1. Volume 12, Issue 1, Pages 58-67 (July 2005)
Hepatocyte Growth Factor Gene Transfer to Alveolar Septa for Effective Suppression of Lung Fibrosis 
Masaki Watanabe, Masahito Ebina, Frank M. Orson, Akira Nakamura, Kazuo Kubota, Daizo Koinuma, Ken-ichi Akiyama, Makoto Maemondo, Shinya Okouchi, Minoru Tahara, Kunio Matsumoto, Toshikazu Nakamura, Toshihiro Nukiwa 
Molecular Therapy 
Volume 12, Issue 1, Pages (July 2005)
DOI: /j.ymthe
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

2. FIG. 1
Lung-selective distribution of hHGF gene by MAA-PEI. (A) Scintillation-scanning of 99mTc-MAA in a mouse body 1 h after intravenous administration. (B) The radioactivity of the organs 6 h after intravenous administration of 99mTc-MAA (n = 5). Adjustment was made for the decrease of the radioactivity due to the decay of 99mTc. (C) The expression of hHGF in the mouse lungs 72 h after intravenous administration of pCAG.hHGF (0–10 μg) with (black bars, n = 5 each) and without (white bars, n = 5 each) MAA-PEI (NS, no significant difference). (D) Time course (days) of hHGF expression in the mouse lung by pCAG.hHGF (1 μg) with MAA-PEI (black boxes) was compared with that by pCAG.hHGF alone (10 μg, white boxes). With administration of MAA-PEI + pCAG.hHGF (1 μg) three times (indicated by arrows), the levels of hHGF (black triangles) increased until day 9. MAA-PEI + pCAG.null administration was examined as a negative control (black circles). n = 3–5 per group. (E) The organ distribution on day 3 with 1 μg of pCAG.hHGF with MAA-PEI (black bars, n = 5) was compared with that with 10 μg pCAG.hHGF without MAA-PEI (white bars, n = 6) (*P < 0.05). (F) ELISA of serum TNF-α (black bars) and IL-6 (white bars) 5 h after intravenous administration of adenoviral vector containing pCAG.null (1 × 109 pfu) or pCAG.null with and without MAA-PEI. n = 6–8 per group (*P < 0.05).
Molecular Therapy  , 58-67DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

3. FIG. 2
Distribution of transfected pulmonary cells expressing hHGF. (A) hHGF mRNA in the transfected mouse lungs was detected by RT-PCR. Lane 1, a mouse lung transfected with MAA-PEI + pCAG.hHGF (1 μg); lane 2, a mouse lung transfected with pCAG.hHGF (10 μg) alone; lane 3, a control lung transfected with MAA-PEI + pCAG.null; lanes 4 and 5, without the reverse transcription using the same samples as in lanes 1 and 2, respectively. (B) In situ PCR of hHGF cDNA revealed stained nuclei in the transfected pulmonary cells 72 h after the administration of MAA-PEI + pCAG.hHGF (1 μg). (C–E) In situ RT-PCR of hHGF without DNase treatment revealed transfected pulmonary cells by staining both their cytoplasm and their nuclei, distributed diffusely in the mouse lung 72 h after the administration of MAA-PEI + pCAG.hHGF (1 μg). No reactive staining was observed in airway epithelial cells. (F) Amplification of β-actin by in situ RT-PCR of all pulmonary cells as a positive control. (G) As a negative control, no reaction by in situ RT-PCR for hHGF was observed without gene transfection of hHGF. Scale bars: (B–D, F, and G) 20 μm; (E) 5 μm.
Molecular Therapy  , 58-67DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

4. FIG. 3
Endogenous expression of HGF and phosphorylation of c-Met in the lung tissues. (A) Immunohistochemistry for HGF using an anti-human HGF antibody that cross-reacts with mouse HGF revealed that the alveolar macrophages (arrowheads) faintly expressed endogenous mHGF in the lungs without transfection. (B) Three days after intravenous injection of MAA-PEI + pCAG.hHGF (1 μg), immunohistochemistry demonstrated high expression of HGF in the alveolar epithelial cells (arrows) and alveolar capillary endothelial cells (open arrowheads) as well as alveolar macrophages (arrowheads). Scale bars: (A and B) 20 μm. (C) The endogenous mouse HGF in the mouse lungs with and without injury by bleomycin (BLM) was detected by ELISA using an antibody that does not cross-react with human HGF. pCAG.null (Null) or pCAG.hHGF (hHGF) bound to MAA-PEI complex was administered once (on day 1) for day 3 and twice (on days 1 and 4) for day 7. The endogenous mouse HGF was increased by administration of MAA-PEI + pCAG.hHGF (1 μg) and also by bleomycin injury on day 3, not continued to day 7. Control lungs (PBS + PBS), white bars; bleomycin-injured lungs with injection of MAA-PEI + pCAG.null (BLM + Null), black bars; MAA-PEI + pCAG.hHGF-transfected lungs with (BLM + HGF), hatched bars, and without bleomycin injury (PBS + HGF), gray bars. n = 3 per group (*P < 0.05). (D) Immunoblot analysis showed an HGF-triggered elevation of phospho-Met (p-Met, 140 kDa) in the mouse lungs on days 3 and 7 after bleomycin injury (injured on day 0) with and without administration of MAA-PEI + pCAG.hHGF (1 μg) (HGF) or pCAG.hHGF (10 μg) only (p.HGF) on days 1 and 4.
Molecular Therapy  , 58-67DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

5. FIG. 4
The effects of hHGF gene transfer on bleomycin-induced lung injury. (A) The increase of TNF-α (black bars) and IL-6 (white bars) in mouse lungs 7 days after the administration of bleomycin was attenuated by the injection of MAA-PEI + pCAG.hHGF on days 1 and 4 (*P < 0.05). (B) The hydroxyproline content in the mouse lungs 21 days after bleomycin injury was reduced by pCAG.hHGF (10 μg) (dotted bars) and by MAA-PEI + pCAG.hHGF (1 μg) (hatched bars), which were administered on days 1, 4, and 7, but not by pCAG..null (*P < 0.05). (C–F) Photomicrographs of bleomycin-injured lungs with and without transfection of hHGF gene using MAA-PEI complex (Elastica-Masson staining). (C) The massive fibrosis after bleomycin injury was not changed by the administration of MAA-PEI + pCAG.null. The lesions close to the pleura mimic honeycombing lesions, which are frequently observed in the lungs of patients with idiopathic pulmonary fibrosis. (D) The distribution of fibrotic lesions was decreased by the administration of MAA-PEI + pCAG.hHGF (1 μg), although (F) fibrous lesions close to the central airways remained. (F) No fibrotic lesions were distributed in the peripheral lungs after the transfection of hHGF. Scale bars: (C and E) 100 μm; (D and F) 50 μm.
Molecular Therapy  , 58-67DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

6. FIG. 5
Antiapoptotic effects of hHGF on A549 cells and mouse alveolar epithelial cells after bleomycin injury. (A–C) Flow-cytometric DNA histograms of PI-stained A549 cells with no treatment (NT; A), with 24 h incubation in bleomycin (50 μg/ml) (BLM; b), or with incubation that also included 1 μg/ml rhHGF (BLM + rhHGF; C). (D) The percentages of apoptotic cells expressed as the sub-G1 population of DNA histograms, examined in triplicate. (E) rhHGF (1 μg/ml) increased the cell number of A549 cells that had been decreased after 48 h incubation with bleomycin. (F) The suppressive effect of rhHGF on the caspase-3 activity of A549 cells after 48 h incubation with bleomycin. (G and H) Apoptotic cells in the lungs 3 days after bleomycin injury, revealed by the TUNEL assay (G), were reduced substantially by the administration of MAA-PEI + pCAG.hHGF (1 μg) (H). Scale bars: (G and H) 20 μm. (I) Percentages of apoptotic cells revealed by the TUNEL assay in the mouse lungs on days 3 and 7 after bleomycin injury were remarkably decreased by administration of MAA-PEI + pCAG.hHGF (1 μg) (hHGF, gray bars) but not by pCAG.null (null, black bars) (*P < 0.05). n = 3 per group.
Molecular Therapy  , 58-67DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

7. FIG. 6
The effects of hHGF on regeneration by bone marrow-derived stem cells after bleomycin-injury. (A-C) After transplantation of GFP-positive bone marrow, the lungs were injured by bleomycin with or without hHGF gene transfection. (A) Immunohistochemistry for GFP showed that most of the alveolar macrophages were GFP-positive (shown in brown) in the lungs before lung injury by bleomycin. (B) The GFP-positive cells were increased in the lungs 14 days after bleomycin-injury, including subsets of both epithelial cells and endothelial cells. (C) The increase of GFP-positive cells by bleomycin-injury was inhibited by additional intravenous administration of MAA-PEI + pCAG.hHGF (1 μg) on days 1, 4, and 7, although some GFP-positive alveolar epithelial cells and capillary endothelial cells were found (indicated by arrows). Scale bars: (A-C) 20 μm. (D-I) The lungs 14 days after bleomycin injury contained the alveolar capillary endothelial cells positive for both GFP (in green, D) and thrombomodulin (in red, E) (merged to yellow in F). The alveolar epithelial cells positive for both GFP (in green, G) and cytokeratin (in red, H) (merged to yellow in I) were also found in these injured lungs. (J) The numbers of GFP-positive/thrombomodulin-positive alveolar capillary endothelial cells and GFP-positive/cytokeratin-positive alveolar epithelial cells 14 days after bleomycin injury were compared with those inhibited by additional intravenous injection of MAA-PEI + pCAG.hHGF (1 μg) on days 1, 4, and 7 (*P < 0.05). n = 3 per group. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Molecular Therapy  , 58-67DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions