Bone Abstracts

The process of vascular calcification shares many similarities with that of skeletal mineralisation, and involves the phenotypic trans-differentiation of vascular smooth muscle cells (VSMCs) to osteoblastic and chondrocytic cells within a calcified environment. Various microRNAs (miRs) are known to regulate cell differentiation, however their role in mediating VSMC calcification has yet to be fully understood.

Murine VSMCs were cultured for up to 28 days in calcifying medium containing phosphate. Calcium deposition and gene expression of chondrocyte markers (aggrecan, collagen types II and X), osteoblast markers (osteocalcin, Runx2) and regulators of calcification (Ank, Enpp1, Pit-1) were significantly elevated by 7 days in VSMCs (P<0.05), confirming the chondro-osseous phenotype associated with vascular calcification. miR-microarray analysis revealed the significant down-regulation of a wide range of miRs by 9 days of culture, including miR-199b (270-fold), miR-29a (168-fold), miR-221 (108-fold), miR-222 (81-fold) and miR-31 (40-fold).

Following this microarray analysis, subsequent studies investigated the specific role of the miR-221/222 family in VSMC calcification. qPCR data confirmed the down-regulation of miR-221 (30%; P<0.01) and miR-222 (15.7%; P<0.05). VSMCs were transfected with mimics of miR221 (50 nM) and miR222 (50 nM), individually and in combination. Interestingly, an increase in calcium deposition was observed in the combined treatment (sevenfold; P<0.01) but not in individual miR treatments. These data suggest that miR-221 and miR-222 work concomitantly to alter the trans-differentiation of VSMCs and increase the rate of calcification in vitro.

The miR-221/222 family is known to target PTEN, a phosphatase involved in cell cycle regulation, in cancer cells. Western blot analysis confirmed a reduction in PTEN expression in calcifying VSMCs following transfection with miR-221 and miR-222 mimics in combination. Increased PTEN expression through miR-221/222 down-regulation may induce the phenotypic transition of VSMCs to osteoblastic and chondrocytic cells during calcification.