Hyperphosphatemia-induced nanocrystals upregulate the expression of bone morphogenetic protein-2 and osteopontin genes in mouse smooth muscle cells in vitro

Kidney Int. 2011 Feb;79(4):414-22. doi: 10.1038/ki.2010.390. Epub 2010 Oct 13.

Abstract

Vascular calcification, which contributes to cardiovascular disease in patients with uremic hyperphosphatemia, is associated with vascular cell expression of osteogenic genes, including bone morphogenetic protein (BMP)-2 and osteopontin (OPN). High inorganic phosphate levels in vitro stimulate the osteogenic conversion of smooth muscle cells; however, the mechanism governing this is not clear. We found that high-phosphate medium increased the expression of BMP-2 and OPN in mouse smooth muscle cells in culture. However, this effect was lost in the presence of the mineralization inhibitor, pyrophosphate, suggesting a contribution of calcium phosphate crystals. Addition of 1-2 mmol/l phosphate alone to growth medium was sufficient to induce nanosized crystals after 1 day at 37 °C. Isolated crystals were about 160 nm in diameter and had a calcium to phosphate ratio of 1.35, consistent with the hydroxyapatite precursor octacalcium phosphate. Nanocrystal formation increased fourfold in the absence of serum, was blocked by fetuin-A, and was dependent on time and on the concentrations of phosphate and calcium. Purified synthetic hydroxyapatite nanocrystals and isolated high-phosphate-induced nanocrystals, but not nanocrystal-free high-phosphate medium, also induced BMP-2 and OPN. Thus, our results suggest that BMP-2 and OPN are induced by calcium phosphate nanocrystals, rather than soluble phosphate. This mechanism may contribute, in part, to hyperphosphatemia-related vascular cell differentiation and calcification.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • Bone Morphogenetic Protein 2 / genetics*
  • Calcinosis / etiology
  • Calcinosis / metabolism
  • Calcinosis / pathology
  • Calcium Phosphates / metabolism
  • Calcium Phosphates / pharmacology
  • Cell Differentiation
  • Cell Line
  • DNA Primers / genetics
  • Hyperphosphatemia / complications
  • Hyperphosphatemia / genetics*
  • Hyperphosphatemia / metabolism*
  • In Vitro Techniques
  • Mice
  • Microscopy, Electron, Scanning
  • Myocytes, Smooth Muscle / drug effects
  • Myocytes, Smooth Muscle / metabolism*
  • Myocytes, Smooth Muscle / pathology
  • Nanoparticles / chemistry*
  • Nanoparticles / ultrastructure
  • Osteogenesis / genetics
  • Osteopontin / genetics*
  • Up-Regulation / drug effects

Substances

  • Bmp2 protein, mouse
  • Bone Morphogenetic Protein 2
  • Calcium Phosphates
  • DNA Primers
  • Spp1 protein, mouse
  • Osteopontin
  • calcium phosphate