CAMP STIMULATES OSTEOBLAST-LIKE DIFFERENTIATION OF CALCIFYING VASCULAR CELLS - POTENTIAL SIGNALING PATHWAY FOR VASCULAR CALCIFICATION

The role of the cAMP signaling pathway in vascular calcification was i nvestigated using calcifying vascular cells (CVC) derived from primary aortic medial cell cultures. We previously showed that CVC have fibro blastic morphology and express several osteoblastic differentiation ma rkers. After confluency, they aggregate into cellular condensations, w hich later mature into nodules where mineralization is localized. Here , we investigated the effects of cAMP on CVC differentiation because i t plays a role in both osteoblastic differentiation and vascular disea se. Dibutyryl-cAMP or forskolin treatment of CVC for 3 days induced os teoblast-like ''cuboidal'' morphology, inhibited proliferation, and en hanced alkaline phosphatase activity, all early markers of osteoblasti c differentiation. Isobutylmethylxanthine and cholera toxin had the sa me effects. Treatment of CVC with pertussis toxin, however, did not in duce the morphological change or increase alkaline phosphatase activit y, although it inhibited CVC proliferation to a similar extent. cAMP a lso increased type I procollagen production and gene expression of mat rix gamma-carboxyglutamic acid protein, recently shown to play a role in in vivo vascular calcification. cAMP inhibited the expression of os teopontin but did not affect the expression of osteocalcin and core bi nding factor, Prolonged cAMP treatment enhanced matrix calcium-mineral incorporation but inhibited the condensations resulting in diffuse mi neralization throughout the monolayer of cells. Treatment of CVC with a protein kinase A-specific inhibitor, KT5720, inhibited alkaline phos phatase activity and mineralization during spontaneous CVC differentia tion. These results suggest that the cAMP pathway promotes in vitro va scular calcification by enhancing osteoblast like differentiation of C VC.