STUDIO DEI MECCANISMI PATOGENETICI COINVOLTI NELLA PROGRESSIONE DELLE CALCIFICAZIONI VASCOLARI INDOTTE DA ELEVATI LIVELLI DI FOSFORO
Creators: name: VOLPI, ELISA MARIA
Publisher: name: Universit� degli Studi di Milano
Year: 2012

description: Vascular Calcification (VC), induced by a high-phosphate, 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. 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 VC. The first aim of these studies was to characterize the combined effects of AA and P on VC. 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 ?-actin protein content and core-binding factor alpha-1 (Cbf?1/RUNX2) messenger RNA (mRNA) expression. 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 ?-actin protein content and in a 4-fold increase of Cbf?1/RUNX2 mRNA expression. 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. The second aim of this study was to investigate the effects of lanthanum chloride (LaCl3) on the progression of high Pi-induced Ca deposition in rat VSMCs. In fact, lanthanum is a Ca-free P-binder used in the treatment of hyperphosphatemia in patients affected by renal failure. In rat VSMCs, Pi-induced Ca deposition was inhibited by LaCl3, with a maximal effect at 100?M. Furthermore, we studied the effects on VC of calcium sensing receptor (CaSR) agonists. Gadolinium chloride, neomycin, spermine, and the calcimimetic calindol significantly inhibited Pi-induced VC. To investigate the hypothesis that LaCl3 reduces the progression of VC by interacting with the CaSR, we performed a concentration-response curve of LaCl3 in presence of a sub-effective concentration of calindol (10nM). Interestingly, this curve was shifted to the left, compared to the curve in the presence of LaCl3 alone. Additionally we analysed ?-actin and SM-22? protein content after LaCl3 treatment observing that LaCl3 maintains the proteins levels as high as controls?. We also studied the Cbf?1/RUNX2 and Matrix Gla Protein (MGP) mRNA expression. LaCl3 prevents the over expression of Matrix Gla Protein mRNA observed in cells treated with Pi alone, while it doesn?t affect the Cbf?1/RUNX2 mRNA increase induced by Pi. In conclusion, we demonstrated that lanthanum chloride effectively reduces the progression of high phosphate-induced VC. In addition, LaCl3 cooperates with the calcimimetic calindol in decreasing Ca deposition in this in vitro model. Moreover LaCl3 is able to prevent the VSMC dedifferentiation preserving the expression of smooth muscle cell lineage markers. These results suggest the potential role of lanthanum in the treatment of VC induced by high Pi.