Expression of syntaxin 6-cyto in mice blocks VEGF164-induced angiogenesis and permeability. (A,G) Mice in each group (all of which were Nu/Nu mice) were given 2 injections under anesthesia. The first set of injections (1st) was PBS, syntaxin 6-cyto, or syntaxin 16-cyto. The second (2nd) was PBS or Ad-VEGF164, and given 2 days later at the site of first injection as shown in panel A. At 7 days after the first set of injections, animals were euthanized and the ears were either photographed or removed for further processing. (B) Western blot analysis of syntaxin 6-cyto and syntaxin 16-cyto expression in mouse ear lysates prepared 5 days after Ad-VEGF164 treatment as described in panel A. (C) Quantification of VEGF164 levels in ear extracts using the Quantikine Mouse VEGF immunoassay kit (R&D Systems). Values represent mean (± SD) for n = 10 ears, P ≤ .005). (D) Representative images showing gross appearance of angiogenesis in mock, syntaxin 6-cyto– or syntaxin 16-cyto–injected mouse ears, 5 days before Ad-VEGF164 administration. (E,G) Ears were collected and processed for detailed histologic assessment. (E) Representative images showing staining of 1-μm Epon spur sections stained with Toludine blue. Vertical line marks extent of edema developed after different treatments. (F-G) Immunohistochemical staining of mouse ear sections stained with antibodies against CD31 (F) or VEGFR2 (G). Red arrowheads mark blood vessels developed after different treatments. (H,I) In an experiment similar to that described in panel C, mice treated 5 days before with Ad-VEGF164, were injected with Evans blue dye. (H) Mouse ears were photographed 30 minutes after injection, and representative images are shown. (I) Quantification of dye extravasation, carried out in another group of mice that were euthanized. Ears were removed and Evans blue dye was extracted and measured spectrophotometrically. Quantitated values represent mean (± SD) for n = 10 ears per data point, P ≤ .03.
