Activated Notch4 Inhibits Angiogenesis: Role of β1-Integrin Activation

Abstract

Notch4 is a member of the Notch family of transmembrane receptors that is expressed primarily on endothelial cells. Activation of Notch in various cell systems has been shown to regulate cell fate decisions. The sprouting of endothelial cells from microvessels, or angiogenesis, involves the modulation of the endothelial cell phenotype. Based on the function of other Notch family members and the expression pattern of Notch4, we postulated that Notch4 activation would modulate angiogenesis. Using an in vitro endothelial-sprouting assay, we show that expression of constitutively active Notch4 in human dermal microvascular endothelial cells (HMEC-1) inhibits endothelial sprouting. We also show that activated Notch4 inhibits vascular endothelial growth factor (VEGF)-induced angiogenesis in the chick chorioallantoic membrane in vivo. Activated Notch4 does not inhibit HMEC-1 proliferation or migration through fibrinogen. However, migration through collagen is inhibited. Our data show that Notch4 cells exhibit increased β1-integrin-mediated adhesion to collagen. HMEC-1 expressing activated Notch4 do not have increased surface expression of β1-integrins. Rather, we demonstrate that Notch4-expressing cells display β1-integrin in an active, high-affinity conformation. Furthermore, using function-activating β1-integrin antibodies, we demonstrate that activation of β1-integrins is sufficient to inhibit VEGF-induced endothelial sprouting in vitro and angiogenesis in vivo. Our findings suggest that constitutive Notch4 activation in endothelial cells inhibits angiogenesis in part by promoting β1-integrin-mediated adhesion to the underlying matrix.

We thank Mina Bissell and Nancy Boudreau for providing the avian retroviral vector CK and Nancy Boudreau for advice on the chick CAM assay. We also thank Kelly McNagny for the Q2bn cell line, John Harlan for the 8A2 antibody, John A. Wilkins for the B44 antibody, and Louis F. Reichardt for the TASC antibody. The V2E9 antibody, developed by Alan F. Horwitz, was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, Iowa. Thanks are also due to Penny Costello for assistance with the setup of the chick CAM assay and Linda Hughes for immunohistochemical staining of CAM sections.

This research was supported by grants to A.K. from the Heart and Stroke Foundation of British Columbia and the Yukon and the National Cancer Institute of Canada with funds from the Canadian Cancer Society and the Canadian Breast Cancer Foundation (BC Chapter) and to L.L. from the Stowers Institute for Medical Research. B.L. was supported by a Doctoral Research Award from the Heart and Stroke Foundation of Canada. K.G.L. was supported by a Doctoral Research Award from the Canadian Institutes of Health Research and a Predoctoral Fellowship Award from the Department of the Army (DAMD17-01-1-0164). The U.S. Army Medical Research Acquisition Activity, Fort Detrick, Md., is the awarding and administering acquisition office. A.K. is a Clinician-Scientist of the Canadian Institutes of Health Research and a Scholar of the Michael Smith Foundation for Health Research.