KCa3.1 channel blockade attenuates microvascular remodelling in a large animal model of bleomycin-induced pulmonary fibrosis

Figures

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  Figure 1. Alveolar capillaries in saline and bleomycin treated lung segments. (A) The panels show representative histological sections of either saline-infused (left-side images), or bleomycin-infused (right-side images), lung segments taken from the same sheep. The top two panels show sections that were immuno-stained with antibodies against either CD34 or collagen type IV to detect capillaries in parenchyma. Arrows show large and dilated capillaries in bleomycin treated segment. The third panel shows representative Masson’s trichrome-stained sections that were used to determine fibrotic fraction. Primary antibodies were omitted in the negative control (fourth panel). Graphs show the relationship between vascular density and fibrotic fraction in bleomycin infused lung segments immuno-stained with CD34 (r = 0.67, p = 0.048) (B) and collagen type IV (r = 0.64, p = 0.067) (C) (n = 9 sheep). Scale bars = 50 µm.

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  Figure 2. Effects of senicapoc and pirfenidone on remodelling of CD34 stained capillaries induced by bleomycin. (A) The panels show representative anti-CD34 immuno-stained sections of lung segments from sheep that were treated with either vehicle, senicapoc or pirfenidone. The lung segments from individual sheep were infused with either saline (left-side images), or bleomycin (right-side images). (B) Shows the vascular density data between the different treatment groups. Vascular density was determined by the percentage of the total area of CD34-positive vascular area per field of lung parenchyma. Ten images were taken from fibroproliferative areas for each lobe from each sheep (n = 10) and the values for each of the ten images were averaged for each lobe. Data show individual values plus mean. Significance was determined using a paired t test between lobes from the same treatment group, and an unpaired t test between lobes from different groups, **p < 0.01, ***p < 0.001, ns-not significant. Data represent mean (±SEM). Scale bars = 50 µm.

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  Figure 3. Effects of senicapoc and pirfenidone on remodelling of collagen type IV stained capillaries induced by bleomycin. (A) The panels show representative anti-collagen type IV immuno-stained sections of lung segments from sheep that were treated with either vehicle, senicapoc or pirfenidone. The lung segments from individual sheep were infused with either saline (left-side images), or bleomycin (right-side images). (B) Shows the vascular density data between the different treatment groups. Vascular density was determined by the percentage of the total area of anti-collagen type IV-positive vascular area per field of lung parenchyma. Ten images were taken from fibroproliferative areas for each lobe from each sheep (n = 10) and the values for each of the ten images were averaged for each lobe. Data show individual values plus mean. Significance was determined using a paired t test between lobes from the same treatment group and an unpaired t test between lobes from different groups, *p < 0.05, ***p < 0.001. Data represent mean (±SEM). Scale bars = 50 µm.

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  Figure 4. Senicapoc, but not pirfenidone, down regulates VEGF expression that was increased by bleomycin. (A) The panels show representative anti-VEGFA immuno-stained sections of lung segments from sheep that were treated with either vehicle, senicapoc or pirfenidone. The lung segments from individual sheep were infused with either saline (left-side images), or bleomycin (right-side images). (B) Shows the data for the percentage VEGF-positive area per total field area of lung parenchyma for the different treatment groups. Data show individual values plus mean, (n = 10 sheep). Significance was determined using a paired t test between lobes from the same treatment group and an unpaired t test between lobes from different groups, *p < 0.05, **p < 0.01, ns-not significant. Data represent mean (±SEM). Scale bars = 50 µm.

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  Figure 5. Senicapoc reduces alveolar capillary endothelial proliferation in vivo. (A) Representative immunofluorescence photomicrograph of lung cryosections from differentially treated lung segments double stained with Ki-67 and CD34 antibodies. CD34 stained vessels together with Ki-67 signals indicate endothelial cell proliferation (arrows indicate Ki-67 overlapping with CD34 immunofluuorescence). Arrowheads indicate proliferating non-endothelial cells. (B) Shows data for CD34/Ki-67 positive cells from the different treatment groups (average/10 fields in each differentially treated segments, n = 10 sheep), **p < 0.01, ns-not significant. Data represent mean (±SEM). Sal-Veh = saline-vehicle lobe; BLM-Veh = bleomycin-vehicle lobe; Sal-Sen = saline-senicapoc lobe; BLM-Sen = bleomycin-senicapoc lobe. Scale bars = 50 µm.

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  Figure 6. Pirfenidone does not reduce alveolar capillary endothelial proliferation in vivo. (A) Representative immunofluorescence photomicrograph of lung cryosections from differentially treated lung segments double stained with Ki-67 and CD34 antibodies. CD34 stained vessels together with Ki-67 signals indicate endothelial cell proliferation (arrows indicate Ki-67 overlapping with CD34 immunofluuorescence). Arrowheads indicate proliferating non-endothelial cells. (B) Shows data for CD34/Ki-67 positive cells from the different treatment groups (average/10 fields in each differentially treated segments, n = 10 sheep), **p < 0.01, ***p < 0.001, ns-not significant. Data represent mean (±SEM). Sal-Veh = saline-vehicle lobe; BLM-Veh = bleomycin-vehicle lobe; Sal-Pirf = saline-pirfenidone lobe; BLM-Pirf = bleomycin-pirfenidone lobe. Scale bars = 50 µm.

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  Figure 7. Induction of pulmonary fibrosis and therapeutic intervention in sheep. (A) Schematic diagram depicting the bleomycin and saline treatments administered to individual caudal lung segments in sheep. One lung segment received bleomycin, while the contralateral segment in the same sheep received saline as internal control. (B) Diagram showing the timeline for treatment of tested drugs (senicapoc or pirfenidone) or vehicle (methylcellulose), and euthanasia of experimental animals.