Combined effects of ascorbic acid and phosphate on rat VSMC osteoblastic differentiation

Background. Ascorbic acid (AA) supplementation has been suggested to afford erythropoietin hyporesponsiveness and high levels of ferritin in haemodialysis (HD) patients. However, little is known about the possible side effects of this policy on vascular calcification (VC). VC, induced by a high-phosphate and uraemic milieu, is characterized by a passive deposition of calcium-phosphate (Ca-P) and an active transformation of vascular smooth muscle cells (VSMCs) in osteoblastic-like cells. The aim of these studies was to characterize the combined effects of AA and P on VC. Materials and methods. Rat VSMCs were challenged with inorganic P (Pi) and AA, and Ca deposition analysis was performed to quantify VC. To investigate VSMC osteoblastic differentiation, we analysed alpha-actin protein content and core-binding factor alpha-1 (Cbf alpha 1/RUNX2) messenger RNA (mRNA) expression. Results. When incubated with 5 mM Pi, VSMCs showed a significant increase in Ca deposition compared to control cells. Interestingly, the addition of AA in the calcification medium resulted in a dose-dependent increase in Pi-induced Ca deposition. At the same time, the combined effect of AA and Pi on VSMCs resulted in the reduction of alpha-actin protein content and in a 4-fold increase of Cbfa1/RUNX2 mRNA expression. Conclusions. We demonstrated that AA combined with Pi increases Ca deposition in rat VSMCs. The role of AA as cofactor in osteoblastic differentiation was demonstrated by phenotypic changes in VSMCs and enhanced bone mineralization key gene expression. These in vitro preliminary data suggest a potential role for AA combined with Pi in worsening VC.