Inhibition of Galectin-3 Alleviates Cigarette Smoke Extract-Induced Autophagy and Dysfunction in Endothelial Progenitor Cells

Abstract

Endothelial progenitor cells (EPCs) have the potential to repair damaged blood vessels and promote angiogenesis. Smoking, an important risk factor for cardiovascular diseases, is associated with impaired functions of EPCs. However, the underlying mechanisms remain unclear. The aim of the study was to investigate the effects of cigarette smoke extract (CSE) on autophagy and dysfunction of EPCs and the involvement of galectin-3 in its effects. EPCs were treated with 8% CSE for 24 h (without affecting cell viability). EPC functions were assessed by tube formation and migration capacity and intracellular ROS and eNOS expression. Autophagy was assessed by autophagic protein expression by Western blotting and immunofluorescence microscopy and autophagosome accumulation by transmission electron microscopy. Galectin-3 expression was measured by real-time PCR, Western blotting, and immunofluorescence microscopy, while phospho-AMPK and phospho-mTOR were measured by Western blotting. EPCs were transfected by shRNA-Gal-3 or shRNA-NC before treatment with CSE to examine the effects of galectin-3 on CSE-induced autophagy and dysfunction of EPCs. CSE-treated EPCs showed decreased tube formation and migration ability and eNOS expression but increased oxidative stress. CSE also induced autophagy which was characterized by a decrease in p62 protein, an increase in LC3B-II/I ratio, and accumulation of autophagosomes. CSE upregulated galectin-3 expression on EPCs. Inhibition of galectin-3 abrogated CSE-induced autophagy and dysfunction of EPCs. CSE activated phospho-AMPK and inhibited phospho-mTOR, and inhibition of galectin-3 abolished CSE’s effect on activating phospho-AMPK and inhibiting phospho-mTOR. In conclusion, our results suggest that galectin-3 mediates CSE-induced EPC autophagy and dysfunction, likely via the AMPK/mTOR signaling pathway.