Vps15 is critical to mediate autophagy in AngII treated HUVECs probably by PDK1/PKC signaling pathway

Abstract

Aims: Vps15 is an important regulator on the activity of class III PI3K in autophagy induction. AngII plays a positive role of autophagy in the early protection of endothelial cells. In this study, the expression of Vps15 was knocked down using the specific shRNA to investigate the effects of Vps15 on cell autophagy, senescence and apoptosis in HUVECs stimulated by AngII. The associated cell signaling pathway was also explored.

Materials and methods: MDC staining was applied to show autophagic bodies. Cell senescence was detected using β-galactosidase staining. Cell apoptosis was examined by flow cytometry using Annexin V-FITC/PI staining. And western blot was used to evaluate the ratio of LC3-II/I and the activation of associated cell signaling pathway.

Key findings: Cell autophagy induced by AngII was inhibited in HUVECs transfected with Vps15-shRNA, while cell senescence and apoptosis were enhanced. Rescue experiment revealed that cell autophagy was activated after Vps15 reexpression, while cell senescence and apoptosis were inhibited. Moreover, the phosphorylations of PDK1 and PKC substrates were increased after AngII treatment, which were decreased by Vps15 knockdown. Pretreatment of cells with the inhibitor for PDK1 or PKC attenuated cell autophagy after AngII stimulation, yet promoted cell senescence and apoptosis. The phosphorylations of both PDK1 and PKC were inhibited in cells pretreated with PDK1 inhibitor. Only the activation of PKC was inhibited when the inhibitor for pan-PKC was used.

Significance: These results suggested that Vps15 was critical to the protective autophagy in HUVECs induced by AngII, and PDK1/PKC signaling pathway was probably involved.

Keywords: Angiotensin II; Autophagy; HUVEC; Senescence; Vps15.