Vascular and valvular calcifications are a form of extra-skeletal ossification. The pathogenesis of extra-skeletal ossification is unknown, yet recent studies show that this highly regulated process involves the differentiation of mature vascular cells into osteochondrogenic cells and recapitulates embryonic endochondral ossification. Histone deacetylases (HDAC's) have a major role in chondrocytes and osteoblasts differentiation and maturation.
In order to assess the role of HDACs in vascular calcifications we used the MC3T3-E1 cell differentiation system. These cells have similar gene expression profile to heart valve cells, and under specific culture conditions can be induced to differentiate and mineralize into osteoblasts. We analyzed the expression levels of different HDACs during bone differentiation using qRT-PCR. This analysis showed that the expression levels of HDAC's 1, 3 and 4 were up-regulated, while HDAC 7 levels did not significantly change. Next, we induced differentiation of MC3T3-E1 cells into osteoblasts in the presence of TSA, an organic compound that's selectively inhibit class I mammalian HDAC's. We observed that TSA strongly inhibited bone differentiation. This was documented by the considerable decrease in alizarin red calcium staining and in alkaline phosphatase staining, and by the almost complete abolition of the bone marker genes Runx2, Osterix, Osteocalcin, BMP2 and Col1A1 expression, using qRT-PCR.
In conclusion TSA in a potent inhibitor of bone differentiation in the MC3T3-E1 cell model system of valvular cells. Further experiments are being conduct in vivo to determine the effect of TSA in valvular and vascular ossification.
