calcification / pathology*


  • Culture Media
  • Cytokines
  • Phosphates
  • Receptors, Tumor Necrosis Factor, Type I
  • TNFRSF1A protein, human
  • Tumor Necrosis Factor-alpha
  • Calcium