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



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.



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.



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.
Department of Clinical Research, University of Bern, Switzerland.
Parisa
P
Aghagolzadeh
Department of Clinical Research, University of Bern, Switzerland; National Centre of Competence in Research (NCCR) Kidney.ch, Switzerland.
Matthias
M
Bachtler
Department of Clinical Research, University of Bern, Switzerland; National Centre of Competence in Research (NCCR) Kidney.ch, Switzerland.
Rakesh
R
Bijarnia
Department of Clinical Chemistry, University Hospital Bern, Inselspital, Bern, Switzerland.
Christopher
C
Jackson
Department of Nephrology, Royal Melbourne Hospital, Melbourne, Victoria, Australia.
Edward R
ER
Smith
National Centre of Competence in Research (NCCR) Kidney.ch, Switzerland; Division of Molecular & Systems Toxicology, Department of Pharmaceutical Sciences, University of Basel, Basel, Switzerland.
Alex
A
Odermatt
Department of Clinical Research, University of Bern, Switzerland.
Ramin
R
Radpour
Department of Clinical Research, University of Bern, Switzerland; National Centre of Competence in Research (NCCR) Kidney.ch, Switzerland; Department of Clinical Chemistry, University Hospital Bern, Inselspital, Bern, Switzerland. Electronic address: andreas.pasch@insel.ch.
Andreas
A
Pasch
D017209:Apoptosis; D002118:Calcium / Q000097:blood; D002470:Cell Survival; D002478:Cells, Cultured; D003470:Culture Media; D016207:Cytokines / Q000378:metabolism; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D020868:Gene Silencing; D006801:Humans; D007249:Inflammation; D009131:Muscle, Smooth, Vascular / Q000166:cytology*; D032389:Myocytes, Smooth Muscle / Q000166:cytology* / Q000473:pathology; D010710:Phosphates / Q000097:blood / Q000737:chemistry; D010766:Phosphorylation; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*; D061205:Vascular Calcification / Q000473:pathology*
D003470:Culture Media; D016207:Cytokines; D010710:Phosphates; D047888:Receptors, Tumor Necrosis Factor, Type I; C506235:TNFRSF1A protein, human; D014409:Tumor Necrosis Factor-alpha; D002118:Calcium
CPP; Calciprotein particles; Inflammation; Mineralization; TNF-α; Vascular smooth muscle cells