Articles in PresS. Am J Physiol Lung Cell Mol Physiol (April 22, 2016). doi:10.1152/ajplung.00065.2016
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Surfactant dysfunction during over-expression of TGF-β1 precedes profibrotic
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lung remodeling in vivo
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Elena Lopez-Rodriguez1, Caroline Boden1, Mercedes Echaide3, Jesus Perez-Gil3, Martin Kolb4,
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Jack Gauldie4, Ulrich A. Maus2,5, Matthias Ochs1,5,6 and Lars Knudsen1,5.
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Institute of Functional and Applied Anatomy, Hannover Medical School, Hannover,
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Germany 2
Department of Experimental Pneumology, Hannover Medical School, Hannover, Germany 3
Department of Biochemistry and Molecular Biology, Faculty of Biology, Universidad
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Complutense de Madrid, Madrid, Spain 4
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Firestone Institute of Respiratory Health, McMaster University, Hamilton, Canada
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Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH),
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Member of the German Center for Lung Research (DZL) 6
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REBIRTH Cluster of Excellence
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Corresponding Author: Lars Knudsen, Prof MD Institute of Functional and Applied Anatomy, Hannover Medical School, Carl-Neuberg-Str-1 30625 Hannover Germany Phone number: +49 511 532 2888 Fax number: +49 511 532 166741 e-mail:
[email protected]
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Running title: In vivo effects of TGF-β1 in the lung
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Keywords:
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TGF-β1, TTF-1, AE2C polarity, surfactant, pulmonary fibrosis
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Copyright © 2016 by the American Physiological Society.
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ABSTRACT
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Transforming growth factor beta1 (TGF-β1) is involved in regulation of cellular proliferation,
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differentiation and fibrogenesis, inducing myofibroblast migration and increasing
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extracellular matrix synthesis. Here, TGF-β1 effects on pulmonary structure and function
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were analysed. Adenoviral mediated gene-transfer of TGF-β1 in mice lungs was performed
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and evaluated by design-based stereology, invasive pulmonary function testing and detailed
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analyses of the surfactant system one and two weeks after gene-transfer. After one week
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decreased static compliance was linked with a dramatic alveolar derecruitment without
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edema formation or increase in the volume of septal wall tissue or collagen fibrils.
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Abnormally high surface tension correlated with down-regulation of surfactant proteins B
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and C. TTF-1 expression was reduced and using PLA (proximity ligand assay) technology, we
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found Smad3 and TTF-1 forming complexes in vivo, which are normally translocated into the
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nucleus of the alveolar epithelial type II cells (AE2C), but in the presence of TGF-β1 remain in
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the cytoplasm. AE2C show altered morphology, resulting in loss of total apical surface area
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per lung and polarity. These changes of AE2C were progressive 2 weeks after gene-transfer
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and correlated with lung compliance. While static lung compliance remained low, the
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volume of septal wall tissue and collagen fibrils increased two weeks after gene-transfer. In
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this animal model, the primary effect of TGF-β1 signalling in the lung is downregulation of
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surfactant proteins, high surface tension, alveolar derecruitment and mechanical stress
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which precede fibrotic tissue remodelling and progressive loss of AE2C polarity. Initial TTF-1
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dysfunction is potentially linked to down-regulation of surfactant proteins.
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INTRODUCTION
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Transforming growth factor beta 1 (TGF-β1) is a cytokine with a pivotal role in development
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of lung fibrosis (1, 47). TGF-β1 has been previously related to fibroblast migration,
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extracellular matrix deposition and collagen accumulation (22). Molecular pathways of TGF-
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β1 start with the activation of two receptors, followed by numerous signalling cascades, one
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of which includes activation of members of the Smad family. Complexes of Smad2, Smad3
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and Smad4 translocate to the nucleus and are responsible, together with other transcription
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factors, of the expression of different genes in the cell (30, 31). Adenoviral transfection of
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active TGF-β1 has already been studied in rats and mice, where development of a fibrotic
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phenotype after a minor inflammatory phase showed a prolonged deposition of collagen
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and recruitment of myofibroblast (2, 12, 40, 48). On the other hand, TGF-β1 effects have
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also been deeply studied in vitro, especially concerning alveolar epithelial cells and
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surfactant regulation. Surfactant proteins B (SP-B) and C (SP-C) are physiologically important
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components of pulmonary surfactant, playing a key role on the maintenance of opened
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alveoli, reducing surface tension and avoiding alveolar collapse (45, 62). The effect of TGF-β1
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over-expression in the regulation of SP-B has been described in alveolar epithelial-like cells,
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NCI H441 and A549 cells (24, 27, 32), but never before in an in vivo model. SP-B is essential
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for surfactant activity, and SP-B conditional knock-out models showed increased surface
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tension leading to abnormal mechanical stress in the alveolar epithelium (36). Moreover, SP-
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B levels are reduced in patients suffering different lung diseases such as lung fibrosis (15). In
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addition to surfactant impairment, TGF-β1 has also been directly related to mechanical
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stress (7, 17) in the lung, and closely linked to epithelial-to-mesenchymal transition (EMT) (6,
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21, 28, 35, 57, 59) leading to loss of epithelial integrity (with deficiencies on cadherins and
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integrins to maintain proper cell-cell and cell-basal membrane adhesions) and remodelling of
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the lung tissue components (such as increased deposition of collagen) (3, 11, 46). The order
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of the events triggering and enhancing remodelling of the lung is still unclear, but there is
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growing evidence that surfactant dysfunction and alveolar collapse may be events preceding
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fibrotic remodelling in the lung (29). As a result alveolar epithelial cells show diverse
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phenotypes, from cells suffering ER stress and apoptosis, through those one migrating and
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transitioning to a wound-healing phenotype to those displaying a partial EMT-like process
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(46). Moreover, since alveolar epithelial type II cells (AE2C) hyperplasia is a feature of
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idiopathic pulmonary fibrosis (IPF) (9), of special interest would be to analyse both the
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temporal development and the effects of TGF-β1 on AE2C in vivo. Studying early stages in
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development of fibrotic phenotype we aim to help elucidating the mechanisms undergoing
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changes in AE2C, surfactant function, mechanical stress and chronic lung injury, thought to
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be one of the triggering events leading to lung fibrotic remodelling.
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MATERIALS AND METHODS
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Animals: 80 male C57BL/6 mice (8 weeks old; 20-25g) were obtained from Charles River
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Laboratories GmbH (Sulzfeld, Germany). The study protocol was approved by the authorities
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of Lower Saxony LAVES (Niedersächsisches Landesamt für Verbraucherschutz und
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Lebensmittelsicherheit, which houses the German equivalent of an institutional animal care
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and use committee) following European Animal Welfare Regulations (approval TVA
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12/0799).
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Adenoviral transfection and sample collection: 108 plaque forming units of AdTGFβ1223-225
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(48) or empty control vector were instilled intra-tracheally. Mice were sacrificed 1 week and
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2 weeks after adenoviral transfection.
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Invasive pulmonary function testing: Animals were anesthetized with intraperitoneal (ip)
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injection of 80mg/kg Ketamin (Anesketin®, Albrech GmbH, Aulendorf, Germany) and 5mg/kg
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Xylazin (Rompun® 2%, Bayer, Leverkusen, Germany), diluted in physiological NaCl. Following
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the cessation of pain reflexes, a cannula was surgically inserted in the trachea. The animal
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was then connected to the FlexiVent and an additional ip injection of the same anaesthesia
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solution was applied in order to maintain the animal deep in narcosis during the mechanical
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ventilation. Additionally, 0.8mg/kg Pancuroniumbromid (Pancuronium-Actavis 2mg/ml,
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Actavis, München, Germany) was ip injected to prevent spontaneous breathing. Following a
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5-10 minutes stabilization period ventilating the animal at PEEP 3cmH2O, de-recruitability
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tests were conducted as described before (29). First two recruitment manoeuvres (deep
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inflation of the lung consisting of a 3 seconds increasing ramp to 30cmH2O followed by 3
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seconds pressure hold to standardize the volume history) were applied. Then forced
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oscillation technique (FOT), consisting of baseline ventilation for 5 minutes with 30 seconds
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intervals of 8 seconds broadband, low-amplitude of 17 sinusoidal frequencies (0.5-19 Hz)
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perturbations was conducted. Respiratory impedance was calculated for each frequency by
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Fourier Transform and fit to the constant phase model to compute tissue elastance (H).
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Tissue hysteresivity is calculated dividing tissue damping (G) (data not shown) by tissue
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elastance (H). The Flexiware software calculates therefore tissue hysteresivity (G/H) values
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within the 5 minutes of FOT, we calculated an average per animal and plotted the average
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per group. Finally, 3-pressure controlled quasi-static pressure-volume (PV) loops were
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performed and quasi-static compliance (Cst) was calculated. The reported H values are the
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mean value of all the animals per group, while Cst values are mean values from 3
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measurements in each animal. Inflation of the lung before fixation was performed by deep
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inflation of the lung (up to 30cmH2O) and subsequently decreased to 10cmH2O, pressure at
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which the trachea was closed. Since mechanical differences between healthy controls and
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empty vector controls (EV control) are not recognizable, next assays were performed with
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both healthy and EV controls and referred from now on as control group to reduce the
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number of animal experiments.
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Tissue preparation and fixation: vascular perfusion-fixation was performed right after
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inflation of the lungs to 30 cm H2O during the deflation limb at a constant airway opening
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pressure of 10 cm H2O using the FlexiVent. Perfusion of the lungs was carried out with 0.9%
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NaCl and 0.25% Heparin at a constant pressure of 25cmH2O and followed by perfusion with
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the fixation solution (1.5% glutaraldehyde, 1.5% paraformaldehyde in 0.15M Hepes buffer).
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Then the whole lung-heart block was excised and further fixed by immersion in the same
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fixing solution for 24h at 4°C. Afterwards all tissue not belonging to the lungs was eliminated
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and lung volume was determined by fluid displacement. Sampling of the fixing lungs was
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performed following Systematic Uniform Random Sampling as described before (52) and
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lung sections were embedded in Technovite 8100 (Kulzer Hereaus, Wehrheim, Germany)
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(43) and further stained with 0.1% toluidine or 0.2% orceine for stereological analysis at light
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microscopy level. Lung cubes were also randomly sampled, embedded in Epon (Serva,
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Heidelberg, Germany) and contrasted with 1% Osmium tetraoxyde (EMS, Hatfield, PA, USA)
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and 4% uranyl acetate (Serva, Heidelberg, Germany) for stereological analysis under electron
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microscopy (19, 34, 37). Following the same procedure, another set of lungs were
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instillation-fixed with 10% formalin and sampled before embedding in Paraffin (Paraplast®,
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Leica, Wetzlar, Germany) and used for immunohistochemistry and proximity ligand assay.
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Histological sections and staining: Formalin-fixed paraffin-embedded tissue was stained
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with a Masson-Goldner staining kit (Merck, Darmstadt, Germany) for visualization of
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connective tissue (green).
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Design-based stereology in light microscopy and electron microscopy: A list of
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recommended and determined stereological parameter (34, 37) is given in table 1. At the
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light microscopic level, a newCAST-system (Viopharm A/S, Horsholm, Denmark) was used to
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perform systematic uniform random area sampling and to superimpose an appropriate test
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system on the fields of view. Generally a point grid was used and points hitting the structure
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of interest or the reference volume were counted so that the ratio of the counted points
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resulted in the volume fraction of the given structure within the reference space. Since the
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volume of the reference space (volume of the lung) is known, volume fractions were
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converted to total volumes in all cases. At light microscopy level from 8 animals per group 6
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to 8 sections each and at least 60 fields of view to obtain 100-200 counting events per lung
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were analysed. At electron microscopy level a transmission electron microscope (Morgagni,
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Eindhoven, Netherlands) equipped with a digital camera (Olympus Soft Imaging Systems,
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Münster, Germany) was used to obtain representatives fields of view from 5 animals per
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group. 5 to 7 sections per lung and at least 60 fields of view were analysed to obtain 100-200
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counting events per lung, providing appropriate precision of the stereological parameters.
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For superimposing a test system STEPanizer© stereology tool (53) was used.
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Western-Blot: for BAL sample preparation (n=5 per group), equal volumes of BAL samples
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were taken and mixed with loading buffer (2% SDS, 10% glycerol, 0.1% bromophenol blue,
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10% β-mercaptoethanol in 12.5 µ Tris-HCl (pH 6.8)) and boiled for 10 min. 20µl of BAL
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samples were loaded in the 16% polyacrylamide (PA) electrophoresis gel for detecting SP-B
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and SP-C and 10% PA gels to detect proSP-B and proSP-C. Proteins were transferred into
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Immun-Blot PVDF membrane (BioRad Laboratories, München, Germany) and following 2h of
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blocking (TBS-0.1% Tween 20, 5% non-fat milk, pH 7.4) at room temperature (RT), primary
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antibodies were incubated over night at 4°C. Table 2 shows the list of primary antibodies
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used for western-blot from BAL samples.
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After washing with TBS-T (TBS-0.1% Tween 20), secondary antibody (1:2000, polyclonal
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swine anti-rabbit, DAKO, Glostrup, Denmark) was incubated for 1h at RT. Blot membranes
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were developed with ECL Prime Western blotting detection reagent (Amersham Bioscience,
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Buckinghamshire, UK) and band density of exposed films (Amersham Bioscience,
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Buckinghamshire, UK) was analysed by densitometry using Image J (Rasband, W.S., ImageJ,
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U. S. National Institutes of Health, Bethesda, Maryland, USA).
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For lung homogenate (LH) samples (n=5 per group), little pieces of lung tissue (approx.
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15mg) were mixed with lysis buffer (50mM Tris-HCl pH 7.5, 150mM NaCl, 1% Triton, 0.5%
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Na-deoxycholate, 5mM EDTA and protease inhibitor cocktail (complete mini, Roche,
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Mannheim, Germany). A TissueLyser II (Qiagen, Hilden, Germany) was used to disrupt the
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tissue. Soluble proteins were isolated from the supernatant of the disrupted tissue after
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centrifugation (10000 xg, 10min, 4°C). Total protein content was determined using BCA
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protein assay (Thermo Scientific, Illinois, USA) and total 20 µg of proteins per well in loading
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buffer were loaded in 12% PA gels. Proteins were transferred into Immun-Blot PVDF
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membrane (BioRad Laboratories, München, Germany) and following 2h of blocking (TBS-
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0.1% Tween 20, 5% bovine serum albumin (BSA), pH 7.4) at room temperature (RT), primary
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antibodies were incubated over night at 4°C. A list of primary antibodies is shown in table 3.
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After washing with TBS-T (TBS-0.1% Tween 20), secondary antibodies (1:2000, polyclonal
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swine anti-rabbit-HRP or 1:1000 polyclonal rabbit anti-goat-HRP, DAKO, Glostrup, Denmark)
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were incubated for 1h at RT. Blot membranes were developed with ECL Prime Western
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blotting detection reagent (Amersham Bioscience, Buckinghamshire, UK) and band density
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of exposed high performance ECL X-ray films (Amersham Bioscience, Buckinghamshire, UK)
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was analysed by densitometry using Image J (Rasband, W.S., ImageJ, U. S. National Institutes
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of Health, Bethesda, Maryland, USA).
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RT-PCR: Total cell RNA was isolated from frozen lung tissue using ISOLATE RNA Mini Kit
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(Bioline GmbH, Berlin, Germany) and quantified by spectrometry at 260 nm. Reverse
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Transcription was performed using 1 µg of total RNA and the iScript™ cDNA Synthesis Kit
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(BioRad Laboratories, München, Germany) in a thermocycler (MasterCycler, Eppendorf,
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Hamburg, Germany). Real Time PCR was performed using iQ™ SYBR® Green Supermix
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(BioRad Laboratories, München, Germany) at a final primer concentration of 100 nm and
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50ng cDNA. Antibody-mediated hot-start iTaq™ DNA polymerase was activated after an
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initial 3 minutes denaturation step at 95°C. Denaturation of DNA was performed by holding
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further 95°C for 10 seconds. Annealing and extension temperature was set to 59°C (30
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seconds) for 40 cycles in a BioRad CFX96 RT-PCR system (BioRad Laboratories, München,
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Germany). Primer design was performed using the GeneFisher software from the University
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Bielefeld (Bielefeld, Germany) (13) and primer sequences are shown in table 4. RT-PCR data
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is represented as normalized expression (∆∆Cq) which corresponds to the relative quantity
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of the target gene normalized to the quantity of the reference gene (GAPDH). Bars
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correspond to the mean and standard error of n=6 animals per group.
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Captive Bubble Surfactometer (CBS): surfactant membranes
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samples and were assayed at 20mg/ml phospholipid concentration in a CBS as described
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before (44).
were isolated from BAL
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Proximity Ligand Assay (PLA) and quantification: allow us to detect and quantify protein
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interactions using two specific primary antibodies for the proteins of interest and two
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different secondary antibodies which contain unique DNA strands, which are ligated, when
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closed enough (
