Furin Regulates the Alveolarization of Neonatal Lungs in a Mouse Model of Hyperoxic Lung Injury

Abstract

Despite advances in treatment options, such as corticosteroid administration and less invasive respiratory support, bronchopulmonary dysplasia (BPD) remains an important prognostic factor in preterm infants. We previously reported that furin regulates changes in lung smooth muscle cell phenotypes, suggesting that it plays a critical role in BPD pathogenesis. Therefore, in this study, we aimed to evaluate whether it regulates the alveolarization of immature lungs through activating alveolarization-driving proteins. We first examined furin expression levels, and its functions, using an established hyperoxia-induced BPD mouse model. Thereafter, we treated mice pups, as well as primary myofibroblast cell cultures, with furin inhibitors. Finally, we administered the hyperoxia-exposed mice pups with recombinant furin. Immunofluorescence revealed the co-expression of furin with alpha-smooth muscle actin. Hyperoxia exposure for 10 d decreased alveolar formation, as well as the expression of furin and its target, IGF-1R. Hexa-D-arginine administration also significantly inhibited alveolar formation. Another furin inhibitor, decanoyl-RVKR-chloromethylketone, accumulated pro-IGF-1R, and decreased IGF-1R phosphorylation in myofibroblast primary cultures. Finally, recombinant furin treatment significantly improved alveolarization in hyperoxia-exposed mice pups. Furin regulates alveolarization in immature lungs. Therefore, this study provides novel insights regarding the involvement of furin in BPD pathogenesis, and highlights a potential treatment target for ameliorating the impact of BPD.

Keywords: alveologenesis; bronchopulmonary dysplasia; proprotein convertase.

Figure 1

Furin expression in developing mouse lungs. Furin-positive cells (arrow head) were detected in P10 mice pups via immunohistochemistry using an anti-furin polyclonal antibody and observed under an optical microscope (a). Furin immunoreactivity was also detected in vascular smooth muscle cells (arrow head), but absent in airway epithelial cells (b). Furin expression in developing mouse lungs was downregulated following mice pup exposure to 85% O₂ for 10 d; Scale bar, 10 μm (c). Number of furin-positive cells in air- or 85% O₂-exposed mice pups was assessed (n = 5 animals in each group, * p < 0.05) (d). Furin was confirmed to be co-expressed with α-smooth muscle actin in cells isolated from the peripheral lungs of P1 mice; Scale bar, 20 μm (e).

Figure 2

Downregulated furin expression in hyperoxia-exposed developing mouse lungs. C57BL6J mice pups and their mothers were exposed to 85% oxygen within 12 h of birth to P10. Hyperoxia exposure impaired alveolar development compared to the control on fixed sections of lungs in each treatment group, ((a); control, (b); hyperoxia), and the changes in hyperoxic mice pups were confirmed via Lm (c) and TVD (d) measurements (control, n = 7; hyperoxia, n = 5; * p < 0.05). qRT-PCR revealed a reduction in mRNA levels of furin in the lungs of mice exposed to hyperoxia (e). Immunoblotting of whole lung lysates from each group showed decreased furin immunoreactivity in hyperoxia-exposed mouse lungs (f) and densitometry results is shown (g); Scale bar, 50 μm (a). Lm, mean linear intercept; TVD, tissue volume density.

Figure 3

Downregulated expression of a furin target protein in hyperoxia-exposed mouse lungs. Immunoreactivity of IGF-1R, a protein cleaved and matured by furin, was confirmed using whole lung lysates from hyperoxia-exposed mouse lungs. IGF-1R expression was downregulated in hyperoxia-exposed mouse lungs compared to the control on P10 (a), and densitometry results is shown (b).

Figure 4

Impaired alveolar development using a furin inhibitor, hexa-D-arginine (D6R), in developing mouse lungs. Mice pups were intraperitoneally injected a peptide furin inhibitor, D6R, four times every 2 d from P2, and the pups were euthanized at P10. Lung morphology was examined ((a); control, (b); D6R-treated), and changes in D6R-treated mouse lungs were confirmed via Lm (c) and TVD (d) measurements; Scale bar, 50 μm (control, n = 7; D6R, n = 6; * p < 0.05). Lm, mean linear intercept; TVD, tissue volume density.

Figure 5

Furin inhibition accumulates IGF-1R proprotein and decreases its signaling. Subconfluent primary myofibroblast cultures of P1 mice pup lungs were serum-starved for 24 h under treatment with a furin inhibitor, decanoyl-Arg-Val-Lys-Arg-chloromethylketone (dek-RVKR-CMK), and then allowed to recover for another 24 h with continued inhibitor treatment. After cell harvesting, the immunoreactivity of the pro-IGF-1R and phospho-IGF-1R was determined in cell lysates using specific antibodies and immunoblotting.

Figure 6

Recombinant furin treatment is associated with improved alveolar development in developing mouse lungs exposed to hyperoxia. Exposure to 85% O₂ for 10 d was associated with decreased alveolar formation (a,b), determined based on increased MLI (d) and decreased TVD (e) values. Treatment with recombinant furin improved alveolar development in hyperoxia-exposed mouse lungs (c–e, control, n = 7; hyperoxia, n = 6; furin treatment, n = 6, * p < 0.05). Scale bar, 50 μm. MLI, mean linear intercept; TVD, tissue volume density.