The mice were originally engineered by microinjection of a DNA fragment into the nuclei of one-cell mouse embryos. The DNA fragment containing an altered porcine TGFβ1 cDNA associated with an albumin promoter to ensure the preferential expression of the active form of TGFβ1 from the liver, with resultant high circulating levels 18. Mouse embryos were obtained from mating of F1 hybrid mice (C57BL6 × CBA background). Two lines of mice (line 18 and line 25) were purchased (Nancy Sanderson, National Institute Of Health, Bethesda, MD, USA).

The transgene was expressed in both sexes of the line 18 mice. The mice were bred by crossing a positive with a wild type animal. In the line 25 mice, only the male mice expressed the transgene. Therefore, the mice were bred by setting up a harem consisting of a positive male animal and three F1 females. Mice were housed under pathogen-free conditions and husbanded according to Home Office regulations.

On day 0 mice were given intraperitoneal bleomycin (BLM) or phosphate buffer solution (PBS) in three divided doses (0.5 ml volume) over a course of 5 days. They were observed on a daily basis and sacrificed on day 42. Mice were divided into 6 groups (n = 8/group).

Mouse-tail snips, measuring approximately 0.25 cm, were incubated with proteinase K overnight at 55°C and DNA was extracted the following day using Phenol/Chloroform/Isoamylalcohol method followed by washing-step in 70% ethanol.

TGFβ1 quantification was performed using a PAI-1/Luciferase assay (PAIL). PAIL assay is a quantitative bioassay based upon active TGFβ's ability to stimulate the expression of Plasminogen Activator Inhibitor 1 (PAI-1) 19. The assay uses mink lung epithelial cells (MLEC's) (a kind gift from Dr Dan Rifkin, New York University Medical Center, New York), which have been stably transfected with a gene for Luciferase activity and its expression is regulated and promoted by a truncated PAI-1 promoter construct. TGFβ1 therefore regulates Luciferase activity via PAI-1 promoter. Luciferase activity in MLEC cell lysates was measured in a luminometer.

Liver and lung tissue sections were stained with haematoxylin and eosin and Masson's trichrome, which determines collagen deposition and localization. Lung fibrosis was graded histologically by an established scoring system 20.

Immunohistochemical analysis was performed as previously described 21. In brief, paraffin sections were stained with rabbit anti-reticulin (Sigma, UK), rabbit anti-TGFβ1 (Santa Cruz, CA, USA) and its receptors (TGFβ1R1, TGFβ1R2, TGFβ1R3) (Santa Cruz, CA, USA) (1:100). Antibody binding was visualized using a biotinylated secondary antibody, avidine conjugated peroxidase (ABC method; Vector Laboratories) and 3,3' diaminobenzidine tetrachloride (DAB) as a substrate and hematoxylin as counterstain.

For collagen determination we employed a hydroxyproline assay technique. Briefly after death, lungs were removed and weighed. 6 M hydrochloric acid was added to each sample, then sealed and placed in an oven overnight at 110°C. Excess acid was removed by evaporation and hydrolyzed samples were dissolved in 1 ml of PBS. The samples were aliquoted adding Chloramine T reagent equally to each sample. After 20 minutes of mixing, 1 ml of p-DAB reagent (p-dimethyl-amino-benzaldehyde) was added and the mixture incubated at 60°C. The colour signals were measured in a spectrophotometer at 550 nm, and compared to a standard curve.

RNA isolation, cDNA synthesis, in vitro transcription and microarray analysis were performed as previously reported 22. Arrays were scanned with a confocal scanner (Affymetrix). All in vitro time points were microarrayed in duplicate.

Image files were obtained through Affymetrix GeneChip software (MAS5). Subsequently robust multichip analysis (RMA) was performed 2324. Expression data was further probed to identify those genes whose expression is altered 25. Expression data following injury was compared to control and a signal log ratio of 0.6 or greater (equivalent to a fold change in expression of 1.5 or greater) was taken to identify significant differential regulation. Using normalised RMA values, Unsupervised Average Linkage Hierarchical Cluster Analysis was performed 26. Functional annotation of differentially expressed genes was curated via the publicly available Onto-Compare and Gene-Ontology (GO) databases 27.

Following breeding, TGFβ1 expression was confirmed by PCR amplification in Tr+ TGFβ1 transgenic mice (Figure 1a). To determine the effect of the TGFβ1 transgene in these mice, serum levels of both total and active TGFβ1 were determined. The Tr+ transgenic mice had higher levels of total TGFβ1 (2.2 ng/ml, SEM 0.23) compared to Tr- wild types (1.58 ng/ml, SEM 0.39), though this comparison did not reach statistical significance (p = 0.16) (Figure 1b); while, Tr+ transgenic mice had higher plasma levels of active TGFβ1 (mean 98.1 pg/ml, SEM 16.1) compared to Tr- wild types (mean 9.37, SEM 6.6) (p < 0.01). Individually, the line 18 mice had a similar level of active TGFβ1 (mean 87 pg/ml, SEM 19.2) to the line 25 mice (105.5 pg/ml, SEM 24.5) (Figure 1c).

Having demonstrated altered DNA and protein expression in TGFβ1-transgenic mice we sought to determine the effect of TGFβ1 overexpression on tissue phenotype.

Tr+ transgenic mouse livers were histologically abnormal as early as 1 month, though the most marked changes were seen from 3 months onwards. This consisted of extensive cellular degeneration, vacuolisation, fibrosis and architectural disruption (Figure 2a), compared to Tr- wild type mouse liver (Figure 2b). Staining for the pre-collagen, reticulin signalling was higher in transgenic mice tissue than wild type, confirming the presence of ongoing tissue fibrosis (Figure 2c–d). Tissue changes were most pronounced in the line 25 mice, but also present in line 18 mice, while wild type mice had normal liver architecture and normal reticulin levels.

These data demonstrate overexpression of TGFβ1 in Tr+ transgenic mice and detail the alterations in phenotype, providing a model for the assessment of the contribution of this important effector cytokine to the fibrotic milieu in vivo.

Overexpression of TGFβ1 in the liver leads to a severe liver fibrosis. Fibrotic liver phenotype presented at 1 month with the injury being most severe from 3 months onwards. Of note was the finding of enhanced reticulin deposition in the fibrotic tissue versus Tr- wild type mice. These data provide evidence that overexpression of TGFβ1 in mouse liver promotes de novo fibrosis, even in the absence of other pro-fibrotic stimuli.

Having demonstrated the molecular effect of TGFβ1 overexpression and its effect on mouse liver we examined the impact of Tr+ TGFβ1 transgenic expression on mouse lung. Of note was the finding that transgenic mouse lungs (Figure 3a) showed no evidence of de novo fibrosis at any time point studied.

To determine the putative mechanism underpinning this tissue specific finding we characterised the expression of TGFβ1 in transgenic mouse lung. Figure 3b shows staining for active TGFβ1 in lung tissue, providing evidence that whilst TGFβ1 is present in the lung it does not produce a fibrotic response. Having determined the presence of active TGFβ1 in these lungs, expression of TGFβ1 receptors was investigated. Of note was the finding that, Tr+ transgenic mouse lung was found to contain an abundance of both Type I (Figure 3c) and type II (Figure 3d) TGFβ1 receptors.

These data demonstrate that de novo tissue fibrosis in response to TGFβ1 overexpression is tissue specific. In the setting of the lung, active TGFβ1 does not produce a fibrotic phenotype despite an abundance of both type I and type II receptors. The data presented herein lend weight to the hypothesis that TGFβ1 contributes to lung fibrosis in vivo through the interplay with other factors, and is not sufficient, in itself to drive lung fibrosis.

The data generated in the histological studies identified that TGFβ1 overexpression is insufficient to establish pulmonary fibrosis. However we determined the effect of a second insult with bleomycin on TGFβ1- and WT-transgenic mice.

To determine the molecular events subserving the TGFβ1 mediated exacerbation of lung fibrosis we utilised an oligonucleotide microarray based strategy to identify altered key transcripts. Specifically, we probed the molecular contribution to the repetitive injury, namely the expression changes induced by TGFβ1 overexpression and the expression changes that result from bleomycin exposure in these Tr+ transgenic mice. Affymetrix Mouse Genome 430_2 microarrays were used to determine gene expression levels in lung tissue from a) untreated Tr+ TGFβ1 transgenic mice b) Tr- wild type mice treated with bleomycin and c) bleomycin treated Tr+ TGFβ1 transgenic mice, to identify the overall pattern of gene expression in this experiment. Significant changes in gene expression were associated with these tissue cohorts (-0.6 < SLR > 0.6, and p < 0.05) (Figure 4a). Distinct patterns of coordinate gene expression were observed throughout the exposures, with substantial transcriptomic effects in terms of both up and downregulation of gene expression separating the sample groups.

Of the 45,101 gene sequences represented on the Affymetrix Mouse Genome 430_2 oligonucleotide microarray, 6.2% (2812 genes) were found to be significantly altered in all three groups. Exposure of Tr+ TGFβ1 transgenic mice to bleomycin elicited a major gene expression response with a total of 3.9% significantly altered transcripts (1724 genes) in compare to untreated Tr+ TGFβ1 transgenic mice. To probe molecular basis of TGFβ1 exacerbation of lung injury the expression profiles of both bleomycin-treated WT and TGFβ1 transgenic mice were compared, showing 640 significant gene expression changes between these groups (1.4% of the transcripts represented on the microarray) (Figure 4b).

To further annotate the transcriptomic differences between the study groups' ontological classification of molecular function was investigated by using Gene Ontology database (Figure 4c). By using Gene Ontology database, Bleomycin TR+ vs TR+ altered transcripts were classified and grouped in functional families in correlation with their significant role in fibrosis development. Altered fibrosis-associated genes, which drive angiogenesis, inflammatory response, immune response and apoptosis, in response to bleomycin in TR+ mice were found dysregulated, as also previously reported 282930. In addition, we found a large number of significantly altered genes that function in the regulation of cellular morphogenesis, development and gene transcription. Bleomycin-exposed TR+ TGFβ1 transgenic mice show overall altered gene expression profile which correlate with cellular morphogenesis and gene transcription, enhanced cellular functions which trigger fibrosis development.

Tables 1 and 2 highlight the genes whose mRNA levels were most strikingly altered in bleomycin injured and Tr+ TGFβ1 transgenic mice.

Table 1 indicates a large number of altered genes, which are largely recognized as mediators of immunological function. Further annotation of these upregulated genes identified a large number of genes involved in cytokine signalling. Further, the transcripts whose expression was found to be altered in response to bleomycin exposure included a large number of extracellular matrix and matrix regulation associated genes, key effectors molecules in the development of tissue fibrosis.

Having demonstrated that overexpression of TGFβ1, whilst initiating severe fibrosis in mouse liver, does not cause de novo lung fibrosis; we determined the effect of escalating doses of bleomycin on TGFβ1 and WT transgenic mice.

Exposure to 1500 IU of bleomycin resulted in 5 mice (Tr+ line 25) developing lung fibrosis compared to only 2 in the Tr- wild type group. 4500 IU of bleomycin showed fibrosis in all 8 mice of the Tr+ line 25, compared to 6 mice in the Tr+ line 18 group and 5 mice in the Tr- wild type group. These data determine the dose response nature of lung injury following exposure to bleomycin.

Lung fibrosis induced by 4500 IU bleomycin in the Tr- wild type group was a mild patchy lung injury (Figure 5a). Tr+ transgenic mice, following exposure to comparable and smaller doses of bleomycin demonstrated marked lung injury hallmarked by grossly thickened alveolar walls, inflammation, fibroblast proliferation and collagen deposition in a peribronchial, interstitial and sub pleural distribution (Figure 5b).

To validate these tissue observations the fibrosis score in bleomycin treated mice was determined as described. The Tr+ transgenic-bleomycin group had greater fibrosis scores (mean 1.88, SEM 0.27) than the Tr- wild type-bleomycin group (mean 0.875, SEM 0.295) (p < 0.05). Tr+ line 25-bleomycin group had the highest mean score (mean 2.0, SEM 0.32) (p < 0.05) (Figure 5c) while the Tr+ line 18-bleomycin group also had a score of 1.75 (SEM 0.45). The PBS vehicle Tr- wild type group had scores of 0 (n = 8). These data further demonstrate the exacerbation of bleomycin elicited lung injury in mice overexpressing TGFβ1.

Finally, we determined the tissue distribution of TGFβ1 in lung tissue from both Tr- wild type and Tr+ transgenic mice following exposure to bleomycin. Immunostaining for TGFβ1 demonstrated marked expression of TGFβ1 in both wild type (Figure 6a) and Tr+ transgenic (Figure 6b) mice following bleomycin exposure. Of note is the particularly strong expression in TGFβ1 transgenic mice, suggesting that bleomycin exposure elicits a more pronounced TGFβ1 response in Tr+ transgenic versus wild type mouse lung.

Having determined the fibrotic response induced by exposure to 4500 IU of bleomycin in both wild type and TGFβ1 transgenic mouse lung tissue we further investigated collagen production in lung tissue following exposure to bleomycin.

De novo collagen production was assessed using hydroxyproline assay. The lung hydroxyproline content was higher in the Tr+ transgenic-bleomycin group (mean 3.3 μg/mg, SEM 0.11) than in the Tr- wild type-bleomycin group (mean 2.4 μg/mg, SEM 0.33)(p < 0.05) or the unwounded PBS group (mean 1.76 μg/mg, SEM 0.16) (Figure 6).

In summary, the fibrotic response in Tr+ transgenic mice is dominated firstly by immune mediators reacting to bleomycin exposure and causing lung injury and secondly by genes (TGFβ1) contributing to the deposition of extracellular matrix. These data lend further weight to the hypothesis that pulmonary fibrosis is a result of combined injury from both endogenous and exogenous mediators and provides important evidence for the interplay of these factors in the development of tissue fibrosis. Further analysis of these transcriptomic alterations will reveal the exact mechanism of the synergistic lung injury induced by TGFβ1 overexpression and bleomycin injury.