Angiogenesis, the formation of new blood vessels from existing vasculature, is a multi-step process that plays a central role in embryogenesis and pathological phenomena. Vascular Endothelial Growth Factor (VEGF) is a key regulator of angiogenesis and is important for the degradation of extracellular matrix (ECM), as well as the subsequent proliferation, migration, and survival of endothelial cells. ECM components, including fibrins, collagens, and laminins, form a lamina around existing vasculature that must be degraded in order to form new vessels.
VEGF signaling via VEGFR-2 induces the expression of endothelial cell-derived matrix metalloproteases (MMPs), including MMP2 , MMP9 , and MT1-MMP , which degrade the matrix to allow for endothelial sprouting. MMPs are thus essential for angiogenesis, and their loss from either endothelium or inflammatory cells has been associated with severe angiogenic defects. At the same time, increased MMP activity has significant vascular consequences. MMPs are antagonized by Tissue Inhibitors of Matrix Metalloproteases (TIMPs), and a pathological increase in endothelial MMP over TIMP activity has been proposed to contribute to vessel wall thickening, abdominal aortic aneurysm formation, varicose veins, hypertension and preeclampsia [4–6]. MMPs are not, however, strictly proangiogenic; other more recently elucidated functions of this protein family include the mediation of vascular regression, as well as the generation of ECM fragments with antiangiogenic properties.
MMP2 and MMP9 belong to the class of gelatinases with collagen and fibrin as substrates and are expressed as inactive pro-proteins. MMP2 and MMP9 are processed to activate their metalloprotease activity. MT1-MMP is a member of the membrane associated or membrane-type MMPs [7–9]. In the vasculature, MT-MMPs are thought to be involved in the localized pericellular release of bioactive growth factors such as bFGF, VEGF, and TGF-beta, processing of pro-MMP2 into an activated MMP2, and degradation of ECM components including collagens and fibrins [10, 11]. MT1-MMP has been shown to be the most potent fibrinolytic MMP and has a critical role in the creation of channels in the ECM, into which endothelial cells migrate during sprouting angiogenesis [12, 13]. MT1-MMP function is also required for proper lumenization of the new vessel .
Notch signaling is an evolutionarily conserved pathway that regulates cell fate decisions. Notch proteins, Notch 1 through 4, act as receptors and their ligands Jagged (Jagged-1, 2) and Delta-like (Dll-1, 3, 4), are all transmembrane proteins . Upon ligand binding, the cytoplasmic domain of Notch is released by proteolytic cleavage via presenilin/γ-secretase , translocates to the nucleus, and interacts with the transcriptional repressor CSL (CBF1/Su(H)/Lag2), converting it to a transcriptional activator . An essential role for Notch signaling in arterial differentiation and vascular remodeling has been demonstrated by genetic studies of mice with targeted mutations in either Notch (Notch1
, and Notch1
), or Notch ligands (Jagged1
), reviewed in Shawber et al. . In addition, targeted activation of Notch4 specifically in endothelial cells disrupted vascular remodeling, resulting in embryonic lethality . These studies demonstrate that proper levels of Notch signaling are essential for patterning of the vasculature during a period of embryonic development that is known to be critically dependent on VEGF [14, 18].
Signaling via both VEGF and Notch is indispensable in vascular development, and it has become evident that these two pathways are interconnected. Heterozygous deficiency of Dll4 results in embryonic lethality with profound vascular defects, including defective arterial branching from the aorta and arterial regression, processes that also depend on VEGF signaling [19–21] suggesting Dll4 and VEGF work in concert. In cultured human arterial endothelial cells, VEGF, but not bFGF, induced expression of Notch1 and Dll4 . Furthermore, expression of Dll4 reduced VEGF/VEGFR-2 signaling, likely via downregulation of VEGFR-2 expression in cultured endothelial cells . A role for Notch signaling in tumor angiogenesis was originally hypothesized from the observation that VEGF induced Dll4 in the angiogenic endothelium of tumor xenografts  and blocking Dll4 functions resulted in dysregulated non-productive angiogenesis [24, 25].
In this study, we demonstrate that Notch mediates VEGF-induced MMP activity in endothelial cells. A Notch antagonist, called Notch1 decoy, blocked VEGF activation of Notch/CSL signaling, VEGF-induced HUVEC morphogenesis on both collagen and fibrin gels, and VEGF-induced fibrinolysis. Notch signaling upregulated the expression of MMP9 and MT1-MMP, and activated MMP2 and MMP9 in endothelial cells. Accordingly, we found that the Notch1 decoy-mediated suppression of HUVEC morphogenesis occurred via inhibition of MMP activity. Finally, Notch1 decoy suppressed endothelial MMP9 expression in an in vivo neovascularization model in mice. These data demonstrate that Notch directly regulates the endothelial cell response to VEGF via induction of MMPs.