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Connective tissue growth factor regulates transition of primary bronchial fibroblasts to myofibroblasts in asthmatic subjects
Cytokine xxx (xxxx) xxx–xxx
Contents lists available at ScienceDirect
Cytokine journal homepage: www.elsevier.com/locate/cytokine
Short communication
Connective tissue growth factor regulates transition of primary bronchial fibroblasts to myofibroblasts in asthmatic subjects Katarzyna Wójcik-Pszczołaa,c, Bogdan Jakiełaa, Hanna Pluteckaa, Paulina Koczurkiewiczc, ⁎ Zbigniew Madejab, Marta Michalikb, Marek Sanaka, a b c
Department of Molecular Biology and Clinical Genetics, 2nd Department of Internal Medicine, Jagiellonian University Medical College, Kraków, Poland Department of Cell Biology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland Department of Pharmaceutical Biochemistry, Faculty of Pharmacy, Jagiellonian University Medical College, Kraków, Poland
A R T I C L E I N F O
A B S T R A C T
Keywords: Connective tissue growth factor Transforming growth factor-beta Asthma Remodelling
Fibroblast to myofibroblast transition (FMT) contributes to bronchial wall remodelling in persistent asthma. Among other numerous factors involved, transforming growth factor type β (TGF-β) plays a pivotal role. Recently it has been demonstrated that connective tissue growth factor (CTGF), a matricellular protein, combines with TGF-β in the pathomechanism of many fibrotic disorders. However, it is not clear whether this interaction takes place in asthma as well. Primary cultures of human bronchial fibroblasts from asthmatic and non-asthmatic subjects were used to investigate the impact of CTGF and TGF-β1 on the fibroblast to myofibroblast transition. The combined activity of TGF-β1 and CTGF resulted in an average of 90% of FMT accomplished in cell lines derived from asthmatics. In this group FMT was highly dependent on the presence of CTGF produced by the cells, as shown by gene silencing experiments with the specific siRNA. Results support the important role of CTGF biosynthesis in the asthmatic bronchi amplifying FMT. This is evidenced by inhibition of TGF-β1-induced FMT following CTGF silencing in asthmatic bronchial fibroblasts. CTGF is produced by fibroblasts and contributes to the FMT phenomenon in positive loop-back, inducing and boosting TGF-β1 triggered FMT. Thus, CTGF is a promising target for pharmacological intervention in secondary prevention of bronchial remodelling in asthma.
1. Introduction Remodelling of asthmatic airways is frequently observed in patients with moderate to severe asthma [1]. This complex process follows chronic inflammation and leads to irreversible narrowing of the bronchial tree. Remodelling of airways depends on numerous cell types. Histological examination of affected subjects reveals structural abnormalities of epithelium with subepithelial fibrosis, increased angiogenesis, and smooth muscle cells proliferation, as well as accumulation of extracellular matrix, suggesting the involvement of fibroblasts and other mesenchymal cells [2]. Increased airway smooth muscle (ASM) mass in asthmatic bronchi may result from the cell proliferation and hypertrophy or influx of blood-derived mesenchymal progenitors, as well as transdifferentiation of epithelial cells or fibroblasts into a contractile phenotype. Therefore, it is plausible that fibroblasts to myofibroblasts transition (FMT) can contribute both to overproduction of extracellular matrix and smooth
⁎
muscle hyperplasia in asthmatic bronchi [3]. Various stimuli, such as growth factors, pro-inflammatory cytokines, mechanical tension and mesenchymal-epithelial interactions may induce a phenotypic switch of fibroblasts, resulting in a gradual increase of α-smooth muscle actin (αSMA) expression. α-SMA is a protein of contractile apparatus and the lineage marker for ASM [4]. Myofibroblasts can be experimentally induced from fibroblasts by transforming growth factor type β (TGF-β), a cytokine readily produced by many cell types. Connective tissue growth factor (CTGF) is another profibrotic protein involved in wound healing and numerous other pathologies. CTGF is a matricellular protein which cooperates with TGF- β in the progression of kidney, pancreas, retina, and skin fibrosis [5]. Increased levels of this growth factor have also been found in the lung tissue and plasma of asthmatics [6,7]. Our study was designed to investigate if CTGF can participate in FMT of airways. For this purpose, we used in vitro models of primary fibroblast cultures taken from asthmatic and non-asthmatic subjects.
Corresponding author at: Department of Medicine, Jagiellonian University Medical College, 8 Skawinska Str., 31-066 Krakow, Poland. E-mail address: [email protected] (M. Sanak).
http://dx.doi.org/10.1016/j.cyto.2017.09.002 Received 12 February 2017; Received in revised form 1 September 2017; Accepted 5 September 2017 1043-4666/ © 2017 Elsevier Ltd. All rights reserved.
Please cite this article as: Wójcik-Pszczola, K., Cytokine (2017), http://dx.doi.org/10.1016/j.cyto.2017.09.002
Cytokine xxx (xxxx) xxx–xxx
K. Wójcik-Pszczoła et al.
Fig. 1. CTGF promotes TGF-β1-induced FMT. (A,B) Representative immunofluorescence microphotography showing cells with α-SMA positive stress fibers derived from AS and NA HBFs. (C) Fraction of myofibroblasts following TGF-β1 and/or CTGF treatment in AS and NA HBFs cultures. (D) ELISA measurements of α-SMA protein in AS and NA HBFs cultures after TGF-β1 and/or CTGF treatment. Values represented as means with SD, * p < 0.05.
2. Material and methods
Western blot. Simultaneously, the mRNA abundance of ACTA2 in total cellular RNA was measured by Real-time Quantitative Reverse Transcription PCR (Real-Time qRT-PCR) using GAPDH as the internal housekeeping transcript. Detailed online descriptions of molecular and statistical methods are presented as further supporting information.
Bronchial biopsies were obtained from 8 asthmatic (AS) and 5 nonasthmatic (NA) subjects. Details of the fibroblast cultures used to establish primary cell lines, characterized by CTGF expression, are described elsewhere [8]. The patients’ characteristics are presented in supplementary material. Approval for the study was given by the Jagiellonian University Ethics Committee (KBET/211/B/2013 and KBET 122.6120.69.2015) and informed consent was obtained from all fibroblast donors. The human bronchial fibroblasts (HBFs) were cultured in DMEM (Sigma Aldrich) and supplemented with 10% foetal bovine serum (FBS; Gibco) at standard tissue culture conditions (37 °C, 5% CO2, 95% humidity). The experimental cultures’ HBFs were seeded at low density (5000 cells/cm2) in serum-free DMEM and supplemented with 0.1% bovine serum albumin (BSA; Sigma-Aldrich) both with, and without human recombinant TGF-β1 (5 ng/ml; BD Biosciences) and human recombinant CTGF (20 ng/ml.; Sigma-Aldrich). Inhibition of CTGF gene expression in HBFs was achieved using CTGF-specific siRNA complementary to nucleotides 1272–1290 of CTGF(CCN2) mRNA (NM_001901.2; 150 nM, Sigma Aldrich). As a control, non-targeting siRNA (60 nM, Santa Cruz Biotechnology) was used. For the transfection, each oligonucleotide was encapsulated in Lipofectamine2000 reagent (0.3% final concentration, Invitrogen). After 24 h of exposure to liposomes, cells were washed with DMEM and cultured in the supplemented DMEM medium for the following 24 h. FMT was ascertained by immunocytochemical analysis using mouse anti-α-SMA Mab to visualize α-SMA positive stress fibers in HBFs. The number of myofibroblasts were counted for the entire culture surface. Whole cells lysate was used to quantify α-SMA protein by ELISA and
3. Results and discussion Our results support the enhanced FMT of bronchial fibroblasts derived from asthmatics, a phenomenon which seems to be maintained for at least several passages of primary cells in vitro. Following co-stimulation with TGF- β1 and CTGF, this transition is extremely efficient and renders a contractile phenotype of 90% of cells on average. However, in response to TGF- β1 stimulation, CTGF is produced by HBFs as autocrine and paracrine growth factor [8]. Therefore, we evaluated HBFs response to the recombinant CTGF. An increase of α-SMA positive myofibroblasts was observed, however, the magnitude of this response differed between AS and NA cell lines. This was also noticeable after concurrent stimulation with CTGF and TGF-β1, causing the highest percentage of FMT in AS HBFs, while the effect was fourfold less in NA cells (91 ± 5% stimulated cells from AS vs. 23 ± 7% from NA; Fig. 1A and C). Quantification of α-SMA using ELISA fluorescence did not reveal any differences between AS cells stimulated with TGF-β1 alone or in combination with CTGF, although CTGF alone did not induce α-SMA (Fig. 1D). This was in contrast with results of NA cells stimulation responding similarly to CTGF + TGF-β1 than to CTGF alone (Fig. 1D). Thus, AS HBFs showed an inherent alteration of sensitivity to CTGF induced FMT in contrast to NA. Since we previously observed that HBFs can produce [8] and release CTGF following TGF-β1 stimulation (Supplementary Fig. 1), the 2
Cytokine xxx (xxxx) xxx–xxx
K. Wójcik-Pszczoła et al.
Fig. 2. Silencing of CTGF expression in AS HBFs leads to inhibition of TGF-β1-induced transition into myofibroblasts. (A) Abundance of ACTA2 transcripts encoding α-SMA after control or specific anti-CTGF siRNA treatment in TGF-β1 induced AS HBFs. GAPDH transcript was used as the internal control. Each data-point represents results of single HBF cultures studied in duplicate. α-SMA protein level after CTGF silencing and TGF-β1 treatment in AS HBFs, as measured by ELISA (B, 8 cell lines in triplicates) and Western Blot (C, 4 cell lines). (D) Percentage of myofibroblasts after CTGF silencing and TGF-β1 treatment in AS HBFs (8 cell lines in duplicates). Values represented as means with SD, * p < 0.05.
asthmatic HBFs. This observation will require replication using larger study groups, however, our conclusions are supported by other reports describing the similar efficacy of antisense CTGF oligonucleotides and anti-CTGF antibodies for prevention of FMT in rat kidney and human corneal fibroblasts used as models for TGF-β dependent fibrosis [11,12]. CTGF, as the matricellular protein, may interact with other proteins and modulate their function. It is plausible, that almost complete inhibition of TGF-β-induced FMT by CTGF silencing may result from the disruption of these interactions. In the kidney fibrosis model, CTGF inhibited Smad7. Decrease in this inhibitory protein promotes and intensifies TGF-β signalling through pSmad2/3 or Smad4 proteins [13]. Our data shed light on FMT signalling in bronchial asthma when the role of CTGF enhancing TGF-β signalling is considered [14]. Inhibition of FMT by CTGF targeting can decrease irreversible bronchial remodelling in asthmatics and has greater potential therapeutic feasibility than anti-TGF- β therapy. The latter is released by numerous structural and inflammatory cells of the lung and has important immunomodulatory properties. Also, due to the systemic distribution and abundance of inactive TGF- β form in plasma anti-TGF-β, intervention does not seem plausible. Topical intrabronchial intervention using anti-CTGF molecules seems more promising to curtail airway remodelling. Also, a similar approach recently demonstrated inhibition of ASM proliferation using a recombinant polypeptide with matrillin, entrapping extracellular CTGF [15]. These arguments all recapitulate the role of CTGF in triggering bronchial remodelling, and warrant further investigations of CTGF antagonists inhibitors or expression silencers for prevention of irreversible damage to the asthmatic lung.
possibility for therapeutic intervention by CTGF expression silencing was tested next. The novelty of the results presented is that CTGF silencing alone can reduce FTM by 60% despite simultaneous TGF-β1 stimulation. After CTGF-specific siRNA treatment, a significant decrease in the abundance of ACTA2 transcripts in TGF-β1 stimulated HBFs were observed, whereas control siRNA had no effect (Fig. 2A). This result was confirmed using both ELISA and Western Blot quantification of α-SMA protein (Fig. 2B and C). Another confirmation was that silencing of CTGF caused a statistically significant decrease in the number of myofibroblasts in AS HBFs (from 71 ± 5%) after stimulation with TGF-β1 to 22 ± 8% following siRNA transduction (Fig. 2D). Thus, this potent effect is coherent for the assessment of ACTA2 mRNA expression, cellular α-SMA level and the fraction of myofibroblasts in HBF populations. No treatment strategies for preventing remodelling of the asthmatic lung have yet been validated. Complexity of this process precludes any single remedy, nevertheless from a clinical point of view, airways smooth muscle hyperplasia seems to be the major mechanism contributing to the loss of vital capacity in moderate to severe asthmatics. It is widely accepted that both TGF-β and CTGF are involved in FMT observed in asthmatics [8,9]. Increased levels of these growth factors have been described in the lung tissue and plasma of asthmatics [6,7], moreover, CTGF was proposed as a marker of fibrosis [5]. Our observation of elevated CTGF synthesis and secretion of fibroblasts from asthmatic bronchi in response to TGF-β1 are in line with other studies [10] (although Burgess et al. used asthmatic smooth muscle cells model). We tested CTGF for incentive TGF-β1-induced FMT of bronchial fibroblasts and the combination suggested additive activity. However, fibroblasts also produce CTGF in response to TGF-β1 stimulation. CTGF alone does not seem capable of increasing FMT in asthmatics and requires concurrent stimulation with TGF-β1. We showed that inhibition of FMT using CTGF transcript silencing was highly effective in
Conflict of interest statement None declared.
3
Cytokine xxx (xxxx) xxx–xxx
K. Wójcik-Pszczoła et al.
295–300. [8] K. Wójcik, P. Koczurkiewicz, M. Michalik, M. Sanak, Transforming growth factorβ1-induced expression of connective tissue growth factor is enhanced in bronchial fibroblasts derived from asthmatic patients, Pol. Arch. Int. Med. 122 (2012) 326–332. [9] M. Michalik, M. Pierzchalska, A. Legutko, M. Ura, A. Ostaszewska, J. Soja, et al., Asthmatic bronchial fibroblasts demonstrate enhanced potential to differentiate into myofibroblasts in culture, Med. Sci. Monit. 15 (2009) 194–201. [10] J.K. Burgess, P.R. Johnson, Q. Ge, W.W. Au, M.H. Poniris, B.E. McParland, et al., Expression of connective tissue growth factor in asthmatic airway smooth muscle cells, Am. J. Respir. Crit. Care Med. 167 (2003) 71–77. [11] H. Yokoi, M. Mukoyama, T. Nagae, K. Mori, T. Suganami, K. Sawai, et al., Reduction in connective tissue growth factor by antisense treatment ameliorates renal tubulointerstitial fibrosis, J. Am. Soc. Nephrol. 15 (2004) 1430–1440. [12] Q. Wang, W. Usinger, B. Nichols, J. Gray, Seeley TW XuL, et al., Cooperative interaction of CTGF and TGF-β in animal models of fibrotic disease, Fibrogenesis Tissue Repair 4 (2011) 4. [13] M.R. Mason, Fell-Muir lecture: connective tissue growth factor (CCN2) – a pernicious and pleiotropic player in the development of kidney fibrosis, Int. J. Exp. Pathol. 94 (2013) 1–16. [14] J.G. Abreu, N.I. Ketpura, B. Reversade, E.M. Robertis, Connective-tissue growth factor (CTGF) modulates cell signalling by BMP and TGF-β, Nat. Cell Biol. 4 (2002) 599–604. [15] W. Gao, L. Cai, X. Xu, J. Fan, X. Xue, X. Yan, et al., Anti-CTGF single-chain variable fragment dimers inhibit human airway smooth muscle (ASM) cell proliferation by down-regulating p-Akt and p-mTOR levels, PLoS One 9 (2014) e113980.
Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cyto.2017.09.002. References [1] C. Bergeron, M.K. Tulic, Q. Hamid, Airway remodelling in asthma: from benchside to clinical practice, Can. Respir. J. 17 (2010) 85–93. [2] A. Shifren, C. Witt, C. Christie, M. Castro, Mechanisms of remodelling in asthmatic airways, J. Allergy (Cairo) 2012 (2012) 316049. [3] L. Bara, A. Ozier, J.-M. Tunon de Lara, R. Marthan, P. Berger, Pathophysiology of bronchial smooth muscle remodelling in asthma, Eur. Respir. J. 36 (2010) 1174–1184. [4] B. Hu, S.H. Phan, Myofibroblasts, Curr. Opin. Rheumatol. 25 (2013) 71–77. [5] A. Leask, S.K. Parapuram, X. Shi-Wen, D.J. Abraham, Connective tissue growth factor (CTGF, CCN2) gene regulation: a potent clinical bio-marker of fibroproliferative disease? J. Cell Commun. Signal 3 (2009) 89–94. [6] E. Ozyilmaz, S. Canbakan, N. Capan, A. Erturk, M. Gulhan, Correlation of plasma transforming growth factor beta 1 with asthma control test, Allergy Asthma Proc. 30 (2009) 35–40. [7] M. Kato, T. Fujisawa, D. Hashimoto, M. Kono, N. Enomoto, Y. Nakamura, et al., Plasma connective tissue growth factor levels as potential biomarkers of airway obstruction in patients with asthma, Ann. Allergy Asthma Immunol. 113 (2014)
4
Contents lists available at ScienceDirect
Cytokine journal homepage: www.elsevier.com/locate/cytokine
Short communication
Connective tissue growth factor regulates transition of primary bronchial fibroblasts to myofibroblasts in asthmatic subjects Katarzyna Wójcik-Pszczołaa,c, Bogdan Jakiełaa, Hanna Pluteckaa, Paulina Koczurkiewiczc, ⁎ Zbigniew Madejab, Marta Michalikb, Marek Sanaka, a b c
Department of Molecular Biology and Clinical Genetics, 2nd Department of Internal Medicine, Jagiellonian University Medical College, Kraków, Poland Department of Cell Biology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland Department of Pharmaceutical Biochemistry, Faculty of Pharmacy, Jagiellonian University Medical College, Kraków, Poland
A R T I C L E I N F O
A B S T R A C T
Keywords: Connective tissue growth factor Transforming growth factor-beta Asthma Remodelling
Fibroblast to myofibroblast transition (FMT) contributes to bronchial wall remodelling in persistent asthma. Among other numerous factors involved, transforming growth factor type β (TGF-β) plays a pivotal role. Recently it has been demonstrated that connective tissue growth factor (CTGF), a matricellular protein, combines with TGF-β in the pathomechanism of many fibrotic disorders. However, it is not clear whether this interaction takes place in asthma as well. Primary cultures of human bronchial fibroblasts from asthmatic and non-asthmatic subjects were used to investigate the impact of CTGF and TGF-β1 on the fibroblast to myofibroblast transition. The combined activity of TGF-β1 and CTGF resulted in an average of 90% of FMT accomplished in cell lines derived from asthmatics. In this group FMT was highly dependent on the presence of CTGF produced by the cells, as shown by gene silencing experiments with the specific siRNA. Results support the important role of CTGF biosynthesis in the asthmatic bronchi amplifying FMT. This is evidenced by inhibition of TGF-β1-induced FMT following CTGF silencing in asthmatic bronchial fibroblasts. CTGF is produced by fibroblasts and contributes to the FMT phenomenon in positive loop-back, inducing and boosting TGF-β1 triggered FMT. Thus, CTGF is a promising target for pharmacological intervention in secondary prevention of bronchial remodelling in asthma.
1. Introduction Remodelling of asthmatic airways is frequently observed in patients with moderate to severe asthma [1]. This complex process follows chronic inflammation and leads to irreversible narrowing of the bronchial tree. Remodelling of airways depends on numerous cell types. Histological examination of affected subjects reveals structural abnormalities of epithelium with subepithelial fibrosis, increased angiogenesis, and smooth muscle cells proliferation, as well as accumulation of extracellular matrix, suggesting the involvement of fibroblasts and other mesenchymal cells [2]. Increased airway smooth muscle (ASM) mass in asthmatic bronchi may result from the cell proliferation and hypertrophy or influx of blood-derived mesenchymal progenitors, as well as transdifferentiation of epithelial cells or fibroblasts into a contractile phenotype. Therefore, it is plausible that fibroblasts to myofibroblasts transition (FMT) can contribute both to overproduction of extracellular matrix and smooth
⁎
muscle hyperplasia in asthmatic bronchi [3]. Various stimuli, such as growth factors, pro-inflammatory cytokines, mechanical tension and mesenchymal-epithelial interactions may induce a phenotypic switch of fibroblasts, resulting in a gradual increase of α-smooth muscle actin (αSMA) expression. α-SMA is a protein of contractile apparatus and the lineage marker for ASM [4]. Myofibroblasts can be experimentally induced from fibroblasts by transforming growth factor type β (TGF-β), a cytokine readily produced by many cell types. Connective tissue growth factor (CTGF) is another profibrotic protein involved in wound healing and numerous other pathologies. CTGF is a matricellular protein which cooperates with TGF- β in the progression of kidney, pancreas, retina, and skin fibrosis [5]. Increased levels of this growth factor have also been found in the lung tissue and plasma of asthmatics [6,7]. Our study was designed to investigate if CTGF can participate in FMT of airways. For this purpose, we used in vitro models of primary fibroblast cultures taken from asthmatic and non-asthmatic subjects.
Corresponding author at: Department of Medicine, Jagiellonian University Medical College, 8 Skawinska Str., 31-066 Krakow, Poland. E-mail address: [email protected] (M. Sanak).
http://dx.doi.org/10.1016/j.cyto.2017.09.002 Received 12 February 2017; Received in revised form 1 September 2017; Accepted 5 September 2017 1043-4666/ © 2017 Elsevier Ltd. All rights reserved.
Please cite this article as: Wójcik-Pszczola, K., Cytokine (2017), http://dx.doi.org/10.1016/j.cyto.2017.09.002
Cytokine xxx (xxxx) xxx–xxx
K. Wójcik-Pszczoła et al.
Fig. 1. CTGF promotes TGF-β1-induced FMT. (A,B) Representative immunofluorescence microphotography showing cells with α-SMA positive stress fibers derived from AS and NA HBFs. (C) Fraction of myofibroblasts following TGF-β1 and/or CTGF treatment in AS and NA HBFs cultures. (D) ELISA measurements of α-SMA protein in AS and NA HBFs cultures after TGF-β1 and/or CTGF treatment. Values represented as means with SD, * p < 0.05.
2. Material and methods
Western blot. Simultaneously, the mRNA abundance of ACTA2 in total cellular RNA was measured by Real-time Quantitative Reverse Transcription PCR (Real-Time qRT-PCR) using GAPDH as the internal housekeeping transcript. Detailed online descriptions of molecular and statistical methods are presented as further supporting information.
Bronchial biopsies were obtained from 8 asthmatic (AS) and 5 nonasthmatic (NA) subjects. Details of the fibroblast cultures used to establish primary cell lines, characterized by CTGF expression, are described elsewhere [8]. The patients’ characteristics are presented in supplementary material. Approval for the study was given by the Jagiellonian University Ethics Committee (KBET/211/B/2013 and KBET 122.6120.69.2015) and informed consent was obtained from all fibroblast donors. The human bronchial fibroblasts (HBFs) were cultured in DMEM (Sigma Aldrich) and supplemented with 10% foetal bovine serum (FBS; Gibco) at standard tissue culture conditions (37 °C, 5% CO2, 95% humidity). The experimental cultures’ HBFs were seeded at low density (5000 cells/cm2) in serum-free DMEM and supplemented with 0.1% bovine serum albumin (BSA; Sigma-Aldrich) both with, and without human recombinant TGF-β1 (5 ng/ml; BD Biosciences) and human recombinant CTGF (20 ng/ml.; Sigma-Aldrich). Inhibition of CTGF gene expression in HBFs was achieved using CTGF-specific siRNA complementary to nucleotides 1272–1290 of CTGF(CCN2) mRNA (NM_001901.2; 150 nM, Sigma Aldrich). As a control, non-targeting siRNA (60 nM, Santa Cruz Biotechnology) was used. For the transfection, each oligonucleotide was encapsulated in Lipofectamine2000 reagent (0.3% final concentration, Invitrogen). After 24 h of exposure to liposomes, cells were washed with DMEM and cultured in the supplemented DMEM medium for the following 24 h. FMT was ascertained by immunocytochemical analysis using mouse anti-α-SMA Mab to visualize α-SMA positive stress fibers in HBFs. The number of myofibroblasts were counted for the entire culture surface. Whole cells lysate was used to quantify α-SMA protein by ELISA and
3. Results and discussion Our results support the enhanced FMT of bronchial fibroblasts derived from asthmatics, a phenomenon which seems to be maintained for at least several passages of primary cells in vitro. Following co-stimulation with TGF- β1 and CTGF, this transition is extremely efficient and renders a contractile phenotype of 90% of cells on average. However, in response to TGF- β1 stimulation, CTGF is produced by HBFs as autocrine and paracrine growth factor [8]. Therefore, we evaluated HBFs response to the recombinant CTGF. An increase of α-SMA positive myofibroblasts was observed, however, the magnitude of this response differed between AS and NA cell lines. This was also noticeable after concurrent stimulation with CTGF and TGF-β1, causing the highest percentage of FMT in AS HBFs, while the effect was fourfold less in NA cells (91 ± 5% stimulated cells from AS vs. 23 ± 7% from NA; Fig. 1A and C). Quantification of α-SMA using ELISA fluorescence did not reveal any differences between AS cells stimulated with TGF-β1 alone or in combination with CTGF, although CTGF alone did not induce α-SMA (Fig. 1D). This was in contrast with results of NA cells stimulation responding similarly to CTGF + TGF-β1 than to CTGF alone (Fig. 1D). Thus, AS HBFs showed an inherent alteration of sensitivity to CTGF induced FMT in contrast to NA. Since we previously observed that HBFs can produce [8] and release CTGF following TGF-β1 stimulation (Supplementary Fig. 1), the 2
Cytokine xxx (xxxx) xxx–xxx
K. Wójcik-Pszczoła et al.
Fig. 2. Silencing of CTGF expression in AS HBFs leads to inhibition of TGF-β1-induced transition into myofibroblasts. (A) Abundance of ACTA2 transcripts encoding α-SMA after control or specific anti-CTGF siRNA treatment in TGF-β1 induced AS HBFs. GAPDH transcript was used as the internal control. Each data-point represents results of single HBF cultures studied in duplicate. α-SMA protein level after CTGF silencing and TGF-β1 treatment in AS HBFs, as measured by ELISA (B, 8 cell lines in triplicates) and Western Blot (C, 4 cell lines). (D) Percentage of myofibroblasts after CTGF silencing and TGF-β1 treatment in AS HBFs (8 cell lines in duplicates). Values represented as means with SD, * p < 0.05.
asthmatic HBFs. This observation will require replication using larger study groups, however, our conclusions are supported by other reports describing the similar efficacy of antisense CTGF oligonucleotides and anti-CTGF antibodies for prevention of FMT in rat kidney and human corneal fibroblasts used as models for TGF-β dependent fibrosis [11,12]. CTGF, as the matricellular protein, may interact with other proteins and modulate their function. It is plausible, that almost complete inhibition of TGF-β-induced FMT by CTGF silencing may result from the disruption of these interactions. In the kidney fibrosis model, CTGF inhibited Smad7. Decrease in this inhibitory protein promotes and intensifies TGF-β signalling through pSmad2/3 or Smad4 proteins [13]. Our data shed light on FMT signalling in bronchial asthma when the role of CTGF enhancing TGF-β signalling is considered [14]. Inhibition of FMT by CTGF targeting can decrease irreversible bronchial remodelling in asthmatics and has greater potential therapeutic feasibility than anti-TGF- β therapy. The latter is released by numerous structural and inflammatory cells of the lung and has important immunomodulatory properties. Also, due to the systemic distribution and abundance of inactive TGF- β form in plasma anti-TGF-β, intervention does not seem plausible. Topical intrabronchial intervention using anti-CTGF molecules seems more promising to curtail airway remodelling. Also, a similar approach recently demonstrated inhibition of ASM proliferation using a recombinant polypeptide with matrillin, entrapping extracellular CTGF [15]. These arguments all recapitulate the role of CTGF in triggering bronchial remodelling, and warrant further investigations of CTGF antagonists inhibitors or expression silencers for prevention of irreversible damage to the asthmatic lung.
possibility for therapeutic intervention by CTGF expression silencing was tested next. The novelty of the results presented is that CTGF silencing alone can reduce FTM by 60% despite simultaneous TGF-β1 stimulation. After CTGF-specific siRNA treatment, a significant decrease in the abundance of ACTA2 transcripts in TGF-β1 stimulated HBFs were observed, whereas control siRNA had no effect (Fig. 2A). This result was confirmed using both ELISA and Western Blot quantification of α-SMA protein (Fig. 2B and C). Another confirmation was that silencing of CTGF caused a statistically significant decrease in the number of myofibroblasts in AS HBFs (from 71 ± 5%) after stimulation with TGF-β1 to 22 ± 8% following siRNA transduction (Fig. 2D). Thus, this potent effect is coherent for the assessment of ACTA2 mRNA expression, cellular α-SMA level and the fraction of myofibroblasts in HBF populations. No treatment strategies for preventing remodelling of the asthmatic lung have yet been validated. Complexity of this process precludes any single remedy, nevertheless from a clinical point of view, airways smooth muscle hyperplasia seems to be the major mechanism contributing to the loss of vital capacity in moderate to severe asthmatics. It is widely accepted that both TGF-β and CTGF are involved in FMT observed in asthmatics [8,9]. Increased levels of these growth factors have been described in the lung tissue and plasma of asthmatics [6,7], moreover, CTGF was proposed as a marker of fibrosis [5]. Our observation of elevated CTGF synthesis and secretion of fibroblasts from asthmatic bronchi in response to TGF-β1 are in line with other studies [10] (although Burgess et al. used asthmatic smooth muscle cells model). We tested CTGF for incentive TGF-β1-induced FMT of bronchial fibroblasts and the combination suggested additive activity. However, fibroblasts also produce CTGF in response to TGF-β1 stimulation. CTGF alone does not seem capable of increasing FMT in asthmatics and requires concurrent stimulation with TGF-β1. We showed that inhibition of FMT using CTGF transcript silencing was highly effective in
Conflict of interest statement None declared.
3
Cytokine xxx (xxxx) xxx–xxx
K. Wójcik-Pszczoła et al.
295–300. [8] K. Wójcik, P. Koczurkiewicz, M. Michalik, M. Sanak, Transforming growth factorβ1-induced expression of connective tissue growth factor is enhanced in bronchial fibroblasts derived from asthmatic patients, Pol. Arch. Int. Med. 122 (2012) 326–332. [9] M. Michalik, M. Pierzchalska, A. Legutko, M. Ura, A. Ostaszewska, J. Soja, et al., Asthmatic bronchial fibroblasts demonstrate enhanced potential to differentiate into myofibroblasts in culture, Med. Sci. Monit. 15 (2009) 194–201. [10] J.K. Burgess, P.R. Johnson, Q. Ge, W.W. Au, M.H. Poniris, B.E. McParland, et al., Expression of connective tissue growth factor in asthmatic airway smooth muscle cells, Am. J. Respir. Crit. Care Med. 167 (2003) 71–77. [11] H. Yokoi, M. Mukoyama, T. Nagae, K. Mori, T. Suganami, K. Sawai, et al., Reduction in connective tissue growth factor by antisense treatment ameliorates renal tubulointerstitial fibrosis, J. Am. Soc. Nephrol. 15 (2004) 1430–1440. [12] Q. Wang, W. Usinger, B. Nichols, J. Gray, Seeley TW XuL, et al., Cooperative interaction of CTGF and TGF-β in animal models of fibrotic disease, Fibrogenesis Tissue Repair 4 (2011) 4. [13] M.R. Mason, Fell-Muir lecture: connective tissue growth factor (CCN2) – a pernicious and pleiotropic player in the development of kidney fibrosis, Int. J. Exp. Pathol. 94 (2013) 1–16. [14] J.G. Abreu, N.I. Ketpura, B. Reversade, E.M. Robertis, Connective-tissue growth factor (CTGF) modulates cell signalling by BMP and TGF-β, Nat. Cell Biol. 4 (2002) 599–604. [15] W. Gao, L. Cai, X. Xu, J. Fan, X. Xue, X. Yan, et al., Anti-CTGF single-chain variable fragment dimers inhibit human airway smooth muscle (ASM) cell proliferation by down-regulating p-Akt and p-mTOR levels, PLoS One 9 (2014) e113980.
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