Calcific
aortic valve disease (CAVD) is characterized by valvular
fibrosis and calcification and driven by differentiating valvular interstitial cells (VICs). Expression data from patient biopsies suggest that
transforming growth factor (TGF)-β1 is implicated in CAVD pathogenesis. However, CAVD models using isolated VICs failed to deliver clear evidence on the role of TGF-β1. Thus, employing cultures of aortic valve leaflets, we investigated effects of TGF-β1 in a tissue-based three-dimensional (3-D) CAVD model. We found that TGF-β1 induced phosphorylation of
Mothers against decapentaplegic homolog (SMAD) 3 and expression of SMAD7, indicating effective downstream signal transduction in valvular tissue. Thus, TGF-β1 increased VIC contents of rough endoplasmic reticulum, Golgi, and secretory vesicles as well as tissue levels of
RNA and
protein. In addition, TGF-β1 raised expression of proliferation marker
cyclin D1, attenuated VIC apoptosis, and upregulated VIC density. Moreover, TGF-β1 intensified myofibroblastic VIC differentiation as evidenced by increased α-smooth muscle actin and
collagen type I along with diminished
vimentin expression. In contrast, TGF-β1 attenuated phosphorylation of SMAD1/5/8 and upregulation of β-
catenin while inhibiting osteoblastic VIC differentiation as revealed by downregulation of
osteocalcin expression,
alkaline phosphatase activity, and extracellular matrix incorporation of
hydroxyapatite. Collectively, these effects resulted in blocking of valvular tissue calcification and associated disintegration of
collagen fibers. Instead, TGF-β1 induced development of
fibrosis. Overall, in a tissue-based 3-D CAVD model, TGF-β1 intensifies expressional and proliferative activation along with myofibroblastic differentiation of VICs, thus triggering dominant
fibrosis. Simultaneously, by inhibiting SMAD1/5/8 activation and canonical Wnt/β-
catenin signaling, TGF-β1 attenuates osteoblastic VIC differentiation, thus blocking valvular tissue calcification. These findings question a general phase-independent CAVD-promoting role of TGF-β1.NEW & NOTEWORTHY Employing aortic valve leaflets as a tissue-based three-dimensional disease model, our study investigates the role of
transforming growth factor (TGF)-β1 in calcific
aortic valve disease pathogenesis. We find that, by activating
Mothers against decapentaplegic homolog 3, TGF-β1 intensifies expressional and proliferative activation along with myofibroblastic differentiation of valvular interstitial cells, thus triggering dominant
fibrosis. Simultaneously, by inhibiting activation of
Mothers against decapentaplegic homolog 1/5/8 and canonical Wnt/β-
catenin signaling, TGF-β1 attenuates apoptosis and osteoblastic differentiation of valvular interstitial cells, thus blocking valvular tissue calcification. These findings question a general phase-independent calcific
aortic valve disease-promoting role of TGF-β1.