Transforming Growth Factor-β1 Promotes Fibrosis and Attenuates Calcification in a Tissue-Based Three-Dimensional CAVD Model

 

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Objectives: Expression data from patient biopsies suggest that transforming growth factor (TGF)-β1 is implicated in CAVD pathogenesis. However, studies examining isolated valvular interstitial cells (VICs) as CAVD model 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.

Methods: Tissue culture: tricuspid aortic roots were isolated from hearts of 6 to 8 months old sheep. The aortic valve leaflets were excised, fixed on plastic rings and each cultured under different media conditions (M1: DMEM + 10% FCS, M2: M1 + 10 mM β-glycerol phosphate + 1.5 mM CaCl2, M3: M2 + 5 ng/mL TGF-β1) for 4 weeks. Stainings: for alizarin red staining cryosections were incubated for 2 min at RT with a 2% alizarin red-S solution. For ALP staining cryosections were incubated for 30 min at 37°C with a NBT/BCIP solution. For DAPI staining sections were incubated for 10 min at RT with a DAPI solution. Immunoblot: proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes via tank blot. Blotted membranes were probed with antibodies from cell signaling and sigma.

Results: TGF-β1 induced the phosphorylation of transcription factor mothers against decapentaplegic homolog (SMAD)3 (more than twofold increase, p < 0.01) indicating effective activation of downstream signaling in valvular tissue. Thus, TGF-β1 upregulated cell density along with protein tissue content (each with more than twofold increase, p < 0.01). Moreover, TGF-β1 intensified the myofibroblastic differentiation of VICs as evidenced by increased tissue expression of -smooth muscle actin (p < 0.05) and collagen type I (p < 0.001) along with diminished expression of vimentin (p < 0.01). In contrast, TGF-β1 inhibited the osteoblastic differentiation of VICs as revealed by downregulation of osteopontin expression (p < 0.05), alkaline phosphatase (p < 0.001) activity and ECM incorporation of hydroxyapatite (p < 0.001). Collectively, these TGF-β1-mediated effects resulted in macroscopically detectable blocking of valvular tissue calcification and development of distinct fibrosis.

Conclusion: In aortic valve leaflets employed as tissue-based 3-D CAVD model TGF-β1 intensifies expressional and proliferative activation along with myofibroblastic differentiation of VICs thus triggering dominant fibrosis. Simultaneously, TGF-β1 inhibits the osteoblastic differentiation of VICs thus blocking the calcification of valvular tissue. These findings question a general phase-independent CAVD-promoting role of TGF-β1.