The poly (ADP-ribose) polymerase inhibitor olaparib attenuates established pulmonary fibrosis in a large animal model

Figures

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  Fig. 1. Induction of pulmonary fibrosis and therapeutic intervention in sheep. (A) Schematic diagram showing localised delivery of bleomycin (BLM) and saline to individual caudal lung lobes in sheep. While one lung lobe received 3 U BLM, the contralateral lobe in the same animal received saline as an internal control. (B) Timeline (weeks) showing bleomycin injury (blue arrows), olaparib and vehicle treatments, bronchoalveolar lavage fluid (BAL) sampling (green arrows) and necropsy (red arrows)

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  Fig. 2. Plasma PK profile (over 24 h) following oral olaparib administration in sheep. Mass spectrometric assessment of plasma olaparib levels; the data are expressed as the means ± SEM (n = 6 sheep)

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  Fig. 3. PARP1 lung expression. (A) Representative micrographs showing PARP1+ immunostaining (brown stained cells) in saline- and BLM-exposed lung lobes of vehicle control and olaparib-treated sheep. (B) PARP1+ cell counts in saline- and BLM-exposed lungs; the data are expressed as the means ± SEM (n = 6/group); *p < 0.05, **p < 0.01. Scale bars = 50 μm

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  Fig. 4. Lung histopathology. (A) Representative H&E staining of saline- and BLM-exposed lung lobes from vehicle only treated (control) and olaparib treated sheep. (B-C) Histopathology scoring data for (B) inflammation and (C) fibrosis as assessed on H&E-stained sections from the differentially treated lung lobes. The data are expressed as the means ± SEM (n = 6/group); *p < 0.05, **p < 0.01

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  Fig. 5. Collagen deposition in the lungs. (A) Masson’s trichrome staining of BLM-exposed lung lobes in vehicle-only treated (control) and olaparib-treated sheep. (B) Graphs showing the evaluation of collagen content; Masson’s trichrome blue-stained collagen area percentage (upper panel) and total hydroxyproline content (lower panel); data are expressed as the means ± SEM (n = 6/group), *p < 0.05, **p < 0.01

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  Fig. 6. BAL inflammatory cells in differentially treated lung lobes at baseline (0), week two (2) and week six (6) after 2nd BLM/saline exposure. (A) neutrophil numbers, (B) lymphocyte numbers, (C) macrophage numbers, (D), total leukocyte numbers, (E) neutrophil percentage, (F) lymphocyte percentage, and (G) macrophage percentage. data expressed as means ± SEM (n = 6/group), *p < 0.05, **p < 0.01, ***p < 0.001.The cell numbers are shown as ×104 cells/mL BAL fluid. The data are expressed as the means ± SEM (n = 6/group), *p < 0.05, **p < 0.01, ***p < 0.001

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  Fig. 7. Effect of olaparib on the migration of primary sheep lung fibroblasts. (A) Photomicrographs of wound scratch taken at 0 and 24 h (4x magnification). (B) Graph showing wound closure percentage in cells at 24 h following incubation in the presence of increasing concentrations of olaparib (50-200mM). The data are expressed as means ± SEM (n = 3 sheep), ***p < 0.001