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. 2017 Aug 15;196(4):479–493. doi: 10.1164/rccm.201603-0568OC

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Copyright © 2017 by the American Thoracic Society

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Figure 6. — The effect of vascular endothelial growth factor (VEGF)-A₁₆₅b on the development of pulmonary fibrosis. (A) Lung phenotype of the MMTV-VEGF-A₁₆₅b transgenic (TG) mouse. Immunohistochemical staining for VEGF-A₁₆₅b in the lung of TG mice with quantification of VEGF-A₁₆₅b expression by ELISA, in whole-tissue lysates and bronchoalveolar lavage fluid (BALF). VEGF-A₁₆₅b expression was increased in the alveolar type II (ATII) cells of the TG mouse lung (d and f, indicated by arrows) compared with the wild-type (WT) lung (c and e). Isotype IgG staining was used as a negative control (a and b). Images were taken at ×100 magnification; scale bars = 10 μm, (n = 3, n = 1 shown). VEGF-A₁₆₅b expression was significantly up-regulated in whole-tissue lysates of TG mice compared with WT mice (*P < 0.05; unpaired Student’s t test with Welch correction; n = 4). The expression of VEGF-A₁₆₅b was below the limit of detection of the ELISA in all BALF samples of WT mice but present in detectable levels in all BALF samples of TG mice. There was no statistical difference between these groups (P = 0.055; unpaired Student’s t test with Welch correction; n = 4). (B) Overexpression of VEGF-A₁₆₅b in ATII cells ameliorates the development of bleomycin (BLM)-induced pulmonary fibrosis. Representative images of Masson trichrome staining with lung fibrosis score and quantitative reverse transcription–polymerase chain reaction (RT-PCR) of fibronectin (FN) and pro-collagen-1α mRNA levels (bottom). Masson trichrome staining of mouse lung sections, 21 days after oropharyngeal (OP) instillation of BLM to MMTV-VEGF-A₁₆₅b TG mice (j–l) or littermate control animals (g–i). The development of BLM-induced pulmonary fibrosis was ameliorated in TG mice. Saline control subjects are shown in a–f (n = 6 per group; n = 3 shown). Scale bar = 100 μm; original magnification, ×10. The lung fibrosis score of BLM-treated WT mice was significantly greater than saline-treated WT mice (^****P < 0.0001; n = 6 per group). The lung fibrosis score of BLM-treated TG mice was significantly lower than BLM-treated WT mice (***P < 0.001; n = 6 per group). FN mRNA was significantly up-regulated in the lungs of WT BLM-treated mice compared with WT saline-treated control animals (**P < 0.01; n = 6 analyzed) and in TG BLM-treated mice compared with TG saline-treated control animals (*P < 0.05; n = 6). There was no statistical difference between BLM-treated WT and TG mice (P = 0.09; n = 6). In contrast, pro-collagen-1α mRNA levels were significantly reduced in BLM-treated TG mice compared with BLM-treated WT littermates (*P < 0.05; n = 6). Data are presented as means with SEM. Statistical analysis: analysis of variance with Holm-Šidák multiple comparisons test used throughout. (C) IP instillation of rhVEGF-A₁₆₅b ameliorates the development of pulmonary fibrosis. Masson trichrome staining of lung sections with lung fibrosis score and quantitative RT-PCR of FN and pro-collagen-1α mRNA levels (bottom). Representative images of Masson trichrome staining of mouse lung sections, 21 days after OP instillation of BLM with or without additional IP rhVEGF-A₁₆₅b instillation. (a–c) OP saline control (no differences between early IP VEGF-A₁₆₅b [EV] or late IP VEGF-A₁₆₅b [LV] plus saline). (d–f) OP BLM with saline IP. (g–i) Early IP VEGF-A₁₆₅b treatment with subsequent BLM OP instillation. (j–l) BLM OP with subsequent late IP VEGF-A₁₆₅b instillation. Early and late rhVEGF-A₁₆₅b instillation ameliorated the development of lung fibrosis (n = 6 per group; n = 3 shown). Scale bar = 100 μm; original magnification, ×10. By qRT-PCR, FN and pro-collagen-1α mRNA was significantly up-regulated in the lungs of BLM-treated mice compared with saline-treated control animals (*P < 0.05; **P < 0.01; n = 5). In contrast, FN and pro-collagen-1α expression in mice treated with either early (EV) or late (LV) rhVEGF-A₁₆₅b and BLM was not significantly different from saline control (n = 6). The lung fibrosis score in both early (EV, **P < 0.01) and late (LV, *P < 0.05) IP rhVEGF-A₁₆₅b treatment groups was significantly reduced compared with BLM OP–saline IP–treated mice; n = 5 or 6 per group; data are presented as means with SEM. Statistical analysis: analysis of variance with Holm-Šidák multiple comparisons test used throughout. CTRL = control; n/s = not significant.

Figure 6. — The effect of vascular endothelial growth factor (VEGF)-A₁₆₅b on the development of pulmonary fibrosis. (A) Lung phenotype of the MMTV-VEGF-A₁₆₅b transgenic (TG) mouse. Immunohistochemical staining for VEGF-A₁₆₅b in the lung of TG mice with quantification of VEGF-A₁₆₅b expression by ELISA, in whole-tissue lysates and bronchoalveolar lavage fluid (BALF). VEGF-A₁₆₅b expression was increased in the alveolar type II (ATII) cells of the TG mouse lung (d and f, indicated by arrows) compared with the wild-type (WT) lung (c and e). Isotype IgG staining was used as a negative control (a and b). Images were taken at ×100 magnification; scale bars = 10 μm, (n = 3, n = 1 shown). VEGF-A₁₆₅b expression was significantly up-regulated in whole-tissue lysates of TG mice compared with WT mice (*P < 0.05; unpaired Student’s t test with Welch correction; n = 4). The expression of VEGF-A₁₆₅b was below the limit of detection of the ELISA in all BALF samples of WT mice but present in detectable levels in all BALF samples of TG mice. There was no statistical difference between these groups (P = 0.055; unpaired Student’s t test with Welch correction; n = 4). (B) Overexpression of VEGF-A₁₆₅b in ATII cells ameliorates the development of bleomycin (BLM)-induced pulmonary fibrosis. Representative images of Masson trichrome staining with lung fibrosis score and quantitative reverse transcription–polymerase chain reaction (RT-PCR) of fibronectin (FN) and pro-collagen-1α mRNA levels (bottom). Masson trichrome staining of mouse lung sections, 21 days after oropharyngeal (OP) instillation of BLM to MMTV-VEGF-A₁₆₅b TG mice (j–l) or littermate control animals (g–i). The development of BLM-induced pulmonary fibrosis was ameliorated in TG mice. Saline control subjects are shown in a–f (n = 6 per group; n = 3 shown). Scale bar = 100 μm; original magnification, ×10. The lung fibrosis score of BLM-treated WT mice was significantly greater than saline-treated WT mice (^****P < 0.0001; n = 6 per group). The lung fibrosis score of BLM-treated TG mice was significantly lower than BLM-treated WT mice (***P < 0.001; n = 6 per group). FN mRNA was significantly up-regulated in the lungs of WT BLM-treated mice compared with WT saline-treated control animals (**P < 0.01; n = 6 analyzed) and in TG BLM-treated mice compared with TG saline-treated control animals (*P < 0.05; n = 6). There was no statistical difference between BLM-treated WT and TG mice (P = 0.09; n = 6). In contrast, pro-collagen-1α mRNA levels were significantly reduced in BLM-treated TG mice compared with BLM-treated WT littermates (*P < 0.05; n = 6). Data are presented as means with SEM. Statistical analysis: analysis of variance with Holm-Šidák multiple comparisons test used throughout. (C) IP instillation of rhVEGF-A₁₆₅b ameliorates the development of pulmonary fibrosis. Masson trichrome staining of lung sections with lung fibrosis score and quantitative RT-PCR of FN and pro-collagen-1α mRNA levels (bottom). Representative images of Masson trichrome staining of mouse lung sections, 21 days after OP instillation of BLM with or without additional IP rhVEGF-A₁₆₅b instillation. (a–c) OP saline control (no differences between early IP VEGF-A₁₆₅b [EV] or late IP VEGF-A₁₆₅b [LV] plus saline). (d–f) OP BLM with saline IP. (g–i) Early IP VEGF-A₁₆₅b treatment with subsequent BLM OP instillation. (j–l) BLM OP with subsequent late IP VEGF-A₁₆₅b instillation. Early and late rhVEGF-A₁₆₅b instillation ameliorated the development of lung fibrosis (n = 6 per group; n = 3 shown). Scale bar = 100 μm; original magnification, ×10. By qRT-PCR, FN and pro-collagen-1α mRNA was significantly up-regulated in the lungs of BLM-treated mice compared with saline-treated control animals (*P < 0.05; **P < 0.01; n = 5). In contrast, FN and pro-collagen-1α expression in mice treated with either early (EV) or late (LV) rhVEGF-A₁₆₅b and BLM was not significantly different from saline control (n = 6). The lung fibrosis score in both early (EV, **P < 0.01) and late (LV, *P < 0.05) IP rhVEGF-A₁₆₅b treatment groups was significantly reduced compared with BLM OP–saline IP–treated mice; n = 5 or 6 per group; data are presented as means with SEM. Statistical analysis: analysis of variance with Holm-Šidák multiple comparisons test used throughout. CTRL = control; n/s = not significant.