JCS000679 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Transverse section of a large artery and vein in the peritoneum recovered from an MT1-MMP^+/lacZ mouse. LacZ staining (red) is confined to the adventitial layer of the artery (white arrows) whereas CD31 staining (green) was restricted to the artery and associated vein endothelium (white arrowheads). Bar, 0.25 mm.

• Supplemental Figure 2 -

  Fig. S2. (A) CD31-positive sprout number was determined in 3D explants of rat tissue embedded in type I collagen gels in the absence or presence of BB-94 (10 μM), TIMP-1 (1.0 μg/ml) or TIMP-2 (1.0 μg/ml) for 10 days. Significance levels were determined by F-test (n=8). (B) DQ collagen degradation assay. As illustrated in the schematics on the left side of the panel, explants were embedded in 3D gels of type I collagen. Following a 7-day culture period, outgrowing neovessels (shown as red tube in the cartoon) were confronted with an interfacing 3D gel of type I collagen impregnated with DQ collagen for an additional 2 days (shown as green layer). As vessel outgrowth infiltrated the zone of DQ collagen, a fluorescent signal was generated. The two panels to the right (upper row) show respective phase contrast and fluorescent micrographs of an invading neovessel (white arrows indicate neovessel tip cell infiltrating the layer of DQ collagen). In the lower set of panels, explants were cultured as above for 7 days and DQ collagen-impregnated gels applied in the presence of BB-94 (10 μM) for an additional 2 days. Under these conditions, neovessels were unable to penetrate the DQ collagen layer and no fluorescent signal was generated.

• Supplemental Figure 3 -

  Fig. S3. In 3D explants of rat tissue, type IV collagen-positive (green) neovessels are frequently surrounded by SMA-positive (red) mural cells (panels a-c). Arrows highlight the neovessel tip. In panel d, the boxed area shown in panel c has been expanded. The double-headed arrows in panel d border neovessel tip cells that are not invested with SMA-positive cells and are largely negative for type IV collagen staining. Bar, 0.25 mm.

• Supplemental Figure 4 -

  Fig. S4. Smooth muscle cell localization in 3D HUVEC co-cultures. HUVEC and PKH-labeled VSMCs were embedded in 3D collagen gels and stimulated with a growth factor cocktail for 7 days (white arrows mark PKH-positive VSMCs). The merged image in panel c demonstrates that VSMCs align themselves along the surface of the endothelial cell stalks. Bar, 0.25 mm.

• Supplemental Figure 5 -

  Fig. S5. Relative collagen content of mouse peritoneal and retinal tissues of 8-week-old wild-type C57BL/6 mice. The collagen content of the tail is shown as an example of a type I collagen-rich tissue. Hydroxyproline content was determined as described (Sabeh et al., 2004) and used to calculate relative type I collagen content.