Vascular endothelial (VE) cadherin is a key component of endothelial adherens junctions (AJs) and plays an important role in maintaining vascular integrity. Endocytosis of VE-cadherin regulates junctional strength and a decrease of surface VE-cadherin reduces vascular stability. However, disruption of AJs is also a requirement for vascular sprouting. Identifying novel regulators of endothelial endocytosis could enhance our understanding of angiogenesis. Here, we evaluated the angiogenic potential of (CKLF-like MARVEL transmembrane domain 4) CMTM4 and assessed in which molecular pathway CMTM4 is involved during angiogenesis. Using a 3D vascular assay composed of GFP-labeled HUVECs and dsRED-labeled pericytes, we demonstrated in vitro that siRNA-mediated CMTM4 silencing impairs vascular sprouting. In vivo, CMTM4 silencing by morpholino injection in zebrafish larvae inhibits intersomitic vessel growth. Intracellular staining revealed that CMTM4 colocalizes with Rab4+ and Rab7+ vesicles, both markers of the endocytic trafficking pathway. CMTM4 colocalizes with both membrane-bound and internalized VE-cadherin. Adenovirus-mediated CMTM4 overexpression enhances the endothelial endocytic pathway, in particular the rapid recycling pathway, shown by an increase in early endosomal antigen-1 positive (EEA1+), Rab4+, Rab11+ , and Rab7+ vesicles. CMTM4 overexpression enhances membrane-bound VE-cadherin internalization, whereas CMTM4 knockdown decreases internalization of VE-cadherin. CMTM4 overexpression promotes endothelial barrier function, shown by an increase in recovery of transendothelial electrical resistance (TEER) after thrombin stimulation. We have identified in this study a novel regulatory function for CMTM4 in angiogenesis. CMTM4 plays an important role in the turnover of membrane-bound VE-cadherin at AJs, mediating endothelial barrier function and controlling vascular sprouting.

Additional Metadata
Keywords Adherens junctions, Angiogenesis, CMTM4, Endothelial cells, VE-cadherin
Persistent URL dx.doi.org/10.1007/s10456-018-9638-1, hdl.handle.net/1765/109776
Journal Angiogenesis
Citation
Chrifi, I, Louzao-Martinez, L, Brandt, M.M, Van Dijk, C.G.M, Bürgisser, P.E, Zhu, C, … Cheng, C. (2018). CMTM4 regulates angiogenesis by promoting cell surface recycling of VE-cadherin to endothelial adherens junctions. Angiogenesis. doi:10.1007/s10456-018-9638-1