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Relaxin deficiency in mice is associated with an age-related progression of pulmonary fibrosis ¹

CHRISHAN S. SAMUEL^*2, CHONGXIN ZHAO^*, ROSS A. D. BATHGATE^*, COURTNEY P. BOND, MATTHEW D. BURTON, LAURA J. PARRY^*,, ROGER J. SUMMERS, MIMI L. K. TANG, EDWARD P. AMENTO^# and GEOFFREY W. TREGEAR^*

^* Howard Florey Institute of Experimental Physiology and Medicine and
Department of Zoology, University of Melbourne, Victoria, Australia;
Department of Pharmacology, Monash University, Clayton, Victoria, Australia;
Department of Immunology, Murdoch Children’s Research Institute, Royal Children’s Hospital, Parkville, Victoria, Australia; and
^# Molecular Medicine Research Institute, Sunnyvale, California, USA

²Correspondence: Howard Florey Institute, University of Melbourne, Victoria, Australia. E-mail: c.samuel{at}hfi.unimelb.edu.au

SPECIFIC AIMS

The peptide hormone relaxin is known for its actions on the female reproductive system and its potential ability to inhibit short-term collagen production from tissues and cell culture models. In the present study, the long-term effects of relaxin deprivation on lung morphology, collagen content, and function were examined in relaxin gene knockout mice. The anti-fibrotic properties of relaxin in lung tissue were also investigated.

PRINCIPAL FINDINGS

1. Relaxin-deficient mice develop an age-related progression of lung fibrosis with significantly increased tissue weight, collagen content, and alveolar congestion
Male and female relaxin knockout (RLX-/-) and relaxin wild-type (RLX+/+) mice were assessed for changes in lung wet weight, collagen content, and collagen concentration as they aged (3–19 months of age). Whereas significant increases (P<0.05) in phenotype were detected earlier in male RLX-/- mice, both male and female RLX-/- mice had significantly larger (P<0.05) lung weights by 9 months of age onward than lung tissue from age-matched RLX+/+ mice (Fig. 1 ). The collagen content of male and female RLX-/- mouse lungs (as determined by hydroxyproline analysis) was progressively higher at each time measured than that found in RLX+/+ lung tissues (Fig. 1) . Consistent with the increase in tissue weight, the increase in collagen was statistically significant (P<0.05) from 9 months of age on and was associated with a significant increase (P<0.05) in collagen concentration (collagen content as a percentage of the wet weight tissue). By 12 months of age, the lung collagen content of RLX-/- male and female mice was 65% and 33%, respectively, greater than levels measured in age-matched RLX+/+ animals. Histological studies of RLX-/- lung tissues demonstrated differences in tissue structure at 9 months of age onward. At 9 and 12 months of age, lung tissues from RLX-/- mice were progressively distorted with congestion of alveolar structure, a thickening of the bronchiole tubule epithelium, and a buildup of collagen fibers around the bronchioles and arterioles. Quantitative histological analysis confirmed the hydroxyproline data by demonstrating a significantly increased (P<0.05) collagen staining per field in RLX-/- mouse tissues than the staining measured in lung tissue sections from age-matched RLX+/+ mice.

View larger version (32K): [in this window] [in a new window]   Figure 1. The total lung weights (A) and total collagen content (which was converted from the hydroxyproline values) (B) from RLX+/+, RLX+/-, and RLX-/- male mice (3–12 months of age) and female mice (3–19 months of age). Numbers in parentheses represent number of samples. *P < 0.05 compared with aged-matched RLX+/+ values.

2. The increased fibrosis seen in RLX-/- mice was associated with significant changes in lung function
To determine whether the changes observed in lung structure in RLX-/- mice were associated with changes in lung function, various parameters of lung function were measured in older conscious mice by whole-body plethysmography. Baseline measurements of lung function revealed that 12-month-old RLX-/- mice had increased pressure and force of breath, as indicated by a significantly higher peak expiratory flow (PEF)(P<0.05) and shorter end inspiratory pause (EIP) (P<0.05), than 12-month-old RLX+/+ animals. The pattern of expiration was different in RLX-/- mice, demonstrating a more rapid exhalation, which is consistent with the reduced EIP and increased PEF. As changes in PEF may be associated with differences in airway reactivity in an inverse manner and fibrosis may influence airway resistance, methacholine (a bronchoconstrictor) -induced airway reactivity was examined in 12-month-old RLX+/+ and RLX-/- mice. There was a trend toward reduced airway reactivity in RLX-/- mice, but this decrease was not statistically significant.

3. Relaxin treatment reverses pulmonary fibrosis in RLX-/- mice
To determine whether treatment with relaxin could reverse or prevent progression of fibrosis, male RLX-/- mice (at 9 and 12 months of age) were treated with a fixed dose of 0.5 mg·kg^-1·day^-1 recombinant human relaxin (rH2) for 14 days. rH2 has been shown to be bioactive in mice. Circulating serum rH2 levels were determined in each mouse by radioimmunoassay and were measured at 20–40 ng/mL by day 14 of infusion. At 9 months of age (when fibrosis was first apparent), relaxin treatment alone significantly decreased lung wet weight (P<0.05), collagen content (P<0.05), and collagen concentration (P<0.05) of RLX-/- mice to the level seen in RLX+/+ mouse tissues (Fig. 2 A). At 12 months of age (when fibrosis was well established), rH2 treatment again significantly decreased (P<0.05) lung collagen content. However, levels of collagen measured after rH2 treatment were still significantly higher (P<0.05) than that observed in RLX+/+ lung tissues. Histologically, the treatment of 9-month-old RLX-/- mice with rH2 reduced extracellular collagen deposition and alveolar congestion to that seen in RLX+/+ mouse lung tissues. However, at 12 months of age, lung collagen and structure of rH2-treated mice were only somewhat restored to that observed in RLX+/+ mouse lung tissues (Fig. 2B ). Quantitative analysis of the collagen staining within rH2-treated RLX-/- mouse lung tissues showed a similar trend to total collagen content as measured by hydroxyproline analysis (Fig. 2C ) The effects of rH2 on muscle contraction were examined in vitro. Application of rH2 to precontracted lung strips induced a relaxation response, but this response was not significantly different between vehicle-treated and rH2-treated RLX-/- mice or RLX+/+ mice.

View larger version (40K): [in this window] [in a new window]   Figure 2. Relaxin-induced reversal of fibrosis. Total collagen content (A) from lungs of RLX+/+ mice, RLX-/- mice, RLX-/- mice treated with vehicle alone, and RLX-/- mice treated with 0.5 mg·kg-1·day-1 rH2 for 14 days. *P < 0.05 compared with age-matched untreated- and vehicle-treated RLX-/- mouse lung collagen levels. #P < 0.05 compared with age-matched RLX+/+ mouse lung collagen levels. Masson trichrome staining of collagen within the lung of 12-month-old RLX-/- mice (B) treated with vehicle (a, c) or recombinant relaxin (b, d) and from age-matched RLX+/+ samples (e, f). At this age, rH2 treatment significantly decreased collagen deposition around the bronchioles and arterioles, but not to the extent seen in RLX+/+ mouse tissues. Bar = 0.1 mm. Quantitative histological analysis of the collagen (blue) staining was performed on lung tissues from RLX+/+ and RLX-/- mice before and after rH2 treatment (C). The collagen-stained area was calculated as a % of the total area within random fields from each tissue section analyzed and expressed as the average percentage collagen per field. *P < 0.05 compared with age-matched RLX+/+ and rH2-treated RLX-/- mouse lung collagen-stained levels.

4. The mouse lung is a site for RLX and RLX receptor mRNA expression
Mice have two relaxin genes, designated mouse relaxin-1 (M1 relaxin), which is absent in relaxin knockout mice, and mouse relaxin-3 (M3 relaxin). M1 relaxin gene expression was detected only in RLX+/+ male and female lung tissues, whereas M3 relaxin mRNA expression was detected in both RLX+/+ and RLX-/- mouse lungs by RT-PCR. RLX receptor (LGR7) gene transcripts were demonstrated in the lungs of RLX-/- and RLX+/+ mice, suggesting that the mouse lung may serve as both a target for relaxin activity and a source of local relaxin production. At all ages studied, there was no significant difference in M3 relaxin mRNA expression between RLX-/- and RLX+/+ mouse lung samples as determined by real-time PCR, suggesting that M3 relaxin does not play a pivotal role in the progressive changes to collagen deposition observed in the lung of RLX-/- mice.

CONCLUSIONS AND SIGNIFICANCE

Relaxin is known for its potential ability to inhibit collagen deposition when applied to tissues and cells that have been subjected to short-term overexpression of collagen by various agents such as TGF-ß, IL-1ß, bleomycin, and experimental bromoethylamine. However, its potency as an anti-fibrotic agent is often varied depending on the dose of relaxin added, the time of relaxin incubation, and the severity of collagen expression when relaxin is added. In this study, we demonstrate that deletion of the M1 relaxin gene from mice results in a progressive age-related phenotype of pulmonary fibrosis: in the absence of M1 relaxin, interstitial collagen within the lung slowly accumulates to levels seen with artificially induced collagen stimulation in lung tissue. The increased lung fibrosis was measured in male and female mice and was characterized by a significantly increased lung weight, collagen content and concentration, alveolar congestion, and bronchiole tubule epithelium from 9 months of age on. These findings extend our previous data demonstrating that reproductive tissues of RLX-/- mice (including the testis, prostate, vagina, and nipple) were associated with a progressive increase in collagen deposition and support the usefulness of the relaxin knockout mouse model as a tool to improve our understanding of organ-specific fibrosis.

The progressive fibrosis observed in RLX-/- mouse lungs was associated with altered lung function. At 12 months of age (when fibrosis was well established in this model), RLX-/- mice had significantly altered aspects in their breathing patterns compared with that observed in RLX+/+ animals. RLX-/- mice had significantly greater force and pressure of expiration than RLX+/+ mice, which is consistent with an increased lung recoil on expiration resulting from increased fibrosis. These changes in lung function were not associated with a significant alteration in airway responsiveness, which is consistent with our knowledge that altered airway responsiveness is associated with allergic inflammatory airway disease rather than pulmonary fibrosis states.

Relaxin’s ability to inhibit or reverse pulmonary fibrosis associated with relaxin deficiency was investigated. Our findings confirm that relaxin can be used effectively as an anti-fibrotic agent. However, the potency of relaxin in inhibiting collagen deposition appeared to be decreased especially when applied to later and more severe stage of fibrosis.

In conclusion, M1 relaxin-deficient mice develop a buildup of the extracellular collagen, leading to the distortion of lung structure and function. Relaxin treatment of these mice at early and established phases of lung fibrosis resulted in a significantly reduced level of collagen deposition and restored lung structure to that observed in normal mice. Thus, relaxin provides an important means to regulate excessive collagen deposition in lung diseases characterized by fibrosis, particularly when applied at an early stage of the disorder.

View larger version (22K): [in this window] [in a new window]   Figure 3. Consequences of M1 relaxin deficiency in the aging lung and the addition of recombinant human relaxin (rH2), back into relaxin gene knockout mice.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0449fje; to cite this article, use FASEB J. (November 1, 2002) 10.1096/fj.02-0449fje

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