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Endothelial tubulogenesis within fibrin gels specifically requires the activity of membrane-type-matrix metalloproteinases (MT-MMPs)

Marc A. Lafleur*, Madeleine M. Handsley, Vera Knäuper, Gillian Murphy and Dylan R. Edwards{ddagger}

School of Biological Sciences, University of East Anglia, Norwich, NR4 7TJ, UK
* Present address: St. Vincent's Institute of Medical Research, 9 Princes Street, Fitzroy, Victoria 3065, Australia



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  		Fig. 1. HUVECs form tubular structures with lumens when cultured within fibrin gels. (A) HUVECs were cultured within fibrin gels in standard serum-containing medium and stimulated with VEGF (25 ng/ml) and FGF-2 (25 ng/ml). The cells were incubated at 37°C and 5% (v/v) CO2 for 3 days, after which time the cells were photographed (bar, 100 µm). (B) HUVECs were treated as above then fixed in glutaraldehyde, embedded in LR resin, sectioned and analysed by transmission electron microscopy. n, nucleus; 1, lumen. Bar, 1 µm. (C) HUVECs were cultured as above, then fixed in glutaraldehyde, embedded in LR resin, serially sectioned, stained with toluidine blue and analysed by light microscopy. i-ix correspond to the order of serial semi-thin sections (bar, 10 µm).

 


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  		Fig. 2. Angiogenic factors and tumour-derived factors increased HUVEC tubulogenesis within fibrin gels. (A) HUVECs were grown within fibrin gels in standard serum-containing medium and stimulated with the angiogenic factors as indicated: VEGF (25 ng/ml), FGF-2 (10 ng/ml), TNF-{alpha} (10 ng/ml), TGF-ß1 (1 ng/ml), EGF (50 ng/ml), TGF-{alpha} (10 ng/ml), HGF/SF (300 U/ml), IL1{alpha} (10 ng/ml), and angiogenin (100 ng/ml). The factors present in the angiogenic cocktail are a combination of all the above listed angiogenic factors. The cells were incubated at 37°C and 5% CO2 (v/v) for 3 days, after which time the cells were photographed and the tubules quantified with the LUCIA G/Comet software. (B) HUVECs were cultured within a fibrin gel as a drop culture in the bottom of a well. Where indicated, equal amounts of U87 glioma cells or MDCK cells, or twice the amount of U87 glioma cells, were also incubated within a separate fibrin gel as a drop culture within the same well as the HUVECs. Standard serum-containing EC medium (with no exogenous angiogenic factors) was added to each well with both gels covered with the same medium, allowing diffusible molecules produced by the cells from one gel to reach the cells in the other gel. Aprotinin (100 µg/ml) was also added to each well in order to prevent gel degradation by soluble serine protease. The cells were incubated at 37°C and 5% CO2 (v/v) for 3 days, after which time the cells were photographed and the tubules quantified with the LUCIA G/Comet software. Quantification of tubular structures was performed by measuring the total length of structures per field. Five fields were quantified per gel and the average and s.d. of each result are shown. * indicates statistically significant differences (P<0.05) compared with the buffer control (** P<0.0001) using the student's t-test assuming equal variance, two tail.

 


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  		Fig. 3. Effect of protease inhibitors on HUVEC and HDMEC tubulogenesis within fibrin gels. HUVECs (A,B,D,E) and HDMEC (C) were grown within fibrin gels in standard serum-containing EC medium and stimulated with VEGF (25 ng/ml) and FGF-2 (25 ng/ml). Protease inhibitors were added both to the gel and to the culture medium at the beginning of the experiment where indicated. aprot, aprotinin; pep, pepstatin. The cells were incubated at 37°C and 5% (v/v) CO2 for 3 days, and the total tube length was calculated as described in the legend of Fig. 2. The average and s.d. of each experiment is shown. * indicates statistically significant differences (P<0.05) compared with the buffer control (** P<0.0001). A significant difference (P<0.05) was also observed between full-length TIMP-2 and wild-type N-TIMP-2 in E.

 


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  		Fig. 4. TaqMan RT-PCR on HUVECs cultured both in 2D and 3D cultures. HUVECs were cultured either within fibrin gels or in 2D cultures in standard serum-containing EC medium and stimulated with VEGF (25 ng/ml) and FGF-2 (25 ng/ml). The cells were incubated at 37°C and 5% (v/v) CO2 for the indicated time period, after which total RNA was extracted from the cells and TaqMan RT-PCR performed on these samples. Results are indicated as relative values that have been normalised to the levels of 18S rRNA. Each data point is the average of duplicate samples. The data shown are from a single experiment that is representative of four separate experiments carried out with different batches of HUVECs.

 


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  		Fig. 5. Protein analysis of MMP-2 and MT1-MMP in HUVECs in 2D and 3D cultures. HUVECs were grown either within fibrin gels or in 2D cultures in standard serum-containing EC medium and stimulated with VEGF (25 ng/ml) and FGF-2 (25 ng/ml). The cells were incubated at 37°C and 5% (v/v) CO2 for the indicated time period, after which total cell lysates were collected. Equal amounts of cell lysates as determined by a BCA protein quantification assay were analysed by gelatin zymography (A) or western analysis for MT1-MMP (B). Duplicate samples from separate cultures were analysed for each treatment. Pro-MMP-2 has a molecular mass of 72 kDa, whereas the intermediate and fully active forms run at 64 kDa and 62 kDa, respectively. Active MT1-MMP has a molecular mass of 60 kDa.

 

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