Role of insulin like growth factor axis in the bleomycin induced lung injury in rats

Experimental and Molecular Pathology 102 (2017) 86–96

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Role of insulin like growth factor axis in the bleomycin induced lung injury in rats Lakshmi Kanth Kotarkonda a,b, Ritu Kulshrestha b,⁎, Krishnan Ravi a a b

Department of Physiology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi 110007, India Department of Pathology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi 110007, India

a r t i c l e

i n f o

Article history: Received 18 October 2016 and in revised form 21 December 2016 Accepted 4 January 2017 Available online 07 January 2017 Keywords: Alveolar epithelial cells Bleomycin Epithelial mesenchymal transition Insulin like growth factor Lung fibrosis Pioglitazone

a b s t r a c t Background: Alveolar epithelial cell injury has been proposed as a causative factor for the onset and progression of pulmonary fibrosis. However, the role of type II alveolar epithelial cells (AECs) in the epithelial mesenchymal transition (EMT) is controversial. Aims: The present study performed in rats instilled with bleomycin investigated a) the expressions of the insulin growth factor (IGF-1) and insulin growth factor binding protein 5 (IGFBP-5) and transforming growth factor (TGF-β1) in the type II AECs, b) the role of type II AECs in EMT and extracellular matrix (ECM) formation and, c) the effect of pioglitazone on all the above parameters. Methods: Male Wistar rats were divided into three Groups: Group I (saline control), Group II (Bleomycin, given as a single intratracheal instillation, 7 U/kg) and Group III (Bleomycin + Pioglitazone (40 mg/kg/day orally, starting 7 days post bleomycin instilled as in Group II). From lung tissues, the protein expressions of IGF-1, IGFBP-5, TGFβ1, surfactant protein C (SP-C, as a marker for type II AECs) and α-smooth muscle actin (α-SMA, as a marker for EMT), were determined on day 7 in Groups I and II and on days 14, 21 and 35 in all the three groups. Results: IGFBP-5 and IGF-1 expressions were reduced significantly and TGF-β1 expression increased significantly in type II AECs in Group II from day 7 till day 35 as compared to Group I. An increase in SP-C and α-SMA expression and their co-localization were seen in the type II AECs undergoing EMT from day 7 till day 35. A concomitant remodeling and laying down of ECM was observed also. In Group III, with pioglitazone, there was a reversal with significant up-regulation in IGFBP-5 and IGF-1 expressions and down-regulation of TGF-β1 in the type II AECs along with a significant decrease in the solid area fraction, EMT and ECM in the lung tissue. Conclusions: IGFBP-5, IGF-1 and TGF-β1 in the type II AECs play a key role in lung injury caused by bleomycin and pioglitazone attenuates the lung injury/fibrosis by restoring IGFBP-5 and IGF-1 and decreasing TGF-β1 expressions in the type II AECs. © 2017 Published by Elsevier Inc.

1. Introduction Pulmonary fibrosis is a progressive, degenerative complex lung disease (Oruqaj et al., 2015) the exact pathophysiological mechanisms for the development of which are not fully understood. There is a general consensus that this disease is due to the combination of alveolar epithelial cell injury which is followed by the release of proinflammatory/ fibrotic cytokines, inflammatory cell infiltration, epithelial mesenchymal transition (EMT) and excessive deposition of extracellular matrix in the lung (Serrano-Mollar, 2012). Presently in idiopathic pulmonary fibrosis (IPF), combination therapies comprising of immunosuppressive agents, anti-oxidants and anti-inflammatory agents are often prescribed

⁎ Corresponding author. E-mail address: [email protected] (R. Kulshrestha).

http://dx.doi.org/10.1016/j.yexmp.2017.01.004 0014-4800/© 2017 Published by Elsevier Inc.

to the patients and have also been reported to be partially successful (Rafii et al., 2013). One possibility for the decrease in efficacy of these drugs whether alone or in combination may be because the treatment is started at a later stage when pulmonary fibrosis has already set in. An effective line of treatment may be targeting the primary factor namely the alveolar epithelial cells (AECs) which undergo apoptosis, alterations in the cytokines and the growth factors in the AECs and the interlink between them and the fibroblasts which ultimately result in pulmonary fibrosis (Sakai and Tager, 2013). In fact, it has been proposed that the aim of future therapy should be to increase the alveolar epithelial regeneration (Rafii et al., 2013) as well as to reverse the process of EMT (Kagalwalla et al., 2012). The present study makes an attempt to investigate this proposal in the bleomycin induced experimental model of lung injury in rats and following up the lung pathology in a sequential manner. Growth factors such as transforming growth factor beta1 (TGF-β1), connective tissue growth factor (CTGF), fibroblast growth factor (FGF),

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insulin-like growth factor-1 (IGF-1) and platelet derived growth factor (PDGF) have been shown to regulate the growth and differentiation of the AECs (Allen and Spiteri, 2002). There is increasing evidence that the IGF axis may play a significant role in pathogenesis of pulmonary fibrosis (Hung et al., 2013; Ahasic et al., 2012; Yasuoka et al., 2006; Krein and Winston, 2002; Aston et al., 1995). In patients with IPF, IGF-1 expression has been shown to be increased in the bronchoalveolar lavage (BAL) fluid (Frankel et al., 2005) suggesting that an imbalance in the IGF axis may play a pivotal role in the progress of this disease. An increase in the IGF-1 has been reported in the BAL cells after bleomycin induced lung injury in the murine model also (Maeda et al., 1996). Such an increase in IGF-1 in the BAL fluid as well as in BAL cells is understandable as IGF-1 has been shown to be increased in the EMT cells, macrophages and myofibroblasts (Hung et al., 2013) and is generally considered to be profibroblastic (Hung et al., 2013). When attempts have been made to determine IGF-1 in AECs from IPF patients, it has been observed that as seen in BAL fluid and BAL cells, there is an increase (Maeda et al., 1996) in IGF-1 in the AECs in the early stage IPF when there is diffuse alveolar damage. However, a decrease in IGF-1 occurs in them in the later stage when there is interstitial fibrosis and honeycombing (Homma et al., 1995). Another study while confirming the increase has not found a decrease in IGF-1 staining of AECs in IPF patients with extensive collagen deposition and honeycombing (Maeda et al., 1996). Thus, even though the function of IGF-1 in the interstitial cells has been defined, there is no definite information on the role of IGF-1 in AECs in pulmonary fibrosis. Insulin-like growth factor binding proteins (IGFBPs 1–10) are a family of proteins that bind IGFs with high affinity. The IGFBPs vary in their tissue expression and also in their response and regulation by other growth factors. The IGFBP-5 is the most conserved of all the IGFBPs. The IGFBPs have been suggested to be involved in the initiation and/or perpetuation of fibrosis by inducing the production of ECM components such as collagen type I and fibronectin (Pilewski et al., 2005). The IGFBPs bind and regulate the access of IGF to its receptor and thereby regulate the biological activity of IGF-1 on target cells. Depending on content, IGFBPs can potentiate or inhibit the biological effects of IGF-1 in a given tissue (Pilewski et al., 2005). There has been no investigation on the expression of IGFBP-5 in AECs and their interaction with IGF-1 expressed in AECs either in samples from IPF patients or in animal models administered with bleomycin. Based upon these reports, for the present study, it has been hypothesized that in the rat, during bleomycin induced lung injury, there will be a decrease in the expression of IGF-1 in the type II AECs. This decrease predisposes to the injury of the type II AECs as IGF-1 is responsible not only for cell proliferation and tissue differentiation but also for protection from apoptosis (Laviola et al., 2007). Reduced IGF-1 expression will hamper the differentiation of type II AECs into type I cells and prevent parenchymal repair (Ghosh et al., 2013; Narasaraju et al., 2006). It has been hypothesized further that along with IGF-1, there will be a decrease in the expression of IGFBP-5 in the type II AECs. Since IGFBP-5, by increasing the epithelial production of the basement membrane protein, laminin, protects the epithelium from injury, its decrease will promote pulmonary fibrosis (Sureshbabu et al., 2011). Additionally, its decrease will make the pulmonary fibrosis worse by causing apoptosis of AECs by reducing the bio-availability of IGF-1. To prove the hypothesis, after intratracheal instillation of bleomycin, lung sections have been examined at various time intervals. In them, the IGF axis has been assessed by determining the expressions of IGF-1 and IGFBP-5 in the type II AECs (SP-C is used as a marker for type II AECs), EMT has been studied by α-SMA expression and the ECM is determined by measuring the solid area fraction as well as by Masson's trichrome staining for collagen. Besides IGF axis, TGF-β1 expression has been investigated in the type II AECs. Subsequently, to assess whether there is an attenuation of the observed changes by a drug, pioglitazone has been administered. Thiazolidinediones (TZDs) like pioglitazone are the synthesized ligands for the peroxisome proliferator-activated receptorγ

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(PPARγ) and in vascular smooth muscle cells, PPARγ agonists have been reported to up-regulate IGF-1 and its receptor (Higashi et al., 2010). Its role in up-regulating the IGF-1 in the type II AECs is yet to be investigated.

2. Materials and methods 2.1. Chemicals and reagents Bleomycin sulphate was purchased as Bleocip from Cipla Limited, Mumbai, India, and Pioglitazone tablets (Pioglar) were purchased from Ranbaxy laboratories Limited, New Delhi, India. The rat antibodies of Surfactant Protein C (SC-13979), IGFBP-5 (SC-13093), IGF-1 (SC-9013) were purchased from Santa Cruz Biotechnology, California, U.S.A. Antibodies of TGF-β1 (SAB4502954) and α-SMA (A5228), and the secondary antibody kits (Extra-2, 3 kit), for immunohistochemistry were purchased from Sigma Chemicals, Missouri, U.S.A. Secondary antibodies for immunofluorescence, goat anti rabbit Alexa Fluor 488 (A11008) and goat anti mouse Alexa Fluor 555 (A21422) were purchased from Molecular probes, Invitrogen, Carlsbad, U.S.A. Hoechst 33,258 dye (TC225) was purchased from Himedia laboratories, Mumbai, India. All other chemicals used were of the highest commercial grade available.

2.2. Animals Seven weeks old male Wistar rats were obtained from Vallabhbhai Patel Chest Institute animal house. The animals were quarantined for minimum of 1 week prior to use. Rats weighing approximately 260 ± 20 g were used in this study. Proper precautionary measures were followed to minimize any suffering during the animal experimentation. Animal experiments were performed with the regulations specified by the Institute's Animal Ethical Committee and conform to the National guidelines on the care and use of laboratory animals, India.

2.3. Rat model for lung fibrosis The animals were randomly divided into three Groups with six rats in each group: Group I: Saline control, Group II: Bleomycin and Group III: (Bleomycin + Pioglitazone). In Group II, induction of lung injury was done by a single intratracheal instillation of bleomycin sulphate (7 U/kg) in a volume of 100 μl saline (0.9%). Group I rats received a single dose of intratracheal saline (100 μl of 0.9% saline). In Group III, the pioglitazone drug treatment was given orally starting from 7 days after instillation of bleomycin and administered daily till the completion of the study. For intratracheal instillation, rats were anesthetized with ketamine hydrochloride (50 mg/kg, intra muscular). After inducing anesthesia, the skin overlying the trachea was cleaned with ethanol, and a small incision was made in the skin under local anesthesia (1% xylocaine). The trachea was exposed and using a 1 ml sterile syringe and 24 gauge needle, bleomycin was instilled. An equivalent volume of sterile saline was instilled intratracheally for the saline control Group. For better dispersion of the instillation into the lung, the animal's head was kept at an angle of 30 °C from the dissection table. The wound was closed with proper suturing of the skin and muscles. Betadine was applied to the wound until it healed. Pioglitazone was given orally as gavage in the conscious rat. For this purpose, the pioglar tablets were powdered and dissolved in distilled water and then administered daily for a duration of 28 days at a dose of 40 mg/kg/day. Histopathology, morphometric changes, immunohistochemistry, immunofluorescence, Masson's trichrome were performed and assessed with image analysis software NIS-Ar elements in all Groups on day 7 in Group I and Group II and on days 14, 21 and 35 in Groups I, II and III.

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2.4. Assessment of histopathology by morphometry Lung tissues from the euthanized rats were fixed in neutral buffered 10% formalin solution and embedded in paraffin. Paraffin sections of 4 μm thickness of lung tissues were stained with Hematoxylin and Eosin (H&E) and Masson's trichrome stain for the evaluation of degree of lung fibrosis. The parenchymal areas were measured in 10 non-coinciding fields of having maximum fibrotic areas at 10 × magnification. Subsequently, quantification of the selected region of interest (ROI) was done by Nikon Eclipse 90i microscope with the exclusion of bronchioles and blood vessels. Then, the solid area fraction, was calculated as a ratio of total area of the ROI and the parenchymal area and was obtained in percentage.

preparations were quantified based on the protocol described below. The diaminobenzidene (DAB)-labelled images with hematoxylin were opened in image J software, and by colour deconvolution option (Nguyen, 2013; Ruifrok and Johnston, 2001), Hematoxylin DAB images were selected for quantification. Individually cells that were positive for chromogens were marked and their intensity was obtained using analyse option and then measure option in the software. The results of the mean intensity were plotted in the graph. As darker areas have lower intensity values, it results in an inverse correlation between the amount of antigen and its numerical value. Since standard red-greenblue (RGB) colour images acquired from bright field microscopy have a maximum intensity of 250, the intensity of positively stained cells was subtracted from 250 to derive a reciprocal intensity which is directly proportional to the chromogen intensity (Nguyen, 2013).

2.5. Immunohistochemistry (IHC) The paraffin-embedded sections were deparaffinized with xylene and rehydrated in graded ethanol and in water. Quenching of endogeneous peroxidase was done with 3% H2O2 for 10 min at room temperature (RT) followed by antigen retrieval by boiling tissue sections in 10 mM citrate buffer (pH 6.0) for 15 min in the microwave at 700 watts. After cooling to RT, sections were incubated with 4% bovine serum albumin in PBS, for 40 min at RT, to block non-specific binding of immunoglobulins. After washing with PBS, sections were then incubated with primary antibodies against SP-C, α-SMA, TGF-β1, IGFBP-5 and IGF-1. Sections were incubated without the primary antibody and they served as negative controls. The control and treated sections were then incubated in the humidity chamber overnight at 4 °C. After washing 3 times with PBS (pH 7.4), the sections were incubated with secondary antibody for 1 h at RT as per kit instructions (Extra3, which included biotinylated monoclonal anti rabbit, clone RG-16, extravidin peroxidase and Extra2 kit, which included biotinylated goat antimouse, extravidin peroxidase) for the chromogen stain. After 3 washes with PBS, the sections were treated with extravidin peroxidase for 30 min at RT. After wash in PBS, the sections were visualized with Nova red substrate (NovaRED Substrate Kit, SK4800, Vector labs, USA) which was used as the colour reagent, and Mayer's hematoxylin which was used as the counter stain. Finally, stained slides were dehydrated and mounted with water-soluble mounting DePeX and the mounted slide preparations were examined with light microscope (Nikon 90i eclipse, Japan) and images were captured with NIS-Ar elements 3.0 software.

2.8. Collagen analysis by Masson's trichrome staining Masson's trichrome staining of lung sections was performed to determine interstitial collagen secreted by active fibroblasts. First, the lung sections were brought to distilled water, and then incubated in Bouin's fixative for overnight at 60 °C. Subsequently, the sections were stained in Weigert's hematoxylin for 10 min and then washed in running water for 10 min. Thereafter, sections were stained in Biebrich scarlet-acid fuchsin solution for 15 min and washed in distilled water and then differentiated in phosphomolybdic-phosphotungstic acid solution for 10–15 min until the collagen turned pale blue. Sections were counterstained with aniline blue for 5 min, differentiated in 1% acetic water for 4 min and dehydrated, cleared and mounted with DePeX. 2.9. Statistics Statistical analysis was performed using Graph pad prism (version 5.0). The results were expressed as means ± S.E.M. (standard error of mean). For morphometric analysis, comparison between Groups I and II on days 7, 14, 21 and 35 and comparisons among Groups I, II and III on days 14, 21 and 35 were done by one way ANOVA followed by Dunnett's test. Protein expressions by IHC were compared similarly using one way ANOVA followed by Tukey's multiple comparison tests between and among these groups. A probability level of P-value ≤0.05 was considered significant.

2.6. Immunofluorescence (IF)

3. Results

Paraffin-embedded sections were deparaffinized with xylene and rehydrated in graded ethanol and in water. Antigen retrieval was done by boiling the slides in 10 mM citrate (pH 6.0) for 15 min in the microwave at 700 watts, and then cooled to RT. Negative Controls sections were incubated without the primary antibody. Control and treated lung tissue sections with primary antibodies α-SMA and SP-C, were incubated in the humidity chamber overnight at 4 °C. The next day, after washing with PBS, the sections were incubated with fluorochrome-conjugated secondary antibodies (Alexa Fluor 488, Alexa Fluor 555, diluted in 1% PBSA). Negative controls were processed in parallel by addition of PBS buffer instead of the primary antibodies. The slides were then incubated with Hoechst, a nuclear dye, for 10 min at RT and after washing with PBS, fluorescent labelled slides were mounted with fluoromount aqueous medium (S3023, Dako, Denmark).

3.1. Bleomycin induced lung injury

2.7. IHC quantification The stained IHC images of random and non-contiguous fields from the parenchymal regions were acquired with a Nikon 90i microscope. Ten fields per lung section (40× magnifications) were obtained. Fields that contained a large airway or blood vessel were rejected. IHC slide

In Group I, the lung architecture was well preserved. The alveolar walls were intact with minimal inflammatory cells in the interstitium (Fig. 1A). In contrast, bleomycin instillation resulted in an ascending grade of fibrosis from day 7 to day 35 in Group II (Fig. 1B). In Group I, since there was no significant change in the solid area fraction on all the four time points, it was averaged and was 28.9 ± 0.4%. In Group II, it increased to 47.4 ± 0.8, 53.2 ± 0.7, 59.3 ± 1.2 and 57.7 ± 1.2% on days 7, 14, 21 and 35 respectively (Fig. 1B). This increase was statistically significant. On day 7, there was infiltration of inflammatory cells such as lymphocytes and macrophages (as evidenced by morphology) in the pulmonary interstitium. This inflammation persisted and continued till day 14 in the cellular phase. In addition to inflammatory cells, from among the cells undergoing transition to EMT/myofibroblasts, cells having spindle shaped morphology were seen on this day which continued till day 35 (Fig. 2B). Their lineage as type II AECs was confirmed by studying the SP-C expression and their partial participation in EMT by α-SMA expression (Fig. 2 A and B) individually and collectively by colocalizing SP-C and α-SMA in the type II AECs (Fig. 2 A and B). A

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Fig. 1. Histopathology (A) and morphometry (B and C) of the lung in Group I (saline), Group II (bleomycin) and Group III (bleomycin + pioglitazone). Compared to Group I, note the progressive remodeling of alveolar architecture in A and increase in solid area shown in A and B in Group II. Note also the resolution of architectural remodeling and decrease in solid area fraction with pioglitazone treatment in Group III starting from day 14 shown in A and in C. All the images were taken at 10× magnification and the scale bar is 50 μm. The solid area fraction of Group I in B, represents the mean of the values on days 7, 14, 21 and 35 after saline instillation. In B, ***p b 0.0001, significantly different from Group I values. Within Group II, ≠≠≠p b 0.0001, significantly different from day 7 value, ×××p b 0.0001, ××p b 0.001 significantly different from day 14 value. In C, ***p b 0.0001, significantly different from corresponding Group I and ≠≠≠p b 0.0001, significantly different from corresponding Group II values.

progressive increase in the thickness of the alveolar septae and distortion of alveolar architecture was seen from day 7 till day 35. During the first week following a single bleomycin instillation, the response was primarily characterized by lung inflammation. Inflammatory cells in the alveolar interstitium and alveolar spaces were consistently observed along with a few fibroblasts (Fig. 1A). During the following two weeks, alveolar and interstitial fibrosis became progressively more prominent with partial to complete effacement of the alveoli (Fig. 1A). By five weeks post-bleomycin instillation, the changes were more heterogeneous with fibrosis interspersed with small, partially collapsed alveoli and alveoli lined by bronchoalveolar epithelial cells (Fig. 1A).

3.2. Effect of pioglitazone on bleomycin induced lung injury In Group III, with pioglitazone treatment after bleomycin instillation, there was a substantial improvement in the lung architecture. The alveoli were well defined with clear cut boundaries and the thickness of the alveolar septae was reduced. The parenchymal remodeling started becoming evident on day 14 (which is 7 days of pioglitazone therapy) and it continued on days 21 and 35 (Fig. 1 A and C). The corresponding solid area fraction in this Group was 44.7 ± 1.1, 44.9 ± 0.8 and 39.3 ± 1.2% respectively (Fig. 1C). Additionally, the excessive collagen deposition observed in Group II was found to be decreasing progressively in this group starting from day 7 and continuing till day 35. An example is shown in Fig. 3.

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Fig. 2. Immunofluorescence for SP-C and α-SMA in type II alveolar epithelial cells (AECs) in Group I (saline), Group II (bleomycin) and Group III (bleomycin + pioglitazone) showing their participation in epithelial mesenchymal transition (EMT). In A, note the localization of SP-C (green) in the first column in all the three groups. Note the increasing appearance of α-SMA (red) in the second column and the co-localization of SP-C and α-SMA in the fourth column in the type II AECs in Group II suggesting the participation of type II AECs in EMT. Note the progressive decrease in the α-SMA in the second column in Group III and the progressive increase in SP-C in the fourth column. The third column (Hoechst) in all the three groups shows the nuclear staining of cells. All the images were taken at 60 × magnification and the scale bar is 10 μm. B. A representative example for the change in cell morphology in Group II (bleomycin) and its resolution in Group III (bleomycin + pioglitazone) on day 35. Closed arrows show the normal type II AECs in Group I, spindle shaped cells in Group II and again their resolution to normal type II AECs in Group III. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 3. Masson's trichrome staining for collagen in Group I (saline), Group II (bleomycin) and Group III (bleomycin + pioglitazone). Compared to Group I, note the progressive collagen deposition in lung interstitium in Group II and its resolution in Group III. All the images were taken at 20× magnification and the scale bar is 50 μm.

3.3. Bleomycin induced change in TGF-β1 with and without treatment with pioglitazone In Group I, there was weak or negligible expression of TGF-β1 in the type II AECs as well as in the other interstitial cells (Fig. 4).

With bleomycin induced injury, in Group II, an increased expression of TGF-β1 was noted in the type II AECs, EMT cells, alveolar interstitial macrophages and interstitial fibroblast/myofibroblasts. This process which started on day 7 became more marked on succeeding days and persisted on day 35 (Fig. 4).

Fig. 4. Immunohistochemistry for TGF-β1 in Group I (saline), Group II (bleomycin) and Group III (bleomycin + pioglitazone). Compared to Group I, note the increased expression of TGFβ1 in the type II alveolar epithelial cells (AECs) in Group II. Note also the comparative decrease in TGF-β1 in type II AECs in Group III with pioglitazone treatment. Closed arrows in Groups I, II and III show TGF- β1 expressions in a few AECs. All the images were taken at 40× magnification and the scale bar is 50 μm.

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With pioglitazone treatment, in Group III, there was an attenuation of TGF-β1 expression in both type II AECs and the interstitial cells. A decrease was noted on day 14 itself which became more prominent on day 35 (Fig. 4). 3.4. Bleomycin induced change in EMT with and without treatment with pioglitazone In Group I, there was weak or negligible expression of α-SMA and a strong expression of SP-C in the type II AECs. The type II AECs were confined to the alveolar lining. With bleomycin induced injury, in Group II, along with SP-C, there was an increased expression of α-SMA in the type II AECs. The cells showing positivity for α-SMA and SP-C were prominent in the interstitium suggesting EMT. Additionally, some of the type II AECs showed morphological changes exhibiting spindle shape with tapered ends. An example is shown in Fig. 2B. This process started on day 7, became more marked on day 21 and persisted till day 35 (Fig. 2A). Further, immunofluorescence staining revealed that both SP-C and α-SMA were co-localized in some of these cells (Fig. 2A). With pioglitazone, there was a reversal in EMT. In Group III, there was a decrease in the number of cells showing positivity for α-SMA, an increase in the expression of SP-C and the return of the cell morphology to normal type II AECs (Fig. 2B). 3.5. Bleomycin induced change in IGFBP-5 with and without treatment with pioglitazone A strong expression of IGFBP-5 was observed in type II AECs in Group I (Fig. 5A) where as in Group II, with bleomycin alone, there was a significant reduction in the intensity of IGFBP-5 expression in the type II AECs which was evident on day 7, 14 and 21 and was less marked on day 35 (Fig. 5A). In Group I, the IGFBP-5 expression was 122.5 ± 2.7, 123.7 ± 3.0, 122.7 ± 5.9 and 121.4 ± 4.8 arbitrary units on days 7, 14, 21 and 35 respectively and the average was 122.5 ± 2.7 arbitrary units. The corresponding values in Group II were 95.92 ± 2.7, 96.35 ± 6.4, 102.3 ± 1.9 and 112.9 ± 2.3 arbitrary units respectively (Fig. 5B). In Group II, an increased expression of IGFBP-5 was noted in the other interstitial cells - the EMT cells, alveolar interstitial macrophages and interstitial fibroblast/myofibroblasts (Fig. 5A) at all the time intervals. Comparison of IGFBP-5 specifically in the type II AECs between Group I and Group II revealed that in Group II, the IGFBP-5 expression decreased significantly on day 7, 14, 21. Thereafter, there was an increase and it returned towards that in Group I (Fig. 5B). With pioglitazone treatment, in Group III, there was an improvement in the IGFBP-5 expression in the type II AECs. In Group III, the IGFBP-5 expression was 140.5 ± 3.6, 132.6 ± 1.9, 138.5 ± 2.6 arbitrary units on days 7, 14, 21 and 35 respectively and it returned towards that in Group I (Fig. 5 A and C). 3.6. Bleomycin induced change in IGF-1 with and without treatment with pioglitazone In Group I, these was a strong expression of IGF-1 in type II AECs (Fig. 6A), while it was minimal in other interstitial cells. In contrast, in Group II, with bleomycin alone, there was a reduction in the intensity of IGF-1 expression in the type II AECs which started becoming evident on day 7 and continued till day 35 (Fig. 6A). Simultaneously an increased expression of IGF-1 was noted in the other interstitial cells - the EMT cells, alveolar interstitial macrophages and interstitial fibroblast/ myofibroblasts (Fig. 6A) at all the time intervals. Quantification of IGF-1 specifically in the type II AECs revealed that compared to Group I, the IGF-1 expression decreased significantly on days 7, 14, 21 and 35 in Group II and the decrease was maximum on day 7 (Fig. 6B). In Group I, the IGF-1 expression was 160.6 ± 2.9, 161.6 ± 3.6, 161.1 ± 4.9, and 159 ± 5.4, arbitrary units on days 7, 14,

21 and 35 respectively and the average was 160.6 ± 2.8 arbitrary units. The corresponding values in Group II were 87.22 ± 3.1, 112.9 ± 5.7, 121.1 ± 6.1 and 128.5 ± 4.2 arbitrary units respectively (Fig. 6B). With pioglitazone treatment, starting from day 14, there was a significant increase in IGF-1 expression which returned towards that in the saline group on days 21 and 35 (Fig. 6C). In Group III, the IGF-1 expression was 138.6 ± 1.7, 161.1 ± 2.3, 147.4 ± 1.0 arbitrary units on days 14, 21 and 35 respectively. 4. Discussion The results of the present study demonstrate that after bleomycin instillation, there is a significant down-regulation of IGF-1 and its binding protein IGFBP-5 in the type II AECs. Simultaneously, there is a significant up-regulation of TGF-β1 in type II AECs resulting in their injury, transition to mesenchymal cells and deposition of extra-cellular matrix. Treatment with pioglitazone not only up-regulates the IGF-1 and IGFBP5 expressions in type II AECs, but also reduces the TGF-β1, EMT and extracellular matrix deposition. Several attempts had been made previously to look at the histopathological changes in the lung following bleomycin instillation (Mouratis and Aidinis, 2011; Walters and Kleeberger, 2008). In them, bleomycin was administered intravenously, intraperitoneally and intratracheally. Mostly, the responses were similar (Lindenschmidt et al., 1986). While in some studies, lung specimens were taken for histological examination at fixed time intervals (Izbicki et al., 2002), in others, they were examined in a sequential manner. In the latter ones, the changes that occurred were categorized into an early cellular phase where there was infiltration of macrophages, neutrophils and lymphocytes into the interstitium, an intermediate phase in which there was inflammation and continued destruction of epithelial cells and a late fibrotic phase when there was excess extracellular matrix formation, failure of tissue repair and fibrosis (till day 35) (Peng et al., 2013). The present results are in agreement with these findings. Additionally, it has been observed that as the days progress, there is a progressive increase in the solid area fraction which gets stabilized on day 21 and persists till day 35. Along with this increase, a parallel increase in collagen deposition has been observed starting from day 7 and progressing till day 35. The results support the finding that bleomycin induced lung injury results in changes in lung mechanics reducing lung tissue elastance and increasing work of breathing (Peng et al., 2013). In fact, a significant correlation has been observed between the collagen formation and the changes in lung mechanics following bleomycin administration (Peng et al., 2013). 4.1. Evidence for type II AECs as the source for the EMT and collagen formation Until a few years ago, there was a general perception that the alveolar epithelium played a passive role in pulmonary fibrosis (Willis et al., 2006). This notion has now been revised and it has been proposed that the continued destruction of the type II AECs and compromised tissue repair contribute significantly to the development of pulmonary fibrosis (Zoz et al., 2011). The injured type II AECs may take part in the reepithelialization or contribute to EMT (Alipio et al., 2011). Evidence in support of the type II AECs participating partially in EMT has been provided in the present study. It has been shown that in the rat, following bleomycin instillation, there is injury to type II AECs, and starting from day 7, there is transformation of type II AECs into mesenchymal cells. That the type II AECs are involved in the EMT process has been demonstrated by the co-presence of SP-C and α-SMA in the same cells. The results are in agreement with the in vitro studies using cell culture (Yamauchi et al., 2010; Tan et al., 2010) and in vivo studies from lung specimens from patients with idiopathic pulmonary fibrosis (Willis et al., 2006).

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Fig. 5. Immunohistochemistry for insulin like growth factor binding protein 5 (IGFBP-5) in Group I (saline), Group II (bleomycin) and Group III (bleomycin + pioglitazone). In A, note the strong expression of IGFBP-5 in the type II alveolar cells (AECs) in Group I (Large inset of the small inset shows dark staining of three cells) and its weak expression in them in Group II shown in B on days 7, 14 and 21 and is return towards that in Group I on day 35. In Group III, as shown in A and C, there is an improvement in the IGFBP-5 expression in the type II AECs starting from day 14 and returning to that in Group I on days 21 and 35. Closed arrows in Group II show weak expression in type II AECs and open arrows show strong expression in the EMT cells in the interstitium. In B, in Group II, ***p b 0.0001, significantly different from Group I value. The Group I data represents the mean of the reciprocal intensity of 7, 14, 21 and 35 days after saline instillation. Within Group II, ≠≠p b 0.001, significantly different from day 7 value and ≠p b 0.01, significantly different from day 14 value. In C, **p b 0.001, ***p b 0.0001, significantly different from corresponding Group I values. ≠≠≠p b 0.0001, significantly different from corresponding Group II values. All the images were taken at 40× magnification and the scale bar is 10 μm.

4.2. TGF-β1 in EMT in lung fibrosis Among the various cytokines, TGF-β1 has been reported to induce EMT of type II AECs and pleural epithelial cells in lung fibrosis both in vivo and in vitro (Degryse et al., 2011; Kasai et al., 2005). During EMT, besides morphological alteration, there is increased expression of the fibroblast phenotypic markers Fn-EDA and vimentin in the epithelial cells (Kasai et al., 2005). Concomitantly, there is a down-regulation of the epithelial phenotype marker E-cadherin. This down-regulation of E-

cadherin has been proposed to result in the migration of type II AECs from the alveoli to the interstitium. The cells that have undergone EMT become principal foci for the development of fibroblasts and abnormal collagen production bringing out the changes that take place from the cellular phase to the fibrotic phase (Peng et al., 2013). In keeping with this proposed mechanism, in the present study also, following bleomycin instillation, TGF-β1 has been found to be up-regulated from day 7 and it persists till day 35. Even though no effort has been made to investigate the expressions of Fn-EDA, vimentin or E-cadherin,

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Fig. 6. Immunohistochemistry for insulin like growth factor 1 (IGF-1) in Group I (saline), Group II (bleomycin) and Group III (bleomycin + pioglitazone). Note the strong expression of IGF1 in A in the type II alveolar epithelial cells (AECs) in Group I. Note that compared to Group I, there is a significant decrease in IGF-1 in Group II starting from day 7 shown in A and B. Even though a small but significant increase is seen on day 21 and 35 when compared to day 7, it is significantly lower when compared to Group I (shown in B). Note also the reversal in IGF1 with pioglitazone treatment on days 21 and 35 in Group III (shown in C). Closed arrows in Group II show weak expression in type II alveolar epithelial cells and open arrows on days 14, 21 show strong expression in the EMT cells in the interstitium. The Group I data represents the mean of the reciprocal intensity of 7, 14, 21 and 35 days after saline instillation. In B, ***p b 0.0001 significantly different from Group I value. Within Group II, ≠≠≠p b 0.0001 and ≠≠p b 0.001, significantly different from day 7 value. In C, ***p b 0.0001, *p b 0.01 significantly different from Group I value. ≠≠≠p b 0.0001, significantly different from corresponding Group II day 21 value. ≠≠p b 0.001, significantly different from corresponding day 14 and 35 values of Group II. All the images were captured at 40× magnification and the scale bar is 50 μm.

nervertheless, a change in the shape of the cells from cuboidal to spindle shaped fibroblastoid morphology has been observed. 4.3. IGF-1 in lung fibrosis A number of growth factors has been implicated in pulmonary fibrosis. These include platelet derived growth factor, keratinocyte growth factor (KGF), connective tissue growth factor (CTGF), TGF-β1, IGF-1 etc.,. Most studies have reported that in pulmonary fibrosis, there is an up-regulation of the platelet derived growth factor, TGF-β1 (discussed already) and CTGF in the hyperplastic type II AECs (Allen and Spiteri, 2002) promoting fibroblast proliferation and extracellular matrix

deposition (Allen and Spiteri, 2002). Unlike the rest, KGF has been reported to be down-regulated in this disease. In fact, efforts are being made to increase KGF in order to alleviate pulmonary fibrosis (Sakamoto et al., 2011). Just as KGF, IGF-1 also appears to have a protective role in pulmonary fibrosis. For instance, IGF-1 binds to IGF-1 receptor and inhibits apoptosis via the phosphoinositide 3-kinase (PI3K) pathway (Párrizas et al., 1997). It up-regulates the mRNA and protein expression of anti-apoptotic members of the BCL2 family-BCL2 and BCL-X (L). Additionally, it inhibits the apoptosis protein, survivin (Hilmi et al., 2008; Kooijman, 2006). Further, it helps in their differentiation into alveolar type I cells through the activation of non-canonical Wnt pathway (Narasaraju et

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al., 2006) aiding the re-epithelialization process. Thus, it is tempting to postulate that in bleomycin induced lung injury, there is diminished IGF-1 in the type II AECs which activates a cascade of events resulting in pulmonary fibrosis. Evidence in support of this postulation has been provided in the present investigation. Following bleomycin instillation, a decrease in the IGF-1 expression has been observed in the type II AECs. Importantly, this decrease is evident from day 7 itself. The present findings appear to differ partly with a previous finding in which it has been reported that in biopsy samples obtained from patients with IPF, there is an increase in IGF-1 in these cells in the early stages. However, in the later stage, it has been reported to be decreasing (Homma et al., 1995). When splice variants of IGF-1 gene has been investigated, it has been shown that while there is down-regulation of IGF-IA mRNA, there is an increase in the expression of IGF-1B transcripts in the bronchoalveolar cells from IPF subjects (Bloor et al., 2001). It is quite possible that the decrease observed in the present study may be due to a decreased expression of IGF-1A and it is IGF-1A which may have the protective role. However, the present study has not been designed to address this issue as IGF-1 antibody used for immunohistochemistry detects both IGF-1A and IGF-1B in the rat. Nevertheless, the results indicate that the unhindered IGF-1B may promote the AEC hyperplasia and fibrogenic process as proposed by other investigators (Bloor et al., 2001) by its increased expression in the cells in the interstitium such as myofibroblasts and macrophages. 4.4. IGFBP-5 in lung fibrosis The IGF signalling axis requires the coordinated function of two ligands, IGF-1 and IGF-2, two cell surface receptors (IGF-1R, IGF-2R), ten high affinity binding proteins (IGFBP1–10) and binding protein proteases (Pollak, 2012; Mohan and Baylink, 2002). While there are studies investigating the role of IGF-1 in pulmonary fibrosis, not much information is available regarding the role of IGF-2. The IGFBPs regulate IGF-1 activity and its bio-availability. It is generally believed that these binding proteins inhibit the activity of IGF-1. However, it has been proposed that IGFBP-1, -3, and -5, may enhance IGF-1 activity also (2). Among these, IGFBP-5 by binding to its putative receptor has been proposed to amplify the IGF-1 signalling pathway (Mohan and Baylink, 2002). There is no direct evidence linking IGF-1 and IGFBP-5 in bleomycin induced lung injury. Just as IGF-1A and IGF-1B have dual functions, IGFBP-5 also has dual effects 1) a protective role in type II AECs by increasing their adherence to the basement membrane, as any loss to it leads to fibrosis (Strieter, 2008) and preventing apoptosis of fibroblasts (Sureshbabu et al., 2012) and, 2) a promoter role in the differentiation of fibroblast into myofibroblasts enhancing EMT and collagen formation (Sureshbabu et al., 2012). Thus, a decrease in IGF-1 in the broncholavage fluid on days 14–35 reported by other investigators may be due to the protective role of IGFBP-5 as mentioned above or there could be a decrease in IGFBP-5 itself which may decrease the bioavailability of IGF1 by decreasing their binding and promoting their degradation. The present results support the latter propositions. For instance, it has been observed in the present study that the IGFBP-5 parallels the changes in IGF-1 in the type II AECs both down-regulated as early as 7 days after bleomycin instillation and showing a tendency for an increase on day 35. With down-regulation in IGFBP-5 and IGF-1, the condition is favourable for apoptosis through the enhanced TGF-β1 release, and the subsequent EMT and fibroblast formation. The latter effects are compounded by the increased IGFBP-5 and IGF-1 in the other alveolar interstitial cells as described earlier. 4.5. Effect of pioglitazone treatment Thiazolidinediones (TZDs) such as pioglitazone are high-affinity ligands for peroxisome proliferator-activated receptor (PPAR) gamma and are used mainly as insulin-sensitizing drugs in type 2 diabetes mellitus. Of late, they are being investigated for their potential in

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alleviating cardiac and pulmonary fibrosis (Elrashidy et al., 2012; Aoki et al., 2009). In the present study, it has been observed that pioglitazone treatment for 7, 14 and 28 days produces a marked reversal in the lung fibrosis caused by bleomycin instillation. There is not only a significant decrease in the solid area fraction, EMT and collagen, but also an increase in the IGFBP-5 and IGF-1 in the type II AECs. A decrease in the number of cells showing IGFBP-5 and IGF-1 expressions in the pulmonary interstitium has been observed. Additionally, pioglitazone treatment produces a decrease in the TGF-β1 expression. Indeed, an attenuation of TGF-β signalling in lung epithelium has been reported recently to provide protection from bleomycin-induced fibrosis in the mice (Degryse et al., 2011). Thus, pioglitazone, by promoting IGFBP-5 and IGF-1 may protect the type II AECs from injury, promote their survival by decreasing TGF-β1, cause re-epithelialization and transformation of type II AECs to type I AECs. At the same time, it ameliorates pulmonary fibrosis by reducing EMT and collagen formation by antagonizing the actions of TGF-β1 and reducing the actions of profibrotic functions of IGFBP-5 and IGF-1 in the cells of the mesenchymal compartment. The present results are comparable with a previous study in rats where pioglitazone was administered seven days after bleomycin administration. It was reported that pioglitazone produced effects at the initial inflammatory phase as well as at the late fibrotic phase (Aoki et al., 2009). The responses were explained by the suppression of pro-inflammatory and fibrotic cytokines such as TNF-α in the alveolar macrophages (Aoki et al., 2009). Even though mRNA levels of the various cytokines have not been estimated in the present study, Northern blot analysis in MRC-5 cells (human lung fibroblasts) has revealed that pioglitazone inhibits TGF-β induced procollagen 1 and CTGF expressions which supports the above conclusions (Aoki et al., 2009). To the best of our knowledge, the present study is the first one which shows the up-regulation of IGFBP-5 and IGF-1 in type II AECs after pioglitazone and suggests that the IGF axis may be another pathway through which pioglitazone produces its therapeutic effect in pulmonary fibrosis. Conflict of interest There is no conflict of interest for the authors. Acknowledgement RK is grateful to Council of Scientific and Industrial Research (CSIR) (37 (1527)/12/EMR-II dated 02/04/2012), Government of India, for funding the project. LK is thankful for the senior research fellowship from this project. References Ahasic, A.M., Zhai, R., Su, L., Zhao, Y., Aronis, K.N., Thompson, B.T., et al., 2012. IGF1 and IGFBP3 in acute respiratory distress syndrome. Eur. J. Endocrinol. 166:121–129. http://dx.doi.org/10.1530/EJE-11-0778. Alipio, Z.A., Jones, N., Liao, W., Yang, J., Kulkarni, S., Sree Kumar, K., et al., 2011. Epithelial to mesenchymal transition (EMT) induced by bleomycin or TFG(b1)/EGF in murine induced pluripotent stem cell-derived alveolar type II-like cells. Differentiation 82: 89–98. http://dx.doi.org/10.1016/j.diff.2011.05.001. Allen, J.T., Spiteri, M.A., 2002. Growth factors in idiopathic pulmonary fibrosis: relative roles. Respir. Res. 3, 13. Aoki, Y., Maeno, T., Aoyagi, K., Ueno, M., Aoki, F., Aoki, N., et al., 2009. Pioglitazone, a peroxisome proliferator-activated receptor gamma ligand, suppresses bleomycin-induced acute lung injury and fibrosis. Respiration 77:311–319. http://dx.doi.org/10. 1159/000168676. Aston, C., Jagirdar, J., Lee, T.C., Hur, T., Hintz, R.L., Rom, W.N., 1995. Enhanced insulin-like growth factor molecules in idiopathic pulmonary fibrosis. Am. J. Respir. Crit. Care Med. 151:1597–1603. http://dx.doi.org/10.1164/ajrccm.151.5.7537587. Bloor, C.A., Knight, R.A., Kedia, R.K., Spiteri, M.A., Allen, J.T., 2001. Differential mRNA expression of insulin-like growth factor-1 splice variants in patients with idiopathic pulmonary fibrosis and pulmonary sarcoidosis. Am. J. Respir. Crit. Care Med. 164: 265–272. http://dx.doi.org/10.1164/ajrccm.164.2.2003114.

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