The combination of a candidate gene approach, column chromatography, and mass spectrometry identifies several fibroblast-derived proteins essential for endothelial cell sprouting and lumen formation. Furthermore, proteins responsible for EC lumen formation increase matrix stiffness, which correlates with EC lumenogenesis.

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Pathological angiogenesis associated with wound healing often occurs subsequent to an inflammatory response that includes the secretion of cytokines such as tumor necrosis factor (TNF). Controversy exists on the angiogenic actions of TNF, with it being generally proangiogenic in vivo, but antiangiogenic in vitro. We find that whereas continuous administration of TNF in vitro or in vivo inhibits angiogenic sprouting, a 2-to 3-day pulse stimulates angiogenesis by inducing an endothelial "tip cell" phenotype. TNF induces the known tip cell genes plateletderived growth factor B (PDGFB) and vascular endothelial cell growth factor receptor-2 (VEGFR2), while at the same time blocking signaling through VEGFR2, thus delaying the VEGF-driven angiogenic response. Notch signaling regulates tip cell function, and we find that TNF also induces the notch ligand jagged-1, through an NFB-dependent mechanism. Enrichment of jagged-1 in tip cells was confirmed by immunofluorescent staining as well as by laser capture microdissection/quantitative reversetranscription-polymerase chain reaction IntroductionNeovascularization, or the formation of new blood vessels, is a critical component of many physiologic as well as pathologic conditions, including development, reproduction, wound healing, diabetic retinopathy, and tumor growth. During wound healing, new vessel growth by angiogenesis is a necessary early step in rebuilding tissue, however the coordination of angiogenesis with the resolution of the acute inflammatory stage is not well understood. The earliest events after tissue damage include the generation of a fibrin clot and the bursting of platelets to release numerous growth factors. Fibrin provides a provisional matrix that promotes the accumulation of blood-derived monocytes that then differentiate into tissue macrophages. Activated macrophages synthesize several cytokines, including tumor necrosis factor (TNF), which activate local endothelial cells (ECs) and promote leukocyte recruitment. After 3 to 4 days, when the initial infection has been cleared, there is a switch toward tissue repair and concomitant with this is the acceleration of angiogenesis. 1,2 TNF is a major inflammatory mediator that induces multiple changes in EC gene expression including induction of adhesion molecules, integrins, and matrix metalloproteinases (MMPs). Its effects on angiogenesis have been the subject of some controversy. For example, TNF blocks EC proliferation and migration in vitro [3][4][5] and has been reported to down-regulate activity 6 and expression 7,8 of vascular endothelial cell growth factor receptor-2 (VEGFR2). On the other hand, TNF has also been shown to up-regulate VEGFR2 expression 9 and promote EC migration. 10 In vivo the situation is no clearer: TNF promotes angiogenesis in the cornea, 3,11 whereas loss of TNFR1 (p55 receptor) leads to enhanced angiogenesis in both retina 12 and wounded skin. 13 Further studies with TNF receptor-deficient mice have demonstrated enhanced hind limb angiogenesis after temporary ischemia in T...

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The requirement for notch signaling during vascular development is well-documented but poorly understood. Embryonic and adult endothelial cells (EC) express notch and notch ligands; however, the necessity for cell-autonomous notch signaling during angiogenesis has not been determined. During angiogenesis, EC display plasticity, whereby a subset of previously quiescent cells loses polarity and becomes migratory. To investigate the role of notch in EC, we have used a three-dimensional in vitro system that models all of the early steps of angiogenesis. We find that newly forming sprouts are composed of specialized tip cells that guide the sprout and trunk cells that proliferate and rearrange to form intercellular lumens. Furthermore, we find that notch acts cell-autonomously to suppress EC proliferation, thereby regulating tube diameter. In addition, when notch signaling is blocked, tip cells divide, and both daughter cells take on a tip cell phenotype, resulting in increased branching through vessel bifurcation. In contrast, notch signaling is not required for re-establishment of EC polarity or for lumen formation. Thus, notch is used reiteratively and cell-autonomously by EC to regulate vessel diameter, to limit branching at the tip of sprouts, and to establish a mature, quiescent phenotype.