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Sprouting angiogenesis is a multistep process consisting of basement membrane degradation, endothelial cell (EC) activation, proliferation, invasion, lumen formation, and sprout stabilization. Such complexity is consistent with a requirement for orchestration of individual gene expression alongside multiple signaling pathways. To better understand the mechanisms that direct the transformation of adherent ECs on the surface of collagen matrices to develop multicellular invading sprouts, we analyzed differential gene expression with time using a defined in vitro model of EC invasion driven by the combination of sphingosine-1-phosphate, basic FGF, and VEGF. Gene expression changes were confirmed by real-time PCR and Western blot analyses. A cohort of cell adhesion molecule genes involved in adherens junction and cell-extracellular matrix (ECM) interactions were upregulated, whereas a set of genes associated with tight junctions were down-regulated. Numerous genes encoding ECM proteins and proteases were induced, indicating that biosynthesis and remodeling of ECM is indispensable for sprouting angiogenesis. Knockdown of a highly upregulated gene, a disintegrin and metalloproteinase with thrombospond-in-type repeats-I (ADAMTSI), decreased invasion responses, confirming a role for ADAMTS l in mediating EC invasion. Furthermore, differential expression of multiple members of the Wnt and Notch pathways was observed. Functional experiments indicated that inhibition and activation of the Notch signaling pathway stimulated and inhibited EC invasion responses, respectively. This study has enhanced the molecular road map of gene expression changes that occur during endothelial invasion and highlighted the utility of threedimensional models to study EC morphogenesis. angiogenesis; extracellular matrix; Notch; endothelial cell; sphingosine-l-phosphate; collagen

Sprouting angiogenesis is a multistep process consisting of basement membrane degradation, endothelial cell (EC) activation, proliferation, invasion, lumen formation, and sprout stabilization. Such complexity is consistent with a requirement for orchestration of individual gene expression alongside multiple signaling pathways. To better understand the mechanisms that direct the transformation of adherent ECs on the surface of collagen matrices to develop multicellular invading sprouts, we analyzed differential gene expression with time using a defined in vitro model of EC invasion driven by the combination of sphingosine-l-phosphate, basic FGF, and VEGF. Gene expression changes were confirmed by real-time PCR and Western blot analyses. A cohort of cell adhesion molecule genes involved in adherens junction and cell-extracellular matrix (ECM) interactions were upregulated, whereas a set of genes associated with tight junctions were down-regulated. Numerous genes encoding ECM proteins and proteases were induced, indicating that biosynthesis and remodeling of ECM is indispensable for sprouting angiogenesis. Knockdown of a highly upregulated gene, a disintegrin and metalloproteinase with thrombospondin-type repeats-l (ADAMTS1), decreased invasion responses, confirming a role for ADAMTS1 in mediating EC invasion. Furthermore, differential expression of multiple members of the Wnt and Notch pathways was observed. Functional experiments indicated that inhibition and activation of the Notch signaling pathway stimulated and inhibited EC invasion responses, respectively. This study has enhanced the molecular road map of gene expression changes that occur during endothelial invasion and highlighted the utility of three-dimensional models to study EC morphogenesis. [ABSTRACT FROM AUTHOR]

The objective of this study was to determine the potential mechanisms through which the known anti-angiogenic factor, tissue inhibitor of metalloproteinase-3 (TIMP-3) blocks angiogenesis. Endothelial monolayers were infected with recombinant adenoviruses constructed to deliver TIMP-3 fused to carboxy-terminal S and His tags (TIMP-3-S-His) along with TIMP-1-S-His and green fluorescent protein (GFP) control. Primary endothelial cells were allowed to express constructs for 36-48 hours prior to extraction and tandem affinity purification. Silver staining of final eluates revealed distinct protein interactions. Samples were analyzed by Western blotting and revealed binding of A Disintegrin and Metalloproteinase-17 (ADAM17) and ADAM15, to TIMP-3, but not TIMP-1 or GFP controls (n=4). Analyses of samples using mass spectrometry are underway in large-scale experiments to identify distinct binding partners of TIMP-3. To confirm the involvement of ADAM17 or ADAM15 in mediating angiogenic events, models of primary human endothelial cell invasion into three-dimensional matrices were utilized. Silencing expression of ADAM17 and ADAM15 using small interfering RNA (siRNA) successfully prevented protein expression in invading cell cultures. Knockdown of ADAM17, but not ADAM15 significantly interfered with invasion, resulting in decreased density of invasion and decreased sprout length. Taken together, these data implicate a role for ADAM17 in mediating endothelial sprouting events during angiogenesis. [ABSTRACT FROM AUTHOR]