Extracellular matrix proteins: A positive feedback loop in lung fibrosis?

Matrix Biology 34 (2014) 170–178

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Extracellular matrix proteins: A positive feedback loop in lung fibrosis? Marjolein E. Blaauboer a,b,⁎, Fee R. Boeijen a, Claire L. Emson c, Scott M. Turner c, Behrouz Zandieh-Doulabi a, Roeland Hanemaaijer b, Theo H. Smit d, Reinout Stoop b, Vincent Everts a a Department of Oral Cell Biology, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University Amsterdam, MOVE Research Institute Amsterdam, The Netherlands b TNO Metabolic Health Research, Leiden, The Netherlands c Kinemed Inc., Emeryville, CA, USA d Department of Orthopaedics, VU Medical Center, MOVE Research Institute Amsterdam, The Netherlands

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Article history: Received 5 June 2013 Received in revised form 15 November 2013 Accepted 19 November 2013 Available online 27 November 2013 Keywords: Lung fibrosis Extracellular matrix Elastin Type V collagen Tenascin C Myofibroblast differentiation

a b s t r a c t Lung fibrosis is characterized by excessive deposition of extracellular matrix. This not only affects tissue architecture and function, but it also influences fibroblast behavior and thus disease progression. Here we describe the expression of elastin, type V collagen and tenascin C during the development of bleomycin-induced lung fibrosis. We further report in vitro experiments clarifying both the effect of myofibroblast differentiation on this expression and the effect of extracellular elastin on myofibroblast differentiation. Lung fibrosis was induced in female C57Bl/6 mice by bleomycin instillation. Animals were sacrificed at zero to five weeks after fibrosis induction. Collagen synthesized during the week prior to sacrifice was labeled with deuterium. After sacrifice, lung tissue was collected for determination of new collagen formation, microarray analysis, and histology. Human lung fibroblasts were grown on tissue culture plastic or BioFlex culture plates coated with type I collagen or elastin, and stimulated to undergo myofibroblast differentiation by 0–10 ng/ml transforming growth factor (TGF)β1. mRNA expression was analyzed by quantitative real-time PCR. New collagen formation during bleomycin-induced fibrosis was highly correlated to gene expression of elastin, type V collagen and tenascin C. At the protein level, elastin, type V collagen and tenascin C were highly expressed in fibrotic areas as seen in histological sections of the lung. Type V collagen and tenascin C were transiently increased. Human lung fibroblasts stimulated with TGFβ1 strongly increased gene expression of elastin, type V collagen and tenascin C. The extracellular presence of elastin increased gene expression of the myofibroblastic markers α smooth muscle actin and type I collagen. The extracellular matrix composition changes dramatically during the development of lung fibrosis. The increased levels of elastin, type V collagen and tenascin C are probably the result of increased expression by fibroblastic cells; reversely, elastin influences myofibroblast differentiation. This suggests a reciprocal interaction between fibroblasts and the extracellular matrix composition that could enhance the development of lung fibrosis. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Idiopathic pulmonary fibrosis (IPF) is a severely destructive lung disease, resulting in impaired architecture and function of lung tissue (Selman et al., 2004). The incidence of IPF is estimated to be 5 to 10 per 100,000 (Fernandez Perez et al., 2010) and appears to increase in recent years (Nalysnyk et al., 2012). At the core of the fibrotic process are changes in both the structure and the composition of the extracellular matrix. The deposition of excessive amounts of type I collagen is classically seen as the main problem in fibrosing tissues (Meltzer and Noble, 2008).

⁎ Corresponding author at: Department of Oral Cell Biology, Academic Centre for Dentistry Amsterdam (ACTA), Gustav Mahlerlaan 3004, 1081 LA Amsterdam, The Netherlands. Tel.: +31 20 5980875. E-mail address: [email protected] (M.E. Blaauboer). 0945-053X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.matbio.2013.11.002

Fibroblasts are responsible for the maintenance of the extracellular matrix. During fibrosis development, they differentiate toward the myofibroblastic phenotype, characterized by an increased contractile capacity due to the expression of α smooth muscle actin (αSMA) and by increased release of different types of extracellular matrix proteins (Tomasek et al., 2002; Hinz, 2007; Hinz et al., 2012). Extensive literature exists on how this process is regulated by growth factors, such as transforming growth factor (TGF)β1 (Todd et al., 2012). However, myofibroblast differentiation is also affected by the changes in the composition and architecture of the fibrotic matrix by a) determining which specific attachment sites are available to the cells, b) influencing the mechanical properties of the matrix and c) determining the mechanical loading experienced by the cells during, for example, breathing (Suki and Bates, 2008). The effect of the fibrotic matrix was confirmed recently by seeding lung fibroblasts into decellularized matrix from IPF patients and healthy controls, resulting in an increased expression of myofibroblast markers in IPF matrix (Booth et al., 2012), thus

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indicating that changes in the extracellular matrix composition determine disease progression. Possible candidates for specific proteins within the extracellular matrix regulating fibrotic cellular processes can be derived from our earlier study (Blaauboer et al., 2013). In that study we measured new collagen formation by analysis of deuterated water incorporation into hydroxyproline in mice with bleomycin-induced lung fibrosis. We combined this with microarray analysis and correlating these results allowed us to identify fibrosis-relevant changes in gene expression within this model. Interestingly, three extracellular matrix proteins were strongly correlated to new collagen formation: elastin, type V collagen and tenascin C. Patients with IPF and its histopathological equivalent usual interstitial pneumonia, also have increased levels of elastin (Cha et al., 2010), type V collagen (Parra et al., 2006) and tenascin C (Kuhn and Mason, 1995; Fitch et al., 2011). Therefore, these proteins are attractive candidates in the search for regulatory roles of extracellular matrix proteins in fibrosis. For type V collagen and tenascin, pro-fibrotic mechanisms have been described. Exposure to type V collagen results in inflammatory responses (Braun et al., 2010), that could affect the development of fibrosis, for example via fibrosis-relevant cytokine release by immune cells (Todd et al., 2012). Also tenascin C could play a regulatory role in the development of fibrosis since it increases cell migration (Trebaul et al., 2007) and migration of cells is important for the recruitment of myofibroblasts. These observations emphasize the reciprocal relationship between changes in matrix composition and cellular contributions to fibrosis development. In this study, we aimed to further unravel this reciprocal relationship between cells and matrix during fibrosis development. For this, we first analyzed the expression of elastin, type V collagen and tenascin C at different time points during the development of bleomycin-induced lung fibrosis. Then we addressed the role

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of lung fibroblasts and myofibroblasts in the changed expression of these extracellular matrix proteins. Since it is not known if elastin has similar fibrosis-inducing effects as type V collagen and tenascin C, we investigated the effect of elastin on lung fibroblasts and the differentiation to myofibroblasts. 2. Results 2.1. Elastin, type V collagen, and tenascin C strongly correlate with new collagen deposition in bleomycin-induced lung fibrosis In vivo new collagen formation, as measured by incorporation of deuterated water into hydroxyproline, correlated strongly with gene expression of elastin (r = 0.93, Fig. 1A), the α-1 chain of type V collagen (r = 0.84, Fig. 1B) and tenascin C (r = 0.88, Fig. 1C) during the development of bleomycin-induced lung fibrosis. 2.2. Increased protein deposition of elastin, type V collagen and tenascin C during bleomycin-induced lung fibrosis Resorcin-fuchsin staining in lung sections of healthy control mice indicates the presence of elastin around blood vessels and at the tips of alveolar septae (Fig. 2A). In fibrotic lung sections, elastin was increasingly found in fibrotic areas at all time points (Fig. 2B–D). Type V collagen was present in healthy lung tissue in blood vessel walls and in a thin layer around bronchioles (Fig. 2E). In fibrotic lungs, during the first two weeks after fibrosis-induction by bleomycin instillation type V collagen was increased in fibrotic areas (Fig. 2F and G). At 4 weeks, type V collagen immunostaining was decreased compared to the high levels in the first two weeks (Fig. 2H).

Fig. 1. During bleomycin-induced lung fibrosis, gene expression of elastin, type V collagen, and tenascin C is highly correlated to new collagen formation; the core process of fibrosis. Correlation between new collagen formation as measured by deuterated water incorporation in hydroxyproline and A) elastin gene (ELN) expression, B) gene expression of the α-1 chain of type V collagen (COL5A1), and C) tenascin C gene (TNC) expression. Each point represents data of one experimental animal, lines represent trend-lines from linear regression.

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In healthy lung tissue minimal tenascin C staining was present in only a few alveolar structures (Fig. 2I). Extracellular tenascin C was observed at high levels in fibrotic areas 1 and 2 weeks after bleomycin instillation (Fig. 2J and K). At 4 weeks, extracellular tenascin C levels were reduced (Fig. 2L). From 2 weeks onward, tenascin C was also visible intracellularly, which was especially obvious at the later time points (Fig. 2K and L). In healthy lung tissue αSMA was present in blood vessel walls and around bronchioles (Fig. 2M). At 1 and 2 weeks after bleomycin instillation, αSMA was present in fibrotic areas (Fig. 2N and O). At 4 weeks, αSMA levels were reduced again in fibrotic areas (Fig. 2P). 2.3. Myofibroblast markers are increased by TGFβ1 stimulation in lung fibroblasts Culturing normal human lung fibroblasts (NHLFs) and human fetal lung fibroblasts (HFL1s) in the presence of TGFβ1 to mimic fibrotic conditions resulted in differentiation into the myofibroblastic phenotype, as indicated by a dose-dependent increase in mRNA expression of αSMA (ACTA2) and the α-1 chain of type I collagen (COL1A1) (Fig. 3A–D). For αSMA this was confirmed at the protein level by western blot (Fig. 3E–H).

2.4. Elastin, type V collagen, and tenascin C mRNA expression is increased by TGFβ1 stimulation in human lung fibroblasts Under fibrotic conditions, mimicked by the presence of TGFβ1, mRNA expression of elastin (ELN), the α-1 chain of type V collagen (COL5A1), and tenascin C (TNC) was dose-dependently increased (Fig. 4). Especially elastin mRNA expression was strongly upregulated in NHLFs (up to 24-fold at 10 ng/ml TGFβ1) and HFL1s (up to 62-fold at 10 ng/ml TGFβ1).

2.5. Elastin coating increases TGFβ1-induced mRNA expression of α smooth muscle actin, elastin, and tenascin C in human lung fibroblasts NHLF cells cultured on elastin-coated surfaces had an increased mRNA expression of αSMA (ACTA2) and type I collagen (COL1A1) compared to NHLF cells cultured on collagen coated surfaces in the presence of 10 ng/ml TGFβ1 (Fig. 5A and B), indicating increased myofibroblast differentiation in the presence of extracellular elastin. Extracellular elastin did also increase mRNA expression of elastin (ELN) both in the absence and presence of 10 ng/ml TGFβ1 (Fig. 5C).

Fig. 2. The extracellular matrix proteins are visible at the protein level on histological staining in fibrotic areas of lungs with bleomycin-induced lung fibrosis. In healthy tissue, elastin and type V collagen are present around blood vessels (closed arrow heads). Elastin is also visible at the tips of alveolar septae (open arrow heads) and in the walls of the bronchioles. Tenascin C is not present in healthy tissue, except for very small areas (open arrow). In fibrotic tissue, elastin is increasingly present extracellularly during the time course of bleomycin-induced lung fibrosis, at the same locations compared to in healthy tissue. Fibrotic areas (closed arrows) are extensively stained for elastin. Type V collagen and tenascin C are present extracellularly in fibrotic areas at increased levels 1 and 2 weeks after bleomycin induction and reduce thereafter. αSMA is present in the same fibrotic areas and also temporarily present. From 2 weeks on, tenascin C was also found intracellularly (line arrows). A–D) Elastin staining, blue-purple, E–H) immunohistochemical staining of type V collagen, dark brown, and hematoxylin staining of nuclei, blue-purple, I–L) immunohistochemical staining of tenascin C, brown, and hematoxylin staining of nuclei, blue-purple, M–P) immunohistochemical staining of αSMA, dark brown, and hematoxylin staining of nuclei, blue-purple, in the control mice lungs (A, E, I, and M), and 1 week (B, F, J, and N), 2 weeks (C, G, K, and O), and 4 weeks (D, H, L, and P) after induction of bleomycin-induced lung fibrosis. Scale bar: 100 μm.

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Fig. 3. Myofibroblast markers are increased by TGFβ1 stimulation in lung fibroblasts. mRNA expression of A–B) αSMA (ACTA2), and C–D) the α-1 chain of type I collagen (COL1A1) in NHLF cells (A and C) and HFL-1 cells (B and D) after 24 h of stimulation with 0–10 ng/ml TGFβ1. Protein expression of αSMA was determined by western blot analysis in NHLF cells (E) and HFL-1 cells (F) after 4 days of stimulation with 0–10 ng/ml TGFβ1 and quantified (G and H). Gene expression is shown relative to mRNA expression of the household gene GAPDH. Protein expression is shown relative to expression of β-actin. Data are normalized to 0 ng/ml TGFβ1 and shown as mean ± standard deviation (n = 4 or 5 for mRNA data and n = 3 for protein data). Significant differences with unstimulated control are indicated. *p b 0.05, **p b 0.01, ***p b 0.001.

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Fig. 4. mRNA expression of elastin, type V collagen, and tenascin C is highly upregulated by TGFβ1 stimulation in lung fibroblasts. mRNA expression of, A–B) elastin (ELN), C–D) the α-1 chain of type V collagen (COL5A1), and E–F) tenascin C (TNC) in NHLF cells (A, C, and E) and HFL-1 cells (B, D and F) after 24 h of stimulation with 0–10 ng/ml TGFβ1. Gene expression is shown relative to mRNA expression of the household gene GAPDH. Data are normalized to 0 ng/ml TGFβ1 and shown as mean ± standard deviation (n = 4 or 5). Significant differences with unstimulated control are indicated. *p b 0.05, **p b 0.01, ***p b 0.001.

Fig. 5. mRNA expression of elastin, type V collagen, and tenascin C is increased on elastin coated surfaces. mRNA expression of A) α smooth muscle actin (ACTA2), B) the α-1 chain of type I collagen (COL1A1) and C) elastin (ELN) in NHLF cells growing on either type I collagen or elastin coating after 48 h of stimulation with 0 or 10 ng/ml TGFβ1. Data are shown as mean ± standard deviation (n = 6 for collagen coating, n = 12 for elastin coating). Significant differences between type I collagen and elastin coating are indicated. *p b 0.05, **p b 0.01, ***p b 0.001.

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3. Discussion In this study, we aimed to shed light on the reciprocal relationship between the cells and the changing extracellular matrix following induction of lung fibrosis with bleomycin. In an earlier study, we quantified the core process of fibrosis – new collagen formation – in the bleomycin-induced lung fibrosis model by measuring deuterated water incorporation in hydroxyproline. We used this measure of fibrotic activity in the lung to select fibrosis-relevant genes from microarray results by correlating new collagen formation values with gene expression data from the same mice (Blaauboer et al., 2013). Interestingly, gene expression of three extracellular matrix proteins was strongly correlated to new collagen formation: elastin, the α-1 chain of type V collagen and tenascin C. An upregulation of mRNA expression of elastin and tenascin C is in line with data presented by others: bleomycininduced lung fibrosis increased levels of elastin mRNA in mice (Lucey et al., 1996) and tenascin C mRNA levels in rats (Zhao et al., 1998). Furthermore, the clear correlation of elastin, type V collagen and tenascin C mRNA expression to new collagen formation indicates that the highest levels of mRNA expression of these matrix proteins are present specifically in mice with very active fibrosis development, further emphasizing the relevance of this finding. By histology we confirmed that during the development of fibrosis the levels of elastin, type V collagen and tenascin C protein are increased. Interestingly, whereas elastin staining remained elevated until five weeks after fibrosis-induction, intense immunostaining of both type V collagen and tenascin C was present only in the first few weeks after fibrosis induction and levels decreased from 3 weeks on. In the case of type V collagen, it is unclear if this decrease in immunostaining is the result of a decrease in type V collagen protein levels present in the tissue, or the result of masking of the type V collagen epitope by type I collagen, since type V collagen is often present in tissues as a component buried within type I collagen fibrils (Fichard et al., 1995; Birk, 2001). The transient upregulation of tenascin C reported here is supported by earlier findings in the rat lungs during bleomycin-induced lung fibrosis (Zhao et al., 1998): immuno-histochemical staining of tenascin C in this rat model was strongest in intensity at 8 days, and reduced at 12 days after induction. The patterns of elastin expression in fibrotic lung tissue correspond to earlier descriptions of end-stage lung fibrosis induced by bleomycin (Starcher et al., 1978; Collins et al., 1981; Laurent et al., 1981; Dolhnikoff et al., 1999), by overexpression of TGFβ1 (Sime et al., 1997; Tarantal et al., 2010) and by butylated hydroxytoluene with 70% oxygen (Hoff et al., 1999). In the last model, the abnormal elastic fiber morphology persisted in the mice lungs for at least 6 months. This is in line with the seemingly permanent changes in deposited elastin in the current study. In order to relate matrix deposition to myofibroblast occurrence, we also analyzed αSMA protein expression by immunohistochemistry. Expression patterns of αSMA were comparable to type V collagen and tenascin C: myofibroblasts were visible in fibrotic areas at 1 and 2 weeks after bleomycin induction but clearly reduced in numbers at 4 weeks. This simultaneous appearance in vivo is confirmed by our in vitro data, where mRNA expression of both αSMA and extracellular matrix proteins increases within 24 h in response to TGFβ1-stimulation. Unfortunately, these correlated expression patterns do not provide information on the question whether the extracellular matrix changes are the cause or effect of myofibroblast differentiation. The value of the bleomycin model in comparison with IPF patients has been debated (Chua et al., 2005; Moeller et al., 2008; Degryse and Lawson, 2011). Recently, a comparison was made between gene expression in the single dose bleomycin model and in patients with idiopathic pulmonary fibrosis (Peng et al., 2013). In contrast to the current beliefs, the bleomycin model showed high correspondence to active fibrosis in IPF patients. Interestingly, genes upregulated in both the model and the patient material included elastin, type V collagen and tenascin C. At the protein level, there seem to be more permanent changes in patients

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with IPF than in the rodent bleomycin model: patients, considered to be at the end stage of fibrosis development, have increased expression of type V collagen (Parra et al., 2006) and tenascin C (Kuhn and Mason, 1995; Fitch et al., 2011) within fibroblastic foci and higher levels of tenascin C in their bronchoalveolar lavage fluid (Kaarteenaho-Wiik et al., 1998). In this light, the recent paper by Booth et al. (2012)) analyzing the protein content of IPF lungs by mass spectrometry could provide quantitative information about the extracellular matrix composition. Unfortunately, it is possible that in their dataset elastin and type V collagen were underrepresented, due to difficulties with solubilizing the hydrophobic elastin proteins and the type V collagen intercalating with larger insoluble collagen bundles, making a direct comparison difficult. Using whole lung RNA for the microarray analysis that includes RNA of the many cell types present within the lung allowed us to explore fibrosis-relevant cellular processes in all these cell types in this in vivo model. However, as a consequence these data do not reveal the cell type(s) responsible for the increased expression. To investigate the possible role of fibroblastic cells in the increased expression of elastin, type V collagen and tenascin C during the development of lung fibrosis, we cultured human lung fibroblasts under fibrotic conditions, i.e. in the presence of TGFβ1, to induce myofibroblast differentiation. Under these fibrotic conditions, we found a strong increase in gene expression of elastin, type V collagen and tenascin C. The fact that in the in vitro model human fibroblasts are used, indicates that our results from the in vivo mouse model are relevant for human pulmonary fibrosis. Fibrotic changes in the composition of the extracellular matrix can have a regulatory role during fibrosis development, thereby completing a positive feedback loop explaining the progressive nature of fibrotic diseases. Here, we report that in addition to a reported role for type V collagen and tenascin C (Trebaul et al., 2007; Braun et al., 2010), also elastin has a pro-fibrotic effect, since culturing human lung fibroblasts on elastin-coated culture plates resulted in increased expression of the myofibroblast marker α smooth muscle actin and type I collagen, indicating an increased myofibroblast differentiation in the presence of extracellular elastin. Furthermore, this effect is self-amplifying, since elastin coating also increased mRNA expression of elastin itself. In literature several candidates have been proposed as potential receptors for elastin: the integrin αVβ3 (Bax et al., 2009) and the elastin receptor GLB1 (Duca et al., 2007) in dermal fibroblasts and heparan sulfatecontaining glycosaminoglycans in chondrocytes (Broekelmann et al., 2005). Interestingly, in our microarray GLB1 is upregulated at 1 and 2 weeks after fibrosis induction and positively correlated to collagen deposition (r = 0.72, data not shown). This could indicate a role for this elastin receptor in fibrosis development. Furthermore, gene expression of glb1l2 is negatively correlated to collagen deposition (r = −0.84, data not shown), suggesting that this gene could encode for a decoy receptor. Alternatively, elastin could also influence availability of TGFβ1, thereby influencing the TGFβ1 signaling, without (elastin) receptor involvement. We have shown before that cyclic mechanical stretch, mimicking the in vivo breathing movement, reduces TGFβ1-induced myofibroblast differentiation (Blaauboer et al., 2011). In that study, mRNA levels of the α2 chain of type V collagen and tenascin C were also decreased by cyclic mechanical stretch. Here we found that the gene expression of elastin and the α1 chain of type V collagen were also reduced after cyclic mechanical stretch (Supplemental data). The relevance of the reduced gene expression after mechanical loading is related to the fact that, during the development of lung fibrosis, the lung tissues increase in stiffness (Ebihara et al., 2000; Liu et al., 2010; Booth et al., 2012). This could result in lower cyclic mechanical stretch for the fibroblasts in the lung tissue. The lower cyclic mechanical stretch can increase myofibroblast differentiation both directly, via an increase of α smooth muscle actin and type I collagen expression, and indirectly, via an increase in elastin expression, which, in turn, also increases myofibroblast differentiation.

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In summary, elastin, type V collagen and tenascin C are increasingly expressed in mice suffering from active fibrosis development, as characterized by high levels of new collagen formation. In vitro studies indicate a role for fibroblastic cells in this increased expression. Furthermore, the presence of extracellular elastin further increases myofibroblast differentiation, contributing to disease progression. When fibrotic tissue stiffens, resulting in reduced levels of mechanical stimulation of lung fibroblasts, levels of elastin, type V collagen and tenascin C will further increase, as will their effects on fibroblasts. From these data we conclude that the development of lung fibrosis could depend on a feedback loop due to the reciprocal interaction between fibroblasts and the extracellular matrix composition, in particular elastin, type V collagen and tenascin C. 4. Experimental procedures 4.1. Animal procedures All animal procedures were approved by the TNO Animal Welfare Committee (#2738). Female C57Bl/6J mice (Charles River Laboratories, Germany) 10–12 weeks of age received a single intratracheal instillation of 30 μl bleomycin (Pharmachemie BV, Haarlem, The Netherlands; 1.25 U/ml in PBS). To label new collagen the mice received 35 μl deuterated water (D2O)/g body weight (i.p.) at 7 days before sacrifice and normal drinking water was replaced with 8% deuterated water. Water was refreshed every second day. Mice were sacrificed by CO2 asphyxiation at 1 (n = 8), 2 (n = 8), 3 (n = 8), 4 (n = 6) or 5 (n = 7) weeks after bleomycin treatment. Untreated animals were used as control (t = 0 week, n = 7). After sacrifice, the left lung lobe was fixed with 10% formalin and processed for histology; the cranial lung lobe was stored at −80 °C until determination of D2O incorporation in hydroxyproline while the caudal lobe was snap-frozen in liquid nitrogen for microarray analysis. 4.2. Microarray analysis Gene expression data were retrieved from the microarray analysis dataset published in Blaauboer et al. (2013) and accessible online through GEO Series accession number GSE37635 (http://www.ncbi. nlm.nih.gov/geo/query/acc.cgi?acc=GSE37635). 4.3. Kinetic analysis Deuterated water incorporation into hydroxyproline was used as a measure for new collagen formation and the analysis is described earlier in Blaauboer et al. (2013). 4.4. Histology and immune-histochemical staining Formalin-fixed tissues were embedded in paraffin, sectioned in 5 μm sections, and stained for extracellular matrix proteins. To deparaffinize, the slides were immersed in xylene and rehydrated in decreasing ethanol concentrations. To investigate the expression of elastin, sections were incubated for 1 h in resorcin-fuchsin solution (Electron Microscopy Sciences, Hatfield, PA, USA), differentiated in 96% and 100% ethanol, followed by xylene. The expression of type V collagen, tenascin C, and αSMA protein was assessed by immuno-histochemical staining. During the deparaffinization process, in between the 100% ethanol steps, endogenous peroxidase in the lung tissue was blocked (20 min at room temperature; 0.3% H O in methanol). For type V collagen staining, the sections were incubated after deparaffinization in a citrate-buffer (10 mM tri-sodium citrate dehydrate in H O; pH = 6) at 100 °C for 10 min for antigen retrieval. For all immunostainings, a specific protein binding was blocked for 15 min with bovine serum albumin (BSA, 1% in PBS) at room temperature. The sections were incubated with primary antibodies against type

V collagen (polyclonal rabbit anti-type V collagen with biotin label (Acris antibodies, Herford, Germany) diluted 1:200 in 1% BSA in PBS, overnight at 4 °C), tenascin C (polyclonal rabbit anti-tenascin C (Millipore, Amsterdam, The Netherlands) diluted 1:200 in 1% BSA in PBS, 2 h at room temperature) or αSMA (monoclonal mouse antiαSMA (clone ASM-1, Monosan, Uden, The Netherlands) diluted 1:800 in 1% BSA in PBS, overnight at 4 °C). As unspecific antibody for type V collagen and tenascin C, Dako Universal Negative Control Rabbit (Dako, Heverlee, Belgium) was used diluted 1:100 in 1% BSA in PBS. Hereafter, bound immunoglobulin was detected by incubating for 45 min (tenascin C staining) or 60 min (type V collagen staining) at room temperature with the labeled polymer from the EnVision anti-rabbit Kit (Dako). Anti-αSMA antibodies were detected for 1 h at room temperature with HRP-bound rabbit-anti-mouse IgG (Dako) diluted 1:300 in 1% BSA in PBS. This was followed by an incubation for 8 min in DAB substrate solution (Vector, Burlingame, CA, USA). Then nuclei were stained using Hematoxylin Mayer solution. Sections were dehydrated again using increasing ethanol concentrations, followed by xylene incubation. Finally the sections were covered with depex or malinol and a coverslip. All sections were photographed with a CRi Nuance FX Multispectral Camera (Quorum Technologies, Ontario, Canada). 4.5. Cell culture Primary normal human lung fibroblast (NHLF) cells were obtained from Lonza Walkersville, Inc. (Walkersville, MD, USA). Human fetal lung (HFL-1) fibroblasts were obtained from ATCC (ATCC, Wesel, Germany). NHLF cells were cultured in Dulbecco's minimal essential medium (D-MEM; Invitrogen, Paisley, UK) and HFL-1 cells were cultured in F12K medium (ATCC), both supplemented with 10% fetal clone serum (FCS; HyClone, South Logan, UT, USA) and 1% antibiotic–antimycotic solution (100 U/ml penicillin, 100 μg/ml streptomycin, and 250 ng/ml amphotericin B (PSA), Sigma-Aldrich, St. Louis, MO, USA) in an incubator set at 37 °C, 95% humidity, and 5% CO2. Once grown to confluency, cells were trypsinized using 0.5% trypsin (Sigma-Aldrich) and 0.1% EDTA (Merck, Darmstadt, Germany) in PBS (Invitrogen). 4.6. TGFβ1-response experiments Cells from passage 6 (NHLF cells) or passage 16 (HFL-1 cells) were seeded in D-MEM with 10% FCS and 1% PSF at a density of 50.000 cells/cm2 in a 24 well plate. After allowing the cells to attach for 24 h, medium was replaced by D-MEM with 1% FCS and 1% PSA and 24 h later myofibroblast differentiation was induced by replacing the medium with D-MEM supplemented with 1% FCS and 1% PSA containing 0 to 10 ng/ml recombinant human TGFβ1 (PeproTech EC, London, UK). Fibroblasts were cultured in the presence or absence of TGFβ1. mRNA samples were collected after 24 h and protein samples after 96 h. 4.7. Elastin and collagen coating For each experiment, NHLF cells from passages 6 to 10 were seeded at a density of 3.000 or 10.000 cells/cm2 on BioFlex six-well culture plates coated with type I collagen or elastin. After allowing the cells to attach for 24 h, medium was replaced by D-MEM with 1% FCS and 1% PSA and 24 h later myofibroblast differentiation was induced by replacing the medium with D-MEM supplemented with 1% FCS and 1% PSA containing 0 or 10 ng/ml recombinant human TGFβ1. Fibroblasts were cultured for 48 h in the presence or absence of TGFβ1 and mRNA samples were collected. 4.8. Quantitative real-time PCR mRNA was isolated from cell culture experiments using an RNeasy Mini Kit for RNA extraction (Qiagen, Hilden, Germany). The mRNA concentration was measured using a Synergy HT microplate reader

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(BioTek Instruments, Winooski, VT, USA). mRNA was reverse-transcribed to complementary DNA (cDNA) using the MBI Fermentas cDNA synthesis kit (Vilnius, Lithuania). mRNA expression of αSMA, COL1A1, COL5A1, tenascin C (TNC), elastin (ELN) and the housekeeping gene GAPDH was analyzed by real-time PCR performed on an ABI PRISM 7000 (Applied Biosystems, Foster City, CA, USA). GAPDH mRNA expression was determined using TaqMan® Rodent GAPDH Control Reagents (Applied Biosystems). All other genes were analyzed using unique TaqMan® Assays-on-Demand™ Gene Expression kits (Table 1; Applied Biosystems) specific for human.

4.9. Western blotting Cells were lysed with 1% Triton X-100 (Serva, Heidelberg, Germany) in water containing complete protease inhibitors (Roche Diagnostics, Mannheim, Germany). Protein content was measured with the BCA Protein Assay (Thermo Scientific, Pierce, Rockford, IL, USA). Equal amounts of protein were denatured and separated on a NuPAGE 4–12% Bis–Tris gel (Life Technologies, Novex, Carlsbad, CA, USA) under reducing conditions. Proteins were transferred to a nitrocellulose membrane using the iBlot system (Life Technologies, Novex). Background was blocked for 1 h at room temperature with 1% BSA in PBS containing 0.1% Tween 20 (Sigma-Aldrich; blocking buffer). Blots were incubated with anti-αSMA monoclonal antibody (clone 1A4, Dako) diluted 1:500 in blocking buffer for 1 h at room temperature and subsequently overnight at 4 °C. Then the blots were rinsed 3 times with blocking buffer followed by a 1 h incubation step with HRP-labeled goat anti-mouse antibody (Pierce, Rockford, IL, USA) diluted 1:10,000 in blocking buffer. Chemiluminescent signal was detected using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) and the Biospectrum AC Imaging System and VisionWorksLS software (UVP, Cambridge, UK). Gamma settings were optimized for clarity of the bands. After chemiluminescent detection, blots were washed with blocking buffer and incubated for 1 h at room temperature with mouse anti-β-actin antibody (1:5000, Sigma) followed by 3 wash steps in blocking buffer. An HRP labeled secondary goat anti-mouse antibody (Pierce) diluted 1:10,000 in blocking buffer was incubated for 1 h and stained with DAB solution (Vector). Density of the bands was measured using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

4.10. Statistics Statistical analyses were performed using SPSS (version 20, IBM Corporation, Armonk, NY, USA). Results of the in vitro experiments were evaluated using two-way ANOVA or with Bonferroni-adjusted t-tests for multiple comparisons between groups. Using Microsoft Office Excel (2007, Microsoft Corporation, Redmond, WA, USA) Pearson's correlation coefficients were calculated over all data points of all time points to correlate new collagen formation to single gene data. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.matbio.2013.11.002.

Table 1 Gene product, Gene, GenBank ID, and Assay-on-Demand™ used in this study. Gene product

Gene

GenBank ID

Assay-on-Demand™

α-Smooth muscle actin α1 chain type I collagen α1 chain type V collagen Elastin Tenascin C

ACTA2 COL1A1 COL5A1 ELN TNC

NM_001613.2 NM_000088.3 NM_000093.3 NM_000501.2 NM_002160.3

Hs00426835_g1 Hs01076777_m1 Hs00609088_m1 Hs00355783_m1 Hs01115665_m1

177

Acknowledgments The authors would like to thank Joline Attema and Jessica Snabel for technical assistance with the animal experiments, Elly de Wit for help with the histology and Marjan van Erk for the analysis of the microarray data.

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