CD93 promotes β1 integrin activation and fibronectin fibrillogenesis during tumor angiogenesis
Roberta Lugano, Kalyani Vemuri, Di Yu, Michael Bergqvist, Anja Smits, Magnus Essand, Staffan Johansson, Elisabetta Dejana, Anna Dimberg
Roberta Lugano, Kalyani Vemuri, Di Yu, Michael Bergqvist, Anja Smits, Magnus Essand, Staffan Johansson, Elisabetta Dejana, Anna Dimberg
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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 6

CD93 and MMRN2 regulate fibronectin fibrillogenesis in endothelial cells.

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CD93 and MMRN2 regulate fibronectin fibrillogenesis in endothelial cells...
(A) CD93 (green) and MMRN2 (red) colocalization in confluent HDBECs. High-magnification picture shows CD93-MMRN2 colocalizing in fibrillary structures. Scale bar: 20 μm. (B) CD93 (green) and fibronectin (red) costaining in control or MMRN2-silenced (siMMRN2_3) confluent HDBECs. Scale bars: 20 μm. (C) MMRN2 (green) and fibronectin (red) costaining in control or CD93-downregulated cells (siCD93_1). (D) Immunofluorescent staining of fibronectin (green) and F-actin (red) in control, siCD93-treated, and siMMRN2-treated HDBECs cultured under confluent conditions on gelatin-coated surfaces. Scale bars: 20 μm. (E) Real-time qPCR for fibronectin in control (Mock and siCtrl), CD93-downregulated (siCD93_1 and siCD93_2), and MMRN2-silenced cells (siMMRN2_3 and siMMRN2_6). Values represent mean ± SEM (3 independent experiments); 1-way ANOVA with Dunnett’s multiple-comparisons test. (F) Fibronectin (green) and F-actin (red) in brain endothelial cells isolated from WT and CD93–/– mice and cultured on gelatin-coated surfaces. Scale bars: 20 μm. (G) Rescue of fibronectin fibrillogenesis disrupted by CD93 downregulation. Immunofluorescent staining for fibronectin (green) and F-actin (red) in HDMECs stably expressing WT-CD93 insensitive to siRNA knockdown (WT-CD93 HDMEC) or untransfected cells (Control HDMEC) after endogenous CD93 silencing (siCD93_1 and siCD93_2). Scale bars: 20 μm. (H) Rescue of fibronectin fibrillogenesis upon MMRN2 downregulation (siMMRN2) in the presence or absence of conditioned medium derived from control cells (+Mock CM or +siCtrl CM). Immunofluorescent staining for fibronectin (green) and F-actin (red). Scale bars: 20 μm.