Runx2/Cbfa1, but not loss of myocardin, is required for smooth muscle cell lineage reprogramming toward osteochondrogenesis

Abstract

Vascular calcification is a major risk factor for cardiovascular morbidity and mortality. Smooth muscle cells (SMCs) may play an important role in vascular cartilaginous metaplasia and calcification via reprogramming to the osteochondrogenic state. To study whether SM lineage reprogramming and thus matrix calcification is reversible and what the necessary regulatory factors are to reverse this process, we used cells isolated from calcifying arterial medias of 4-week-old matrix Gla protein knockout mice (MGP−/− SMCs). We found that vascular cells with an osteochondrogenic phenotype regained SMC properties (positive for SM22α and SM α-actin) and down-regulated osteochondrogenic gene expression (Runx2/Cbfa1 and osteopontin) upon culture in medium that favors SMC differentiation. Over time, the MGP−/− SMCs no longer expressed osteochondrogenic proteins and became indistinguishable from wild-type SMCs. Moreover, phenotypic switch of the restored SMCs to the osteochondrogenic state was re-induced by the pro-calcific factor, inorganic phosphate. Finally, loss- and gain-of-function studies of myocardin, a SM-specific transcription co-activator, and Runx2/Cbfa1, an osteochondrogenic transcription factor, revealed that upregulation of Runx2/Cbfa1, but not loss of myocardin, played a critical role in phosphate-induced SMC lineage reprogramming and calcification. These results are the first to demonstrate reversibility of vascular SMCs to an osteochondrogenic state in response to local environmental cues, and that myocardin-enforced SMC lineage allocation was not sufficient to block vascular calcification. On the other hand, Runx2/Cbfa1 was found to be a decisive factor identified in the process.

5-aza-2´-Deoxycytidine, a DNA Methyltransferase Inhibitor, Facilitates the Inorganic Phosphorus-Induced Mineralization of Vascular Smooth Muscle Cells

“…Our previous study also showed that an increased ALP expression contributes to the promotion of vascular calcification in the presence of trichostatin A (TSA), a representative histone deacetylase (HDAC) inhibitor 40) . Accordingly, in the present study, the increased ALP expression induced by 5-azadC promoted the mineralization of HASMCs, while suppression of the ALP expression inhibited the development of Pi-induced vascular calcification with or A B C lar calcification 41) . Therefore, the upregulation of SM lineage genes was not associated with the 5-aza-dCpromoted mineralization of HASMCs in the present study.…”

Runx2 deletion in smooth muscle cells inhibits vascular osteochondrogenesis and calcification but not atherosclerotic lesion formation

“…Given previous studies suggesting a role for Runx2 in osteochondrogenic differentiation of SMCs in vitro 34,35 and in vivo, 12,21,33 we were surprised that deletion of Runx2 in SMCs did not change the overall content of chondrocyte-like cells in the intima. Our studies show that while SMC expression of Runx2 was not required for initial chondrogenesis, it was necessary for maturation of chondrocytes to a promineralizing, hypertrophic state, leading to endochondral ossification.…”

Characterization of Pax3-expressing cells from adult blood vessels

“…This might be due to absence of Runx2 upregulation (supplementary material Fig. S3B), which was shown to be crucial for vascular smooth muscle cells to express osteochondrogenic markers (Speer et al, 2010).…”