TIMP3 attenuates vascular calcification by restoring autophagy in vascular smooth muscle cells through the STAT1/FOXO1 pathway.

Abstract

BACKGROUND: Vascular calcification, characterized by abnormal calcium phosphate minerals deposition in the vascular system, is closely associated with adverse cardiovascular outcomes, including chronic kidney disease (CKD). Autophagy, a conserved cellular degradation mechanism, has been shown to attenuate vascular smooth muscle cell (VSMC) calcification. Tissue inhibitor of metalloproteinases-3 (TIMP3) has been implicated in cardiovascular pathophysiology. However, the role of TIMP3 and molecular mechanisms in vascular calcification remains unknown. METHODS: A CKD mouse model and β-glycerophosphate (β-GP)-stimulated VSMCs was used to investigate the role of TIMP3 in vascular calcification. Vascular calcification was assessed using von Kossa staining, Alizarin Red S staining, and calcium quantification. Autophagy flux was evaluated via LC3 immunofluorescence and Western blotting for LC3II/I and p62. The involvement of the STAT1/FOXO1 signaling pathway was investigated using immunofluorescence and Western blotting. RESULTS: TIMP3 expression was significantly reduced in calcified arteries of CKD mice and β-GP-stimulated VSMCs. TIMP3 overexpression attenuated calcium deposition and Runx2 upregulation, while promoting autophagy flux, as indicated by increased LC3II/I ratio and decreased p62. Inhibition of autophagy with 3-methyladenine (3-MA) abolished the protective effects of TIMP3. Mechanistically, TIMP3 overexpression suppressed β-GP-induced STAT1 activation and restored FOXO1 expression, indicating that the STAT1/FOXO1 pathway mediates TIMP3-induced autophagy and calcification attenuation. In vivo, TIMP3 overexpression significantly reduced aortic calcification, decreased calcium deposition, and enhanced autophagy markers in CKD mice. CONCLUSION: TIMP3 attenuates vascular calcification by restoring autophagy and reducing the osteogenic phenotypic transformation in VSMCs through inhibition of the STAT1/FOXO1 signaling axis. These findings highlight TIMP3 as a potential therapeutic target for vascular calcification in CKD and associated cardiovascular diseases.