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

Abstract

Thrombospondin 2 (THBS2) is reported to participate in the development of calcific aortic valve disease (CAVD), while the effects are not elucidated completely. The study aimed to explore the role and mechanism of THBS2 in CAVD. Differentially expressed genes related to stenosis and sclerosis were screened through Limma package based on data from Gene Expression Omnibus (GEO), and the functional enrichment analysis was performed by the Database for Annotation, Visualization and Integrated Discovery (DAVID) database. The immunoreactivity of THBS2 in CAVD and normal samples was detected through immunohistochemistry. Valve interstitial cells (VICs) were transfected with short hairpin RNA against THBS2 (shTHBS2) and THBS2 overexpression plasmid and treated with LY294002 (Akt inhibitor) and induced osteogenic differentiation. The expression of THBS2 in CAVD and normal samples and the levels of THBS2, osteocalcin, Runx2, SPARC, COL1A2, COL1A1, SPP1, CTGF, MMP-2, MMP-13, Akt, p-Akt, p65, p-p65, and nuclear p65 in VICs were tested by qRT-PCR and Western blot. ALP activity was assessed using colorimetry. Calcic nodule formation was measured by Alizarin Red staining. THBS2 and PI3K-Akt pathway were differentially enriched in stenosis samples when compared with those in sclerosis samples. THBS2 expression was upregulated in CAVD and positively correlated with ALP activity, calcic nodule formation, osteogenic differentiation-related (osteocalcin, Runx2, SPARC, COL1A2, COL1A1, SPP1, and CTGF) and extracellular matrix– (ECM–) related (MMP-2 and MMP-13) factors in the process of osteogenic differentiation. ShTHBS2 suppressed ALP activity, calcic nodule formation, and osteogenic differentiation/ECM-related molecules while upregulating p-Akt/Akt, p-p65/p65, and nuclear p65 expressions in VICs during osteogenic differentiation. However, THBS2 overexpression had the opposite effect to shTHBS2, and LY294002 reversed the effect of shTHBS2. Collectively, overexpressed THBS2 induces the osteogenic differentiation of VICs via inhibiting Akt/NF-κB pathway to promote the development of CAVD.