Abstract
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.
Highlights
Angiogenesis is a critical process that occurs during embryonic development, is re-activated in wound healing, and is often disturbed in disease
We investigated the angiogenic potential of CMTM4 and assessed in which molecular pathways CMTM4 is involved in endothelial cells (ECs)
The function of CMTM4 was investigated in vitro using Short interference RNA (siRNA)-mediated silencing in human umbilical vein endothelial cells (HUVECs). Both quantitative real-time PCR (qPCR) and Western blot analysis confirmed efficient silencing of CMTM4 in cells transfected with two different siRNA sets specific for CMTM4, compared to untreated cells and cells transfected with a pool of non-targeting siRNA sequences (Fig. 1a, b)
Summary
Angiogenesis is a critical process that occurs during embryonic development, is re-activated in wound healing, and is often disturbed in disease. Acquiring and maintaining vascular homeostasis is crucial for the formation and stabilization of new blood vessels during angiogenesis. In order for the endothelial cells (ECs), that form the inner lining of new blood vessels, to function in an integrated manner, specific junctions consisting of cell adhesion molecules are required. From these junctions, adherens junctions (AJs) are critical for cell–cell adhesion and are tightly regulated by complex molecular. Vascular endothelial (VE) cadherin is the main cell adhesion molecule found in endothelial AJs and plays an important role in maintaining endothelial barrier function [2, 3]. VE-cadherin-deficient mice suffer from severe vascular defects, including impaired vascular sprouting, limited organization of ECs into large mature vessels, and vessel regression, resulting in death at mid-gestation [5, 6]
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