Effect of metformin on autophagy in human umbilical vein endothelial cells

Abstract

Objective To observe the effects of metformin on the autophagy of endothelial cells and explore the underlying mechanism. Methods Human umbilical vein endothelial cells (HUVECs) were treated with 0, 2.5, 5, 10, 20 and 40 mmol/L metformin for 24 h or with 5 mmol/L metformin for 24, 48 and 72 h, and cell survival after the treatment was assessed using CCK-8 assay. The formation of autophagosomes in HUVECs treated with metformin and transforming growth factor-β1 (TGF-β1), alone or in combination, was observed with transmission electron microscopy, and the expression levels of autophagy-related molecules LC3II and Beclin-1 were detected using immunofluorescence assay and Western blotting. The effects of XMU-MP-1 (an inhibitor of Hippo signaling pathway) and verteporfin (a blocker of Hippo signal transcription factor YAP) in the absence or presence of metformin or TGF-β1 on the expression levels of LC3II, YAP, pYAP and CTGF (the target protein of pYAP) were detected using Western blotting. Results CCK-8 assay showed that compared with the control cells, HUVECs treated with 10, 20 and 40 mmol/L metformin for 24 h exhibited significantly reduced cell survival rates (all P < 0.01), and we therefore used 5 mmol/L metformin for 24 h in the subsequent experiments. The results of electron microscopy, immunofluorescence assay and Western blotting showed that treatment with TGF-β1 significantly increased the number of autophagosomes, enhanced the expression of LC3II and Beclin-1 (P < 0.05), lowered the expression of pYAP (P < 0.01), and up-regulated CTGF expression in HUVECs (P < 0.01). These changes were significantly blocked by metformin. The expression of levels LC3II and CTGF were also inhibited in the cells by treatment with verteporfin and TGF-β1 (P < 0.01). XMU-MP-1 showed similar effects to TGF-β1 and significantly up-regulated the expression of LC3II (P < 0.01), decreased the expression of pYAP (P < 0.01), and enhanced the expression of CTGF (P < 0.01); such effects of XMU-MP-1 were also significantly blocked by metformin (all P < 0.05). The expression levels of CTGF and LC3II were obviously down-regulated in the cells treated with both verteporfin and XMU-MP-1 (P < 0.01). Conclusion Metformin may protect the veins by inhibiting TGF-β1-induced autophagy and blocking the activation of YAP signaling in the endothelial cells.

Keywords

• vascular injury
• endothelial cells
• autophagy
• transforming growth factor-β1
• metformin
• yes-associated protein