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First published online 3 April 2007
doi: 10.1242/jcs.000679

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Crosstalk between neovessels and mural cells directs the site-specific expression of MT1-MMP to endothelial tip cells

¹ Division of Cancer Cell Research, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokane-dai, Minato-ku, Tokyo 108-8639, Japan
² Division of Immunology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokane-dai, Minato-ku, Tokyo 108-8639, Japan
³ Mouse Genome Technology Center, Mitsubishi Kagaku Institute of Life Sciences, Tokyo 194-8511, Japan
⁴ Laboratory for Animal Resources and Genetic Engineering, Center for Developmental Biology, RIKEN, Kobe 650-0047, Japan
⁵ National Institute for Basic Biology, Okazaki 444-8585, Japan
⁶ Institute on Aging and Adaptation, Shinshu University Graduate School of Medicine, Japan
⁷ Daiichi Fine Chemical Corporation, 530 Chokeiji, Takaoka, Toyama 933-8511, Japan
⁸ Department of Molecular Virology and Oncology, Cancer Research Institute, Kanazawa University, Japan
⁹ Division of Molecular Medicine and Genetics, Department of Internal Medicine, University of Michigan Comprehensive Cancer Center, Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA

View larger version (62K): [in this window] [in a new window]   Fig. 1. Establishment of MT1-MMP+/lacZ mouse strain to monitor Mmp14 transcription. (A) Schematic representation of targeting Mmp14. Exons 1-5 encoding the catalytic domain of MT1-MMP were targeted and the lacZ gene, encoding -galactosidase fused with a nuclear localization signal (NLS) was cloned in-frame with a phosphoglycerate kinase (PGK)-gpt/neo resistance gene cassette. (B) Vascular bed Mmp14 expression. Peritoneal tissue whole-cell mounts with associated segments of the musculus rectus abdominis were isolated from 3-day-old mice [MT1+/+ (left panel) and MT1+/lacZ (right panel)] and stained for -gal in combination with CD31. Whereas -gal staining was not observed in MT1-MMP+/+ tissues, lacZ-positive cells were found throughout the stroma and also in association with perivascular cells in tissues of MT1+/lacZ mouse (see below). Bars, 0.25 mm for large panels, 0.1 mm for insets. (C) Transverse sections of peritoneum and associated musculus rectus abdominus muscle from the MT1+/lacZ mice were stained both for -gal (red) and CD31 (green). Within the vascular bed, -gal staining was confined to perivascular cells, with limited or no staining associated with the CD31-positive endothelial cells. Bar, 0.25 mm.

View larger version (89K): [in this window] [in a new window]   Fig. 2. MT-MMP expression during neovessel formation in vivo and ex vivo. (A) Neocapillary formation was induced in vivo in type I collagen gel implants containing a VEGF slow-release polymer. Neovessels are indicated by arrows. Bar, 1 mm. (B) Whole-cell mount of neovessels infiltrating the collagen gel implant stained for -gal and CD31. In neovessels, nuclear -gal staining was predominately associated with the advancing tip of the blood vessels (arrows, left panel), which are pointed at by arrows in the accompanying tracing (right panel). Bar, 0.25 mm. (C) Muscle explants derived from MT1-MMP+/lacZ mice were embedded in 3D collagen gels and neovessel formation was induced with a growth factor cocktail. In the left panel, the advancing tip of CD31-positive neovessels (arrowheads) exhibit strong -gal staining (arrows). Following staining for type IV collagen (right panel), the advancing endothelial cell tips (arrows) were largely poor of type IV collagen, whereas the neovessel stalk is strongly positive of type IV collagen (arrowheads). Bars, 0.25 mm. (D) Neovessel number per explant was quantified at day 7 by phase-contrast microscopy and the numbers of tubes with either tip-specific (i.e. within two cells of the neovessel tip) or pan-vessel (i.e. neovessels staining with two or more endothelial stalk cells) -gal staining was determined (*P<0.01, F-test).

View larger version (44K): [in this window] [in a new window]   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).

View larger version (54K): [in this window] [in a new window]   Fig. 4. Tek expression during ex vivo angiogenesis. (A) Tissue fragments isolated from Tek GFP mice were embedded in 3D collagen gels and neovessel formation was assessed by phase-contrast (left panel) and fluorescence microscopy (right panel). The GFP signal was restricted to the stalk region of the growing neovessel (white arrows) whereas the endothelial tip cells were GFP-negative (yellow arrows). (B) Higher magnification of yellow boxed areas from upper two panels. Bar, 0.1 mm.

View larger version (47K): [in this window] [in a new window]   Fig. 5. VSMC-dependent regulation of endothelial cell MT1-MMP expression. (A) MT1-MMP expression was monitored in 3D cultures with HUVECs alone or with a combination of HUVECs and VSMCs. With HUVECs alone, MT1-MMP (green) was stained uniformly along the length of growing neovessels (white arrows, left panel). Under these conditions, only low amounts of type IV collagen (red) were detected. By contrast, in HUVEC-VSMC co-cultures, MT1-MMP expression was restricted to the advancing endothelial tip cells (white arrows, right panel) of the capillary stalk positive for type IV collagen, which is comprised of an endothelial cell tube decorated with vascular smooth muscle cells (see supplementary material Fig. S3). Bar, 0.25 mm. (B) In HUVEC-VSMC co-cultures, neovessel stalks express only low levels of MT1-MMP in contrast with strong staining for type IV collagen (panels a-c). By contrast, in the presence of sTek (30 µg/ml), neovessel stalks express heightened levels of MT1-MMP concomitant with low levels of type IV collagen (panels d-f). White arrows indicate areas of MT1-MMP expression. Bar, 0.125 mm.

View larger version (67K): [in this window] [in a new window]   Fig. 6. Tek-dependent regulation of MT1-MMP expression during ex vivo angiogenesis. (A) MT1-MMP transcription in MT1-MMP+/lacZ explants (as monitored by lacZ activity) was assessed in the absence or presence of sTek. Wheras lacZ activity is confined to endothelial tip cells in the absence of sTek (white arrow, left panel), addition of sTek induced widespread MT1-MMP expression in growing neovessels (white arrows, right panel). Mural cells and fibroblasts stain positive for smooth muscle actin (SMA). Bar, 0.25 mm. (B) Total number neovessels (blue bars) and of neovessels displaying endothelial tip-cell-specific MT1-MMP expression (red bars) was determined in explant cultures in the absence or presence of sTek after a 7-day culture period. Whereas the total number of neovessels was unaffected by sTek, the number of capillary structures exhibiting tip-specific MT1-MMP induction was significantly reduced in the eight experiments performed (P=0.0045, F-test). (C) In murine explants, treatment with sTek (right panel) induced widespread expression of MT1-MMP (green, indicated by yellow arrows) and the concomitant decrease in type IV collagen staining (red) compared with control explants (left panel). Bar, 0.25 mm.

View larger version (94K): [in this window] [in a new window]   Fig. 7. Model for site-specific MT1-MMP expression during neovessel formation. MT1-MMP-expressing endothelial tip cells are maintained in an immature state as they drive invasion and proliferation. By contrast, endothelial stalk cells assume a quiescent state as perivascular mural cells signal neighboring endothelial cells to suppress MT1-MMP expression and increase type IV collagen deposition.