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Cell matrix interactions in lung fibrosis

Blaauboer, M.E.

2013

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Chapter 3

Extracellular matrix proteins: a positive

feedback loop in lung fibrosis?

submitted for publication

Marjolein E. Blaauboer

Fee R. Boeijen, Claire L. Emson, Scott M. Turner, Roeland Hanemaaijer, Theo H. Smit,

Reinout Stoop, Vincent Everts

Chapter 3

42

Abstract

Lung fibrosis is characterized by excessive deposition of extracellular matrix. This affects not

only 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 labelled 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 to 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 with

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.

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

43

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, Cid‐Ruzafa, Rotella, & Esser, 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 & Noble, 2008).

Fibroblasts are responsible for 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 (Hinz, 2007; Hinz

et al., 2012; Tomasek, Gabbiani, Hinz, Chaponnier, & Brown, 2002). Extensive literature

exists on how this process is regulated by growth factors, such as transforming growth

factor (TGF)β1 (Todd, Luzina, & Atamas, 2012). However, myofibroblast differentiation is

also affected by the extracellular matrix. Cell surface receptors like integrins allow cells to

chemically and mechanically probe their environment and to respond to the composition

of the extracellular matrix (Huang & Ogawa, 2012; Wells, 2013). The composition and

architecture of the matrix determine which specific attachment sites are available to the

cells and also influence the mechanical properties of the matrix and determine the

mechanical loading experienced by the cells during, for example, breathing (Suki & Bates,

2008). Via this route, changes in the extracellular matrix during the development of fibrosis

will influence cell behavior such as myofibroblast differentiation. This was confirmed 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 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 with 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 (Fitch, Howie, & Wallace, 2011; Kuhn & Mason,

Chapter 3

44

1995). Therefore, these proteins are attractive candidates in the search for regulatory roles

of extracellular matrix proteins in fibrosis.

Direct effects of type V collagen on fibrosis‐related pathways have been described in

the literature. Exposure to this extracellular matrix protein 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). This is in line with

the fact that patients with IPF are more often hypersensitive to type V collagen (Bobadilla

et al., 2008). Furthermore, immunization of rabbits with type V collagen was shown to result

in scleroderma‐like symptoms in the skin (Bezerra et al., 2006) and increased remodeling in

the lung (Teodoro et al., 2004).

Also tenascin C could play a regulatory role in the development of fibrosis since it

increases cell migration (Trebaul, Chan, & Midwood, 2007) and migration of cells is

important for the recruitment of myofibroblasts. This was confirmed by observations that

interstitial cells express tenascin C after myocardial infarction and myofibroblasts appear in

tenascin C positive areas (Imanaka‐Yoshida et al., 2001). Furthermore, tenascin C knockout

mice have a decreased recruitment of myofibroblasts after myocardiac wounding (Tamaoki

et al., 2005) or during the progression of hepatitis to liver fibrogenesis (El‐Karef et al., 2007).

Finally, fragments of tenascin C resulting from its breakdown, were shown to inhibit

fibroblast migration (Trebaul et al., 2007).

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 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.


45

Experimental Procedures

Animal procedures

All animal procedures were approved by TNO Animal Welfare Committee (#2738). Female

C57Bl/6J mice (Charles River Laboratories, Germany) 10 to 12 weeks of age received

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) /

gram 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 wk, 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.

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).

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).

Histology and immune‐histochemical staining

Formalin‐fixed tissues were embedded in paraffin, sectioned in 5 μm sections, and stained

extracellular matrix proteins. To deparaffinize, the slides were immersed in xylene and

rehydrated in decreasing ethanol concentrations.

To investigate 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 and tenascin C 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 minutes at room

temperature; 0.3% H₂O₂ in methanol). For type V collagen staining, the sections were

incubated after deparaffinization in a citrate‐buffer (10mM tri‐sodium citrate dehydrate in

H₂O; pH = 6) at 100°C for 10 minutes for antigen retrieval. For both stainings, aspecific

protein binding was blocked for 15 minutes with bovine serum albumin (BSA, 1% in PBS) at

Chapter 3

46

room temperature. The sections were incubated overnight at 4°C with polyclonal rabbit

anti‐type V collagen antibodies with biotin label (Acris antibodies, Herford, Germany) or 2

h at room temperature with polyclonal rabbit anti‐tenascin C (Millipore, Amsterdam, The

Netherlands) diluted 1:200 in 1% BSA in PBS. As unspecific rabbit antibody 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 minutes (tenascin C

staining) or 60 minutes (type V collagen staining) at room temperature with the labeled

polymer from the EnVision anti‐rabbit Kit (Dako), followed by an incubation for 8 minutes

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

and a coverslip.

All sections were photographed with a CRi Nuance FX Multispectral Camera (Quorum

Technologies, Ontario, Canada).

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).

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% PSA at a density of 50,000 cells/cm2 in a 24 wells 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 20 ng/ml recombinant human TGFβ1

(PeproTech EC, London, UK). Fibroblasts were cultured for 24 h in the presence or absence

of TGFβ1 and mRNA samples were collected.

Elastin and collagen coating

For each experiment, NHLF cells from passage 6 to 10 were seeded at a density of 3,000 or

10,000 cells/cm2 on BioFlex type I collagen or elastin‐coated six‐well culture plates (Flexcell

International Corp, McKeesport, PA, USA). After allowing the cells to attach for 24 h,


47

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.

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 (Biotek Instruments, Winooski, VT, USA). mRNA was reverse‐

transcribed to complementary DNA (cDNA) using a High Capacity RNA‐to‐cDNA Kit (Applied

Biosystems, Foster City, CA, USA). mRNA expression of elastin (ELN), the α1 chain of type V

collagen (COL5A1), tenascin C (TNC), αSMA (ACTA2), and the α1 chain of type I collagen

(COL1A1) and the housekeeping gene GAPDH was analyzed by Real‐Time PCR performed on

a 7500 Fast Real‐Time PCR system (Applied Biosystems). GAPDH mRNA expression was

determined using TaqMan® Rodent GAPDH Control Reagents (Applied Biosystems). All

other genes were analyzed using unique TaqMan® Assays‐on‐DemandTM Gene Expression

kits (Table 1; Applied Biosystems) specific for human.

Table 1: Gene product, Gene, GenBank ID, and Assay‐on‐DemandTM used in this study.

Gene product Gene GenBank ID Assay‐on‐DemandTM

α‐smooth muscle actin ACTA2 NM_001613.2 Hs00426835_g1

α1 chain type I collagen COL1A1 NM_000088.3 Hs01076777_m1

α1 chain type V collagen COL5A1 NM_000093.3 Hs00609088_m1

elastin ELN NM_000501.2 Hs00355783_m1

tenascin C TNC NM_002160.3 Hs01115665_m1

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.

Chapter 3

48

Results

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, Figure 1A), the

α1 chain of type V collagen (r = 0.84, Figure 1B) and tenascin C (r = 0.88, Figure 1C) during

the development of bleomycin‐induced lung fibrosis.

Figure 1: During bleomycin‐induced lung fibrosis, gene expression of elastin, type V collagen, and

tenascin C is highly correlated with 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 α1 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.


49

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 (Figure 2A). In fibrotic lung

sections, elastin was increasingly found in fibrotic areas at all time points (Figure 2B‐D).

Type V collagen was present in healthy lung tissue in blood vessel walls and in a thin

layer around bronchioles (Figure 2E). In fibrotic lungs, during the first two weeks after

fibrosis‐induction by bleomycin instillation type V collagen was increased in fibrotic areas

(Figure 2F and 2G). At 4 weeks, type V collagen immunostaining was decreased compared

to the high levels in the first two weeks (Figure 2H).

In healthy lung tissue minimal tenascin C staining was present in only a few alveolar

structures (Figure 2I). Extracellular tenascin C was observed at high levels in fibrotic areas 1

and 2 weeks after bleomycin instillation (Figure 2J and 2K). At 4 weeks, extracellular

tenascin C levels were reduced (Figure 2L). From 2 weeks onward, tenascin C was also visible

intracellularly, which was especially obvious at the later time points (Figure 2K and 2L).

Elastin, type V collagen, and tenascin C mRNA expression is increased by TGFβ1 stimulation

in human 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 α1 chain of type I collagen (COL1A1) (Figure 3A‐D). Under these

fibrotic conditions, mRNA expression of elastin (ELN), the α1 chain of type V collagen

(COL5A1), and tenascin C (TNC) was dose‐dependently increased as well (Figure 3E‐J).

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).

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 (Figure 4A and 4B), 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 (Figure 4C).

Chapter 3

50


51

Figure 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, localized at higher levels 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. 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, in control mice lungs (A, E, I), and 1 week (B, F, J), 2

weeks (C, G, K), and 4 weeks (D, H, L) after induction of bleomycin‐induced lung fibrosis. Scale bar:

100 µm.

Chapter 3

52

Figure 3: mRNA expression of

elastin, type V collagen, and

tenascin C is highly upregulated

by TGFβ1 stimulation in lung

fibroblasts.

mRNA expression of A‐B) αSMA

(ACTA2), C‐D) the α1 chain of type

I collagen (COL1A1), E‐F) elastin

(ELN), G‐H) the α1 chain of type V

collagen (COL5A1), and I‐J)

tenascin C (TNC) in NHLF cells (A,

C, E, G, and I) and HFL‐1 cells (B,

D, F, H, and J) after 24 h of

stimulation with 0 to 20 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 < 0.05,

** p < 0.01, *** p < 0.001.


53

Figure 4: 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 α1 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 < 0.05, ** p < 0.01, *** p < 0.001.

Chapter 3

54

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. We

found increased gene expression of elastin, type V collagen and tenascin C in mice with

active fibrosis and confirmed this finding at the protein level in histological sections.

Furthermore, fibroblastic cells increased expression of these extracellular matrix proteins

under fibrotic conditions in vitro, suggesting a contribution of myofibroblasts in the

increased levels found in vivo. Finally, we uncovered a regulatory role for elastin in

myofibroblast differentiation, emphasizing the possibility that a positive feedback loop

plays a role in the progressive nature of lung fibrosis in patients.

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 with new collagen

formation: elastin, the α1 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: bleomycin‐

induced lung fibrosis increased levels of elastin mRNA in mice (Lucey et al., 1996) and

tenascin C mRNA levels in rats (Zhao et al., 1998). To our knowledge, we are the first to

report increased type V collagen mRNA levels during bleomycin‐induced lung fibrosis. This

indicates that increased staining of type V collagen protein could at least in part be the result

of new collagen production, besides the possibility of increased epitope availability of type

V collagen protein already present in the tissue, due to increased remodeling of the tissue.

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 further confirmed the presence of elastin, type V collagen and tenascin C

at the protein level. In healthy lung tissue, elastin was present in blood vessels walls,

bronchiolar walls and alveolar septae, as described before (Mariani et al., 1997). During the

development of fibrosis elastin levels increased. The patterns of expression in healthy and

fibrotic lung tissue correspond to earlier descriptions of end‐stage lung fibrosis induced by

bleomycin (Collins et al., 1981; Dolhnikoff et al., 1999; Laurent et al., 1981; Starcher et al.,

1978), 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


55

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, where levels of

elastin were increasing until five weeks after fibrosis‐induction. Earlier, it was suggested

that elastin proteins level in liver fibrosis were regulated at the level of degradation by

MMP12 (Pellicoro et al., 2012). Here, we also find a clear elevation in mRNA levels of elastin,

suggesting that in bleomycin‐induced lung fibrosis elastin is regulated at least in part at the

transcriptional level or at the posttranscriptional level. This posttranscriptional level is an

interesting candidate, since it has been reported before that TGFβ1 regulates the stability

of elastin mRNA (Kähäri et al., 1992).

Type V collagen and tenascin C were present at low levels in healthy lung tissue. This

corresponds to reports in the literature, that there is normally little type V collagen (Parra

et al., 2006) and almost no tenascin C (Kaarteenaho et al., 2010) present in healthy lung

tissue. The small spots of tenascin C we observed in healthy lung sections might be areas

with minor damage to the tissue, occurring during normal use of the lungs, since expression

of tenascin C has been related to tissue damage (Chiquet‐Ehrismann and Chiquet, 2003).

Interestingly, 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 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 (Birk, 2001; Fichard et

al., 1995). The transient upregulation of tenascin C reported here is supported by earlier

findings in 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.

In patients with pulmonary fibrosis expression of type V collagen and tenascin C seems

to be more permanent than in the rodent bleomycin model. These patients are considered

to be at the end stage of fibrosis development. Even though, in patients with usual

interstitial pneumonia, the histopathological equivalent of IPF, expression of type V collagen

within fibroblastic foci is increased and the most important predictor of survival, with higher

levels of type V collagen being related to a lower chance of survival (Parra et al., 2006). Also,

tenascin C is present throughout the matrix of the fibroblastic focus in IPF patients (Kuhn

and Mason, 1995) and in patients with usual interstitial pneumonia (Fitch et al., 2011), who

also have higher levels of tenascin C in their bronchoalveolar lavage fluid (Kaarteenaho‐Wiik

et al., 1998).

It is attractive to speculate about the meaning of this difference in time‐dependence

of expression of type V collagen and tenascin C during IPF and the development of

bleomycin‐induced lung fibrosis. Both type V collagen and tenascin C could have a

regulatory role during the development of pulmonary fibrosis: type V collagen increases

Chapter 3

56

inflammation and tenascin C increases myofibroblast recruitment. Therefore, the transient

appearance of these extracellular matrix proteins in the rodent model versus the prolonged

presence in human lung fibrosis patients could explain why lung fibrosis in the rodent

bleomycin model is resolving (Moore et al., 2013), while patients have not been shown to

improve.

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. This increased expression of these extracellular matrix proteins

could be phenotypical for myofibroblasts, suggesting a fundamental role in different types

of fibrosis. Such is confirmed by reports in the literature describing increases in gene

expression in response to TGFβ1 in different cell types: for elastin in neonatal rat lung

fibroblasts (McGowan and McNamer, 1990), for the α2 chain of type V collagen in human

lung fibroblasts (Blaauboer et al., 2011) and for tenascin C in chick embryo fibroblasts

(Pearson et al., 1988) and the alveolar epithelial cell line A549 (Fitch et al., 2011). Moreover,

transdifferentiation of hepatic stellate cells into myofibroblasts by culturing on plastic also

increased expression of elastin (Kanta et al., 2002). 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 (Braun et al., 2010; Trebaul et al., 2007), 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. Thus, the effect of an increased presence of

elastin in the fibrotic lung on myofibroblast differentiation is one more example of how

changes in the extracellular matrix contribute to the progressive development of lung

fibrosis.


57

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 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

increases in stiffness (Booth et al., 2012; Ebihara et al., 2000; Liu et al., 2010). 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.

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. Reduced levels of mechanical

stimulation of lung fibroblasts due to stiffening of fibrotic tissue will further increase levels

of elastin, type V collagen and tenascin C, and 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.

Chapter 3

58

Acknowledgements

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.


59

Supplemental data

Supplemental Figure 1: TGFβ1‐induced mRNA expression of elastin and type V collagen is reduced

by cyclic mechanical stretch.

mRNA expression of A) elastin (ELN) and B) the α1 chain of type V collagen (COL5A1) in NHLF cells after

cyclic mechanical stretch. NHLF cells from passage 6 were seeded at a density of 3,000 or 10,000

cells/cm2 on BioFlex type I collagen coated six‐well culture plates (BioFlex, Flexcell International Corp,

McKeesport, PA, USA). 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 cells were subjected to 48 h of cyclic mechanical strain in a

Flexercell FX4000 apparatus (Flexcell International Corp) placed in an incubator set at 37°C, 95%

humidity and 5% CO2. Cyclic mechanical loading was applied in a sinusoidal pattern with a frequency

of 0.2 Hz and a maximum elongation of 10%. Control cells not receiving cyclic mechanical loading were

placed in the same incubator next to the loading station of the Flexercell apparatus. During mechanical

loading, 50% of the samples were stimulated with 10 ng/ml TGFβ1. After mechanical loading mRNA

samples were collected and analyzed as described in the Experimental Procedures. Gene expression

is shown relative to mRNA expression of the household gene GAPDH. Data are shown as mean ±

standard deviation (n = 6). Significant differences with static control are indicated. * p < 0.05, ** p <

0.01, *** p < 0.001.

Chapter 3

60

References

Bezerra, M.C., Teodoro, W.R., De Oliveira, C.C., Velosa, A.P.P., Ogido, L.T.I., Gauditano, G.,

Parra, E.R., Capelozzi, V.L., Yoshinari, N.H., 2006. Scleroderma‐like remodeling induced

by type V collagen. Arch. Dermatol. Res. 298, 51–7.

Birk, D.E., 2001. Type V collagen: heterotypic type I/V collagen interactions in the regulation

of fibril assembly. Micron. 32, 223–237.

Blaauboer, M.E., Emson, C.L., Verschuren, L., Van Erk, M., Turner, S.M., Everts, V.,

Hanemaaijer, R., Stoop, R., 2013. Novel combination of collagen dynamics analysis and

transcriptional profiling reveals fibrosis‐relevant genes and pathways. Matrix Biol.

Blaauboer, M.E., Smit, T.H., Hanemaaijer, R., Stoop, R., Everts, V., 2011. Cyclic mechanical

stretch reduces myofibroblast differentiation of primary lung fibroblasts. Biochem.

Biophys. Res. Commun. 404, 23–7.

Bobadilla, J.L., Love, R.B., Jankowska‐Gan, E., Xu, Q., Haynes, L.D., Braun, R.K., Hayney, M.S.,

Munoz del Rio, A., Meyer, K., Greenspan, D.S., Torrealba, J., Heidler, K.M., Cummings,

O.W., Iwata, T., Brand, D., Presson, R., Burlingham, W.J., Wilkes, D.S., 2008. Th‐17,

monokines, collagen type V, and primary graft dysfunction in lung transplantation. Am.

J. Respir. Crit. Care Med. 177, 660–8.

Booth, A.J., Hadley, R., Cornett, A.M., Dreffs, A. a, Matthes, S. a, Tsui, J.L., Weiss, K.,

Horowitz, J.C., Fiore, V.F., Barker, T.H., Moore, B.B., Martinez, F.J., Niklason, L.E.,

White, E.S., 2012. Acellular normal and fibrotic human lung matrices as a culture

system for in vitro investigation. Am. J. Respir. Crit. Care Med. 186, 866–76.

Braun, R.K., Martin, A., Shah, S., Iwashima, M., Medina, M., Byrne, K., Sethupathi, P.,

Wigfield, C.H., Brand, D.D., Love, R.B., 2010. Inhibition of bleomycin‐induced

pulmonary fibrosis through pre‐treatment with collagen type V. J. Heart Lung

Transplant. 29, 873–80.

Cha, S.I., Groshong, S.D., Frankel, S.K., Edelman, B.L., Cosgrove, G.P., Terry‐Powers, J.L.,

Remigio, L.K., Curran‐Everett, D., Brown, K.K., Cool, C.D., Riches, D.W., 2010.

Compartmentalized expression of c‐FLIP in lung tissues of patients with idiopathic

pulmonary fibrosis. Am. J. Respir. Cell Mol. Biol. 42, 140–148.

Chiquet‐Ehrismann, R., Chiquet, M., 2003. Tenascins: regulation and putative functions

during pathological stress. J. Pathol. 200, 488–499.

Collins, J.F., McCullough, B., Coalson, J.J., Johanson, W.G., 1981. Bleomycin‐induced diffuse

interstitial pulmonary fibrosis in baboons. II. Further studies on connective tissue

changes. Am. Rev. Respir. Dis. 123, 305–12.

Dolhnikoff, M., Mauad, T., Ludwig, M.S., 1999. Extracellular matrix and oscillatory

mechanics of rat lung parenchyma in bleomycin‐induced fibrosis. Am. J. Respir. Crit.

Care Med. 160, 1750–7.


61

Ebihara, T., Venkatesan, N., Tanaka, R., Ludwig, M.S., 2000. Changes in extracellular matrix

and tissue viscoelasticity in bleomycin‐induced lung fibrosis. Temporal aspects. Am. J.

Respir. Crit. Care Med. 162, 1569–76.

El‐Karef, A., Yoshida, T., Gabazza, E.C., Nishioka, T., Inada, H., Sakakura, T., Imanaka‐Yoshida,

K., 2007. Deficiency of tenascin‐C attenuates liver fibrosis in immune‐mediated chronic

hepatitis in mice. J. Pathol. 211, 86–94.

Fernandez Perez, E.R., Daniels, C.E., Schroeder, D.R., St, S.J., Hartman, T.E., Bartholmai, B.J.,

Yi, E.S., Ryu, J.H., 2010. Incidence, prevalence, and clinical course of idiopathic

pulmonary fibrosis: a population‐based study. Chest 137, 129–137.

Fichard, A., Kleman, J.P., Ruggiero, F., 1995. Another look at collagen V and XI molecules.

Matrix Biol. 14, 515–31.

Fitch, P.M., Howie, S.E.M., Wallace, W. a H., 2011. Oxidative damage and TGF‐β

differentially induce lung epithelial cell sonic hedgehog and tenascin‐C expression:

implications for the regulation of lung remodelling in idiopathic interstitial lung

disease. Int. J. Exp. Pathol. 92, 8–17.

Gardner, J.L., Turner, S.M., Bautista, A., Lindwall, G., Awada, M., Hellerstein, M.K., 2007.

Measurement of liver collagen synthesis by heavy water labeling: effects of profibrotic

toxicants and antifibrotic interventions. Am. J. Physiol. Gastrointest. Liver Physiol. 292,

G1695–705.

Hinz, B., 2007. Formation and function of the myofibroblast during tissue repair. J. Invest.

Dermatol. 127, 526–37.

Hinz, B., Phan, S.H., Thannickal, V.J., Prunotto, M., Desmoulière, A., Varga, J., De Wever, O.,

Mareel, M., Gabbiani, G., 2012. Recent developments in myofibroblast biology:

paradigms for connective tissue remodeling. Am. J. Pathol. 180, 1340–55.

Hoff, C.R., Perkins, D.R., Davidson, J.M., 1999. Elastin gene expression is upregulated during

pulmonary fibrosis. Connect. Tissue Res. 40, 145–53.

Huang, C., Ogawa, R., 2012. Fibroproliferative disorders and their mechanobiology.

Connect. Tissue Res. 53, 187–96.

Imanaka‐Yoshida, K., Hiroe, M., Nishikawa, T., Ishiyama, S., Shimojo, T., Ohta, Y., Sakakura,

T., Yoshida, T., 2001. Tenascin‐C modulates adhesion of cardiomyocytes to

extracellular matrix during tissue remodeling after myocardial infarction. Lab. Invest.

81, 1015–24.

Kaarteenaho, R., Sormunen, R., Paakko, P., 2010. Variable expression of tenascin‐C,

osteopontin and fibronectin in inflammatory myofibroblastic tumour of the lung.

APMIS 118, 91–100.

Kähäri, V.M., Olsen, D.R., Rhudy, R.W., Carrillo, P., Chen, Y.Q., Uitto, J., 1992. Transforming

growth factor‐beta up‐regulates elastin gene expression in human skin fibroblasts.

Evidence for post‐transcriptional modulation. Lab. Invest. 66, 580–8.

Chapter 3

62

Kanta, J., Dooley, S., Delvoux, B., Breuer, S., D’Amico, T., Gressner, A.M., 2002. Tropoelastin

expression is up‐regulated during activation of hepatic stellate cells and in the livers of

CCl(4)‐cirrhotic rats. Liver 22, 220–7.

Kuhn, C., Mason, R.J., 1995. Immunolocalization of SPARC, tenascin, and thrombospondin

in pulmonary fibrosis. Am. J. Pathol. 147, 1759–1769.

Laurent, G.J., McAnulty, R.J., Corrin, B., Cockerill, P., 1981. Biochemical and histological

changes in pulmonary fibrosis induced in rabbits with intratracheal bleomycin. Eur. J.

Clin. Invest. 11, 441–8.

Liu, F., Mih, J.D., Shea, B.S., Kho, A.T., Sharif, A.S., Tager, A.M., Tschumperlin, D.J., 2010.

Feedback amplification of fibrosis through matrix stiffening and COX‐2 suppression. J.

Cell Biol. 190, 693–706.

Lucey, E.C., Ngo, H.Q., Agarwal, A., Smith, B.D., Snider, G.L., Goldstein, R.H., 1996.

Differential expression of elastin and alpha 1(I) collagen mRNA in mice with bleomycin‐

induced pulmonary fibrosis. Lab. Invest. 74, 12–20.

Mariani, T.J., Sandefur, S., Pierce, R. a, 1997. Elastin in lung development. Exp. Lung Res. 23,

131–45.

McGowan, S.E., McNamer, R., 1990. Transforming growth factor‐beta increases elastin

production by neonatal rat lung fibroblasts. Am. J. Respir. Cell Mol. Biol. 3, 369–76.

Meltzer, E.B., Noble, P.W., 2008. Idiopathic pulmonary fibrosis. Orphanet J. Rare Dis. 3, 8.

Moore, B., Lawson, W.E., Oury, T.D., Sisson, T.H., Raghavendran, K., Hogaboam, C.M., 2013.

Animal models of fibrotic lung disease. Am. J. Respir. Cell Mol. Biol. in press.

Nalysnyk, L., Cid‐Ruzafa, J., Rotella, P., Esser, D., 2012. Incidence and prevalence of

idiopathic pulmonary fibrosis: review of the literature. Eur. Respir. Rev. 21, 355–61.

Neese, R.A., Misell, L.M., Turner, S., Chu, A., Kim, J., Cesar, D., Hoh, R., Antelo, F., Strawford,

A., McCune, J.M., Christiansen, M., Hellerstein, M.K., 2002. Measurement in vivo of

proliferation rates of slow turnover cells by 2H2O labeling of the deoxyribose moiety

of DNA. Proc. Natl. Acad. Sci. USA 99, 15345–15350.

Neese, R.A., Siler, S.Q., Cesar, D., Antelo, F., Lee, D., Misell, L., Patel, K., Tehrani, S., Shah, P.,

Hellerstein, M.K., 2001. Advances in the stable isotope‐mass spectrometric

measurement of DNA synthesis and cell proliferation. Anal. Biochem. 298, 189–195.

Parra, E.R., Teodoro, W.R., Velosa, A.P.P., De Oliveira, C.C., Yoshinari, N.H., Capelozzi, V.L.,

2006. Interstitial and vascular type V collagen morphologic disorganization in usual

interstitial pneumonia. J. Histochem. Cytochem. 54, 1315–25.

Pearson, C.A., Pearson, D., Shibahara, S., Hofsteenge, J., Chiquet‐Ehrismann, R., 1988.

Tenascin: cDNA cloning and induction by TGF‐beta. EMBO J. 7, 2977–82.

Pellicoro, A., Aucott, R.L., Ramachandran, P., Robson, A.J., Fallowfield, J. a, Snowdon, V.K.,

Hartland, S.N., Vernon, M., Duffield, J.S., Benyon, R.C., Forbes, S.J., Iredale, J.P., 2012.

Elastin accumulation is regulated at the level of degradation by macrophage

metalloelastase (MMP‐12) during experimental liver fibrosis. Hepatology 55, 1965–75.


63

Previs, S.F., Hazey, J.W., Diraison, F., Beylot, M., David, F., Brunengraber, H., 1996. Assay of

the deuterium enrichment of water via acetylene. J. Mass Spectrom. 31, 639–642.

Selman, M., Thannickal, V.J., Pardo, A., Zisman, D.A., Martinez, F.J., Lynch, J.P., 2004.

Idiopathic pulmonary fibrosis: pathogenesis and therapeutic approaches. Drugs 64,

405–30.

Sime, P.J., Xing, Z., Graham, F.L., Csaky, K.G., Gauldie, J., 1997. Adenovector‐mediated gene

transfer of active transforming growth factor‐beta1 induces prolonged severe fibrosis

in rat lung. J. Clin. Invest. 100, 768–76.

Starcher, B.C., Kuhn, C., Overton, J.E., 1978. Increased elastin and collagen content in the

lungs of hamsters receiving an intratracheal injection of bleomycin. Am. Rev. Respir.

Dis. 117, 299–305.

Suki, B., Bates, J.H., 2008. Extracellular matrix mechanics in lung parenchymal diseases.

Respir. Physiol. Neurobiol. 163, 33–43.

Tamaoki, M., Imanaka‐Yoshida, K., Yokoyama, K., Nishioka, T., Inada, H., Hiroe, M.,

Sakakura, T., Yoshida, T., 2005. Tenascin‐C regulates recruitment of myofibroblasts

during tissue repair after myocardial injury. Am. J. Pathol. 167, 71–80.

Tarantal, A.F., Chen, H., Shi, T.T., Lu, C.‐H., Fang, a B., Buckley, S., Kolb, M., Gauldie, J.,

Warburton, D., Shi, W., 2010. Overexpression of transforming growth factor‐beta1 in

fetal monkey lung results in prenatal pulmonary fibrosis. Eur. Respir. J. 36, 907–14.

Teodoro, W.R., Velosa, A.P., Witzel, S.S., Garippo, A.L., Farhat, C., Parra, E.R., Sonohara, S.,

Capelozzi, V.L., Yoshinari, N.H., 2004. Architectural remodelling in lungs of rabbits

induced by type V collagen immunization: a preliminary morphologic model to study

diffuse connective tissue diseases. Pathol. Res. Pract. 200, 681–91.

Todd, N.W., Luzina, I.G., Atamas, S.P., 2012. Molecular and cellular mechanisms of

pulmonary fibrosis. Fibrogenesis Tissue Repair 5, 11.

Tomasek, J.J., Gabbiani, G., Hinz, B., Chaponnier, C., Brown, R.A., 2002. Myofibroblasts and

mechano‐regulation of connective tissue remodelling. Nat. Rev. Mol. Cell Biol. 3, 349–

363.

Trebaul, A., Chan, E.K., Midwood, K.S., 2007. Regulation of fibroblast migration by tenascin‐

C. Biochem. Soc. Trans. 35, 695–7.

Varady, K.A., Roohk, D.J., Hellerstein, M.K., 2007. Dose effects of modified alternate‐day

fasting regimens on in vivo cell proliferation and plasma insulin‐like growth factor‐1 in

mice. J. Appl. Physiol 103, 547–551.

Wells, R.G., 2013. Tissue mechanics and fibrosis. Biochim. Biophys. Acta . in press.

Zhao, Y., Young, S.L., McIntosh, J.C., 1998. Induction of tenascin in rat lungs undergoing

bleomycin‐induced pulmonary fibrosis. Am. J. Physiol. 274, L1049–57.

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