Ihsan Chrifi1, Laura Louzao-Martinez1, Maarten Brandt1, Christian G M van Dijk1, Petra Burgisser1, Changbin Zhu1, Johan M Kros1, Dirk J Duncker1, Caroline Cheng2. 1. From the Division of Experimental Cardiology, Department of Cardiology, Thoraxcenter, Erasmus University Medical Center, Rotterdam, the Netherlands (I.C., M.B., P.B., D.J.D., C.C.); Division of Internal Medicine and Dermatology, Department of Nephrology and Hypertension, University Medical Center Utrecht, the Netherlands (L.L.-M., C.G.M.v.D., C.C.); Netherlands Heart Institute, Utrecht (L.L.-M.); and Department of Pathology, Erasmus University Medical Center, Rotterdam, the Netherlands (C.Z., J.M.K.). 2. From the Division of Experimental Cardiology, Department of Cardiology, Thoraxcenter, Erasmus University Medical Center, Rotterdam, the Netherlands (I.C., M.B., P.B., D.J.D., C.C.); Division of Internal Medicine and Dermatology, Department of Nephrology and Hypertension, University Medical Center Utrecht, the Netherlands (L.L.-M., C.G.M.v.D., C.C.); Netherlands Heart Institute, Utrecht (L.L.-M.); and Department of Pathology, Erasmus University Medical Center, Rotterdam, the Netherlands (C.Z., J.M.K.). c.cheng@erasmusmc.nl.
Abstract
OBJECTIVE: Decrease in VE-cadherin adherens junctions reduces vascular stability, whereas disruption of adherens junctions is a requirement for neovessel sprouting during angiogenesis. Endocytosis plays a key role in regulating junctional strength by altering bioavailability of cell surface proteins, including VE-cadherin. Identification of new mediators of endothelial endocytosis could enhance our understanding of angiogenesis. Here, we assessed the function of CMTM3 (CKLF-like MARVEL transmembrane domain 3), which we have previously identified as highly expressed in Flk1+ endothelial progenitor cells during embryonic development. APPROACH AND RESULTS: Using a 3-dimensional coculture of human umbilical vein endothelial cells-GFP (green fluorescent protein) and pericytes-RFP (red fluorescent protein), we demonstrated that siRNA-mediated CMTM3 silencing in human umbilical vein endothelial cells impairs angiogenesis. In vivo CMTM3 inhibition by morpholino injection in developing zebrafish larvae confirmed that CMTM3 expression is required for vascular sprouting. CMTM3 knockdown in human umbilical vein endothelial cells does not affect proliferation or migration. Intracellular staining demonstrated that CMTM3 colocalizes with early endosome markers EEA1 (early endosome marker 1) and Clathrin+ vesicles and with cytosolic VE-cadherin in human umbilical vein endothelial cells. Adenovirus-mediated CMTM3 overexpression enhances endothelial endocytosis, shown by an increase in Clathrin+, EEA1+, Rab11+, Rab5+, and Rab7+ vesicles. CMTM3 overexpression enhances, whereas CMTM3 knockdown decreases internalization of cell surface VE-cadherin in vitro. CMTM3 promotes loss of endothelial barrier function in thrombin-induced responses, shown by transendothelial electric resistance measurements in vitro. CONCLUSIONS: In this study, we have identified a new regulatory function for CMTM3 in angiogenesis. CMTM3 is involved in VE-cadherin turnover and is a regulator of the cell surface pool of VE-cadherin. Therefore, CMTM3 mediates cell-cell adhesion at adherens junctions and contributes to the control of vascular sprouting.
OBJECTIVE: Decrease in VE-cadherin adherens junctions reduces vascular stability, whereas disruption of adherens junctions is a requirement for neovessel sprouting during angiogenesis. Endocytosis plays a key role in regulating junctional strength by altering bioavailability of cell surface proteins, including VE-cadherin. Identification of new mediators of endothelial endocytosis could enhance our understanding of angiogenesis. Here, we assessed the function of CMTM3 (CKLF-like MARVEL transmembrane domain 3), which we have previously identified as highly expressed in Flk1+ endothelial progenitor cells during embryonic development. APPROACH AND RESULTS: Using a 3-dimensional coculture of human umbilical vein endothelial cells-GFP (green fluorescent protein) and pericytes-RFP (red fluorescent protein), we demonstrated that siRNA-mediated CMTM3 silencing in human umbilical vein endothelial cells impairs angiogenesis. In vivo CMTM3 inhibition by morpholino injection in developing zebrafish larvae confirmed that CMTM3 expression is required for vascular sprouting. CMTM3 knockdown in human umbilical vein endothelial cells does not affect proliferation or migration. Intracellular staining demonstrated that CMTM3 colocalizes with early endosome markers EEA1 (early endosome marker 1) and Clathrin+ vesicles and with cytosolic VE-cadherin in human umbilical vein endothelial cells. Adenovirus-mediated CMTM3 overexpression enhances endothelial endocytosis, shown by an increase in Clathrin+, EEA1+, Rab11+, Rab5+, and Rab7+ vesicles. CMTM3 overexpression enhances, whereas CMTM3 knockdown decreases internalization of cell surface VE-cadherin in vitro. CMTM3 promotes loss of endothelial barrier function in thrombin-induced responses, shown by transendothelial electric resistance measurements in vitro. CONCLUSIONS: In this study, we have identified a new regulatory function for CMTM3 in angiogenesis. CMTM3 is involved in VE-cadherin turnover and is a regulator of the cell surface pool of VE-cadherin. Therefore, CMTM3 mediates cell-cell adhesion at adherens junctions and contributes to the control of vascular sprouting.
Authors: Hong S Lu; Ann Marie Schmidt; Robert A Hegele; Nigel Mackman; Daniel J Rader; Christian Weber; Alan Daugherty Journal: Arterioscler Thromb Vasc Biol Date: 2018-10 Impact factor: 8.311
Authors: Mahnaz Shariatzadeh; Maarten M Brandt; Caroline Cheng; Josianne C Ten Berge; Aniki Rothova; Pieter J M Leenen; Willem A Dik Journal: Eye (Lond) Date: 2018-05-10 Impact factor: 3.775
Authors: Ihsan Chrifi; Laura Louzao-Martinez; Maarten M Brandt; Christian G M van Dijk; Petra E Bürgisser; Changbin Zhu; Johan M Kros; Marianne C Verhaar; Dirk J Duncker; Caroline Cheng Journal: Angiogenesis Date: 2018-08-10 Impact factor: 9.596