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 ␤1-integrin-mediated adhesion to collagen. HMEC-1 expressing activated Notch4 do not have increased surface expression of ␤1-integrins. Rather, we demonstrate that Notch4-expressing cells display ␤1-integrin in an active, high-affinity conformation. Furthermore, using function-activating ␤1-integrin antibodies, we demonstrate that activation of ␤1-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 ␤1-integrinmediated adhesion to the underlying matrix.

No abstract

The effect of lipopolysaccharide (LPS) on endothelial cells is a key component of the inflammatory response seen in Gram-negative sepsis. LPS does not cause death of cultured human endothelial cells. However, when the expression of new proteins is inhibited by cycloheximide, microvascular endothelial cells in culture undergo apoptosis. This finding suggests that LPS induces apoptotic and antiapoptotic pathways, with the antiapoptotic response being dependent on the synthesis of new proteins. Concurrent activation of apoptotic and antiapoptotic pathways has previously been documented for tumor necrosis factor (TNF). In the case of TNF, the antiapoptotic signal has been attributed to at least two cytoprotective proteins: the Bcl-2 homologue, A1, and the zinc-finger protein, A20. In this study, we demonstrate that both these molecules are induced in microvascular endothelial cells by LPS. Enforced overexpression of either A1 or A20 inhibits LPS and cycloheximide-initiated apoptosis. Induction of A1 and A20 does not require synthesis of intermediary proteins, but is dependent on the presence of soluble CD14. In addition, we show that inhibition of signaling by the transcription factor, NF-κB, blocks accumulation of A1 and A20 mRNA. Taken together, our findings suggest that LPS directly induces expression of the cytoprotective proteins, A1 and A20, via a CD14-dependent pathway requiring activation of NF-κB. © 1998 by The American Society of Hematology.