Genome-wide transcriptional response during the development of bleomycin-induced pulmonary fibrosis in sprague-dawley rats.

Pulmonary fibrosis is a common consequence of many lung diseases and a leading cause of morbidity and mortality. The molecular mechanisms underlying the development of pulmonary fibrosis remain poorly understood. One model used successfully to study pulmonary fibrosis over the past few decades is the bleomycin-induced pulmonary fibrosis model. We aimed to identify the genes associated with fibrogenesis using an Affymetrix GeneChip system in a bleomycin-induced rat model for pulmonary fibrosis. To confirm fibrosis development, several analyses were performed, including cellular evaluations using bronchoalveolar lavage fluid, measurement of lactate dehydrogenase activity, and histopathological examinations. Common aspects of pulmonary fibrosis such as prolonged inflammation, immune cell infiltration, emergence of fibroblasts, and deposition of extracellular matrix and connective tissue elements were observed. Global gene expression analysis revealed significantly altered expression of genes (≥ 1.5-fold, p < 0.05.) in a time-dependent manner during the development of pulmonary fibrosis. Our results are consistent with previous results of well-documented gene expression. Interestingly, the expression of triggering receptor expressed on myeloid cells 2 (Trem2) , secreted phosphoprotein 1 (Spp1) , and several proteases such as Tpsab1, Mcpt1, and Cma1 was considerably induced in the lung after bleomycin treatment, despite little evidence that they are involved in pulmonary fibrogenesis. These data will aid in our understanding of fibrogenic mechanisms and contribute to the identification of candidate biomarkers of fibrotic disease development.

INTRODUCTION

Idiopathic pulmonary fibrosis is a consequence of abnormal tissue repair, which is characterized by extensive inflammation in the interstitial and alveolar spaces, proliferation of fibroblasts, and progressive fibrosis leading to the destruction of lung function (Selman . Pulmonary fibrosis involves cytokine networks and cellular interactions among several cell types, which results in increased collagen gene expression and collagen deposition in the lungs (Piguet ; Gharaee-Kermani . Despite many investigations on the pathogenesis of pulmonary fibrosis and its relationship to immune cells, extracellular matrix repair, cytokines, and chemokines, the molecular mechanisms underlying this disease remain unclear (Zhang , 1996; Kuwano ; Gharaee-kermani .

Bleomycin is a useful chemotherapeutic antitumor drug, derived from Streptomyces verticillus; however, as a side effect, bleomycin treatment may lead to pulmonary fibrosis in humans and animals. Therefore, animal models of pulmonary fibrosis induced by the intratracheal instillation of bleomycin have been well established and widely used for studying the mechanisms underlying fibrosis and potential therapeutic agents (Jordana ; Sakanashi ; Gharaee-Kermani ; Moeller . Several studies using microarrays have been performed to evaluate gene expression patterns in the lungs after bleomycin instillation and identify the major genes involved in the progression of pulmonary fibrosis (Kaminski ; Zuo ; Katsuma ; Pottier . These studies have provided information about genes that play central roles in the progression of fibrosis over time in a mouse model. Although genomic approaches using bleomycin-induced pulmonary fibrosis animal models have been useful for understanding the molecular mechanisms underlying fibrosis and for identifying fibrogenic markers, these approaches have limitations related to the vast data sets generated and the difficulty of replicating progressive fibrosis in animal models. Furthermore, recent studies aimed at elucidating fibrogenesis mechanisms and developing therapeutic agents for the treatment of fibrosis have focused on mouse models because of the convenience of using genetically modified animals.

Here, we performed a microarray-based gene expression analysis to reveal distinct gene expression patterns and to identify genes associated with the progression of pulmonary fibrosis in the lungs of Sprague-Dawley (SD) rats at 7, 14, and 28 days after treatment with bleomycin by intratracheal instillation (ITI) . Lung damage and pulmonary fibrogenesis were assessed by cellular evaluation of bronchoalveolar lavage fluid (BALF) , measurement of lactate dehydrogenase (LDH) activity, and histopathological examination of lung tissue.

MATERIALS AND METHODS

Animals and experimental design.

Pathogen-free, 7-week-old, male Sprague-Dawley (SD) rats (Orient Bio Inc., Korea) , weighing 210 to 240 g, were administered bleomycin hydrochloride (Nippon Kayaku, Tokyo, Japan) as a single dose of 2.5 mg/kg body weight, dissolved in 0.1 ml of saline (0.9% NaCl) , via intratracheal instillation (ITI) under isoflurane anesthesia. Control rats received an equal volume of saline. The dose of bleomycin was selected based on previous experiments in our laboratory and is known to cause consistent biochemical and histological damage, but not mortality (Cortijo .

The treated and control rats were randomly assigned to six weight-matched groups (n = 5 for each) : three treated groups (7, 14, and 28 days) , and three corresponding control groups (7, 14, and 28 days) . On days 7, 14, and 28 following ITI bleomycin or saline, the rats were sacrificed by exsanguination via the abdominal aorta, under an overdose of isoflurane anesthesia. Lung injury was evaluated by analysis of bronchoalveolar lavage fluid (BALF) and light microscopic examination (Olympus, Japan) .

All experiments were approved by the Institutional Animal Care and Use Committee and conducted in accordance with Association for Assessment and Accreditation of Laboratory Animal Care international guidelines.

Analysis of bronchoalveolar lavage fluid (BALF) .

After exsanguination, the left lung of each rat was clamped off, the right lung was washed twice with 0.9% saline, and the BALF was collected. The cell-free supernatant obtained by centrifugation (500 g, 10 min, 4℃) of the BALF sample was analyzed for LDH activity, as described below. The right lung was lavaged twice more with 3 ml of saline, and the cell pellet was recovered after centrifugation (500 g, 10 min, 4℃) of this sample. The cell pellets from the rats were combined for each group, resuspended in 1 ml of saline, and used to evaluate cellular parameters.

The cells recovered in the BALF pellet were identified and counted. Total cell number was determined using a cell viability analyzer (Beckman Coulter, Fullerton, CA) . To collect the BAL cells on a slide, a sample was spun at 800 rpm for 5 min in a Cytospin 4 centrifuge (Thermo-Shandon, Pittsburgh, PA) . The cells were stained with Wright-Giemsa Sure stain (Muto Pure Chemicals Co., Japan) and differentially counted (300/rat) as alveolar macrophages (AM) , polymorphonuclear neutrophils (PMN) , and lymphocytes.

The activity of the cytosolic enzyme LDH was used as an indicator of pulmonary damage resulting from general cytotoxicity and was measured by an automatic analyzer (TBA-200FR; Toshiba, Japan) .

Histopathological evaluation.

After the BALF was collected, the right lung was perfused intratracheally with 10%neutral phosphate-buffered formalin (NPBF) and fixed in 10% NPBF, pending further processing. Specimens were dehydrated, embedded in paraffin, sectioned as 3-㎛ slices onto microscope slides, and stained with Masson's Trichrome stain (Sigma-Aldrich, St. Louis, MO) . The extent of pulmonary fibrosis was assessed by microscopic examination, as described below. The lesions were histologically graded by two experienced histopathologists using a blinded, scoring system for the extent and severity of inflammation and fibrosis in the lung parenchyma, as previously outlined (Ashcroft .

RNA extraction.

Total RNA was extracted from a portion of the left lung sample using Trizol reagent (Invitrogen, Carlsbad, CA) and an RNeasy mini kit (Qiagen, Valencia, CA) , according to manufacturers’ instructions. Isolated total RNA was quantified using a NanoDrop spectrophotometer (NanoDrop Technologies, Montchanin, DE) . For microarray experiments, the quality of RNA was evaluated using a 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) .

Gene expression analysis.

Changes in gene expression in the lungs of bleomycin-treated SD rats were analyzed using an Affymetrix rat genome 230 2.0 array. Total RNA samples (3 μg) from each control and bleomycin-treated group were used for cDNA synthesis. All microarray procedures, including synthesis of cDNA and biotin-labeled cRNA, fragmentation of cRNA, hybridization, washing, staining, and scanning, were performed according to the manufacturer;s instructions. Data normalization, statistical analysis, and selection of differentially expressed genes (DEGs) were performed by GenPlex software, version 3.0 (ISTECH Inc., Korea) , after raw image data processing by the MARS 5.0 algorithm. The selection of DEGs in the bleomycin-treated groups was based on a greater than 1.5-fold change in expression relative to the corresponding control group. Statistically significant threshold values were assigned at p < 0.05 according to Student’s t-test. The selected DEGs for days 7, 14, and 28 were combined and analyzed by twodimensional hierarchical clustering and principal components analysis. Classifications of the DEGs by biological function/disease and canonical pathway were performed using Ingenuity Pathway Analysis (IPA) for the data set of significantly altered genes at each time point. The P value, represented as a negative log ratio of the IPA results, is the probability based on Fischer’s exact test. In addition, genes that may be involved in bleomycin-induced pulmonary fibrosis were selected using the IPA knowledge base and public databases. The selected genes were annotated based on the NetAffyxTM and Ingenuity® systems.

Statistical analysis.

All measured values are expressed as means ± standard deviations. Statistical analyses were performed using SAS (Version 4.2.2; Xybion Medical Systems Corp., Cedar Knolls, NJ) . Analysis of variance (ANOVA) was used to assess differences between groups,and Student's t-test was used for individual comparisons. Statistical significance was established at p < 0.05 or p < 0.01.

RESULTS

BALF analysis.

The total and differential cell counts in the BALF of rats at 7, 14, and 28 days after treatment with bleomycin are presented in Table 1. The numbers of total cells, alveolar macrophages, neutrophils, and lymphocytes were significantly increased at 7 and 14 days compared with the control values (P < 0.01) . The total number of bronchoalveolar (BAL) cells was highest at 7 days, and the percentage of neutrophils was higher than that in the control group. The total cell number and differential cell counts decreased thereafter. The number of lymphocytes showed a similar increase, but the absolute values were extremely small compared with the numbers of neutrophils and alveolar macrophages.

Table 1.

Total and differential cell counts and LDH concentration in BAL fluid

Parameter Day	Day 7		Day 14		Day 28
	Control	Bleomycin	Control	Bleomycin	Control	Bleomycin
Total cells (× 104/ml)	7.60 (1.15)	58.97 (11.96) **	6.28 (1.21)	25.08 (8.58) **	9.57 (2.11)	15.44 (3.62) *
Macrophages	7.41 (1.09)	27.02 (6.67) **	6.15 (1.2)	20.4 (6.2) **	9.36 (2.09)	15.00 (3.51) *
Neutrophils	0.09 (0.03)	25.07 (6.15) **	0.05 (0.02)	3.49 (1.89) *	0.09 (0.03)	0.24 (0.10) *
Lymphocytes	0.09 (0.04)	6.88 (1.13) **	0.06 (0.02)	1.16 (0.64) **	0.11 (0.03)	0.20 (0.7) *
LDH (U/l)	1.2 (1.3)	50.2 (20.7) **	4.4 (2.88)	11.6 (13.46)	0.4 (0.5)	0.8 (0.04)

Data are presented as mean ± S.D of groups.

*p < 0.05 vs. control group.

**p < 0.01 vs. control group.

LDH activity in BALF, which represents the extent of lung cell damage, was significantly higher (P < 0.01) at 14 days after bleomycin instillation compared with the control activity (Table 1) . The highest LDH activity occurred at day 7.

Histopathological findings.

Histopathological changes in the lungs were examined on days 7, 14, and 28 after bleomycin or saline treatment. To confirm the histopathology of bleomycin-induced lung inflammation and fibrosis, the overall histopathological grades of lung inflammation and fibrosis were scored (Table 2) . Lung inflammation in the bleomycin-treated rats peaked at day 7 and decreased thereafter. Representative light photomicrographs of lung tissues from of bleomycin-treated animals are shown in Fig. 1. On day 7, marked infiltration by inflammatory cells (predominantly macrophages and neutrophils) in the alveolar regions was apparent in the bleomycin-treated rats. Additionally, extensive hyperplasia of alveolar epithelial cells and proliferation of fibroblasts were observed (Fig. 1B) . On day 14, the bleomycin-treated rats showed moderate deposition of collagen fibers and fibroblasts in the peribronchiolar and alveolar interstitium, with a few inflammatory cells in the fibrotic area (Fig. 1C) . On day 28, diffuse or focal dense fibrosis was observed in the peribronchiolar and subpleural interstitial spaces, with a small number of inflammatory cells (Fig. 1D) . The severity of these changes varied among the rats and ranged from mild to moderate.

Table 2.

Incidence and severity of microscopic findings in the lung

Lung pathology	Control (n = 5)	Bleomycin-treated group
		7 Day (n = 5)	14 Day (n = 5)	28 Day (n = 5)
Inflammatory cell infiltration		+++++ (2) ,++++ (3)	++ (3) ,+ (2)	++ (2) ,+ (3)
Pulmonary fibrosis		+++ (2) ,++ (3)	++++ (3) ,+++ (2)	+++ (3) ,++ (2)

The number of animal with corresponding lesions was represented in the parentheses.

Severity of lesion was indicated as follows;+, minimal;++, mild;+++, moderate;++++, marked;+++++, severe.

+, minimal; inflammation (10％ of the slide) , fibrosis (minimal fibrous thickening of alveolar or bronchiolar walls) .

++, mild; inflammation (10~20％) , fibrosis (mild fibrous thickening of alveolar or bronchiolar walls without obvious damage to the lung architecture) .

+++, moderate; inflammation (20~40％) , fibrosis (increased fibrosis with definite damage to the lung structure and formation of fibrous bands) .

++++, marked; inflammation (40~70％) , fibrosis (severe distortion of the lung structure and large fibrous areas) .

+++++, severe; inflammation (> 70％) , fibrosis (total fibrous obliteration of the field) .

Fig. 1.

Representative photomicrographs of lung tissue from bleomycin-instilled rats. A. Control. B. Experimental animal 7 days after ITI of bleomycin. Note the infiltration of numerous neutrophils, alveolar macrophages, and lymphocytes in the peribronchiolar, perivascular, and alveolar spaces, together with thickening of the alveolar interstitium and proliferation of fibroblasts. C. Experimental animal 14 days after ITI of bleomycin. Note the small number of inflammatory cells and numerous fibroblasts, plus a moderate collagenous deposit with infiltration of neutrophils and monocytes. D. Experimental animal 28 days after ITI of bleomycin. Note the moderate alveolar fibrosis and minimal infiltration of inflammatory cells, mainly of neutrophils and few alveolar macrophages. Interstitial thickening is seen in the alveolar walls. Scale bar = 200 ㎛.

Compared with the expression patterns in the corresponding controls, differentially expressed genes were detected on days 7, 14, and 28 after bleomycin treatment, based on the fold change and statistical significance (≥ 1.5-fold, P < 0.05) . The numbers of significantly altered probes were 2,839; 1,665; and 1,906 on days 7, 14, and 28 after bleomycin treatment, respectively. We combined all of the selected probe sets, which included 4,662 probe sets that were significantly altered for at least one time point, and analyzed the expression patterns using two-dimensional hierarchical clustering based on the Pearson correlation and complete linkage. Cluster analysis showed distinguishable gene expression patterns between the treatment and control groups and yielded three differential clusters among days 7, 14, and 28, indicating time dependence (Fig. 2A) . An unsupervised principal components analysis revealed four major differences between the controls and the three different time-point groups, further indicating that the gene expression profiles change over time (Fig. 2B) . The 10 most significantly up- or down-regulated genes at each time point are presented in Table 3. On days 7 and 14, Trem2, Mmp7, and Spp1 were markedly up-regulated. In contrast, on days 14 and 28, several proteases genes, including Tpsab1, Mcpt1, and Cma1, were up-regulated. Interestingly, Trem2 expression was up-regulated by more than 10-fold at all time points, and Cyp2a3a was down-regulated by more than 10-fold at all time points.

Fig. 2.

Hierarchical clustering (A) and principal components analysis (B) using 4662 selected probe sets.

Table 3.

Genes significantly modulated in the lungs of bleomycin-treated rats

Probe ID	Gene symbol	Gene name	Fold change
7 days after bleomycin treatment
Up-regulated
1382513_at	Trem2_predicted	triggering receptor expressed on myeloid cells 2 (predicted)	82.827
1368766_at	Mmp7	matrix metallopeptidase 7	59.790
1367581_a_at	Spp1	secreted phosphoprotein 1	45.831
1387715_at	Expi	extracellular peptidase inhibitor	35.944
1384442_at	Cldn6_predicted	claudin 6 (predicted)	22.171
1368587_at	Apoc1	apolipoprotein C-I	18.956
1393917_at	Cd163_predicted	CD163 antigen (predicted)	18.320
1385687_at	LOC682044	hypothetical protein LOC682044	17.710
1367794_at	A2m	alpha-2-macroglobulin	17.607
1384329_at	Prss22_predicted	protease, serine, 22 (predicte	15.104
Down-regulated
1393771_at	Scgb3a1	secretoglobin, family 3A, member 1	−19.008
1367985_at	Alas2	aminolevulinic acid synthase 2	−17.644
1387796_at	Alox15	arachidonate 15-lipoxygenase	−17.603
1376980_at	Htr2c	5-hydroxytryptamine (serotonin) receptor 2C	−16.090
1368338_at	Cd52	CD52 antigen	15.683
1370269_at	Cyp1a1	cytochrome P450, family 1, subfamily a, polypeptide 1	15.416
1370355_at	Scd1	stearoyl-Coenzyme A desaturase	14.370
1369136_at	Cyp2a3a	cytochrome P450, family 2, subfamily A, polypeptide 3a	12.412
1375519_at	LOC287167	globin, alpha	12.335
1385759_at	Serpinb10	serine (or cysteine) peptidase inhibitor, clade B (ovalbumin) , member 10	10.382
14 days after bleomycin treatment
Up-regulated
1382513_at	Trem2_predicted	triggering receptor expressed on myeloid cells 2 (predicted)	−65.595
1387162_at	Tpsab1	tryptase alpha/beta 1	−64.258
1370405_at	Mcpt1	mast cell protease 1	−46.476
1368912_at	Trh	thyrotropin releasing hormone	−27.322
1368766_at	Mmp7	matrix metallopeptidase 7	−24.723
1384442_at	Cldn6_predicted	claudin 6 (predicted)	−20.582
1387173_at	Cma1	chymase 1, mast cell	−20.505
1379331_at	Tnn_predicted	tenascin N (predicted)	−18.944
1367581_a_at	Spp1	secreted phosphoprotein 1	−18.240
1375052_at	LOC501015	similar to nuclear localized factor 2	−13.946
Down-regulated
1393771_at	Scgb3a1	secretoglobin, family 3A, member 1	53.032
1369136_at	Cyp2a3a	cytochrome P450, family 2, subfamily A, polypeptide 3a	31.674
1368608_at	Cyp2f4	cytochrome P450, family 2, subfamily f, polypeptide 4	27.077
1371209_at	RT1-CE5	RT1 class I, CE5	9.042
1387839_at	RT1-N1	RT1 class Ib gene, H2-TL-like, grc region (N1)	8.928
1385759_at	Serpinb10	serine (or cysteine) peptidase inhibitor, clade B (ovalbumin) , member 10	8.886
1373368_at	LOC684050	similar to procollagen C-endopeptidase enhancer 2	6.774
1375842_at	LOC688972	similar to Glycophorin	6.175
1370789_a_at	Prlr	prolactin receptor	5.00
1369435_at	Ttpa	tocopherol (alpha) transfer protein	4.651
28 days after bleomycin treatment
Up-regulated
1370405_at	Mcpt1	mast cell protease 1	−48.603
1387162_at	Tpsab1	tryptase alpha/beta 1	−44.757
1387173_at	Cma1	chymase 1, mast cell	−30.695
1382513_at	Trem2_predicted	triggering receptor expressed on myeloid cells 2 (predicted)	−26.319
1396388_at	LOC691835	similar to Potassium channel tetramerisation domain-containing protein 4	−20.559
1370971_at	Myh1	myosin, heavy polypeptide 1, skeletal muscle, adult	−18.842
1374863_at	RGD1562168_predicted	similar to retinoid binding protein 7 (predicted)	−17.563
1371164_at	Mcpt10	mast cell protease 10	−15.911
1373865_at	Snap91	synaptosomal-associated protein, 91 kDa homolog (mouse)	−15.911
1368519_at	Serpine1	serine (or cysteine) peptidase inhibitor, clade E, member 1	−14.869
Down-regulated
1380363_at	Klf7_predicted	Kruppel-like factor 7 (ubiquitous) (predicted)	15.640
1369136_at	Cyp2a3a	cytochrome P450, family 2, subfamily A, polypeptide 3a	11.061
1368608_at	Cyp2f4	cytochrome P450, family 2, subfamily f, polypeptide 4	9.198
1375148_at	LOC691504	similar to Zinc finger protein ZFPM1 (Zinc finger protein multitype 1)	8.712
1369300_at	Ncr1	natural cytotoxicity triggering receptor 1	8.316
1378089_at	MGC108778	Similar to RIKEN cDNA 1810057C19	7.732
1391864_at	Enpp6_predicted	Ectonucleotide pyrophosphatase/phosphodiesterase 6 (predicted)	6.857
1380799_at	Grem2_predicted	gremlin 2 homolog, cysteine knot superfamily (predicted)	6.740
1387631_at	Hpgd	hydroxyprostaglandin dehydrogenase 15 (NAD)	6.074
1369703_at	Epas1	endothelial PAS domain protein 1	5.876

Classification by function, disease/disorder, and canonical pathway.

To understand the overall genomic response to bleomycin, selected probe sets at each time point were imported into the IPA software classification into three categories, namely, biological function, disease/disorder, and canonical pathway, based on the IPA knowledge base. Fig. 3A shows the genes with altered expression at each time point that were classified into the biological function category. The genes significantly altered on day 7 involved mainly cell-to-cell signaling and interactions, antigen presentation, and cellular movement. Fewer genes altered on days 14 and 28 were classified into this category. On day 14, the main dysregulated genes were those involved in lipid metabolism, small molecule biochemistry, cellular function and maintenance, and cellular growth and proliferation; the expression of genes involved in molecular transport, cell death, and cell signaling gradually increased. The genes classified into 11 disease/disorder-associated groups are presented in Fig. 3B. Respiratory disease comprised the highest number of altered genes, and many dysregulated genes associated with inflammatory diseases and genetic disorders were identified on day 7. The majority of genes with altered expression on day 28 were related to inflammatory diseases and genetic disorders, although there were also a high number associated with skeletal/muscular and connective tissue disorders. We also examined genes of canonical pathways associated with cytokine signaling and cell-mediated immune responses (Fig. 4) . Most of the genes for cytokines, including TREM, IL-10, IL-4, IL-8, and IL-6, were highly dysregulated on day 7 after bleomycin treatment.

Fig. 3.

Functions and diseases affected as a result of altered gene expression in the lungs of belomycin-treated rats. A. Top functions. B. Top diseases and disorders. Bars represent -log (p-value) for the disproportionate representation of the affected genes among the total number of genes in the selected function/disease category. Yellow line represents the threshold (p=0.05) level.

Fig. 4.

Analysis of canonical pathways associated with cytokine signaling and cell-mediated immune response in the lungs of bleomycin-treated rats. Bars represent -log (p-value) for the disproportionate representation of affected genes among the total number ofgenes in the selected function/disease category. Yellow line represents the threshold (p = 0.05) level.

Altered gene expression involved in the development of fibrosis.

Bleomycin-induced pulmonary fibrosis in the rodent is a well-established model for histopathological, biochemical, and molecular biology studies, and numerous studies have revealed the regulation of genes associated with pulmonary fibrosis in bleomycin-treated, transgenic, and knock-out mice. To compare our gene expression data with the results of previous studies, we extracted 52 genes associated with common fibrogenesis mechanisms and bleomycin-induced pulmonary fibrosis, using the IPA knowledge base and public databases. As listed in Table 4, the expression profiles of the following genes were significantly modulated: fibrogenic cytokines and cytokine receptors such as Il-6, Il-13ra1, Il-13ra2, Tgfb1, and Tgfb2; fibrogenic chemokines and chemokine receptors such as Ccl2, Ccl3, Ccr1, and Ccr2; growth factors such as Egr1 and Ctgf; extracellular matrix components such as Actg2, Eln, Fn1, Col1a1, and Col1a3; and several proteases, including Ctsk, Mmp2, and Mmp12.

Table 4.

Modulation of fibrosis-related genes in the lungs of bleomycin-treated rats

Probe set ID	Gene symbol	Gene name	Fold change
			7 day	14 day	28 day
1386869_at	Actg2	actin, gamma 2, smooth muscle, enteric	2.885	1.736	1.731
1374207_at	Angpt2	angiopoietin 2	1.319	1.073	1.522
1369122_at	Bax	Bcl2-associated X protein	1.959	1.600	1.115
1377759_at	Bid	BH3 interacting domain death agonist	2.188	1.294	1.131
1382783_at	Blm	Bloom syndrome homolog (human)	1.807	1.187	1.253
1390386_at	Casp3	caspase 3, apoptosis related cysteine protease	1.938	1.360	1.031
1370131_at	Cav1	caveolin, caveolae protein 1	–2.469	1.349	–4.636
1370118_at	Ccl17	chemokine (C-C motif) ligand 17	6.033	2.922	2.542
1367973_at	Ccl2	chemokine (C-C motif) ligand 2	7.651	7.096	4.366
1370620_at	Ccl22	chemokine (C-C motif) ligand 22	8.574	2.879	3.207
1369815_at	Ccl3	chemokine (C-C motif) ligand 3	1.421	1.632	2.391
1369983_at	Ccl5	chemokine (C-C motif) ligand 5	–1.232	–3.072	–2.507
1370083_at	Ccr1	chemokine (C-C motif) receptor 1	1.295	2.001	1.630
1387742_at	Ccr2	chemokine (C-C motif) receptor 2	3.201	1.777	3.926
1370034_at	Cdc25b	cell division cycle 25 homolog B (S. pombe)	–3.997	–2.057	–1.177
1387391_at	Cdkn1a	cyclin-dependent kinase inhibitor 1A (p21, Cip1)	6.769	2.970	2.014
1370864_at	Col1a1	collagen, type I, alpha 1	1.792	2.920	3.944
1370959_at	Col3a1	collagen, type III, alpha 1	1.102	1.270	1.702
1367631_at	Ctgf	connective tissue growth factor	1.469	1.804	1.657
1369947_at	Ctsk	cathepsin K	6.666	7.213	7.603
1387316_at	Cxcl1	chemokine (C-X-C motif) ligand 1	1.390	–1.298	–1.975
1368760_at	Cxcl2	chemokine (C-X-C motif) ligand 2	2.777	1.357	–1.076
1370633_at	Cxcl3	chemokine (C-X-C motif) ligand 3	2.261	1.699	–1.547
1370699_a_at	Egfr /// Pepd	epidermal growth factor receptor /// peptidase D	2.272	1.739	1.316
1368321_at	Egr1	early growth response 1	1.950	1.337	1.748
1388111_at	Eln	elastin	1.168	1.883	2.628
1370234_at	Fn1	fibronectin 1	1.967	1.957	1.923
1387386_at	Foxj1	forkhead box J1	1.212	–2.564	–1.135
1371727_at	Glb1	galactosidase, beta 1	1.788	1.506	1.606
1370331_at	Il11ra1	interleukin 11 receptor, alpha chain 1	–1.558	–1.137	1.138
1370728_at	Il13ra1	interleukin 13 receptor, alpha 1	1.561	1.373	–1.141
1369266_at	Il13ra2	interleukin 13 receptor, alpha 2	2.148	1.786	2.544
1369191_at	Il6	interleukin 6	5.899	3.800	3.031
1387144_at	Itga1	integrin alpha 1	1.315	–2.246	–5.186
1383880_at	Itgav_predicted	integrin alpha V (predicted)	2.941	2.340	1.613
1386879_at	Lgals3	lectin, galactose binding, soluble 3	1.790	1.558	1.291
1387946_at	Lgals3bp	lectin, galactoside-binding, soluble, 3 binding protein	2.029	1.532	1.382
1379612_at	Mapk8	mitogen-activated protein kinase 8	1.507	1.095	1.109
1384427_at	Mdm2	Mdm2 p53 binding protein homolog (mouse)	2.762	1.304	–1.694
1368530_at	Mmp12	matrix metallopeptidase 12	6.566	7.207	7.116
1370301_at	Mmp2	matrix metallopeptidase	1.928	1.359	1.738
1369200_at	Nt5e	5' nucleotidase, ecto	1.422	1.185	1.933
1385434_at	Ptger2	Prostaglandin E receptor 2, subtype EP2	2.695	1.414	1.676
1367780_at	Pttg1	pituitary tumor-transforming 1	1.771	1.650	1.886
1368423_at	Retnla	resistin like alpha	3.994	2.069	2.216
1368519_at	Serpine1	serine peptidase inhibitor, clade E, member 1	6.874	3.257	14.869
1367581_a_at	Spp1	secreted phosphoprotein 1	45.831	18.240	8.895
1370082_at	Tgfb1	transforming growth factor, beta 1	2.130	1.287	1.176
1376425_at	Tgfb2	Transforming growth factor, beta 2	1.104	2.117	1.004
1367712_at	Timp1	TIMP metallopeptidase inhibitor 1	6.731	2.685	1.692
1370752_a_at	Tp53	tumor protein p53	1.550	1.599	1.505
1389234_at	Vwf	Von Willebrand factor homolog	–1.859	–1.317	–1.216

Bold; p < 0.05 compared with control group.

DISCUSSION

In the present study, we confirmed bleomycin-induced pulmonary fibrosis in a SD rat model using histopathological examination and BALF analysis. Consistent with previous findings, the histopathological results showed markedly increased inflammation, with infiltrated immune cells extending into the alveolar regions and proliferation of epithelial cells and fibroblasts at day 7; moderate deposition of collagen at day 14; and dense fibrosis in the peribronchiolar and interstitial spaces with reduced inflammation foci at day 28. LDH activity was highest on day 7 after bleomycin treatment. Based on these results, we investigated the gene expression profiles associated with fibrogenic mechanisms in the lungs of this rat model, using well-documented findings and the IPA knowledge base for comparisons.

During the development of fibrosis, IL-4, IL-5, and IL-13 secreted by CD4+T helper type 2 cells (TH2s) play important roles in several cytokine-deficient mice (Wynn . Early microarray-based gene expression profiling revealed that TH1 and TH2 cytokines induced markedly different genomic responses to pathogen-induced inflammation in mice, and differences in the induction of several genes associated with fibrogenic mechanisms have been shown in TH2 polarized mice (Hoffmann ; Sandler . In our study, direct gene induction of Il-4, Il-5, and Il-13, which are the primary TH2 cytokines, was not observed; however, the expression of genes related to the IL-4 signaling pathway was significantly altered on day 7 after bleomycin treatment (Fig. 4) . IL-4 is a profibrotic cytokine that directly stimulates the in vitro synthesis of extracellular matrix (ECM) proteins, including type I and type III collagen, and fibronectin (Fertin ; Doucet . Our data demonstrate significant induction of genes encoding collagen type I-α1 (Col1a1) and type III-α1 (Col3a1) and fibronectin, consistent with previous findings.

Among the TH2 cytokines, IL-13 is one of the most powerful fibrogenic cytokines. Selected expression of IL-13 in the lungs of mice caused severe subepithelial fibrosis (Zhu . Furthermore, antibody-mediated inhibition of IL-13 attenuated collagen deposition in mice with bleomycininduced pulmonary fibrosis (Belperio . Although the induction of Il-13 was not detected in our study, we did find the induction of IL-13 receptor genes such as Il-13ra1 and Il-13ra2. IL-13Rα2 is an inhibitory decoy receptor of IL-13 and thereby attenuates the progression of fibrosis; it has been considered to serve mainly as a negative-feedback mechanism for the TH2 response (Wynn, 2004). However, a recent study has demonstrated that IL-13 signaling through IL-13Rα2 is involved in the induction of TGF-β1 production and collagen deposition (Fichtner-Feigl . Moreover, increases in IL-13Rα1 and IL-13Rα2 in cultured fibroblast cells from an idiopathic interstitial pneumonia patient have been reported (Jakubzick .

In addition to the induction of genes related to TH2 cytokines, Tgfb1 and Tgfb2 were significantly up-regulated on days 7 and 14, respectively. TGF-β is a major regulator of the ECM, and the induction of TGF-β is tightly associated with the development of fibrosis (Kaminski ; Sime . Of the three TGF-β isoforms, TGF-β1 is the main ECM regulator and is stored in the cell in an inactive form, bound by latency-associated protein (LAP) . For TGF-β1 activation, the LAP-TGF-β1 complex must be cleaved through a process catalyzed by integrin-αvβ6 or proteolytic enzymes such as cathepsins or matrix metalloproteinases (MMPs) (Munger ; Gorelik and Flavell, 2002). In the present study, two genes linked with the dissociation of the LAP-TGF-β1 complex, i.e., Mmp2 and integrin-αv (Itgav) , were up-regulated in the lungs of SD rats after bleomycin treatment. Activated TGF-β modulates fibrogenic effects through SMAD protein, which triggers the transcription of target genes such as Col1a1, Col1a3, and connective tissue growth factor (Ctgf) (Roberts ; Perbal, 2004). Although an elevated SMAD expression level was not detected, the induction of Ctgf, Col1a1a, and Col1a3, which are downstream of the TGF-β1-SMAD3 signaling pathway, were observed. Ctgf is an important fibrogenic modulator and regulates fibroblast proliferation, cellular adhesion, chemotaxis, and ECM deposition (Perbal, 2004). On the other hand, it has been reported that TGF-β1 stimulates pulmonary fibrosis via Bcl2-associated X protein (Bax) , BH3 interacting domain death agonist (Bid) , and MMP12 (Kang . TGF-β1 is an in vivo stimulator of Bax, Bid, Timp-1, and Mmp-12, and the Bid- and Bax-mediated apoptosis pathways are important in the pathogenesis of pulmonary inflammation and fibrosis. In addition, Mmp12, which is downstream of TGF-β1 via the Bid- and Baxmediated pathways, is a main contributor to the pathogenesis of TGF-β1- and bleomycin-induced pulmonary fibrosis (Kang . Elevated expression levels of Bid, Bax, and Timp-1 were observed on day 7 in our study, and significantly increased Mmp12 expression levels were maintained up to day 28 after bleomycin treatment.

The CC-chemokine family is known to be strongly involved in the development of fibrosis via cooperation with profibrotic cytokines such as IL-13 and TGF-β (Ma ; Murray . In our study, several CC-chemokine genes, including Ccl2, Ccl3, Ccl17β and Ccl22, and CC-chemokine receptor genes, including Ccr1 and Ccr2, were significantly up-regulated. In particular, CCL2 and CCL3 have been identified as essential mediators of fibrosis, and several CCL2/CCL3 inhibition studies have shown marked reduction of fibrosis in murine models (Smith ; Lloyd . Furthermore, findings from studies using CCR1- and CCR2-deficient mice have confirmed the importance of the CCL2/CCL3 signaling pathway in fibrosis development (Tokuda ; Moore .

Taken together with the induction of well-known genes associated with pulmonary fibrogenesis, our data show that several genes such as triggering receptor expressed on myeloid cells 2 (Trem2) , Mmp7, Mmp12, cathepsin K (Ctsk) , and secreted phosphoprotein 1 (Spp1) are significantly increased more than 6-fold and that these expression levels persist up to 28 days after bleomycin treatment. Among the selected genes, Trem2 showed the highest induction on days 7 and 14 after bleomycin treatment. The molecular function of Trem2 is not well documented; however, a previous study reported high and persistent induction of Trem2 expression in stainless steel welding hume-induced lung injury in SD rats (Oh . Spp1 is a key molecule in the development of angiotensin II-induced cardiac fibrosis and remodeling (Matsui . Recent studies have shown that Spp1 has a pivotal role in smoking-related interstitial lung diseases, which are characterized by the accumulation of macrophages and Langerhans cells, and in fibrotic remodeling (Prasse . The overexpression of Ctsk in silica-induced pulmonary fibrosis in mice has also been reported (van den Brule . In addition, gene expression levels of some proteases such as tryptase alpha/beta 1 (Tpsab1) , mast cell protease 1 (Mcpt1) , and chymase 1 mast cell (Cma1) were markedly increased on day 14 after bleomycin treatment. Although these proteases have not been reported to be associated with pulmonary fibrosis, the remarkable induction of these genes with to the emergence of fibroblasts suggests that they may contribute to the development of fibrosis via degradation of the ECM.

In summary, we have investigated changes in gene expression associated with the development of bleomycin-induced pulmonary fibrosis in SD rats. The genes found to be significantly modulated during the development of pulmonary fibrosis correlated well with the histopathological changes and previously published findings. Our data will aid in an understanding of the underlying mechanisms involved in the development of fibrosis and will contribute to efforts aimed at identifying candidate biomarkers for fibrotic disease.