Predisposition for disrepair in the aged lung

Abstract

Introduction: Idiopathic pulmonary fibrosis (IPF) is a devastating progressive lung disease with an average survival of only 3 to 5 years. The mechanisms underlying the initiation and progression of IPF are poorly understood, and treatments available have only modest effect on disease progression. Interestingly, the incidence of IPF is approximately 60 times more common in individuals aged 75 years and older, but the mechanism by which aging promotes fibrosis is unclear. The authors hypothesized that aged lungs have a profibrotic phenotype that render it susceptible to disrepair after injury.

Methods: Young and old mice were treated with bleomycin to examine disrepair in the aged lung. In addition, uninjured young and old mouse lungs were analyzed for transforming growth factor-beta 1 (TGF-β1) production, extracellular matrix composition and lung fibroblast phenotype. Lung fibroblasts were treated with a DNA methyltransferase inhibitor to examine the potential epigenetic mechanisms involved in age-associated phenotypic alterations.

Results: The lungs of old mice showed worse fibrosis after bleomycin-induced injury compared with the lungs from young mice. At baseline, aged lungs expressed a profibrotic phenotype characterized by increased mRNA expression for fibronectin extracellular domain A (Fn-EDA) and the matrix metalloproteinases (MMPs) MMP-2 and MMP-9. Old lungs also expressed higher levels of TGF-β receptor 1 and TGF-β1 mRNA, protein and activity as determined by increased Smad3 expression, protein phosphorylation and DNA binding. Lung fibroblasts harvested from aged lungs showed reduced expression of the surface molecule Thy-1, a finding also implicated in lung fibrosis; the latter did not seem related to Thy-1 gene methylation.

Conclusion: Altogether, aged lungs manifest a profibrotic phenotype characterized by enhanced fibronectin extracellular domain A and MMP expression and increased TGF-β1 expression and signaling and are populated by Thy-1-negative fibroblasts, all implicated in the pathogenesis of lung fibrosis.

FIGURE 1

Aging increases bleomycin-induced lung injury and fibrosis. Three-month-old and 24-month-old C57BL/6 mice were treated with 3.5 units/kg of bleomycin or PBS intratracheally. Lungs were harvested at 14 days. (A) Histologic sections were stained with Masson’s trichrome to evaluate for collagen deposition. Magnification is 2× (inset 10×). (B) Graph represents the intensity of Masson’s trichrome staining in young (open bar) and old (close bar) lungs at 14 days after bleomycin treatment as analyzed by ImageJ 1.42 software. One of the lungs from young mice was used as a standard sample (value set as 1). *P < 0.05 compared with young group.

FIGURE 2

Aging increases collagen mRNA expression and deposition after bleomycin-induced lung injury. Three-month-old and 24-month-old C57BL/6 mice were treated with 3.5 units/kg of bleomycin (close bars) or PBS (open bars) intratracheally. Lungs were harvested at 14 days and processed for quantitative PCR and hydroxyproline content analysis. Graphs depict type 1 collagen mRNA expression (A) and hydroxyproline content (B) in the lungs harvested from young and old animals 14 days after treatment. One of the samples from the young group was used as standard sample for quantitative PCR analysis. *P < 0.05 and #P = 0.05. PBS, phosphate buffer saline-treated group; Bleo, bleomycin-treated group.

FIGURE 3

Aging is associated with an increase in TGF-β1 expression. Lungs were harvested from uninjured 3-month-old and 24-month-old C57BL/6 mice and analyzed for TGF-β1 expression. (A) Graph depicts TGF-β1 mRNA expression measured by quantitative PCR. (B) Graph depicts relative densitometry analysis of Western blots evaluating TGF-β1 protein. The inactive form of TGF-β1 migrated at 55 kDa, and the active form migrated at 12.5 kDa in SDS-PAGE gel. One of the samples from young group was used as standard sample for quantitative PCR analysis. β-actin protein expression was used for normalization for protein expression. *P < 0.05 compared with young group.

FIGURE 4

TGF-βR1 and Smad3 expression increase with age. Lungs were harvested from uninjured 3-month-old and 24-month-old C57BL/6 mice and analyzed for TGF-βR1, TGF-βR2 and Smad3 expression. (A) Lung samples were evaluated for TGF-βR1, TGF-βR2 and Smad3 by RT-PCR as depicted in the upper panel showing an RT-PCR gel electrophoresis. Graphs represent quantitative PCR analysis for Smad3, TGF-βR1 and TGF-βR2 mRNA expression in young (open bars) and old (closed bars) lungs. One of the samples from the young group was used as standard sample for quantitative PCR analysis. (B) Lung samples were evaluated for TGF-βR1, TGF-βR2, Smad3 and phospho-Smad3 protein expression using Western blot. Upper panel shows representative Western blot gel. Graphs represent relative densitometric analysis of samples from young (open bar) and old (close bars) lungs. β-actin protein expression was used for normalization. *P < 0.05 compared with young group.

FIGURE 5

Aged lungs show increased TGF-β1/Smad3 signaling. Lungs were harvested from uninjured 3-month-old and 24-month-old C57BL/6 mice. (A) Top gel [electrophoretic mobility shift assay (EMSA)] showing increased Smad3 DNA binding in noninjured old lungs compared with young lungs. For competition reactions, 100-fold molar excess of nonradiolabeled Smad3/4 (100 × Smad) or mutated Smad3/4 (100× mSmad) oligonucleotide was added to the reaction mixture containing nuclear extracts isolated from the lungs of old mice. (B) Bottom gel showing an SDS-PAGE gel loaded with nuclear extracts and stained with coomassie blue to indicate equal loading, including protein loading for competitive reaction (last 2 lanes, upper gel). (C) Representative reverse transcriptase PCR gel and quantitative PCR of whole lung showing increased PAI-1 mRNA expression in old lungs (close bars) compared with young lungs (open bars). *P < 0.05 compared with young group.

FIGURE 6

Aging is associated with alterations in fibronectin-EDA splice variant expression in lung. (A) Lungs were harvested from uninjured 3–month-old and 24-month-old C57BL/6 mice. Graphs depict quantitative PCR for mRNA expression of Fn. Insert depicts mRNA expression of Fn-EDB. (B) Graph depicts Western blot analysis (for protein expression) in young and old lungs for total Fn. (C and D) Three-month-old (open bars) and 24-month-old (close bars) C57BL/6 mice were treated with 3.5 units/kg of bleomycin or PBS intratracheally, and lungs were harvested at 7 and 14 days tested for Fn-EDA mRNA (C) and protein (D) expression. One of the samples from the young group was used as a representative example of quantitative PCR analysis. β-actin protein expression was used for normalization. P < 0.001 across all groups by 1-way analysis of variance. P < 0.05 compared with PBS-treated young group.

FIGURE 7

MMP-9 mRNA expression and activity are increased in aged lungs. Lungs were harvested from uninjured 3-month-old and 24-month-old C57BL/6 mice for MMP mRNA expression and activity. (A) Graphs depict quantitative PCR analysis of young and old lungs for MMP-2, MMP-9, TIMP-2 and TIMP-1 mRNA expression. (B) Representative gel for gelatin zymography analysis of MMP-2 and MMP-9 activity. Graphs show densitometry analysis of gelatin zymography from young (open bars) and old (close bars) lungs. *P < 0.05 compared with young group.

FIGURE 8

Aging is associated with increase in number of Thy-1–negative fibroblasts in the lung, and this is not influenced by DNA methylation. Lungs were harvested from uninjured 3-month-old and 24-month-old C57BL/6 mice, and primary fibroblasts were isolated for analysis of Thy-1. (A and B) Phase contrast microscopy of passage 3 young (A) and old (B) primary lung fibroblasts in culture. Magnification, 20×. (C) Quantitative PCR to assess Thy-1 expression in lung fibroblasts isolated from young (open bars) and old (closed bar) mice and in lung fibroblasts from old mice treated with 5′AZA to induce DNA demethylation (gray bar). P < 0.001 as analyzed by 1-way analysis of variance. (D) Representative histogram of flow cytometry for Thy-1 performed in young (gray line) and old (black line) lungs. (E and F) Percent (E) and mean (F) fluorescent intensity of Thy-1 expressed by lung fibroblasts isolated from young and old lungs as analyzed by flow cytometry. *P < 0.05 compared with young group by t test and post-test analysis.