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

Journal of Cell Science 120, 1607-1614 (2007)
Published by The Company of Biologists 2007
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Research Article

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

Ikuo Yana1, Hiroshi Sagara1, Satoshi Takaki2, Kiyoshi Takatsu2, Kenji Nakamura3, Kazuki Nakao4, Motoya Katsuki5, Shun-ichiro Taniguchi6, Takanori Aoki7, Hiroshi Sato8, Stephen J. Weiss9 and Motoharu Seiki1,*

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

* Author for correspondence (e-mail: mseiki{at}ims.u-tokyo.ac.jp)

Accepted 18 March 2007


   Summary
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 Summary
Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The membrane-anchored matrix metalloproteinase MT1-MMP (also known as Mmp14) plays a key role in the angiogenic process, but the mechanisms underlying its spatiotemporal regulation in the in vivo setting have not been defined. Using whole-mount immunohistochemical analysis and the lacZ gene inserted into the Mmp14 gene, we demonstrate that MT1-MMP vascular expression in vivo is confined largely to the sprouting tip of neocapillary structures where endothelial cell proliferation and collagen degradation are coordinately localized. During angiogenesis in vitro, wherein endothelial cells are stimulated to undergo neovessel formation in the presence or absence of accessory mural cells, site-specific MT1-MMP expression is shown to be controlled by crosstalk between endothelial cells and vascular smooth muscle cells (VSMC). When vessel maturation induced by VSMCs is inhibited by introducing a soluble form of the receptor tyrosine kinase Tek, MT1-MMP distribution is no longer restricted to the endothelial tip cells, but instead distributes throughout the neovessel network in vitro as well as ex vivo. Taken together, these data demonstrate that vascular maturation coordinated by endothelial cell/mural cell interactions redirects MT1-MMP expression to the neovessel tip where the protease regulates matrix remodeling at the leading edge of the developing vasculature.

Key words: MT1-MMP, Angiogenesis, Endothelial cells, Mural cells, Type I collagen


   Introduction
Top
 Summary
 Introduction
Results
 Discussion
 Materials and Methods
 References
 
Angiogenesis, the growth of new vasculature from pre-existing capillaries, is an important component of normal growth, tissue repair and neoplasia (Carmeliet, 2005Go; Davis and Senger, 2005Go). In both normal and tumor stroma, type I collagen is the most abundant component of the extracellular matrix and serves not only as a key scaffolding material but also as a physical barrier for new vessel formation (Chun et al., 2004Go; Seandel et al., 2001Go; Davis and Senger, 2005Go; Saunders et al., 2006Go). As multiple matrix metalloproteinases (MMPs), including MMP-1, MMP-2, MMP-8, MMP-13 and the membrane type 1 MMP MT1-MMP (also known as MMP-14) display type I collagenolytic activities in vitro (Brinckerhoff et al., 1987Go; Ohuchi et al., 1997Go; Hotary et al., 2000Go; Atkinson et al., 2001Go), several of these enzymes have been proposed to play important roles in regulating angiogenesis in vivo (Zhou et al., 2000Go; Chun et al., 2004Go; Ling et al., 2004Go; Zijlstra et al., 2004Go). Recently, however, increasing evidence has begun to accumulate that MT1-MMP serves as the dominant pericellular collagenase during angiogenesis (Zhou et al., 2000Go; Koike et al., 2002Go; Chun et al., 2004Go; Saunders et al., 2006Go). Whereas neovessel formation is severely impaired in tissues rich in type I collagen in Mmp14-null mice (Zhou et al., 2000Go), little is known with regard to the factors that regulate vascular MT1-MMP expression in the in vivo setting. To this end, we have established a transgenic mouse line wherein the lacZ gene (encoding beta-galactosidase) was inserted into the endogenous Mmp14 gene to monitor its transcriptional regulation (MT1-MMP+/lacZ mice). In combination with an ex vivo angiogenesis model using murine tissues cultured under 3-dimensional (3D) conditions, we now demonstrate that MT1-MMP expression is largely confined to the sprouting tip of the developing vasculature, a site where cell proliferation and focal degradation of collagen proceed preferentially. Further, we find that MT1-MMP protein expression is downregulated throughout the vessel stalk by surrounding mural cell populations that act through a network depending on the receptor tyrosine kinase Tek, which contains an Ig and EGF homology domain 2. Our results outline a dynamic cellular strategy that is used by the developing vasculature to confine MT1-MMP activity to endothelial tip cells during neovessel formation.


   Results
Top
 Summary
 Introduction
 Results
Discussion
 Materials and Methods
 References
 
Monitoring transcription of Mmp14 via beta-galactosidase activity
To monitor the expression of the Mmp14 transcript, we employed a recently developed mouse strain harboring a lacZ gene under the control of the endogenous Mmp14 promoter. Homologous recombination of the murine Mmp14 gene locus encoding the pro- and catalytic domains (exons 1-5) with the targeting vector carrying the lacZ gene disrupts expression of MT1-MMP and, at the same time, affords the opportunity to monitor endogenous promoter activity via nuclear lacZ activity (Fig. 1A).


Figure 1
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  		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 beta-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 beta-gal in combination with CD31. Whereas beta-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 beta-gal (red) and CD31 (green). Within the vascular bed, beta-gal staining was confined to perivascular cells, with limited or no staining associated with the CD31-positive endothelial cells. Bar, 0.25 mm.

 
beta-Gal staining of the whole MT1-MMP+/lacZ mouse embryo reveals a pattern of MT1-MMP expression that parallels that observed previously by in situ hybridization (Apte et al., 1997Go). Using this system, the transcription of Mmp14 in stable, non-growing vascular beds was monitored in MT1-MMP+/lacZ mice. As shown in Fig. 1B, lacZ activity was largely negative in vein and capillary endothelium of peritoneal tissues in 14-day-old heterozygous mice. Whereas stromal cell populations as well as the fibroblast-like adventitial cells surrounding large arteries stained strongly positive (see supplementary material Fig. S1), endothelial cells themselves displayed minimal beta-gal staining in both small and large vessels (Fig. 1C).

Next, to determine whether MT1-MMP expression is altered during angiogenesis, MT1-MMP+/lacZ mice were injected with a type I collagen gel containing vascular endothelial growth factor (VEGF) trapped within a slow release polymer. Seven days post-injection, the collagen gel was resected and analysed for lacZ activity followed by immunostaining for CD31 (Fig. 2A,B). Within the vasculature itself, nuclear beta-gal staining was localized specifically to the tip of the elongated CD31-positive tubule (Fig. 2B). Notably, the tip-specific lacZ activity was only observed during the first 7 days of culture in vivo, disappearing at approximately at day 14 when vascular network formation was completed (data not shown). In an ex vivo angiogenesis model using mouse tissue explants embedded in 3D gels of type I collagen, the growing neovasculature likewise displayed focal beta-gal expression in CD31-positive cells that was confined to the nucleus of sprouting tip cells (Fig. 2C). By contrast, only low levels of beta-gal staining were observed in the neovessel stalk (i.e. the region of the endothelial cell tubule lying behind the advancing tip cell), which also stained positively for the basement membrane macromolecule type IV collagen (Fig. 2C). Quantitative analyses of beta-gal-positive endothelial cells in the explant model demonstrated that more than 80% of the nuclear staining detected was confined to the tip cell population (Fig. 2D). As described previously, explants recovered from Mmp14-null mice were unable to mount an angiogenic response ex vivo (Chun et al., 2004Go) (and data not shown). Taken together, these data suggest that Mmp14 is transcribed preferentially within the tip cells of extending neovessels while only low level expression is observed in either mature vessels or the stalk region of growing neovessels.


Figure 2
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  		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 beta-gal and CD31. In neovessels, nuclear beta-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 beta-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) beta-gal staining was determined (*P<0.01, F-test).

 
Collagenolysis and proliferation of endothelial tip cells
Given the preferential expression of MT1-MMP in endothelial tip cells, these results raised the possibility that MT1-MMP-dependent collagenolytic activity (Chun et al., 2004Go; Saunders et al., 2006Go) is similarly restricted to the leading edge of the developing neovessel. As such, murine tissue explants were cultured in native type I collagen gels overlaid with a second layer of type I collagen conjugated with a quenched fluorescence probe (DQ collagen; Fig. 3A and supplementary material Fig. S2). Significantly, fluorescent signal developed solely around the MT1-MMP-expressing tip cells in a fashion consistent with focal collagenolysis (Fig. 3A). As expected, focal collagenolytic activity could not be detected in the presence of BB-94 which blocked in-growth of endothelial cell sprouts into the layer of DQ collagen (supplementary material Fig. S2). Further, as MT1-MMP collagenolytic activity can also control cell proliferation in 3D matrix environments (e.g. Hotary et al., 2003Go), bromodeoxyuridine (BrdU) incorporation was confined to the type IV collagen poor endothelial cells at the neovessel front (Fig. 3B; white arrow). Quantitative analyses demonstrate that BrdU incorporation is almost completely restricted to the endothelial tip cells (Fig. 3C). Thus, collagenolysis and cell proliferation appeared to coincide with the site-specific expression of MT1-MMP at the leading edge of the advancing neovessel.


Figure 3
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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).

 

MT1-MMP and Tek expression are invertly related during angiogenesis
Interactions between endothelial cells and perivascular mural cells (i.e. pericytes or smooth muscle cells) promote blood vessel maturation and stabilization (Korff et al., 2001Go; Lafleur et al., 2001Go; Saunders et al., 2006Go; von Tell et al., 2006Go). As neovessel outgrowths are largely enveloped by mural cells in ex vivo models (Gerhardt et al., 2003Go; Zhu et al., 2002Go) (supplementary material Fig. S3), we considered the possibility that interactions of pericytes, vascular smooth muscle cells (VSMCs) and endothelial cells participate in regulating MT1-MMP expression. The mural-cell-derived soluble factor angiopoietin 1 (Ang-1), is a major ligand for Tek, a cell surface, endothelial-specific receptor tyrosine kinase that plays a central role in vessel maturation (Suri et al., 1996Go; Carmeliet, 2005Go; von Tell et al., 2006Go). To determine whether the Tek axis might impact on the spatiotemporal expression of MT1-MMP, we monitored Tek expression in endothelial cells during angiogenesis. Using tissue explants isolated from transgenic mice that express GFP under the control of the endogenous Tek promoter, the GFP signal was preferentially localized to the neovessel stalks (Fig. 4A,B, white arrows). By contrast, Tek promoter expression was absent in the endothelial tip cells (Fig. 4A,B, yellow arrows), in a fashion consistent with the proposition that MT1-MMP expression is suppressed in the Tek-positive endothelium.


Figure 4
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  		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.

 


Figure 5
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  		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.

 
In order to assess the possible involvement of VSMC in regulating MT1-MMP, we next examined MT1-MMP expression in human umbilical endothelial cells (HUVECs) cultured within a 3D gel of type I collagen in the absence or presence of human aortic VSMC. HUVECs cultured alone expressed MT1-MMP throughout the length of the forming tubules and only small amounts of type IV collagen were detected (Fig. 5A, left panel). By contrast, when HUVECs were co-cultured with VSMC, MT1-MMP expression was restricted to the sprouting tip cells whereas type IV collagen deposition, in coincident fashion, was increased dramatically (Fig. 5A, right panel). Although VSMCs were found to envelop the endothelial cell tubules only at intervals (supplementary material Fig. S4), expression of MT1-MMP was suppressed significantly along the entire length of the neovessel stalks (Fig. 5A). Thus, interactions occurring between the endothelial cells and surrounding VSMC in the neovascular stalk provide a potential cellular basis for the selective expression of MT1-MMP.

To examine directly the possible involvement of Tek signals in mural cell-mediated MT1-MMP suppression, HUVEC-VSMC co-cultures were established in the presence of a recombinant, soluble form of Tek (sTek). Interestingly, when neovessel growth was allowed to proceed in the presence of sTek, the area of the endothelial cell tubules expressing MT1-MMP was significantly extended along the entire length of the vessel stalk (i.e. the total area of MT1-MMP-positive staining increased from 6±4% to 75±21%, n=5), whereas type IV collagen deposition was depressed (Fig. 5B). Likewise, in explants of MT1+/lacZ mouse tissue cultured in the presence of sTek, beta-gal was redistributed throughout the length of the tubules (Fig. 6A). Whereas the total tube number was unaffected by sTek, the number of neovessels expressing endothelial tip cell-specific beta-gal staining was decreased by ~75% (Fig. 6B). Finally, sTek also relaxes MT1-MMP suppression in the explant cultures, such that the protease is more globally expressed across the neovessel surface in concert with a marked diminution in type IV collagen deposition (Fig. 6C). Taken together, these results support a model wherein Tek signals negatively regulate the transcription of Mmp14 in the stalk region and act to confine MT1-MMP expression to the leading edge of the neovasculature.


Figure 6
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  		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.

 

   Discussion
Top
 Summary
 Introduction
 Results
 Discussion
Materials and Methods
 References
 
Despite increasing evidence supporting a critical requirement for MT1-MMP in angiogenic responses (Hiraoka et al., 1998Go; Zhou et al., 2000Go; Lafleur et al., 2002Go; Chun et al., 2004Go; Saunders et al., 2006Go), little is known with regard to the factors controlling the endothelial cellspecific, spatiotemporal expression of MT1-MMP in vivo. Given the low levels of MT1-MMP detected in the endothelium of mature vessels, it seemed plausible to propose that vascular MT1-MMP expression are restricted to neovessel formation. Indeed, we now demonstrate that MT1-MMP is specifically expressed in advancing endothelial tip cells and is subsequently downregulated as the vessel structure stabilizes and matures. As angiogenesis is impaired in the absence of MT1-MMP (Zhou et al., 2000Go; Chun et al., 2004Go; Saunders et al., 2006Go), the site-specific expression of the protease in endothelial tip cells probably plays a crucial role in guiding new blood vessels through the stroma rich in type I collagen. Whereas seemingly intact vascular networks can be formed in Mmp14-null mice during early development, the content of type I collagen of multiple mouse tissues is maintained at low levels in utero and increases most dramatically in the postnatal state (Caulfield and Borg, 1979Go; Mays et al., 1988Go; Sabeh et al., 2004Go).

In vivo, endothelial cells at the advancing tips of developing neovessels have been reported to display properties distinct from those observed in the vessel stalk (e.g. Gerhardt et al., 2003Go). Likewise, in our studies, MT1-MMP expression was largely restricted to CD31-positive endothelial tip cells that expressed collagenolytic activity, proliferated, expressed low levels of Tek and remained free of a mural cell coat. Caution should, however, be ascribed to characterizing tip cells as strictly endothelial in terms of their structural or functional phenotype. During vasculogenesis as well as angiogenesis, endothelial cells can undergo an endothelial-mesenchymal transformation wherein tissue-invasive cells adopt VSMC-like characteristics, including the expression of {alpha}-smooth muscle actin (DeRuiter et al., 1997Go; Frid et al., 2002Go; Ishisaki et al., 2003Go; Liebner et al., 2004Go; Timmerman et al., 2004Go; Paruchuri et al., 2006Go). Hence, because endothelial cells engage transcriptional programs necessary to support invasive activity, the leading cell population might be predicted to assume a plastic phenotype more consistent with the unique requirements of the tip cell population. Further, we have found that, whereas the vessel stalk is ensheathed by pericellular matrix positive for type IV collagen, the advancing front of the neovessel is almost completely devoid of a type IV collagen matrix. Interestingly, a similar inverse pattern of MT1-MMP expression and type IV collagen deposition could be recapitulated by culturing HUVECs under 3D conditions in the absence or presence of VSMCs. Together, these data suggest that crosstalk between endothelial and mural cells may be responsible for regulating both the spatiotemporal expression of MT1-MMP and the maintenance of a stable perivascular basement membrane in vivo.

Mural-cell-derived growth factors such as Ang-1 have been proposed to play central roles in regulating vascular maturation by initiating a Tek-dependent signaling cascade in associated endothelial cells (Carmeliet, 2005Go; von Tell et al., 2006Go). In our study, we found that Tek-mediated signals induce the transcriptional suppression of MT1-MMP in the vessel stalk. These results support a model whereby endothelial tip cell-specific expression of MT1-MMP is accentuated as a consequence of the mural-cell-dependent silencing of MT1-MMP expression within the neovessel stalk (see Fig. 7). Interestingly, recent studies have proposed that mural-cell-derived MMP inhibitors similarly act to suppress endothelial cell MT1-MMP proteolytic activity (Lafleur et al., 2001Go; Saunders et al., 2006Go). Whereas these reports demonstrate that pericytes or smooth muscle cells can regulate MT1-MMP activity at the post-translational level, our data support an additional – but upstream – role for the mural-cell-dependent control of MT1-MMP transcription itself. As MT1-MMP is capable of degrading multiple basement-membrane components, including type IV collagen, laminin and fibronectin (d'Ortho et al., 1997Go; Koshikawa et al., 2000Go; Hotary et al., 2006Go), quenching both MT1-MMP expression and also MT1-MMP enzymatic activity in the vessel stalk may contribute to vascular stability by preventing the unregulated proteolysis of the perivascular matrix.


Figure 7
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  		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.

 

By restricting MT1-MMP activity to the leading edge of the neovasculature, the growing vessel would be in an optimal position to advance itself into the surrounding matrix. Interestingly, MT1-MMP expression by endothelial tip cells also correlated with BrdU incorporation. This finding contrasts with a previous study in which BrdU was incorporated preferentially into the stalk of retinal neovessels (Gerhardt et al., 2003Go). Although we have confirmed these observations in the retina (data not shown), a crucial difference between the retinal environment, the peritoneum and also our ex vivo culture conditions is the content of type I collagen of the respective tissues. Indeed, we find that the retinal environment is poor of type I collagen compared with the tissues studied here (supplementary material Fig. S5). In addition, type I collagen can itself induce MT1-MMP expression (Ellerbroek et al., 2001Go; Lafleur et al., 2006Go) and it seems plausible that the surrounding collagenous environment acts to both modulate protease expression and cell function at the neovascular tip. Consequently, endothelial cell invasion and proliferation may take place in a concomitant fashion as MT1-MMP-dependent changes in cell shape, cytoskeletal tension and migration impact on the proliferative response (Hotary et al., 2003Go; Chun et al., 2004Go; Chun et al., 2006Go). Finally, although our studies have focused on endothelial-cell-derived MT1-MMP, VSMC and also pericytes are additional sources of MT1-MMP activity (Shofuda et al., 1997Go; Filippov et al., 2005Go; Lehti et al., 2005Go). However, the expression level of MT1-MMP in the mural cells that cover the neovascular stalk appears to be low as evaluated by beta-gal staining. It remains to be determined whether the expression of MT1-MMP in migrating VSMC is also regulated following recruitment to the vessel wall via endothelial-cell crosstalk.

Recent studies have suggested that endothelial cell MT1-MMP serves as a potential target for inhibiting tumor angiogenesis (Haas et al., 1998Go; Zhou et al., 2000Go; Sounni et al., 2002Go; Seiki and Yana, 2003Go; Chun et al., 2004Go; Plaisier et al., 2004Go). Our findings, however, predict a narrow window of opportunity for targeting MT1-MMP in the neovasculature as the protease is only transiently expressed within the endothelial tip cells of the growing vessels. Nevertheless, given the immature status of mural-cell-deficient tumor vessels (e.g. Baluk et al., 2005Go), more global patterns of MT1-MMP expression may be maintained within the tumor vasculature, thereby rendering it susceptible to anti-MT1-MMP therapy. Additional studies will be needed to address these issues directly but our findings support a growing body of evidence, suggesting that the spatiotemporal regulation of MT1-MMP expression and activity in endothelial cells by surrounding mural cell populations serves as a key determinant of neovessel formation during angiogenic states.


   Materials and Methods
Top
 Summary
 Introduction
 Results
 Discussion
 Materials and Methods
References
 
Animals, cell lines, reagents and antibodies
C57BL/6 mice and Wistar rats were obtained from CLEA JAPAN (Tokyo, Japan). MT1-MMP+/lacZ mice were generated from heterozygous embryonic stem cells (derived from the E14 embryonic stem cell) line that were injected into C57BL/6 blastocysts. The Institutional Review Board in the University of Tokyo approved all animal studies. Human umbilical endothelial cells (HUVECs) and human vascular smooth muscle cells (VSMCs) were purchased from KURABO (Neyagawa, Osaka, Japan), and cultured in modified MCDB131 medium supplemented with 5% fetal calf serum and calf brain extract. An anti-MT1-MMP mouse monoclonal antibody (mAb) raised against the hemopexin domain of MT1-MMP (anti-HPX) was developed in collaboration with Daiichi-Fine Chemical Co., Ltd (Toyama, Japan). Anti-mouse and rat CD31 monoclonal antibodies (clones MEC13.3 and TLD-3A12, respectively) were purchased from BD Biosciences PharMingen (San Diego, CA) and a polyclonal antibody (clone AB756P) for type IV collagen was obtained from Chemicon (Temecula, CA). Anti-smooth-muscle actin (SMA; clone 1A4) was purchased from Sigma (St Louis, MO). Recombinant murine VEGF and endothelial cell growth supplement were obtained from Peprotech (London, UK) and Calbiochem (La Jolla, CA), respectively. BB-94, a synthetic MMP inhibitor, was a gift from Peter Brown (British Biotech, Oxford, UK), whereas TIMP-1 and TIMP-2 were provided by Daiichi Fine Chemical Co. (Takaoka, Japan).

Detection of beta-gal
Tissues were fixed in cold PBS containing 2% paraformaldehyde and 0.2% glutaraldehyde for 5 minutes, washed, and incubated overnight at 37°C in X-Gal buffer (1.3 mg/ml potassium ferricyanide, 1 mg/ml potassium ferricyanide, 0.2% Triton X-100, 1 mM MgCl2, 1 mg/ml Xgal in PBS pH 7.2). For ex vivo cultures, samples were immersed in a periodate-lysine-paraformaldehyde (PLP) buffer (2% paraformaldehyde, 75 mM lysine, 10 mM sodium periodate, 45 mM sodium phosphate pH 7.4) for 40 minutes, followed by successive washes in PBS containing 1% NP-40 and 1% SDS. To detect beta-gal, samples were incubated with X-Gal solution for 3-24 hours as described above.

In vivo angiogenesis assay
A double-layered matrix composed of an outer layer of type I collagen and an inner sphere of a thermosensitive, slow-release polymer (Ikeda Rika, Tokyo, Japan) impregnated with 50 ng/ml of VEGF was implanted subcutaneously. At 7 or 14 days, the gel was resected, stained for beta-gal and immunostained for CD31. The total number of tubules was determined in three independent tissue fragments for each experiment of five or more performed. The number of beta-gal-positive tip cells or beta-gal-positive tubes was determined in stained samples. Statistical significance was determined by the F test.

Immunohistochemistry
Frozen sections were fixed in PLP buffer for 10 minutes, washed, and endogenous peroxidase activity was quenched by treating the samples with methanol containing 0.3% H2O2. Samples were then blocked with 0.5% BSA in PBS followed by incubation with primary antibodies for 30 minutes at the appropriate concentration. Following multiple washes, samples were incubated with the secondary antibody for 30 minutes, washed and developed in a diaminobenzidine staining system (Nichirei, Japan). In ex vivo cultures, samples were fixed in PLP buffer for 4 hours, washed in PBS, immersed in methanol containing 0.3% H2O2, and subsequently blocked in PBS with 0.3% Triton X-100 containing 1% skimed milk. Samples were then incubated with primary antibodies at the appropriate concentration overnight, followed by multiple washes before incubation with the secondary fluorescent antibody for 90 minutes. Fluorescent images were obtained using a CCD fluorescent microscope (IM70-Cool SNAP, OLYMPUS).

3D culture conditions
Fragments of intracostal muscle or diaphragm were isolated from 1-day-old to 14-day-old C57BL/6 MT1-MMP+/+ mice, MT1-MMP+/lacZ mice or from 21-day-old Wistar rats. Tissues were immersed in MCDB131 supplemented with a mixture of antibiotics and antimycotics (Invitrogen-GIBCO) for 30 minutes. Samples were minced (pieces of 3-5 mm) and embedded in 60-120 µl of neutralized type I collagen at a final concentration of 2.4 mg/ml (type I-A Nitta Gelatine, Osaka, Japan) in 48-well or 96-well plates (Corning). Cultures were incubated in a CO2 (5%) air (95%) atmosphere at 37°C for 30 minutes until the gel was polymerized, and then cultured for 14 days in modified MCDB131 medium supplemented with 20% FBS, VEGF (50 ng/ml), endothelial cell growth supplement (75 µg/ml), and heparin (100 µg/ml). For 3D HUVEC cultures, an aggregate of approximately 5000 cells was suspended in 5 µl of medium and embedded in 70 µl of type I collagen. In 3D co-cultures of HUVECs and VSMCs, a mixture of 3000 HUVECs and 2000 VSMCs was suspended in 5 µl of medium and embedded in collagen gels as described above. Where indicated, VSMCs were labeled with the fluorescent dye, PKH26 (Sigma), according to the manufacturer's instructions. Cultures were maintained for 7 days in the pro-angiogenic medium, which was exchanged every 3 days. BrdU incorporation (48-hour pulse) was performed according to the manufacturer's instructions (Pharmacia). The percentage of BrdU-positive tip cells or BrdU-positive stalks was determined in three independent fragments for each of 14 experiments. Statistical significance was determined by the F test.

Collagen degradation assay
Neocapillary collagenolytic activity was evaluated with a quenched fluorescence-labeled substrate (DQ collagen, Molecular Probes, Eugene, Oregon). Briefly, tissue chunks were embedded in unlabeled collagen, and following a 7-day culture period, a mixture of neutralized DQ collagen and unlabeled type I collagen (3:100 ratio) was poured on top of the embedded rat tissue fragment in the absence or presence of BB-94, TIMP-1 or TIMP-2. Following a 48-hour incubation period, images were collected by fluorescence microscopy.


   Acknowledgments
 
We thank Bob Whittier and Naohiko Koshikawa (IMS University of Tokyo) for helpful discussions, Kunika Nishibashi, Akiko Saka, Akira Matsuda, and Tomoko Nakajima-Andou for excellent technical assistance, and Akiko Okada (Mie University, Japan) for her initial work to establish the MT1-MMP-targeted mouse strain. This work was supported by the Specific Coordination Fund for Promoting Science and Technology, a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and NIH grant R01 CA88308.


   Footnotes
 
Supplementary material available online at http://jcs.biologists.org/cgi/content/full/120/9/1607/DC1


   References
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 Summary
 Introduction
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