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Fibrosis: Altered gene expression in TGF-β stimulated human fibroblasts of the Tenon

Fibrosis: Altered gene expression in TGF-β stimulated human fibroblasts of the Tenon DE GRUYTER Current Directions in Biomedical Engineering 2020;6(3): 20203111 Andreas Brietzke*, Rudolf F. Guthoff, Niels Grabow and Thomas Stahnke Fibrosis: Altered gene expression in TGF-β stimulated human fibroblasts of the Tenon Abstract: Despite decades of research, fibrosis still remains ophthalmology fibrosis is one of the main causes of a significant challenge for medicine in many different fields. postoperative complications and implant failure [2, 3]. Although there is a general model of fibrosis, the causes and Intensive research over the last 20 years has led to a characteristics of the various pathologies are as diverse as the general concept of the molecular biological mechanisms in variety of organs and tissues that can be affected by fibrosis. fibrosis development. Basically, the activation of TGF-β Moreover, fibrosis also impedes the long-term prospects of receptors by the cytokine TGF-β leads, via a complex cellular success in implantation surgery. One possibility to address network, to an increased expression of proteins of the extra this challenge is the development of biocompatible implants cellular matrix (ECM) and to the inhibition of matrix- featuring drug delivery systems loaded with antifibrotic regulating factors [4]. The ECM is defined as a non-cellular pharmaceuticals. Due to diverse regulatory mechanisms in three-dimensional macromolecular network composed of organs, tissues and also cell types, these active substances collagens, elastin, fibronectin, laminins, and several other must consequentially be designed for diverse specific proteins and glycoproteins [5]. Myofibroblasts which derive applications. Compared to fibrosis in organs like lung or from fibroblasts were the primary collagen-producing cells in liver, these mechanisms were poorly addressed in this pathological process [6]. In turn these fibroblasts not ophthalmologic research, but it is known that transforming only differ among organs, they also display heterogenous growth factor beta (TGF-β) plays a key role. phenotypes within single organs [7]. This gene expression study revealed 30 genes being From the eye, fibroblast subpopulations of four different upregulated more than two fold in TGF-β1 treated human tissues (Tenon capsule, sclera, chorioidea and orbital fat) primary tenon fibroblasts (hTF). Furthermore, 15 genes were have been reported so far by STAHNKE et al. [8]. Moreover found to be downregulated more than two fold. Tumor primary fibroblasts from these tissues were isolated and used necrosis factor (TNF), vascular endothelial growth factor A for the development of a fibrosis model to predict (VEGFA) and inhibin beta (INHBE) were particular strongly pharmacological efficacy of antifibrotic pharmaca [9, 10]. regulated in TGF-β1 treated hTFs. This study is a first insight into a molecular characterisation of the previously described fibroblast Keywords: fibrosis, ophthalmic implant, gene expression subpopulations via real-time quantitative PCR of 84 different fibrosis related genes. As a first approach, this study focused https://doi.org/10.1515/cdbme-2020-3111 on the induction of the gene expression in hTFs after stimulation with TGF-β1. On the one hand specific biomarkers might be identified to improve the characterisation of fibrotic processes in the eye, on the other 1 Introduction hand targets which can be inhibited by alternative antifibrotic pharmaceuticals might be identified. Fibrosis is a complex process of pathological wound healing that leads to the loss of physiological integrity and function in various organs and tissues [1]. Especially in 2 Materials and methods ______ 2.1 Cell culture and TGF-β treatment *Corresponding author: Andreas Brietzke: Institute for Biomedical Engineering, Rostock University Medical Center, Human primary fibroblasts from the Tenon capsule were Friedrich-Barnewitz-Straße 4, D-18119 Rostock, e-mail: andreas.brietzke@uni-rostock.de isolated as described previously by STAHNKE et al. [8]. Rudolf Guthoff, Thomas Stahnke: Department of Collection of donor material and its further use was approved Ophthalmology, Rostock University Medical Center, Doberaner by the ethics committee of the University of Rostock Straße 140, D-18057 Rostock (approval ID: A2011 11) and followed the guidelines of the Niels Grabow: Institute for Biomedical Engineering, Rostock Declaration of Helsinki. Cultivation of hTFs was carried out University Medical Center, D-18119 Rostock Open Access. © 2020 Andreas Brietzke et al., published by De Gruyter. This work is licensed under the Creative Commons Attribution 4.0 License. Andreas Brietzke et al., Fibrosis: Altered gene expression in TGF-β stimulated human fibroblasts of the Tenon — 2 in Dulbecco's Modified Eagle Medium (DMEM) - low 3 Results and Discussion glucose (MERCK, Darmstadt, Germany) with 1 mg glucose, 1 % Penicillin/Streptomycin, 10 % fetal calf serum (FCS) and Initially the evaluated fibrosis related genes were classified 3.7 g/L NaHCO  under standard cell culture conditions (37 into functional groups, to simplify the overall °C, 5 % CO2). comprehensibility of the data received. The PCR array For the TGFβ-1 treatment hTFs were seeded into two provides measurement of various cytokines, transcription cell culture flasks and incubated until 70 % confluence in factors, growth factors, regulatory proteins, proteins of signal DMEM with FCS. Before treatment, cells were incubated for transduction, ECM components and remodelling enzymes. 24 h in DMEM without FCS. Following this starving medium was removed and DMEM containing 10 ng/mL TGF-β1 was given to one of the flasks. The same procedure without TGF-β1 was performed on the second flask which served as control. 2.2 Isolation of ribonucleic acids For Isolation of the ribonucleic acids (RNA) 2 mL TRIZOL reagent (Ambion) was given to the cell culture flasks. Suspension with disrupted cell material was then transferred into two 1.5mL tubes and covered with chloroform (100 µL) and -mercaptoehtanol (10 µL). After mixing and centrifugation supernatant was transferred to a new 1.5 mL tube and covered with Trizol (400 µL), chloroform : isoamyl alcohol (40 µL) and -mercaptoehtanol (4 µL). Further purification of RNA was conducted with RNeasy mini kit (Qiagen, Hilden, Germany) including a DNAseI digestion. 2.3 cDNA synthesis and qRT-PCR Synthesis of cDNA was conducted with RT First strand kit (Qiagen). A RNA amount 0.5 µg was given to the RT mix and incubated in a thermos cycler (42°C for 15 min and subsequent 95°C for 95 min). 2 Figure 1: Fold changes of mRNA transcription obtained via qRT- RT-qPCR was carried out with RT Profiler PCR Array PCR, calculated as previously described [11]. a) cytokines, b) tran- for human fibrosis (Qiagen) in Eppendorf realplex scription factors, c) growth factors, d) regulatory proteins, e) signal Mastercycler. PCR mix containing cDNA was directly transduction proteins, f) ECM components, g) remodelling pipetted into the 96 wells of the RT Profiler PCR plate enzymes. Dashed lines indicate a fold change of 2. prepared from the manufacturer. Beside fibrosis related genes In all functional groups we found remarkable alterations of 5 housekeeping genes were detected to normalise Ct values. the mRNA transcription in TGF-β1 treated hTFs. We found For information regarding the plate layout and full gene 30 genes being upregulated more than 2 fold and 15 genes names please visit Qiagen homepage. being downregulated to the same extend. Ct values were first normalised with geometric mean of the housekeeping genes ACTB, B2M, GAPDH, HPRT1 and RPLP0. TGF-β1 treatment group was then normalised with 3.1 Cytokines the untreated control to obtain relative transcription (Ct). Within the cytokines the chemokine CC motif ligands Fold changes were subsequent calculated as described from (CCLs) might play an important role in fibrogenesis. While PFAFFL 2004 [11]. CCL2 (-14.4 fold) is involved in the recruitment of immune cells like monocytes and basophiles [12], CCL3 (14.4 fold) is responsible for neutrophil [13], and CCL11 (34.1 fold) for Andreas Brietzke et al., Fibrosis: Altered gene expression in TGF-β stimulated human fibroblasts of the Tenon — 3 eosinophil [14] recruitment. TGF-1 treated hTFs appear to Upregulation of INHBE (>223.2 fold) is known to occur set rather priority on phagocytising neutrophils and reactive when unfolded and misfolded proteins accumulate in the oxygen species releasing eosinophils then on inflammation endoplasmic reticulum of human fibroblasts [24]. This “ER inducing basophiles. stress” may result from overwhelming activation of cellular TNF-mRNA (>1000 fold), which was near the detection processes via TGF-β1. limit in untreated hTFs was strongly induced. Beside its primary role in the regulation of immune cells, the 3.5 Signal transduction proteins multifunctional cytokine is also responsible for the induction of apoptosis [15, 16]. Upregulation of TNF might here The smad family members (SMADs) are described as key compensate exuberant cell growth. Moreover myofibroblasts signalling factors of the TGF-β/SMAD signalling pathway undergo apoptosis in physiological woundhealing [25]. SMAD3 (-6.4 fold), which we found remarkably downregulated is on the one hand directly involved into the TGF-β induced transcription [26], but it is on the other hand 3.2 Transcription factors triggering the transcriptional repression of MYC induced In contrast to cytokines the transcription factors were poorly transcription [27]. regulated in the TGF-β1 treated hTFs. Nevertheless we found The TGF-β receptors (TGFBRs) 1 and 2 build a transcription factor MYC (2.4 fold) and SNAI1 (2.3 fold) heteromeric complex to bind TGF-β and to induce its upregulated while STAT6 (-2.4 fold) was downregulated. downstream signalling pathway [28]. While TGFBR1 (6.29 MYC is a general amplifier of gene expression and implicated fold) was upregulated the TGFBR2 (-2.5 fold) was found to as well with physiological as with pathological cell growth, be downregulated. Considering that only a stoichiometric proliferation, metabolism, and differentiation [17, 18]. increase in the expression of both receptors resulted in an Upregulation of MYC might be interpreted as marker amplification of the TGF-β1 signal [29], TGFBR2 function for cells with enhanced activity. independent function might be considered here. The similarly upregulated SNAI1 is a highly specific transcription factor. It is a repressor of the adhesion molecule 3.6 ECM components E-cadherin, which promotes cell adhesion and is downregulated in epithelial mesenchymal transition [19]. The upregulation of the myofibroblast marker ACTA2 (29.5 STAT6 expression is known to be induced by IL4 [20], which fold) [30] is perfectly evidencing the differentiation of hTFs we also found upregulated in TGF-β1 treated hTFs (2.8 fold). to the myofibroblast cell-type in our fibrotic cell culture This is an indicator for the involvement of the YAK-STAT model. signalling pathway, which induces genes regarding apoptosis Integrins are a complex group of transmembrane and cell cycle. receptors that mediate ECM adhesion to cell membrane [31]. ITGA1 (8.3), binding collagens, and laminins [32], was the only representative of the integrins, being remarkably 3.3 Growth factors upregulated. The growth factor CTGF (29.3 fold) which we also found strongly induced is undoubtedly a key player in physiologic 3.7 Remodelling enzymes control of cell proliferation, differentiation, and adhesion. Moreover it is critically involved in tissue fibrosis [21]. Matrix metalloproteinases (MMPs) are a family of VEGFA (> -500 fold) is a growth factor responsible for endopeptidases, which are collectively degrading all physiological but also pathological angiogenesis, which is components of the ECM [33]. MMP13 is activated by various known to be accompanied by tissue fibrosis [22]. Its other MMPs and has a central role in the formation and downregulation in hTFs must remain undetermined. remodelling of bone substance [34]. The strong upregulation of MMP13 (52.1 fold) suggests its crucial role in remodelling of ECM in TGF-β1 treated hTFs. 3.4 Regulatory Proteins SERPINE1 (39.5 fold), which was strongly upregulated, The upregulated EDN1 (15.3 fold) is involved in regulates tissue homeostasis and wound healing by inhibiting physiological wound healing but also drives fibroblast plasmin-mediated MMP activation [35]. In models of liver, activation, proliferation, and myofibroblast differentiation in lung and kidney SERPINE1 deficiency attenuates fibrosis tissue fibrosis [23]. [35]. Andreas Brietzke et al., Fibrosis: Altered gene expression in TGF-β stimulated human fibroblasts of the Tenon — 4 [16] Holbrook J, Lara-Reyna S, Jarosz-Griffiths H, McDermott M. Tumour Acknowledgment: necrosis factor signalling in health and disease. F1000Res. 2019; 8 We thank Martina Nerger, Gabriele Karsten, and Colette [17] Sabò A, Kress TR, Pelizzola M, de Pretis S, Gorski MM, Tesi A, Morelli MJ, Bora P, Doni M, Verrecchia A, Tonelli C, Fagà G, Bianchi Leyh for their excellent technical assistance. V, Ronchi A, Low D, Müller H, Guccione E, Campaner S, Amati B. Selective transcriptional regulation by Myc in cellular growth control and lymphomagenesis. Nature. 2014;511(7510): 488-492 Author Statement [18] Nie Z, Hu G, Wei G, Cui K, Yamane A, Resch W, Wang R, Green DR, Financial support by the European Social Fund (ESF) within Tessarollo L, Casellas R, Zhao K, Levens D. c-Myc is a universal the collaborative research between economy and science of amplifier of expressed genes in lymphocytes and embryonic stem cells. Cell. 2012; 151(1): 68-79 the state Mecklenburg-Vorpommern and by the Federal [19] Cano 1, Pérez-Moreno MA, Rodrigo I, Locascio A, Blanco MJ, del Ministry of Education and Research (BMBF) within Barrio MG, Portillo F, Nieto MA. The transcription factor snail controls epithelial-mesenchymal transitions by repressing E-cadherin RESPONSE "Partnership for Innovation in Implant expression. Nat Cell Biol. 2000 Feb;2(2):76-83 Technology" is gratefully acknowledged. The authors state [20] Quelle FW, Shimoda K, Thierfelder W, Fischer C, Kim A, Ruben SM, no conflict of interest. Cleveland JL, Pierce JH, Keegan AD, Nelms K, Paul WE, IHLE JN. Cloning of murine Stat6 and human Stat6, Stat proteins that are tyrosine phosphorylated in responses to IL-4 and IL-3 but are not References required for mitogenesis. Mol Cell Biol. 1995; 15(6): 3336-43 [21] Ramazani Y, Knops N, Elmonem MA, Nguyen TQ, Arcolino FO, van [1] White ES and Mantovani AR. Inflammation, wound repair, and den Heuvel L, Levtchenko E, Kuypers D, Goldschmeding R. fibrosis: reassessing the spectrum of tissue injury and resolution. J Connective tissue growth factor (CTGF) from basics to clinics. Matrix Pathol 2013; 229(2): 141-4. Biol. 2018; 68-69: 44-66 [2] Gedde SJ, Schiffman JC, Feuer WJ, Herndon LW, Brandt JD, Budenz [22] Zhang J, Chu M. Differential roles of VEGF: Relevance to tissue DL. Tube versus Trabeculectomy Study Group (2012): Treatment fibrosis. J Cell Biochem. 2019; doi: 10.1002/jcb.28489 outcomes in the Tube Versus Trabeculectomy (TVT) study after five [23] Swigris JJ, Brown KK The role of endothelin-1 in the pathogenesis of years of follow-up. Am. J. Ophthalmol 2012; 153(5):789-803. idiopathic pulmonary fibrosis. BioDrugs. 2010; 24(1): 49-54 [3] Boswell B A, Korol A, West-Mays J A, Musila L S. Dual function of [24] Dombroski BA, Nayak RR, Ewens KG, Ankener W, Cheung VG, TGFβ in lens epithelial cell fate: implications for secondary cataract. Spielman RS. Gene expression and genetic variation in response to Mol. Biol. of the cell. 2017; 28(7): 907–921. endoplasmic reticulum stress in human cells. Am J Hum Genet. 2010; [4] Schaefer L. Decoding fibrosis: Mechanisms and translational aspects. 86(5): 719-29 Matrix Biology 2018; 68–69: 1-7 [25] Massagué J. TGFβ signalling in context. Nat Rev Mol Cell Biol. [5] Theocharis AD, Skandalis SS, Gialeli C, Karamanos NK. Extracellular 2012;13(10): 616-30 matrix structure. Adv Drug Deliv Rev. 2016; 1;97: 4-27 [26] Zhang Y, Feng XH, Derynck R. Smad3 and Smad4 cooperate with c- [6] Wynn TA. Cellular and molecular mechanisms of fibrosis. J Pathol Jun/c-Fos to mediate TGF-beta-induced transcription. Nature. 1998; 2008; 214: 199–210 394(6696): 909-13 [7] Zeisberg M and Kalluri R. Cellular Mechanisms of Tissue Fibrosis. 1. [27] Frederick JP, Liberati NT, Waddell DS, Shi Y, Wang XF. Transforming Common and organ-specific mechanisms associated with tissue growth factor beta-mediated transcriptional repression of c-myc is fibrosis. Am J Physiol Cell Physiol 2013; 304: C216–C225 dependent on direct binding of Smad3 to a novel repressive Smad [8] Stahnke T, Löbler M, Kastner C, Stachs O, Wree A, Sternberg K, binding element. Mol Cell Biol. 2004; 24(6): 2546-59 Schmitz KP, Guthoff R. Different fibroblast subpopulations of the eye: [28] Franzén P, ten Dijke P, Ichijo H, Yamashita H, Schulz P, Heldin CH, A therpeutic target to prevent postoperative fibrosis in glaucoma Miyazono K. Cloning of a TGF beta type I receptor that forms a therapy. Exp. Eye Res. 2012; 100: 88-97. heteromeric complex with the TGF beta type II receptor. Cell. 1993; [9] Stahnke T, Kowtharapu BS, Stachs O, Schmitz KP, Wurm J, Wree A, 75(4): 681-92. Guthoff RF, Hovakimyan. Suppression of TGF-β pathway by [29] Ebner R, Chen RH, Shum L, Lawler S, Zioncheck TF, Lee A, Lopez pirfenidone decreases extracellular matrix deposition in ocular AR, Derynck R. Cloning of a type I TGF-beta receptor and its effect fibroblasts in vitro. PLoS ONE. 2017; 12 (2): e0172592. on TGF-beta binding to the type II receptor. Science. 1993; [10] Brietzke A, Eickner T, Reske T, Matschegewsky C, Guthoff RF, 260(5112): 1344-8 Grabow N and Stahnke T. Actinomycin D for fibrosis management in [30] Desmoulière A, Chaponnier C, Gabbiani G. Tissue repair, contraction, ophthalmic implant surgery. CDBME. 2019; 5(1): 481-484 and the myofibroblast. Wound Repair Regen. 2005; 13(1): 7-12 [11] Pfaffl MA. Real-time RT-PCR: Neue Ansätze zur exakten mRNA [31] Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P. Quantifizierung. BIOspektrum. 2004; 1: 92-95 "Integrins". Molecular Biology of the Cell (4th ed.). 2002 [12] Bischoff SC, Krieger M, Brunner T, Dahinden CA. Monocyte [32] Krieger M, Scott MP, Matsudaira PT, Lodish HF, Darnell JE, Zipursky chemotactic protein 1 is a potent activator of human basophils. J Exp L, Kaiser C, Berk A. Molecular cell biology (fifth ed.). 2004 Med. 1992; 175(5): 1271-5. [33] Murray GI. Matrix metalloproteinases: a multifunctional group of [13] Wolpe SD, Davatelis G, Sherry B, Beutler B, Hesse DG, Nguyen HT, molecules. J Pathol. 2001; 195(2): 135-7 Moldawer LL, Nathan CF, Lowry SF, Cerami A. Macrophages secrete [34] Leeman MF, Curran S, Murray GI. The structure, regulation, and a novel heparin-binding protein with inflammatory and neutrophil function of human matrix metalloproteinase-13. Crit Rev Biochem Mol chemokinetic properties. J Exp Med. 1988;167(2): 570-81. Biol. 2002; 37(3): 149-66 [14] Ponath PD, Qin S, Ringler DJ, Clark-Lewis I, Wang J, Kassam N, [35] Flevaris P, Vaughan D. he Role of Plasminogen Activator Inhibitor Smith H, Shi X, Gonzalo JA, Newman W, Gutierrez-Ramos JC, and Type-1 in Fibrosis. Semin Thromb Hemost. 2017; 43(2): 169-177 Mackay CR. Cloning of the human eosinophil chemoattractant, eotaxin. Expression, receptor binding, and functional properties suggest a mechanism for the selective recruitment of eosinophils. J Clin Invest. 1996; 97(3): 604–612 [15] Locksley RM, Killeen N, Lenardo MJ. The TNF and TNF receptor superfamilies: integrating mammalian biology. Cell. 2001;104(4): 487- http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Current Directions in Biomedical Engineering de Gruyter

Fibrosis: Altered gene expression in TGF-β stimulated human fibroblasts of the Tenon

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Abstract

DE GRUYTER Current Directions in Biomedical Engineering 2020;6(3): 20203111 Andreas Brietzke*, Rudolf F. Guthoff, Niels Grabow and Thomas Stahnke Fibrosis: Altered gene expression in TGF-β stimulated human fibroblasts of the Tenon Abstract: Despite decades of research, fibrosis still remains ophthalmology fibrosis is one of the main causes of a significant challenge for medicine in many different fields. postoperative complications and implant failure [2, 3]. Although there is a general model of fibrosis, the causes and Intensive research over the last 20 years has led to a characteristics of the various pathologies are as diverse as the general concept of the molecular biological mechanisms in variety of organs and tissues that can be affected by fibrosis. fibrosis development. Basically, the activation of TGF-β Moreover, fibrosis also impedes the long-term prospects of receptors by the cytokine TGF-β leads, via a complex cellular success in implantation surgery. One possibility to address network, to an increased expression of proteins of the extra this challenge is the development of biocompatible implants cellular matrix (ECM) and to the inhibition of matrix- featuring drug delivery systems loaded with antifibrotic regulating factors [4]. The ECM is defined as a non-cellular pharmaceuticals. Due to diverse regulatory mechanisms in three-dimensional macromolecular network composed of organs, tissues and also cell types, these active substances collagens, elastin, fibronectin, laminins, and several other must consequentially be designed for diverse specific proteins and glycoproteins [5]. Myofibroblasts which derive applications. Compared to fibrosis in organs like lung or from fibroblasts were the primary collagen-producing cells in liver, these mechanisms were poorly addressed in this pathological process [6]. In turn these fibroblasts not ophthalmologic research, but it is known that transforming only differ among organs, they also display heterogenous growth factor beta (TGF-β) plays a key role. phenotypes within single organs [7]. This gene expression study revealed 30 genes being From the eye, fibroblast subpopulations of four different upregulated more than two fold in TGF-β1 treated human tissues (Tenon capsule, sclera, chorioidea and orbital fat) primary tenon fibroblasts (hTF). Furthermore, 15 genes were have been reported so far by STAHNKE et al. [8]. Moreover found to be downregulated more than two fold. Tumor primary fibroblasts from these tissues were isolated and used necrosis factor (TNF), vascular endothelial growth factor A for the development of a fibrosis model to predict (VEGFA) and inhibin beta (INHBE) were particular strongly pharmacological efficacy of antifibrotic pharmaca [9, 10]. regulated in TGF-β1 treated hTFs. This study is a first insight into a molecular characterisation of the previously described fibroblast Keywords: fibrosis, ophthalmic implant, gene expression subpopulations via real-time quantitative PCR of 84 different fibrosis related genes. As a first approach, this study focused https://doi.org/10.1515/cdbme-2020-3111 on the induction of the gene expression in hTFs after stimulation with TGF-β1. On the one hand specific biomarkers might be identified to improve the characterisation of fibrotic processes in the eye, on the other 1 Introduction hand targets which can be inhibited by alternative antifibrotic pharmaceuticals might be identified. Fibrosis is a complex process of pathological wound healing that leads to the loss of physiological integrity and function in various organs and tissues [1]. Especially in 2 Materials and methods ______ 2.1 Cell culture and TGF-β treatment *Corresponding author: Andreas Brietzke: Institute for Biomedical Engineering, Rostock University Medical Center, Human primary fibroblasts from the Tenon capsule were Friedrich-Barnewitz-Straße 4, D-18119 Rostock, e-mail: andreas.brietzke@uni-rostock.de isolated as described previously by STAHNKE et al. [8]. Rudolf Guthoff, Thomas Stahnke: Department of Collection of donor material and its further use was approved Ophthalmology, Rostock University Medical Center, Doberaner by the ethics committee of the University of Rostock Straße 140, D-18057 Rostock (approval ID: A2011 11) and followed the guidelines of the Niels Grabow: Institute for Biomedical Engineering, Rostock Declaration of Helsinki. Cultivation of hTFs was carried out University Medical Center, D-18119 Rostock Open Access. © 2020 Andreas Brietzke et al., published by De Gruyter. This work is licensed under the Creative Commons Attribution 4.0 License. Andreas Brietzke et al., Fibrosis: Altered gene expression in TGF-β stimulated human fibroblasts of the Tenon — 2 in Dulbecco's Modified Eagle Medium (DMEM) - low 3 Results and Discussion glucose (MERCK, Darmstadt, Germany) with 1 mg glucose, 1 % Penicillin/Streptomycin, 10 % fetal calf serum (FCS) and Initially the evaluated fibrosis related genes were classified 3.7 g/L NaHCO  under standard cell culture conditions (37 into functional groups, to simplify the overall °C, 5 % CO2). comprehensibility of the data received. The PCR array For the TGFβ-1 treatment hTFs were seeded into two provides measurement of various cytokines, transcription cell culture flasks and incubated until 70 % confluence in factors, growth factors, regulatory proteins, proteins of signal DMEM with FCS. Before treatment, cells were incubated for transduction, ECM components and remodelling enzymes. 24 h in DMEM without FCS. Following this starving medium was removed and DMEM containing 10 ng/mL TGF-β1 was given to one of the flasks. The same procedure without TGF-β1 was performed on the second flask which served as control. 2.2 Isolation of ribonucleic acids For Isolation of the ribonucleic acids (RNA) 2 mL TRIZOL reagent (Ambion) was given to the cell culture flasks. Suspension with disrupted cell material was then transferred into two 1.5mL tubes and covered with chloroform (100 µL) and -mercaptoehtanol (10 µL). After mixing and centrifugation supernatant was transferred to a new 1.5 mL tube and covered with Trizol (400 µL), chloroform : isoamyl alcohol (40 µL) and -mercaptoehtanol (4 µL). Further purification of RNA was conducted with RNeasy mini kit (Qiagen, Hilden, Germany) including a DNAseI digestion. 2.3 cDNA synthesis and qRT-PCR Synthesis of cDNA was conducted with RT First strand kit (Qiagen). A RNA amount 0.5 µg was given to the RT mix and incubated in a thermos cycler (42°C for 15 min and subsequent 95°C for 95 min). 2 Figure 1: Fold changes of mRNA transcription obtained via qRT- RT-qPCR was carried out with RT Profiler PCR Array PCR, calculated as previously described [11]. a) cytokines, b) tran- for human fibrosis (Qiagen) in Eppendorf realplex scription factors, c) growth factors, d) regulatory proteins, e) signal Mastercycler. PCR mix containing cDNA was directly transduction proteins, f) ECM components, g) remodelling pipetted into the 96 wells of the RT Profiler PCR plate enzymes. Dashed lines indicate a fold change of 2. prepared from the manufacturer. Beside fibrosis related genes In all functional groups we found remarkable alterations of 5 housekeeping genes were detected to normalise Ct values. the mRNA transcription in TGF-β1 treated hTFs. We found For information regarding the plate layout and full gene 30 genes being upregulated more than 2 fold and 15 genes names please visit Qiagen homepage. being downregulated to the same extend. Ct values were first normalised with geometric mean of the housekeeping genes ACTB, B2M, GAPDH, HPRT1 and RPLP0. TGF-β1 treatment group was then normalised with 3.1 Cytokines the untreated control to obtain relative transcription (Ct). Within the cytokines the chemokine CC motif ligands Fold changes were subsequent calculated as described from (CCLs) might play an important role in fibrogenesis. While PFAFFL 2004 [11]. CCL2 (-14.4 fold) is involved in the recruitment of immune cells like monocytes and basophiles [12], CCL3 (14.4 fold) is responsible for neutrophil [13], and CCL11 (34.1 fold) for Andreas Brietzke et al., Fibrosis: Altered gene expression in TGF-β stimulated human fibroblasts of the Tenon — 3 eosinophil [14] recruitment. TGF-1 treated hTFs appear to Upregulation of INHBE (>223.2 fold) is known to occur set rather priority on phagocytising neutrophils and reactive when unfolded and misfolded proteins accumulate in the oxygen species releasing eosinophils then on inflammation endoplasmic reticulum of human fibroblasts [24]. This “ER inducing basophiles. stress” may result from overwhelming activation of cellular TNF-mRNA (>1000 fold), which was near the detection processes via TGF-β1. limit in untreated hTFs was strongly induced. Beside its primary role in the regulation of immune cells, the 3.5 Signal transduction proteins multifunctional cytokine is also responsible for the induction of apoptosis [15, 16]. Upregulation of TNF might here The smad family members (SMADs) are described as key compensate exuberant cell growth. Moreover myofibroblasts signalling factors of the TGF-β/SMAD signalling pathway undergo apoptosis in physiological woundhealing [25]. SMAD3 (-6.4 fold), which we found remarkably downregulated is on the one hand directly involved into the TGF-β induced transcription [26], but it is on the other hand 3.2 Transcription factors triggering the transcriptional repression of MYC induced In contrast to cytokines the transcription factors were poorly transcription [27]. regulated in the TGF-β1 treated hTFs. Nevertheless we found The TGF-β receptors (TGFBRs) 1 and 2 build a transcription factor MYC (2.4 fold) and SNAI1 (2.3 fold) heteromeric complex to bind TGF-β and to induce its upregulated while STAT6 (-2.4 fold) was downregulated. downstream signalling pathway [28]. While TGFBR1 (6.29 MYC is a general amplifier of gene expression and implicated fold) was upregulated the TGFBR2 (-2.5 fold) was found to as well with physiological as with pathological cell growth, be downregulated. Considering that only a stoichiometric proliferation, metabolism, and differentiation [17, 18]. increase in the expression of both receptors resulted in an Upregulation of MYC might be interpreted as marker amplification of the TGF-β1 signal [29], TGFBR2 function for cells with enhanced activity. independent function might be considered here. The similarly upregulated SNAI1 is a highly specific transcription factor. It is a repressor of the adhesion molecule 3.6 ECM components E-cadherin, which promotes cell adhesion and is downregulated in epithelial mesenchymal transition [19]. The upregulation of the myofibroblast marker ACTA2 (29.5 STAT6 expression is known to be induced by IL4 [20], which fold) [30] is perfectly evidencing the differentiation of hTFs we also found upregulated in TGF-β1 treated hTFs (2.8 fold). to the myofibroblast cell-type in our fibrotic cell culture This is an indicator for the involvement of the YAK-STAT model. signalling pathway, which induces genes regarding apoptosis Integrins are a complex group of transmembrane and cell cycle. receptors that mediate ECM adhesion to cell membrane [31]. ITGA1 (8.3), binding collagens, and laminins [32], was the only representative of the integrins, being remarkably 3.3 Growth factors upregulated. The growth factor CTGF (29.3 fold) which we also found strongly induced is undoubtedly a key player in physiologic 3.7 Remodelling enzymes control of cell proliferation, differentiation, and adhesion. Moreover it is critically involved in tissue fibrosis [21]. Matrix metalloproteinases (MMPs) are a family of VEGFA (> -500 fold) is a growth factor responsible for endopeptidases, which are collectively degrading all physiological but also pathological angiogenesis, which is components of the ECM [33]. MMP13 is activated by various known to be accompanied by tissue fibrosis [22]. Its other MMPs and has a central role in the formation and downregulation in hTFs must remain undetermined. remodelling of bone substance [34]. The strong upregulation of MMP13 (52.1 fold) suggests its crucial role in remodelling of ECM in TGF-β1 treated hTFs. 3.4 Regulatory Proteins SERPINE1 (39.5 fold), which was strongly upregulated, The upregulated EDN1 (15.3 fold) is involved in regulates tissue homeostasis and wound healing by inhibiting physiological wound healing but also drives fibroblast plasmin-mediated MMP activation [35]. 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Journal

Current Directions in Biomedical Engineering – de Gruyter

Published: Sep 1, 2020

Keywords: fibrosis; ophthalmic implant; gene expression

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