AAV-TMD1 suppresses experimental angiogenesis in vivo. (A) Schematic representation of the construction of pAAV-TMD1 and generation of AAV-TMD1 particles by tripartite transfection of 3 plasmids into 293A cells. (B) Luciferase assay. Equivalent samples were analyzed after inoculation of the mice with AAV-Luc or DPBS. RLU indicates relative light unit. (C) RT-PCR of liver extract after inoculation of mice with AAV-TMD1 or DPBS. TMD1: human TMD1, 381 bp; Thbd: mouse TM, 130 bp; Gapdh: mouse GAPDH, 601 bp. (D) Experimental protocol. FVB mice were divided into 6 groups; 1 was given a single injection of AAV-TMD1, DPBS, or AAV-Luc 4 days before implantation of Matrigel; was given a subcutaneous injection of 200 μL of Pichia-expressed rTMD1 (80 μg/mL) daily from day 4 to day 7, and the others were given Matrigel containing EGF with or without rTMD1 (44 nmol/L). On day 8, the mice were killed and the lung, liver, and Matrigel were collected. The angiogenic index was evaluated by the hemoglobin content in the Matrigel. (E) Statistical analysis of the murine angiogenic assay. The data represent mean ± SD. Similar results were obtained in 3 independent experiments; n represents the number of plugs for each group. ***P < .001 compared with the control. ###P < .001 compared with the DPBS or AAV-Luc. s.c. indicates subcutaneously; and i.v., intravenously.