A total fibrinogen deficiency is compatible with the development of pulmonary fibrosis in mice

Abstract

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-gamma-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.

Figure 1.

The strategy for targeted inactivation of the murine FG-γ 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-γ 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-γ 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-γ 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-γ 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-γ 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-γ chain, hybridizes to an 11.5-kb WT and 6.5-kb disrupted FG-γ 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-γ and with reverse primer 3, located in the NEO gene, leads to a 0.55-kb amplicon, whereas amplification of the wild-type FG-γ allele with forward primer 2 in the FG-γ 5′-flank and reverse primer 4 within the FG-γ 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-γ^+/− mouse DNA; lane 3, FG-γ^-−/− mouse DNA; lane 4, FG-γ^+/+ 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-γ^+/+ mouse; lane 4, plasma from a FGγ^+/− mouse; lanes 5 and 6, plasma from FG-γ^−/− mice. These latter two lanes provide additional support that none of the Fg chains are detected in these plasmas.

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, ×200). 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, ×200). 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, ×200). g: H&E stain of lung tissue from a FG^−/− mice treated with saline demonstrated normal lung architecture (original magnification, ×200). h: Masson’s trichrome stain of lung tissue from FG^−/− mice treated with saline demonstrated normal collagen deposition associated mainly with bronchioles (original magnification, ×200). i: Fn immunohistochemistry of lung tissue from FG^−/− mice treated with saline demonstrated the expected lack of Fn deposition (original magnification, ×200). Results from the three stains performed on lung tissue from WT mice treated with saline were indistinguishable to that observed in FG^−/− mice.

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, ×200).