Viral Ang1 vectors and their effects on lymphatic vessels. (A-B) Schematic structures of the viral Ang1 constructs used in the study. (A) The adenoviral Ang1 vector.29 (B) The AAV vector containing the full-length human Ang1 cDNA. CMV indicates cytomegalovirus promoter and early enhancer; WPRE, Woodchuck hepatitis virus posttranscriptional enhancer element; pA, polyadenylation signal (from Simian virus 40); and ITR, inverted terminal repeats. (C) Comparison of AdAng1 and AAV-Ang1 recombinant protein production in vitro by precipitation using Ang1 antibodies or soluble Tie2-Fc protein. Media from AdLacZ- and AAV-EGFP-infected cells were used as negative controls (CT). (D) Ang1 RNA expression in a Northern blot containing total RNA from mouse ears 6 weeks after infection with AAV-Ang1 or AAV-EGFP. (E,G,I) Lymphatic vessels visualized with fluorescent LYVE-1 (red) whole-mount staining 2 weeks after infection with the adenoviruses. Lymphatic sprouts are indicated with arrowheads in panels E and I. (G) Lymphatics after AdLacZ infection. (F,H,J) LYVE-1-stained lymphatic vessels (green) in ears 6 weeks after AAV transduction. Ang1-induced lymphatic enlargement is shown with an asterisk in panel F. Scale bars: 100 μm.
