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(American Journal of Pathology. 2000;157:703-708.)
© 2000 American Society for Investigative Pathology

Short Communications

A Total Fibrinogen Deficiency Is Compatible with the Development of Pulmonary Fibrosis in Mice

From the W. M. Keck Center for Transgene Research and the Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
In addition to their well-known roles in hemostasis, fibrinogen (Fg) and fibrin (Fn) have been implicated in a number of other physiological and pathophysiological events. One of these involves the fibroproliferative response after acute lung injury, which is the focus of the current study. Mice with a total Fg deficiency (FG^-/-) were generated by breeding heterozygous (FG^+/-) pairs, each of which contained an allele with a targeted deletion of its Fg--chain gene. The resulting FG^-/- animals did not possess detectable plasma Fg. FG^-/- mice were then used to assess the roles of Fg and Fn in a bleomycin-induced acute lung injury model. Intratracheal administration of bleomycin in wild-type and FG^-/- mice resulted in equivalent deposition of interstitial collagen and fibrotic lesions at days 7 and 14 after administration. This indicates that Fg and/or Fn are not essential for the development of bleomycin-induced pulmonary fibrosis.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

Before secretion of the intact Fg, its three chains are assembled in a stepwise manner in the rough endoplasmic reticulum.¹⁷ If genetic defects in the A chain,¹⁸ Bß chain,¹⁹ or chain^20,21 exist, impaired secretion can occur that results in congenital hypofibrinogenemia or afibrinogenemia. Complexes of A/Bß and Bß- chains associate with and A chains in the lumenal space of the endoplasmic reticulum, forming Fg, which is then secreted as the native dimeric protein.²²

A targeted deletion of the A chain in mice led to animals without detectable plasma Fg levels.²³ In the current investigation, we wished to similarly generate mice with a targeted deletion of the -chain gene (FG-), reasoning that these mice would also lack plasma Fg because of the requirement for / and ß/ chains to serve as intermediates for the synthesis of the heterodimeric protein. In addition, not only would the availability of Fg-deficient mice allow analysis of the role of Fg in various disease models, but generation of specific -chain-deficient mice would allow facile and rapid development and phenotypic analyses of transgenic animals containing mutations of the chain, which possibly possess loci for platelet binding,^24-28 leukocyte adhesion,^6,15 Fn polymerization,^21,29-31 and cleavage sites for MMPs.³² Therefore, the -chain gene of Fg was deleted from mice and an analysis of the effect of a Fg deficiency on fibrotic lesion development after acute lung injury is the subject of the current report.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Targeted Inactivation of the Murine FG- Chain

FG- chain-deficient mice were generated by targeted inactivation of the murine chain of Fg by homologous recombination in R1 embryonic stem (ES) cells. The steps involved have been described in earlier reports.^33-35

Southern Blot Analysis of ES Cells

Correctly targeted ES cells were identified by Southern blot analysis of EcoRI digests of purified ES cell genomic DNA. A 0.85-kb fragment located downstream of the 3' homologous flanking region was used as a probe to identify an 11.5-kb WT and 6.5-kb disrupted FG--chain allele. A full description of the methods used has been published.³⁵

Polymerase Chain Reaction (PCR) Analysis of Genomic DNA

FG- genotyping of mice was conducted by PCR analysis of purified genomic DNA from ear-punch tissue. An aliquot of 1 µl of genomic DNA was used in a single PCR reaction. The reaction mixture contained the following three primers: a NEO gene-specific primer (5'-GGTTCTAAGTACTGTGGTTTC-3'), a sequence within the 5'-noncoding flank of the FG- chain (5'-CACAGCGGCTTGTCATTAG-3'), and a sequence internal to the FG- chain (5'-CTGAAAGACCTGTCTTTGC-3'). The PCR reaction consisted of an initial denaturation step (94°C for 3 minutes) and 30 cycles of the following (94°C for 30 seconds, 58°C for 40 seconds, and 72°C for 50 seconds), with a final annealing step at 72°C for 5 minutes. The PCR products, 420 bp for the WT allele and 550 bp for the FG--chain disrupted allele, were resolved on a 1% agarose gel.

Thrombin Clot Time of Plasma

The assay was conducted in a standard fibrometer (BBL Microbiology Systems). Whole blood (0.8 to 0.9 ml) was isolated from the vena cava of anesthetized WT, FG^+/-, and FG^-/- mice and placed into 0.1 ml of a 4% sodium citrate solution. The samples were subjected to centrifugation at 3,000 rpm for 10 minutes in a microcentrifuge and the plasma was transferred to a fresh tube for analysis. A volume of 100 µl of 50 mmol/L Tris-HCl, pH 7.4, was incubated for 3 to 10 minutes at 37°C in a fibrometer cup, after which 100 µl of fresh plasma was added and allowed to incubate an additional 1 minute. After 1 minute, 100 µl of 5 NIH U/ml of human -thrombin (Enzyme Research Laboratories, South Bend, IN) was added and the fibrometer timer was started. Samples were analyzed in duplicate.

Western Blot Analysis of Plasma

Western blot analysis was carried out using standard procedures using a rabbit-anti-human Fg antibody which recognizes all three chains (to differing extents) and cross-reacts with mouse Fg (DAKO, Carpinteria, CA). Plasma samples were prepared as described above for thrombin times and 0.1 µl was loaded onto a 9% sodium dodecyl sulfate gel.

Bleomycin-Induced Pulmonary Fibrosis

WT (n = 5) and FG^-/- (n = 6) anesthetized mice, 4 weeks of age, were administered, intratracheally, a single dose (4.5 U/kg) of bleomycin in 25 µl saline by infusion through the vocal cords using a fiber-optic light source for illuminating the tracheal opening. Control animals of similar genotypes were treated with saline alone. At 7 and 14 days after bleomycin infusion, anesthetized mice were perfused with saline and the left lung excised for determination of the collagen content (14 days), while the right lung was processed for histological analyses (7 and 14 days).

Quantitation of Lung Collagen Content

Total lung collagen content in saline and bleomycin-treated mice was determined by a colorimetric assay measuring hydroxyproline levels of acid hydrolyzed lung tissue.³⁶

Histology and Immunohistochemistry

Tissues were processed, embedded in paraffin, and sectioned at 3 to 4 µm. Hematoxylin and eosin (H&E) staining of tissue was performed to identify the tissue and to detect alterations in cellular architecture. Masson’s trichrome staining was used to identify collagen-rich fibrotic lesions in the lung.³⁷ Fn, in the lung, was identified by immunohistochemistry using a polyclonal goat-anti-mouse fibrin(ogen) antibody (Accurate Chemical, Westbury, NY) and a secondary rabbit-anti-goat IgG (DAKO), followed by goat peroxidase/anti-peroxidase (DAKO). The substrate, 3'-aminoethyl carbazole (AEC, Biomeda, CA), was then used to detect peroxidase activity. The slides were counterstained with hematoxylin.

Statistical Analysis

Where appropriate, values were expressed as the mean ± SEM using a Microsoft Excel program.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Targeted Inactivation of the FG- Gene

A targeting vector was constructed in which the NEO gene replaced all coding regions of the FG- chain (Figure 1a) . This altered gene was inserted in place of the FG--chain gene in the mouse genome by homologous recombination. Two independent correctly targeted ES clones (Figure 1b) were aggregated with Swiss morula stage embryos, yielding seven chimeric animals with 80 to 95% coat color chimerism. Chimeric mice transmitted the inactivated FG- gene to their offspring. Intercrossing of these heterozygous FG--chain-deficient mice (FG^+/-) generated homozygous FG--deficient (FG^-/-) progeny for phenotypic analysis (Figure 1c) . Fg antigen was not detected in FG--chain null offspring as determined by Western blot analysis of plasma (Figure 1d) and immunostaining of liver sections (not shown). All animals used in this study were 50% 129SvJ/50% C57Bl/6.

View larger version (36K): [in this window] [in a new window]   Figure 1. The strategy for targeted inactivation of the murine FG- chain gene by homologous recombination in ES cells. a: The top line represents the targeting vector, pND.457, in which the entire coding sequence of the FG- chain has been replaced with a neomycin phosphotransferase (NEO) gene to allow for positive selection. A cytosine deaminase (CDA) gene has also been inserted downstream of the FG- chain gene for negative selection of homologous recombination events. The middle line illustrates the wild-type (WT) allele, with vertical black extensions originating from the line representing the gene indicating the approximate positions of the 10 Fg- chain exons. On homologous recombination at 5' and 3' flanking locations (blue), the NEO gene replaces an approximate 9.0-kb genomic fragment comprising the sequences encoding the entire mature Fg- chain. The bottom line represents the resulting targeted chromosomal locus (TL). The homologous flanking regions of the targeting vector are shown in blue and include a 5' NotI/EcoRV 5.5-kb fragment of the FG- chain gene and a 3.5-kb NheI/NcoI fragment starting at a position 700 bp from the termination codon. b: Southern blot of EcoRI-digested DNA from ES cells to identify correctly targeted cells with 3'-directed probe 1 (red, see a). This probe, a 0.85-kb NcoI/NsiI 3' fragment immediately downstream of the Fg- chain, hybridizes to an 11.5-kb WT and 6.5-kb disrupted FG- chain allele. A properly targeted stem cell showing both alleles is indicated by the *. c: Genotyping of mouse DNA. Amplification of the disrupted allele was accomplished with forward primer 2 in the 5'-noncoding flanking region of FG- and with reverse primer 3, located in the NEO gene, leads to a 0.55-kb amplicon, whereas amplification of the wild-type FG- allele with forward primer 2 in the FG- 5'-flank and reverse primer 4 within the FG- gene results in a 0.42-kb amplicon. The approximate locations of these primers are indicated in a by magenta arrows. Lane 1, DNA markers (top to bottom: 2.0 kb, 1.5 kb, 1.0 kb, 0.75 kb, 0.5 kb, and 0.25 kb); lane 2, FG-+/- mouse DNA; lane 3, FG---/- mouse DNA; lane 4, FG-+/+ mouse DNA. d: Western analysis of mouse plasma using a polyclonal antibody that recognizes (to differing extents) all three murine Fg chains. Lane 1, purified mouse Fg; lane 2, molecular mass markers (top to bottom: 101 kd, 79 kd, and 50 kd); lane 3, plasma from a FG-+/+ mouse; lane 4, plasma from a FG+/- mouse; lanes 5 and 6, plasma from FG--/- mice. These latter two lanes provide additional support that none of the Fg chains are detected in these plasmas.

A significant (25%) percentage of FG^-/- neonates suffered from hemorrhaging events. The bleeding was primarily abdominal but was also observed in the cranial, neck, and nasal areas. Although these events initially seemed dramatic, many FG^-/- mice were able to resolve the bleeding and recover.

Thrombin Clot Time of Plasma

Thrombin-induced clot times were significantly delayed in FG^-/- mice relative to WT mice (>17 minutes, n = 2, versus 35.5 ± 3.5 seconds, n = 3, respectively). When it was observed that FG^-/- samples did not clot after 17 minutes, timing was stopped. Other samples left unattended never formed a clot. FG^+/- mice demonstrated thrombin clot times that were similar to WT mice (35.9 ± 4.3 seconds, n = 3).

Bleomycin-Induced Pulmonary Fibrosis

WT and FG^-/- mice developed pulmonary fibrosis to a similar extent, as evidenced by the equivalent increase, approximately two-fold, of total lung collagen levels, reflected by the hydroxyproline content 14 days after intratracheal administration of bleomycin relative to saline controls (2.0 µg/mg dry lung, n = 6, versus 3.0 µg/mg dry lung, n = 4, for WT mice, and 1.3 µg/mg dry lung, n = 2, versus 2.4 µg/mg dry lung, n = 5, for FG^-/- mice). H&E staining of lung tissue from WT and Fg^-/- mice receiving bleomycin identified focal areas of interstitial fibrosis (Figure 2, a and d) . Masson’s Trichrome staining of lung tissue from these mice indicated that collagen deposition was associated within the fibrotic lesions to a similar extent in WT and FG^-/- mice (Figure 2, b and e) . In WT mice treated with bleomycin, Fg/Fn co-localized with the collagen-rich lesions (Figure 2c) . FG^-/- saline-treated animals demonstrated normal lung architecture (Figure 2g) as well as significantly diminished collagen levels relative to bleomycin-treated mice (Figure 2h) . Masson’s trichrome staining of lung tissue from WT and FG^-/- mice, as early as 7 days after bleomycin treatment, indicated collagen-rich fibrotic lesions (Figure 3, a and b) . Analysis of H&E-stained lung tissue from 7-day treated mice indicated enhanced levels of neutrophils and macrophages in both WT and FG^-/- relative to their respective saline controls (data not shown).

View larger version (113K): [in this window] [in a new window]   Figure 2. Histology of lung tissue from WT and FG-/- mice 14 days after intratracheal administration of bleomycin or saline control. H&E stain of lung tissue from WT (a) and FG-/- (d) mice treated with bleomycin demonstrated interstitial fibrosis (original magnification, x200). Masson’s trichrome stain of a serial section of lung tissue from WT (b) and FG-/- (e) mice identified collagen deposits (blue) within the fibrotic lesion (original magnification, x200). Fn immunohistochemical stain of a serial section of lung tissue from WT (c) and FG-/- (f) mice demonstrated co-localization of Fn within the collagen-rich lesions in WT mice and not in FG-/- mice (original magnification, x200). g: H&E stain of lung tissue from a FG-/- mice treated with saline demonstrated normal lung architecture (original magnification, x200). h: Masson’s trichrome stain of lung tissue from FG-/- mice treated with saline demonstrated normal collagen deposition associated mainly with bronchioles (original magnification, x200). i: Fn immunohistochemistry of lung tissue from FG-/- mice treated with saline demonstrated the expected lack of Fn deposition (original magnification, x200). Results from the three stains performed on lung tissue from WT mice treated with saline were indistinguishable to that observed in FG-/- mice.

View larger version (132K): [in this window] [in a new window]   Figure 3. Histology of lung tissue from WT and FG-/- mice 7 days after intratracheal administration of bleomycin. Masson’s trichrome stain of lung tissue from WT (a) and FG-/- (b) mice identified collagen deposits (blue) within the fibrotic lesion (original magnification, x200).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In this investigation, mice deficient for the chain of Fg, that consequently possessed a total deficiency of plasma Fg, were generated and the development of bleomycin-induced pulmonary fibrosis assessed. Early inflammatory response, 7 days after bleomycin treatment, seemed to be equivalent between WT and FG^-/- mice. It was also observed that there was no significant difference in the development of collagen-rich pulmonary fibrotic lesions between WT and FG^-/- mice 7 and 14 days after administration of bleomycin. This indicates that fibrin(ogen) or fibrinopeptides may not be required for fibroblast proliferation and possibly other related events such as fibroblast migration after bleomycin treatment.⁴⁰ Other matrix proteins may be involved in supporting the fibrotic process after lung injury. For example, studies of lung biopsies from patients with pulmonary fibrosis have indicated that fibroblasts increase their synthesis of collagen and fibronectin, coordinately, within airspaces.⁴¹ Therefore, although Fn is not essential for the progression and persistence of fibrosis, matrix proteins, other than collagen, may play prominent roles in supporting the lung repair process after injury.

A recent study wherein bleomycin-induced pulmonary fibrosis was investigated in mice deficient for plasminogen (Pg), tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator, and urokinase plasminogen activator receptor demonstrated enhanced fibrotic lesion formation in Pg and activator deficient mice.⁴² Collagen levels were especially enhanced in PG^-/- and TPA^-/- mice, relative to WT mice. In the PG^-/- mice a persistence of Fn deposition was observed that co-localized with collagen-rich fibrotic lesions, thus implicating an involvement of Fn with the progression of the repair process. The current study with FG^-/- mice presents a key investigation that expands an analysis of the involvement of the fibrinolytic process in bleomycin-induced pulmonary fibrosis and suggests another role for plasmin in this process, possibly acting as a regulatory protein of collagen turnover through activation of matrix metalloproteinases, specifically collagenases.

	Acknowledgements

 
The authors wish to thank Mayra Sandoval-Cooper for assistance with the histology; Stacey Raje for assistance with the animal colony; and Drs. Harm HogenEsch and Mark Suckow for assistance with evaluation of the histopathology.

	Footnotes

 
Address reprint requests to Dr. Francis J. Castellino, Department of Chemistry and Biochemistry, 229 Nieuwland Science Hall, University of Notre Dame, Notre Dame, IN 46556. E-mail: castellino.1{at}nd.edu

Supported by grants HL13423 (to F. J. C.) and HL63682 (to V. A. P.) from the National Institutes of Health, a National Scientist Development Award (9630009N) from the American Heart Association (to V. A. P.), the Kleiderer-Pezold endowed professorship (to F. J. C.), and a grant from the W. M. Keck Foundation (to F. J. C.).

Accepted for publication May 23, 2000.

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