Antisense oligonucleotide inhibition of heat shock protein (HSP) 47 improves bleomycin-induced pulmonary fibrosis in rats

Abstract

Background: The most common pathologic form of pulmonary fibrosis arises from excessive deposition of extracellular matrix proteins such as collagen. The 47 kDa heat shock protein 47 (HSP47) is a collagen-specific molecular chaperone that has been shown to play a major role during the processing and/or secretion of procollagen.

Objectives: To determine whether inhibition of HSP47 could have beneficial effects in mitigating bleomycin-induced pulmonary fibrosis in rats.

Methods: All experiments were performed with 250-300 g male Wistar rats. Animals were randomly divided into five experimental groups that were administered: 1) saline alone, 2) bleomycin alone, 3) antisense HSP47 oligonucleotides alone, 4) bleomycin + antisense HSP47 oligonucleotides, and 5) bleomycin + sense control oligonucleotides. We investigated lung histopathology and performed immunoblot and immunohistochemistry analyses.

Results: In rats treated with HSP47 antisense oligonucleotides, pulmonary fibrosis was significantly reduced. In addition, treatment with HSP47 antisense oligonucleotides significantly improved bleomycin-induced morphological changes. Treatment with HSP47 antisense oligonucleotides alone did not produce any significant changes to lung morphology. Immunoblot analyses of lung homogenates confirmed the inhibition of HSP47 protein by antisense oligonucleotides. The bleo + sense group, however, did not exhibit any improvement in lung pathology compared to bleomycin alone groups, and also had no effect on HSP47 expression.

Conclusion: These findings suggest that HSP47 antisense oligonucleotide inhibition of HSP47 improves bleomycin-induced pulmonary fibrosis pathology in rats.

Figure 1

Immunohistochemistry for the detection of HSP47 in rat lungs treated with bleomycin with or without antisense HSP47 oligonucleotides (ODN). (A) A representative lung from a control rat sacrificed at 4 weeks after saline administration; 400×. (B) A representative lung from an animal in the Bleo group and sacrificed at 4 weeks; 400×. (C) A representative lung from an animal in the Bleo+AS group sacrificed at 4 weeks; 400×. (D) A representative lung from an animal in the antisense group sacrificed at 4 weeks; 400×. (E) A representative lungs from an animal in the Bleo+S group and sacrificed at 4 weeks; 400×. Arrows indicate immunopositivity cells to HSP47.

Figure 2

(A) Rat lungs treated with bleomycin was lysed and immunoblotted for HSP47 protein. Levels of HSP47 protein at 2 and 4 weeks were analyzed using Western blotting as described. HSP47 was up-regulated in the Bleo group compared to the Bleo+AS group. (B) HSP47 signal intensities were quantified using an image analyzer based on immunoblot density. HSP47 expression is shown as a percentage of the control. Data is expressed as mean ± SEM. * p < 0.05 vs. control group.

Figure 3

H&E staining of bleomycin-treated rat lungs with and without antisense HSP47 ODN. (A) Lungs from the saline group; 40× (B) Lungs from the Bleo group at 4 weeks following bleomycin treatment exhibit diffuse consolidation of parenchyma with loss of alveolar architecture and increased cell number; 40×. (C) Lungs from the Bleo+AS group at 4 weeks post-injection demonstrates improvement of lesions compared to the Bleo alone group; 40×. (D) Lungs from the AS alone group at 4 weeks post-injection show no significant pulmonary consolidation or fibrosis; 40×. (E) Lungs from the Bleo+S group at 4 weeks post-injection exhibit diffuse consolidation of parenchyma with loss of alveolar architecture and increased cell number; 40×.

Figure 4

Macrograph of lungs treated with bleomycin alone (A) or bleomycin + antisense HSP47 ODN (B) and sacrificed 28 days later. (A) Lungs from a Bleo only treated animal exhibit fibrosis and loss of alveolar architecture. (B) Lungs from a Bleo+AS treated animal exhibit normal morphology and no signs of fibrosis.

Figure 5

Azan staining of lung tissue from each group. (A) Azan staining of the saline group shows minimal collagen content in the lung parenchyma; 100×. (B) Azan staining of the Bleo group shows an increase in the collagen content of lung parenchyma; 100×. (C) Azan staining the Bleo+AS group shows a reduction in collagen fibrils compared to the saline and Bleo groups; 100×. (D) Azan staining the AS group shows minimal collagen in the lung parenchyma; 100×. (E) Azan staining of the Bleo+S group shows an increase in the collagen content of lung parenchyma, comparable to the Bleo group; 100×.

Figure 6

Histopathologic assessment of pulmonary fibrosis by the Ashcroft score. Comparison of lung tissue extracted at day 28 between the control, Bleo, Bleo+AS, AS, and Bleo+S groups (n = 8). Data are expressed as mean ± SEM. * p < 0.05 vs. Bleo+AS group; # p < 0.05 vs. control group.

Figure 7

Hydroxyproline content of lung tissues (mg/g of wet tissue) at 28 days post Bleo injection. Comparison between control, Bleo, and Bleo+AS groups (n = 8). Data are expressed as the means ± SEM. # p < 0.05 vs. control group.