Constitutive lung expression of Ang-1 in animals whose pulmonary circulations were perfused with Adeno-Ang-1. (a) The pattern of Ang-1 expression in rodent lung samples was studied by PCR using primers specific to the virally transduced Ang-1 mRNA (Vector). Tissue was analyzed 10, 20, and 40 days after injection of Adeno-Ang-1, Adeno-lacZ, or PBS alone (PBS-Sham). (b) Northern blot demonstrating that lungs of animals injected with the Adeno-Ang-1 construct had detectable steady-state levels of Ang-1 mRNA, whereas control lung specimens did not. (c) Western blot showing Ang-1 protein in lung tissue infected with Adeno-Ang-1 virus. Ang-1 protein was undetectable in animals injected with either Adeno-lacZ or PBS. (d) Immunoblot analysis of anti-TIE2 immunoprecipitates from rodent lung tissue, demonstrating that constitutive Ang-1 expression induced TIE2 receptor tyrosine phosphorylation but did not modulate TIE2 protein levels. (Upper) Immunoprecipitated (IP) with anti-TIE2 antibody, blotted with anti-phosphotyrosine (P-Tyr) antibody. (Lower) IP with anti-TIE2, blotted with anti-TIE2. (e) Detection of recombinant viral mRNA by in situ hybridization. Lung tissue from rats injected with PBS demonstrated no staining when a virus-specific antisense RNA probe was used. Conversely, virus-specific mRNA was detected in animals infected with Adeno-Ang-1 in lung vessel walls. (Scale bar represents 10 μm.)