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Fig. 3. Characterization of neovascular tip cells. (A) In the ex vivo explant system, neovessels invading surrounding 3D gels impregnated with DQ collagen (left panel, phase-contrast micrograph), express collagenolytic activity as assessed by fluorescence microscopy (right panel). Collagen degradation surrounding the neovascular tip (white arrows) is detected as a green signal. Bar, 0.1 mm. (B) BrdU incorporation specific for endothelial tip cells. After a 48-hour pulse period, neovessels arising from the 3D muscle explants were stained for BrdU (green) and type IV collagen (red). Whereas the neovessel stalks stained strongly for type IV collagen (white arrowheads, left panel), little or no BrdU incorporation was detected in these regions (yellow arrowhead, right panel). Instead, BrdU incorporation (white arrow) was largely confined to endothelial tip cells poor of the type IV collagen. The white arrow shows a specific nuclear signal indicating BrdU incorporation at the neovascular tip cell. The yellow arrowhead indicates a BrdU-negative stalk cell. Bar, 0.1 mm. (C) BrdU incorporation (percent positive cells) was localized to either endothelial tip cells or neovessel stalks and quantified in 14 separate experiments (P=0.00382, F-test).

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