Wrapping HUVECs express high levels of MMP-14 and MMP-9, and inhibition of MMPs interferes with WAT anastomosis. (A) Strong MMP-14 expression (blue) colocalizes precisely with HUVECs (green, GFP transduction), but not with unwrapped parts of host vessels (red, mouse-specific MECA32, which is denoted mMECA32, staining) or pericytes (gray, NG2 staining). Filled arrowheads indicate unwrapped host ECs with weak MMP-14 staining; empty arrowheads indicate strong pericyte staining with weak MMP-14 signal. Whole-mount IHC stain; 20×/0.95 NA water objective at 3.0× digital zoom; scale bar represents 40 μm. (B) MMP-14 expression (blue) by wrapping HUVECs (filled arrowheads) is stronger than that by nonwrapping HUVECs (empty arrowheads) or maturing HUVEC vessels (arrows). Whole-mount IHC stain; 20×/0.95 NA water objective; scale bar represents 120 μm. (C) MMP-9 (blue) expression is strongest on wrapping HUVECs (filled arrowheads) compared with nonwrapping HUVECs (empty arrowheads) and maturing HUVEC vessels (arrows). “Box” shows the detail of the area within the white box. The dashed line divides the main HUVEC vessel in this panel into 2 parts: the one on the right is wrapping around some host vessel segments (empty arrows) and has very strong MMP9 staining, whereas the one on the left has no host vessel segments inside, is apparently maturing, and has much weaker MMP-9 staining. Whole-mount stain; 20×/0.95NA water objective; scale bar represents 120 μm. (D) MMP-14 expression (white bars) on unwrapped host ECs and pericytes is 46% ± 18% (n = 12) and 49% ± 16% (n = 12), respectively, that of wrapping HUVECs. MMP-9 expression (gray bar) by unwrapped host ECs is 59% ± 9% (n = 13) that of wrapping HUVECs. (E) MMP-14 expression by HUVECs changes with segment diameter and wrapping status. The normalized value is 0.67 ± 0.23 (n = 94) for “Individual,” nonwrapping HUVECs, 1.00 ± 0.18 (n = 25) for “Wrapping” HUVECs, and 0.54 ± 0.16 (n = 38) for “Maturing” HUVEC vessels. (F) MMP-9 expression by HUVECs also changes with segment diameter and wrapping status. The normalized value is 0.67 ± 0.20 (n = 153) for “Individual,” nonwrapping HUVECs, 1.000 ± 0.176 (n = 37) for “Wrapping” HUVECs, and 0.569 ± 0.126 (n = 47) for “Maturing” HUVEC vessels. (G) Typical perfusion (visualized by intravenous injection of Rho-dextran, which is shown in red) of implanted HUVEC networks (green, GFP transduction) 2 weeks after implantation when the animal is treated with control IgG, monoclonal antibody against MMP-14 (DX-2400), monoclonal antibody against MMP-9 (DX-2802), or DX-2400 + DX-2802. Intravital confocal images; 10×/0.3 NA air objective; scale bar represents 200 μm. (H) Treatment with DX-2400 and/or DX-2802 delays the onset of WAT anastomosis. Data were generated from the analysis of images, such as those shown in panel G. ***P < .05 between “Control IgG” and each of the other 3 groups. *P < .05 only between “Control IgG” and “DX-2400 + DX-2802.”