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Role of TGF-β1 Signaling in Heart Valve Calcification Induced by Abnormal Mechanical Stimulation in a Tissue Engineering Model.

Curr Med Sci. 2018 Oct;38(5):765-775. Epub 2018 Oct 20
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摘要


A tissue engineering model of heart valve calcification induced in a bioreactor was established to evaluate the calcification induced by abnormal mechanical stimulation and explore the underlying molecular mechanisms. Polyethylene glycol (PEG)-modified decellularized porcine aortic leaflets seeded with human valve interstitial cells (huVICs) were mounted on a Ti-Ni alloy frame to fabricate two-leaflet and threeleaflet tissue engineered valves. The two-leaflet model valves were exposed to abnormal pulsatile flow stimulation with null (group A), low (1000 mL/min, group B), medium (2000 mL/min, group C), and high velocity (3000 mL/min, group D) for 14 days. Morphology and calcification were assessed by von Kossa staining, alkaline phosphatase (ALP) content, and Runx2 immunostaining. Leaflet calcification and mRNA and protein expression of transforming growth factor (TGF)-β1, bone morphogenetic protein 2 (BMP2), Smad1, and MSX2 were measured at different time points. ALP content was examined in two-leaflet valves seeded with BMP2 shRNA plasmid-infected huVICs and exposed to the same stimulation conditions. The results showed that during 14 days of flow stimulation, huVICs on the leaflet surface proliferated to generate normal monolayer coverage in groups A, B, and C. Under mechanical stimulation, huVICs showed a parallel growth pattern in the direction of the fluid flow, but huVICs exhibited disordered growth in the high-velocity flow environment. von Kossa staining, ALP measurement, and immunohistochemical staining for Runx2 confirmed the lack of obvious calcification in group A and significant calcification in group D. Expression levels of TGF-β1, BMP2, and MSX2 mRNA and protein were increased under fluid stimulation. ALP production by BMP2 shRNA plasmid-infected huVICs on model leaflets was significantly reduced. In conclusion, abnormal mechanical stimulation in a bioreactor induced calcification in the tissue engineering valve model. The extent of calcification correlated positively with the flow velocity, as did the mRNA and protein levels of TGF-β1, BMP2, and MSX2. These findings indicate that TGF-β1/BMP2 signaling is involved in valve calcification induced by abnormal mechanical stimulation.

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