Calcium Phosphate Deposition With Normal Phosphate Concentration - Role of Pyrophosphate -

Abstract

Calcium phosphate deposition (CPD) is the hallmark of vascular smooth muscle cell (VSMC) calcification. CPD is a thermodynamically-favored process under physiological conditions. Hydroxyapatite, the most common calcium phosphate in calcified arteries, is passively formed during VSMC calcification, independently on any direct cellular activity. Furthermore, in recent years it has been demonstrated there is an anti-calcifying effect by extracellular pyrophosphate, an endogenous inhibitor of CPD, both in vitro and in vivo, which directly blocks hydroxyapatite formation. We have used the in vitro calcification model without cellular activity, by treating confluent rat aortic VSMC with paraformaldehyde. Fixed cells were incubated with the indicated media to obtain or inhibit calcification. The calcium content was determined colorimetrically. Calcification was observed after 3 weeks (21 days) using a physiological concentration of calcium (1.8 mmol/L) and phosphate (1 mmol/L). Calcium deposition was directly proportional to the amount of phosphate in the media, with a calcification rate of 3.5, 7.5, and 14.3 µg·cm⁻²·day⁻¹, using 1, 2, and 4 mmol/L of phosphate, respectively. Under physiological conditions, pyrophosphate inhibits CPD with an IC₅₀ of ≍200 nmol/L. CPD occurs under a physiological concentration of calcium and phosphate, but this deposition is completely inhibited in the presence of a physiological concentration of pyrophosphate (3-5 µmol/L).