<scp>ESCRT</scp> ‐ <scp>III</scp> Subunit <scp>CHMP4C</scp> Regulates Angiogenesis by Targeting Endocytic Trafficking of <scp>GSK3β</scp>

Angiogenesis is critical for tissue repair in chronic ischemia. Chromatin modified protein 4C (CHMP4C), a subunit of the endosomal sorting complex required for transport -III (ESCRT-III), is involved in endocytic progress and cell proliferation. Recent evidence suggests ESCRT-III plays a vital role in endothelial function. This study aimed to determine the role of endothelial CHMP4C in angiogenesis, as well as the underlying molecular mechanisms. Hind-limb ischemia (HLI) 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 post-ischemia capillary density. In vitro, knockdown of CHMP4C by small interfering RNAs (siRNAs) impaired the angiogenic and proliferative functions of endothelial cells (ECs) and induced G1/S cell cycle arrest under hypoxic conditions. RNA-Seq data and further analysis revealed repression of the Wnt/β-catenin pathway and hyperactivation of GSK3β in CHMP4C-deficiency ECs. Selective inhibition of GSK3β significantly ameliorated the inhibitory effects of CHMP4C deficiency on the Wnt/β-catenin pathway and proliferative functions in vitro. Electron microscopy and immunohistochemical colocalization analysis further revealed that CHMP4C deficiency impedes endocytic trafficking of GSK3β. Overall, these findings reveal that CHMP4C regulates angiogenesis by modulating endocytic trafficking of GSK3β, indentifying it as a potential therapeutic target for ischemic diseases.