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

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  Figure 1. DEGs in stenosis and sclerosis samples were identified through bioinformatics. (a) Volcano plot of DEGs in stenosis and sclerosis samples. (b) Heatmap of DEGs in stenosis and sclerosis samples. (c) and (d) Bar graph of functional enrichment analysis performed through DAVID. Abbreviation: DEGs: differentially expressed genes.

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  Figure 2. THBS2 was high-expressed in CAVD and related to increased ALP activity during the OD of VICs. (a) and (b) The immunoreactivity of THBS2 in AV endothelial cells (a) and calcified AV tissue (b) from normal and CAVD samples was evaluated by immunohistochemistry (scale: 100 μm; magnification: ×200). (c) Relative mRNA expression of THBS2 in CAVD and adjacent normal tissues was assessed through qRT-PCR. GAPDH was a loading control. (d and e) Relative mRNA (d) and protein (e) expressions of THBS2 in VICs during the process of OD were tested by qRT-PCR and Western blot. GAPDH was a loading control. (f) ALP activity of VICs during the process of OD was detected through colorimetry. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 vs. control group. All experiments were repeated independently at least three times. Data were expressed as the means ± standard deviation. Abbreviations: THBS2: thrombospondin 2; CAVD: calcific aortic valve disease; ALP: alkaline phosphatase; OD: osteogenic differentiation; VICs: valve interstitial cells; AV: aortic valve; qRT-PCR: quantitative reverse transcription-polymerase chain reaction; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

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  Figure 3. THBS2 was related to increased calcic nodule formation and the expressions of factors associated with OD and ECM during the OD of VICs. (a) Calcic nodule formation of VICs during the process of OD was observed by Alizarin Red staining (scale: 200 μm; magnification: ×100). (b) and (c) Relative mRNA expressions of osteocalcin, Runx2, SPARC, COL1A2, and COL1A1 (b) and SPP1, CTGF, MMP-2, and MMP-13 (c) in VICs during the process of OD were determined through qRT-PCR. GAPDH was a loading control. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 vs. control group. All experiments were repeated independently at least three times. Data were expressed as the means ± standard deviation. Abbreviations: THBS2: thrombospondin 2; OD: osteogenic differentiation; ECM: extracellular matrix; VICs: valve interstitial cells; 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-; qRT-PCR: quantitative reverse transcription-polymerase chain reaction; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

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  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. #p < 0.05, ##p < 0.01, ###p < 0.001 vs. vector group. All experiments were repeated independently at least three times. Data were performed as the means ± standard deviation. Abbreviations: THBS2: thrombospondin 2; OD: osteogenic differentiation; VICs: valve interstitial cells; shTHBS2: short hairpin THBS2; qRT-PCR:…

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  Figure 5. THBS2 negatively regulated Akt/NF-κB signaling pathway during OD procedure of VICs. (a–c) Representative images of protein bands during Western blot (a) and the levels of p-Akt/Akt (b) and p-p65/p65 (c) of VICs transfected with shTHBS2 and THBS2 overexpression plasmid were assessed by Western blot after the induction of OD. GAPDH was a loading control. (d) Relative expression of p65 in nucleus of VICs transfected with shTHBS2 and THBS2 overexpression plasmid was assessed by Western blot after the induction of OD. Lamin B1 was a loading control. (e) Relative mRNA expressions of THBS2, Akt, and p65 in VICs transfected with shTHBS2 and treated with LY294002 were tested by qRT-PCR after the induction of OD. GAPDH was a loading control. (f) Representative images of protein bands during Western blot. (g and h) The levels of p-Akt/Akt (g) and p-p65/p65 (h) of VICs transfected with shTHBS2 and treated with LY294002 were determined through Western blot after the induction of OD. GAPDH was a loading control. (i) Relative protein expression of THBS2 in VICs transfected with shTHBS2 and treated with LY294002 was determined through Western blot after the induction of OD. GAPDH was a loading…

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  Figure 6. THBS2 mediated the OD of VICs via inhibition of Akt/NF-κB signaling pathway. (a) ALP activity of VICs transfected with shTHBS2 and treated with LY294002 was detected through colorimetry after the induction of OD. (b) Calcic nodule formation of VICs transfected with shTHBS2 and treated with LY294002 was measured by Alizarin red staining after the induction of OD (scale: 200 μm; magnification: ×100). (c) and (d) Relative mRNA expressions of osteocalcin, Runx2, SPARC, COL1A2, and COL1A1 (c) and SPP1, CTGF, MMP-2, and MMP-13 (d) in VICs transfected with shTHBS2 and treated with LY294002 were determined through qRT-PCR after the induction of OD. GAPDH was a loading control. ###p < 0.001 vs. vector group; ∗p < 0.05, ∗∗∗p < 0.01 vs. shTHBS2 group; ∧∧p < 0.01; ∧∧∧p < 0.001 vs. LY294002 group. All experiments were repeated independently at least three times. Data were performed as the means ± standard deviation. Abbreviations: THBS2: thrombospondin 2; OD: osteogenic differentiation; VICs: valve interstitial cells; ALP: alkaline phosphatase; shTHBS2: short hairpin THBS2; Runx2: Runt-related transcription factor-2; SPARC: secreted protein acidic and rich in cysteine; COL1A: collagen type I…