Thrombospondin-1 binds to integrin β3 to inhibit vascular calcification through suppression of NF-κB pathway.

Abstract

Vascular calcification is an important risk factor related to all-cause mortality of cardiovascular events in patients with chronic kidney disease (CKD). Vascular extracellular matrix (ECM) proteins have been demonstrated to regulate vascular calcification. ECM protein thrombospondin 1 (THBS1/TSP-1) plays a critical role in the regulation of vascular diseases. However, whether THBS1 is involved in vascular calcification in CKD patients remains unclear. In this study, RNA sequencing datasets from the Gene Expression Omnibus (GEO) database GSE146638 showed that THBS1 was upregulated in the aortas of CKD rats. Enzyme-linked immunosorbent assay (elisa) revealed that serum THBS1 levels were increased in CKD patients with thoracic calcification. Western blotting and immunofluorescence analysis showed that THBS1 expression was increased in calcified vascular smooth muscle cells (VSMCs) and arteries. THBS1 knockdown exacerbated rat VSMC calcification induced by high phosphorus and calcium, as shown by Alizarin red staining and calcium content assays. Conversely, THBS1 overexpression attenuated VSMC calcification and abdominal aortic calcification in rats with CKD. Moreover, addition of recombinant THBS1 protein inhibited calcification of VSMC_S and human arterial rings. Smooth muscle cell-specific knockout of THBS1 mice treated with vitamin D3 displayed aggravated aortic calcification. Mechanistically, the protein-protein interaction database STRING (http://string-db.org/) analysis and coimmunoprecipitation assays revealed THBS1 bound to integrin β3. Reduction of integrin β3 levels abrogated the protective effect of THBS1 on vascular calcification. RNA-seq analysis revealed that THBS1 overexpression modulated the nuclear factor-kappa B (NF-κB) signaling pathway. Of note, the inhibitory effect of THBS1 overexpression on the NF-κB signal was abolished by knockdown of integrin β3. In conclusion, THBS1 interacts with integrin β3 to inhibit vascular calcification through suppression of NF-κB signal, suggesting a promising therapeutic target for vascular calcification in CKD. © 2025 The Pathological Society of Great Britain and Ireland.