Overexpressed Thrombospondin 2 Induced Osteogenic Differentiation of Valve Interstitial Cells via Inhibition of Akt/NF-κB Signaling Pathway to Promote Calcific Aortic Valve Disease Development

Figure 4

THBS2 mediated OD of VICs. (a) and (b) Relative mRNA (a) and protein (b) expressions of THBS2 in VICs transfected with shTHBS2 and THBS2 overexpression plasmid were evaluated by qRT-PCR and Western blot after the induction of OD. GAPDH was a loading control. (c) ALP activity of VICs transfected with shTHBS2 and THBS2 overexpression plasmid was detected through colorimetry after the induction of OD. (d) Calcic nodule formation of VICs transfected with shTHBS2 and THBS2 overexpression plasmid was observed by Alizarin red staining after the induction of OD (scale: 200 μm; magnification: ×100). (e and f) Relative mRNA expressions of osteocalcin, Runx2, SPARC, COL1A2, and COL1A1 (e) and SPP1, CTGF, MMP-2, and MMP-13 (f) in VICs transfected with shTHBS2 and THBS2 overexpression plasmid were determined through qRT-PCR after the induction of OD. GAPDH was a loading control. , , vs. vector group. All experiments were repeated independently at least three times. Data were performed as the . Abbreviations: THBS2: thrombospondin 2; OD: osteogenic differentiation; VICs: valve interstitial cells; shTHBS2: short hairpin THBS2; qRT-PCR: quantitative reverse transcription-polymerase chain reaction; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; ALP: alkaline phosphatase; Runx2: Runt-related transcription factor-2; SPARC: secreted protein acidic and rich in cysteine; COL1A: collagen type I alpha; SPP1: secreted phosphoprotein 1; CTGF: connective tissue growth factor; MMP-: matrix metalloproteinase-.

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