Retinas were harvested 4 h later and stained with ant-BrdU (Dako North America, Inc., Carpinteria, CA) and VE-Cadherin (BD PharMingen, San Diego, CA) antibodies. while suppressing ectopic pathological neovascularization. Our data demonstrate that Dll4 is usually induced by VEGF as a negative feedback regulator and acts to prevent overexuberant angiogenic sprouting, promoting the timely formation of a well differentiated vascular network. Keywords: angiogenesis, retina, Notch, oxygen-induced retinopathy Notch signaling pathways are evolutionarily conserved and play key functions in cell-fate determination and differentiation in many tissues during embryonic and postnatal development (1). Major components of the Notch pathway are expressed in the vasculature (2), and genetic deletion of certain Notch pathway components, including Notch1, Notch1/Notch4 (3, 4), Jagged1 (5), Delta-like ligand (Dll) 4 (6), Hey1/Hey2 (7), or presenilins (8, 9) results in embryonic lethality associated with vascular remodeling defects. Although most of these genes are expressed in multiple tissue and cell types, Dll4 is largely restricted to the vascular endothelium, suggesting that Dll4 is usually a key ligand for Notch receptors in the developing vasculature (6, 10, 11). During early embryonic development, genetic deletion of even a single Dll4 allele produces severe vascular abnormalities that result in embryonic lethality in most mouse strains (6, 12, 13). Indeed, of the many genes involved in vasculogenesis and angiogenesis, haploid insufficiency has been reported to result in major vascular defects and embryonic lethality only for Dll4 and VEGF-A (14, 15). Unfortunately, early embryonic lethality precludes most experimental manipulations, making it difficult to precisely understand the role of Lidocaine hydrochloride Dll4 during vascular development and in pathological settings. To overcome this limitation, we have studied the effects of Dll4 gene deletion Rabbit Polyclonal to Patched in mice of the outbred ICR strain, in which haploinsufficiency produces only limited embryonic lethality (6, 12). We then compared the vascular phenotype observed in these mutant mice to that obtained in wild-type mice in which Dll4/Notch signaling was selectively inhibited by intravitreal Lidocaine hydrochloride injection of Dll4-Fc or a neutralizing antibody against the extracellular domain name of Dll4. For these experiments, we selected the retina as a model system because the retinal vasculature develops postnatally in a stereotypic manner that is highly organized, temporally and spatially (16). Moreover, the murine model of oxygen-induced ischemic retinopathy (OIR) (17) is usually a well characterized model of pathological neovascularization associated with elevated expression of endogenous proangiogenic factors, including Lidocaine hydrochloride VEGF (18, 19), and thus relevant to pathological angiogenesis associated with diverse disease conditions (20). Finally, the retinal vasculature is usually readily accessible to experimental manipulations, including intravitreal microinjections of experimental brokers. We report that during normal retinal vascular development, and in the OIR model, suppression of Dll4/Notch signaling markedly enhances angiogenic sprouting and promotes the formation of a denser primary capillary network. Consistent with this, we find that Dll4 expression is particularly prominent in the most active regions of vascular growth both during normal development and in the OIR model. We further demonstrate that Dll4 expression in these vessels is usually markedly suppressed by pharmacological inhibition of VEGF and that application of exogenous VEGF up-regulates Dll4 expression in normal retinal vessels. These data indicate that VEGF induces Dll4 expression as part of a negative regulatory loop, in which Dll4 acts as a potent endogenous inhibitor of vascular sprouting. Thus, by appropriately restraining VEGF-induced sprouting angiogenesis, Dll4 acts in concert with VEGF to promote the timely formation and differentiation of qualified vascular networks. Results Dll4 Is usually Highly Expressed in Angiogenic Blood Vessels. The retina of the mouse is usually avascular at birth. By the first postnatal day (P1), vascular sprouts emerge from the central vessels at the optic nerve head and begin to elaborate a primitive vascular plexus that rapidly extends across the retinal.