WAT anastomosis between implanted HUVECs and host (mouse) vasculature in cranial windows and dorsal skinfold chambers. (A) In cranial windows, HUVECs (green, GFP transduction) first wrap around host vessels (red, Alexa647-conjugated mouse-specific CD31, which is denoted mCD31-X647, injected intravenously), which have invaded the implant through angiogenesis, and eventually replace them to tap into the blood flow. Perfused vessels were visualized by intravenous injection of cascade blue-conjugated dextran (denoted CB-dextran, blue) on day 23. White dashed curves indicate the edge of the engrafted gel. Empty arrowheads in the central panels indicate a host vessel within the implant. Filled arrowheads in the central and right-hand panels indicate a host segment wrapped and then completely replaced by HUVECs. The arrow indicates a HUVEC-host junction. Intravital confocal images; 4×/0.13 NA air objective (left column) and 20×/0.4 NA air objective at 2.0× digital zoom (middle and right columns); scale bar represents 500 μm. (B) Overview of host-implant vascular anastomosis in dorsal skinfold chambers. HUVECs are green (GFP transduction), host vessels are red (mCD31-X647 injected intravenously), and blood perfusion is shown by intravenous injection of rhodamine-conjugated dextran (denoted Rho-dextran, light blue). Empty arrowheads track a typical example of WAT anastomosis: significant host vessel angiogenesis and extensive HUVEC wrapping are evident at day 14; more host vessels grow into the implant and wrapping continues at day 19; at day 23, there is obvious host vessel degradation and extensive HUVEC network perfusion; at day 28, host vessel degradation continues and perfusion propagates deeper into the bulk of the implant. Details of the areas within the white boxes in “Day 14” and “Day 23” images are shown in supplemental Figure 2C and Figure 1C, respectively. Intravital confocal images; 10×/0.3 NA air objective (each presented image is a montage of 16 captured images); scale bar represents 500 μm. (C) Detail of the area within the white box in “Day 23” image of panel B. Wrapping HUVECs (green, GFP transduction) “tap” a host vessel (red, mCD31-X647 injected intravenously) for blood flow (light blue, Rho-dextran injected intravenously). Arrows indicate where “tapping” is taking place. The right-hand panel shows the detail within the white box in the left-hand panel. Empty arrowhead points to where HUVECs have opened the host vessel wall to divert the blood flow. Intravital confocal images; 20×/0.4 NA air objective at 2.5× (left and middle columns) and 9.0× digital zoom (right column); scale bar represents 40 μm. (D) Regressing host vessel segments (red, mCD31-X647 injected intravenously; empty arrowheads) inside developing HUVEC vessels (green, GFP transduction) in a 30-day-old implant. Perfused vessels (blue) are visualized by intravenous injection of CB-dextran. Arrow indicates a HUVEC-host junction. Intravital confocal images; 10×/0.3 NA air objective at 1.5× digital zoom; scale bar represents 100 μm. (E) Detail of a HUVEC-host junction at day 48 showing that the HUVEC segment encapsulates the end of the remaining host vessel. Vessel perfusion is marked by biotin staining (blue) of lectin injected intravenously before tissue fixation. “Box” shows the detail of the area within the white box in the “Merged” panel. “Illustration” shows the microanatomy of the junction. Whole-mount IHC stain; 20×/0.95 NA water objective (row 1) and 60×/1.1 NA objective at 2.0× digital zoom (row 2); scale bar represents 100 μm.