Abstract 18931: CMTM3 Mediates Angiogenesis by Regulating Early Endocytosis of Ve-cadherin in Endothelial Cells
Introduction: Endocytosis plays a key role in regulating the vascular stability between endothelial cells (ECs) during angiogenesis and vascular homeostasis, by altering the bio-availability of Ve-cadherin-mediated adherens junctions (AJs). Ve-cadherin endocytosis will decrease the junctional strength thereby promoting neovessels to sprout. Identification of new mediators involved in endothelial endocytosis reveal better understanding of angiogenesis.
Hypothesis: CKLF-like MARVEL Transmembrane domain 3 (CMTM3) involves in angiogenesis by mediating Ve-cadherin uptake in early endosome trafficking.
Methods and Results: In vitro mRNA expression of CMTM3 was significantly upregulated in human umbilical vein ECs (HUVECs) in response to pericytes stimulations (p<0.05 n=3). Using a 3D collagen co-culture model of HUVECs-GFP and pericytes-dsRED, we demonstrated that siRNA-mediated CMTM3 silencing (siCMTM3) in HUVECs-GFP impaired 66.7% of tubule formation compared with sisham-treated group (p<0.05 n=8). However, silencing CMTM3 in pericytes-dsRED before co-culturing with HUVECs-GFP did not affect tubule formation. In vivo morpholino-induced silencing of CMTM3 in developing FLI-GFP expressing zebrafish larvae severely disrupted angiogenic sprouting of the intersegmental vessels from the dorsal aorta. CMTM3 knockdown in HUVECs did not affect proliferation or migration. Double immunofluorescent staining showed that CMTM3 co-localized with early endosome marker (EEA1), clathrin vesicles, and cytosolic Ve-cadherin in HUVECs transduced with recombinant adenoviral CMTM3 vector. CMTM3 overexpression in HUVECs enhanced endothelial endocytosis, showed by an increase in EEA1, clathrin, Rab7, and Rab11 vesicles. In addition, siCMTM3 reduced the number of Ve-cadherin-mediated AJs in vitro. Interestingly, transendothelial electrical resistance (TEER) measurements of HUVECs monolayers demonstrated that CMTM3 promoted loss of endothelial barrier function in thrombin-induced responses.
Conclusions: CMTM3 is a novel vascular modulatory factor in ECs involved in Ve-cadherin internalization by endocytic trafficking. CMTM3 plays a role in mediating vascular sprouting by regulating the cell-cell adhesion proteins at the AJs.
Author Disclosures: I. Chrifi: None. L. Louzao-Martinez: None. M.M. Brandt: None. C.G. van Dijk: None. P. Burgisser: None. C. Zhu: None. J.M. Kros: None. D.J. Duncker: None. C. Cheng: None.
- © 2016 by American Heart Association, Inc.