CD93 promotes β1 integrin activation and fibronectin fibrillogenesis during tumor angiogenesis
Roberta Lugano, … , Elisabetta Dejana, Anna Dimberg
Roberta Lugano, … , Elisabetta Dejana, Anna Dimberg
Published May 15, 2018
Citation Information: J Clin Invest. 2018;128(8):3280-3297. https://doi.org/10.1172/JCI97459.
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Research Article Oncology Vascular biology

CD93 promotes β1 integrin activation and fibronectin fibrillogenesis during tumor angiogenesis

Abstract

Tumor angiogenesis occurs through regulation of genes that orchestrate endothelial sprouting and vessel maturation, including deposition of a vessel-associated extracellular matrix. CD93 is a transmembrane receptor that is upregulated in tumor vessels in many cancers, including high-grade glioma. Here, we demonstrate that CD93 regulates β1 integrin signaling and organization of fibronectin fibrillogenesis during tumor vascularization. In endothelial cells and mouse retina, CD93 was found to be expressed in endothelial filopodia and to promote filopodia formation. The CD93 localization to endothelial filopodia was stabilized by interaction with multimerin-2 (MMRN2), which inhibited its proteolytic cleavage. The CD93-MMRN2 complex was required for activation of β1 integrin, phosphorylation of focal adhesion kinase (FAK), and fibronectin fibrillogenesis in endothelial cells. Consequently, tumor vessels in gliomas implanted orthotopically in CD93-deficient mice showed diminished activation of β1 integrin and lacked organization of fibronectin into fibrillar structures. These findings demonstrate a key role of CD93 in vascular maturation and organization of the extracellular matrix in tumors, identifying it as a potential target for therapy.

Authors

Roberta Lugano, Kalyani Vemuri, Di Yu, Michael Bergqvist, Anja Smits, Magnus Essand, Staffan Johansson, Elisabetta Dejana, Anna Dimberg

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Figure 4

MMRN2 stabilizes CD93 in the filopodia and in the migrating front of HDBECs.

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MMRN2 stabilizes CD93 in the filopodia and in the migrating front of HDB...
(A) Immunofluorescent staining of CD93 (green), phalloidin (red), and nuclei (blue) in control (Mock and siCtrl) and MMRN2-silenced (siMMRN2_3 and siMMRN2_6) HDBECs cultured under nonconfluent conditions. High-magnification images show localization of CD93 in the filopodia. Scale bars: 20 μm. (B) CD93 (green) in the migrating front of control and siMMRN2-silenced cells at 6 hours after production of a double-sided scratch in the cell monolayer. Arrows indicate direction of migration. High-magnification pictures show CD93 localization in the migrating front of control and siMMRN2 cells. Scale bars: 20 μm. (C and D) Rescue experiment performed using a 2-well culture insert enabling coculturing of control cells and siMMRN2-transfected cells during migration. Immunofluorescent staining of CD93 (green), phalloidin (red), and nuclei (blue). Scale bars: 20 μm. CD93 localization was assessed after 6 hours of migration in control and siMMRN2 cells (C) and siMMRN2 cells cocultured with control cells (D). (E) Quantification of CD93 in the migrating front area. Bars represent arbitrary units (AU) of the CD93-positive signal at the migrating front area depicted by dotted line in C and D. *P < 0.05 vs. Mock, #P < 0.05 vs. siCtrl, §vs. siMMRN2_3, ‡vs. siMMRN2_6; 1-way ANOVA with Dunnett’s post-test. Values represent mean ± SEM (4 different areas along the migrating edge in 3 independent experiments). (F and G) Costaining of CD93 (green), MMRN2 (red), and phalloidin (gray) in the migrating front of control and siMMRN2 cells and siMMRN2 cells cultured during 24 hours in conditioned medium derived from control cells (+siCtrl CM). Scale bars: 20 μm. (H) Migration assay in control cells, MMRN2-downregulated cells, and siMMRN2-transfected cells cultured in control conditioned medium (CM). Values represent mean ± SEM (3 independent experiments); *P < 0.05, **P < 0.01; 1-way ANOVA with Dunnett’s multiple-comparisons test.