CHMP4C Deficiency Represses Angiogenesis in Chronic Ischemia by Inhibiting Hypoxia-induced Wnt/β-catenin Signaling Activation via GSK3β

Abstract

Abstract Aims Angiogenesis is critical for tissue repair in chronic ischemia. Chromatin modified protein 4C (CHMP4C) is a subunit of the endosomal sorting complex required for transport -III(ESCRT-III), which is involved in endocytic progress and cell proliferation. Recent evidence suggests ESCRT-III plays a vital role in endothelial functioning. This study aimed to determine the role of endothelial CHMP4C in angiogenesis, as well as the underlying molecular mechanisms. Methods and Results Hind-limb ischemia was surgically induced in both CHMP4C-/- mice and wild-type C57BL/6J mice. Loss of CHMP4C was associated with significant decreases in blood perfusion and capillary density post-ischemia. In vitro, knockdown of CHMP4C by small interfering RNAs(siRNA) impaired the angiogenic and proliferate functions of ECs and induces G1/S cell cycle arrest without apoptotic effects under hypoxic conditions. RNA-Seq data and further analysis revealed the repression of Wnt/β-catenin pathway and hyperactivation of GSK3β in CHMP4C-deficiency ECs. And selective inhibition of GSK3β significantly ameliorated the inhibitory effects of CHMP4C deficiency on the Wnt/β-catenin pathway and proliferate functions in vitro. Electron microscopy and immunohistochemical colocalization analysis revealed that the CHMP4C deficiency impedes endocytic trafficking of GSK3β. Additionally, co-immunoprecipitation and proteomic analysis demonstrated CHMP4C interacted with PP2Ac and regulate GSK3β activity. The interaction between CHMP4C and PP2Ac may be crucial for GSK3β endocytic trafficking. Conclusion CHMP4C deficiency suppresses angiogenesis in chronic ischemia by disrupting Wnt/β-catenin signaling via GSK3β. The mechanism is associated with suspension of GSK3β endocytic trafficking.