Bovine aortic smooth muscle cell (BASMC) cultures undergo mineralization on addition of the organic phosphate donor, β-glycerophosphate (βGP). Mineralization is characterized by apatite deposition on collagen fibrils and the presence of matrix vesicles, as has been described in calcified vascular lesions in vivo as well as in bone and teeth. In the present study, we used this model to investigate the molecular mechanisms driving vascular calcification. We found that BASMCs lost their lineage markers, SM22α and smooth muscle α-actin, within 10 days of being placed under calcifying conditions. Conversely, the cells gained an osteogenic phenotype as indicated by an increase in expression and DNA-binding activity of the transcription factor, core binding factor α1 (Cbfa1). Moreover, genes containing the Cbfa1 binding site, OSE2, including osteopontin, osteocalcin, and alkaline phosphatase were elevated. The relevance of these in vitro findings to vascular calcification in vivo was further studied in matrix GLA protein null (MGP ^−/− ) mice whose arteries spontaneously calcify. We found that arterial calcification was associated with a similar loss in smooth muscle markers and a gain of osteopontin and Cbfa1 expression. These data demonstrate a novel association of vascular calcification with smooth muscle cell phenotypic transition, in which several osteogenic proteins including osteopontin, osteocalcin, and the bone determining factor Cbfa1 are gained. The findings suggest a positive role for SMCs in promoting vascular calcification.

Vascular calcification is a major risk factor for cardiovascular morbidity and mortality. To develop appropriate prevention and/or therapeutic strategies for vascular calcification, it is important to understand the origins of the cells that participate in this process. In this report, we used the SM22-Cre recombinase and Rosa26-LacZ alleles to genetically trace cells derived from smooth muscle. We found that smooth muscle cells (SMCs) gave rise to osteochondrogenic precursor- and chondrocyte-like cells in calcified blood vessels of matrix Gla protein deficient (MGP ^−/− ) mice. This lineage reprogramming of SMCs occurred before calcium deposition and was associated with an early onset of Runx2/Cbfa1 expression and the downregulation of myocardin and Msx2. There was no change in the constitutive expression of Sox9 or bone morphogenetic protein 2. Osterix, Wnt3a, and Wnt7a mRNAs were not detected in either calcified MGP ^−/− or noncalcified wild-type (MGP ^+/+ ) vessels. Finally, mechanistic studies in vitro suggest that Erk signaling might be required for SMC transdifferentiation under calcifying conditions. These results provide strong support for the hypothesis that adult SMCs can transdifferentiate and that SMC transdifferentiation is an important process driving vascular calcification and the appearance of skeletal elements in calcified vascular lesions.

Vascular calcification is highly correlated with cardiovascular disease mortality, especially in patients with ESRD or diabetes. In addition to the devastating effects of inappropriate biomineralization seen in cardiac valvulopathies, calciphylaxis, and idiopathic arterial calcification, vascular calcification is now recognized as a marker of atherosclerotic plaque burden as well as a major contributor to loss of arterial compliance and increased pulse pressure seen with age, diabetes, and renal insufficiency. In recent years, several mechanisms to explain vascular calcification have been identified including (1) loss of inhibition, (2) induction of bone formation, (3) circulating nucleational complexes, and (4) cell death. Alterations in calcium (Ca) and phosphorus (P) balance as seen in patients with ESRD promotes vascular calcification via multiple mechanisms and may explain the alarmingly high levels of cardiovascular disease deaths in these patients. Strategies to control Ca and P levels in patients with ESRD have met with early success in preventing progression of vascular calcification. Whether or not vascular calcification can be reversed is not yet known, but exciting new studies suggest that this may be possible in the future.