Cytoskeleton regulators in the control of corneal fibrosis

i Acknowledgements I would like to express my sincere gratitude to Professor Roger W. Beuerman for his supervision and guidance throughout this project. I am grateful for his comments and advice. I would like to thank all the staff of SERI especially Sia-Wey Yeo, Wing-Sam Lee, Jennifer Ng, Queenie Tan, Candice Ho, Jia Lin, Wan-e Lim and Dr. Amutha Veluchamy Barathi, who assisted me in many ways. My work was supported by the grant: National Research Council of Singapore grant (R739/23/2010) and Singhealth Foundation grant (R835/30/2011). I would like to thank my family for their love and support throughout my endeavours. ii TABLE OF CONTENTS CHAPTER I. INTRODUCTION…….………………………………………………. 1 Cornea structure…………………………………………………… ……… 1.1.1 Corneal stroma structureh …………………………………………… .……. 1.1.2 Corneal stroma keratocytes…………………………………………………. 1.1.3 Cytoskeleton in biology………………………………………………… …. 1.1.4 Cytoskeleton regulators in biology…………………………….……………. 1.2 Corneal fibrosis as an adverse outcome…………………… ………………. 1.3 Corneal stroma keratocytes in corneal fibrosis……………………………… 1.4 Mediators trigger corneal fibrosis……………………… …………………. 1.4.1 TGF-β1 induced fibrosis……………………………………………………. 1.4.2 TGF-β1 induced corneal fibrosis………………………… .………………. 1.5 Cytoskeleton in fibrosis……………………………………………….……. 1.5.1 Cytoskeleton regulators in fibrosis…………………………………………. 1.6 Current treatment of corneal fibrosis………………………….……………. 10 1.7 Answering the Experimental Questions……………………………………. 12 1.8 Hypothesis…………………………………………………………….……. 12 1.8.1 Specific aim…………………………………………………………………. 13 CHAPTER II. MATERIAL AND METHODS…………………………………… 14 2.1 Ethics……………………………………………………………………… . 14 2.2 Anterior Keratectomy Procedure……………………………………………. 14 2.3 Animal Experimental Groups………………………………………… …… 15 2.4 Gold-Chloride Procedure………………………………………………….…. 16 iii 2.5 Cytoskeleton Regulators RT² Profiler™ PCR Array………………………… 16 2.6 Moesin siRNA in vivo delivery…………………………………… ………. 17 2.7 List of laboratory techniques………………………………………… ……. 18 2.7.1 Preparation of tissue…………………………………………………………. 18 2.7.2 Use of exogenous protein……………………………………………………. 18 2.7.3 Histological Evaluation …………………………….………………………. 19 2.7.4 Immunochemistry……………………………………………………… …. 21 2.7.5 Imaging…………………………………………………………………… 22 2.7.6 RNA Extraction and quantification……………………………… ………. 22 2.7.7 Reverse transcription and Polymerase chain reaction…………………… 24 2.7.8 Real time RT-PCR………………………………………………………… 25 2.7.9 Total protein extraction……………………………………………………. 27 2.7.10 BCA protein assay………………………………………………………… 28 2.7. 11 Western blot analysis……………………………………………………… 28 2.8 30 Statistical analysis…………………………………………………………. Chapter III. TGF-β1-induced corneal fibrosis…………………………………… 31 3.1 Phenotypic changes of corneal stroma keratocytes in response to the anterior keratectomy…………………………………………………………………. 31 3.2 Effect of various dosing regimes of topical application of TGF-β1 on the transformation of corneal keratocytes-to-myofibroblasts in corneal fibrosis in vivo……………………………………. ……………………………………. 33 3.3 TGF-β1 induced transformation of corneal stroma keratocytes-tomyofibroblasts in the corneal fibrosis……………………………….………. 39 Up-regulation of cytoskeleton regulators in corneal fibrosis in vivo……… 41 3.4 iv Chapter IV. Moesin and fibrosis………………………………………………………. 46 4.1 Moesin functions in cytoskeleton……………… .……………………………. 46 4.2 Moesin in fibrosis…………………………………………………… .………. 46 4.3 Co-localization of moesin and α-SMA within corneal stroma keratocytes in corneal fibrosis………………………………………………………………… 47 4.4 Characterization of moesin expression in the corneal fibrosis………………… 49 4.5 Role of moesin in the corneal fibrosis in vivo…………………………………. 51 4.5.1 In vivo delivery of moesin siRNA into the cornea after an anterior keratectomy…………………………………………………………………… 51 4.5.2 The transformation of corneal stroma keratocytes-to-myofibroblasts in the corneal fibrosis is moesin-dependent…………………………………………. 56 4.5.3 Corneal haze development in the injured cornea……………………………… 60 Chapter V. Moesin and signaling in corneal fibrosis 63 5.1 Introduction to Smad signaling in fibrosis……………………………………. 63 5.2 Effect of TGF-β1 on the activation of Smad and Smad in the corneal fibrosis………………………………………………………………………… 64 5.3 Effect of moesin on TGF-β1-stimulated activation of Smad2 and Smad3 in corneal fibrosis…………………………………………………………………. 68 Chapter VI. Discussion………………………. 72 6.1 Summary of findings………………………………………………………… 72 6.1.1 Transformation of corneal stroma keratocytes to myofibroblasts in corneal fibrosis…………………………………………………………………………. 72 6.1.2 Effect of TGF-β1 on corneal fibrosis……………………….…………………. 73 6.1.3 Role of moesin in the corneal fibrosis in vivo…………………………………. 73 6.1.4 Signaling pathway relevant to the corneal fibrosis regulated by moesin in Vivo…………………………………………………………………………… 75 6.2 Limitations…………………………………………………………………… 77 v 6.3 Clinical application……………………………………………………………. 79 6.4 Future studies………………………………………………………………… 81 Chapter VII. References……………………………………………………………. 85 Chapter VIII. Appendices………………………………………………………… 131 Appendix A: Experimental animal groups……………………………………………. 132 Appendix B: Moesin siRNA sequences………………………………………………. 132 Appendix C: Antibodies………………………………………………………………. 132 Appendix D: PCR primers……………………………………………………………. 133 Chapter X. Supplementary…………………………………………………………… 134 A. Grants: ……………………………………………………………………………… 134 B. Patents………………………………………………………………………………. 134 C. Presentations at Conferences……………………………………………………… 134 D. Publications…………………………………………………………………………. 136 vi SUMMARY The avascular cornea accounts for about two-thirds of the total refractive power of the eye, therefore maintenance of corneal transparency is critical for focusing light onto the retina. The impact on vision is highlighted by the finding that corneal scar as a contributor to “corneal blindness” is the third leading cause of blindness world-wide. Corneal scar may develop as sequel to infections and a wide spectrum of corneal stromal injuries such as refractive surgery (LASIK, etc.). Currently, corneal transplantation is the major treatment regime for corneal scar. To develop more specific medical therapies or as an adjunct to surgery to prevent scar formation, the regulation of corneal scar must be understood. Fibrosis is the cellular process that leads to the formation of scar. A strong association of fibrosis with “cytoskeleton regulators” suggests this class of proteins that links the intracellular cytoskeleton with the extracellular environment may be a target for therapeutic development; however, as this is a large group of proteins, evidence for involvement of specific members of this class of proteins in the corneal fibrosis has not been developed. Recently, interest in the ERM (ezrin/radixin/moesin) family members has shown that these proteins are the organizer of membrane domains and act as links to the cytoskeleton as well as signalling pathways involved in many cellular processes. Cytoskeleton disruption agents have been shown to prevent fibrosis may involve this family of proteins. Our clinical target is the early phase of corneal wounding, before an opaque corneal scar forms. In response to corneal injury, the transformation of corneal stroma keratocytes-to-myofibroblasts is predominently responsible for the corneal fibrosis. vii To define the in vivo role of specific cytoskeleton regulators in mediating the corneal fibrosis, a mouse model with corneal fibrosis stimulated by the anterior keratectomy was established. TGF-β1 was topically applied to accelerate the corneal fibrosis process since it activates corneal stroma keratocytes to a myofibroblast phenotype expressing α-SMA accompanied by altered expression patterns of ECM components. The main results of the present study are that (1) in the corneal fibrosis, moesin was identified as the most highly induced gene among the 84 cytoskeleton regulator genes using cytoskeleton regulators RT² Profiler™ PCR array (Chapter III 3.4); (2) The upregulation of moesin in the corneal fibrosis was confirmed by RT-PCR (Chapter III 3.4) and western blot (Chapter IV 4.4) in a time-dependent manner; (3) the appearance of myofibroblasts was analyzed by examining α-SMA expression.Dual immunofluorescent staining showed that moesin colocalized with α-SMA within corneal stroma keratocytes in the corneal fibrosis (Chapter IV 4.3); (4) Up-regulation of α-SMA and moesin in the corneal fibrosis was reduced by moesin siRNA (Chapter IV 4.5.1 and 4.5.2); (5) Moesin siRNA reduced corneal opacification in corneal fibrosis (Chapter IV 4.5.3); (6) Activation of Smad and Smad in corneal fibrosis was reduced by moesin siRNA (Chapter V 5.3). In conclusion, moesin siRNA decreased the transformation of corneal stroma keratocytes-to-myofibroblasts in the corneal fibrosis, as defined by the expression of α-SMA, through the reduction of activated forms of Smad and Smad 3. Moesin may be a potential drug target for inhibiting corneal fibrosis and details of moesin-related signaling pathways would be critical for understanding corneal fibrosis. viii LIST OF TABLES 1. Experimental animal groups…………………………………………… 133 2. List of moesin siRNA sequence……………………………………… .133 3. List of antibodies……………………………………………………… 132 4. List of PCR primers…………………………………………………… .132 5. Various dosing regimes for topical application of TGF-β1 on the corneal stroma after the anterior keratectomy…………………………… 35 ix LIST OF FIGURES Histological analysis of corneal wound healing after an anterior keratectomy……………………………………………………………………20 Phenotypic changes of corneal stromal keratocytes in response to the anterior keratectomy .………………………………………… 32 Effect of various dosing regimes for TGF-β1 on the transformation of corneal keratocytes-to-myofibroblasts, as defined by the expression of α-SMA in the corneal stroma after the anterior keratectomy 35 Keratocyte-to-myofibroblast transformation induced by topical application of TGF-β1, as defined by α-SMA expression .40 Results for the response of cytoskeleton regulators to topical application of TGF-ß1 after an anterior keratectomy at PO day analyzed by RT² Profiler PCR-array ……….………………………………………………42 RT-PCR quantification of mRNA levels for cytoskeleton regulators induced by topical application of TGF-ß1 following an anterior keratectomy .44 Dual immunofluorescent staining demonstrated that moesin co-localized with α-SMA within corneal stroma keratocytes in the corneal fibrosis…………… 48 x Up-regulation of moesin in the corneal fibrosis……… ……………………50 Distribution of fluorescein–labeled moesin siRNA after in vivo delivery of moesin siRNA by iontophoresis into the cornea…………….52 10 Effect of moesin siRNA on the expression of moesin in the corneal fibrosis ……………………………………………………………….54 11 Effect of moesin siRNA on the transformation of corneal keratocytes-to-myofibroblasts in the corneal fibrosis ……………….……… 58 12 In vivo slit lamp macroscopic observation of the cornea in group 2-AK and group 5-AKTSi……………………………………………………………61 13 Effect of TGF-β1 on Smad activation …………………………………… 65 14 Effect of TGF-β1 on Smad activation ………………………………… …67 15 Effect of moesin on Smad activation …………………… ……………….69 16 Effect of moesin on Smad activation ………………….………………… 70 121 Sharma A, Mehan MM, Sinha S, Cowden JW, Mohan RR. 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Zhu HY, Riau AK, Beuerman RW. Epithelial microfilament regulators show regional distribution in mouse conjunctiva. Mol Vis. 2010;16:2215-24. Zieske JD, Guimaraes SR, Hutcheon AE. Kinetics of keratocyte proliferation in response to epithelial debridement. Experimental Eye Research 2001; 72: 33–39. 131 Chapter VIII. Appendices Appendix A: Experimental animal groups Appendix B: Moesin siRNA sequence Appendix C: Antibodies Appendix D: PCR primers 132 Appendix A: Experimental animal groups Appendix B: Moesin siRNA sequences Appendix C: Antibodies 133 Appendix D: PCR primers 134 Chapter X. Supplementary A. Grants: 1. 2010 Singhealth Foundation Grant. Recipient of the grant: Zhu Hong-Yuan. 2. 2010 Association for Research in Vision and Ophthalmology (ARVO) International Annual Meeting-International Travel Grant. Recipient of the grant: Zhu Hong-Yuan. 3. 2011 National Medical Research Council Core Grant (Singapore Eye Research Institute Pilot Grant). Recipient of the grant: Zhu Hong-Yuan. 4. 2012 DUKE-NUS Khoo Scholar Grant. Recipient of the grant: Zhu HongYuan. B. Patents: 1. Moesin a new antifibrosis target in the cornea US provisional patent US Application N061/613, 633, March 21,2012 Zhu Hong-Yuan, RW Beuerman 2. High mobility group box protein and anti-inflammaotry target for postsurgical inflammation. 2012 Zhu Hong-Yuan, RW Beuerman C. Presentations at Conferences 1. Zhu Hong-Yuan, RW Beuerman. Molecular Characteristics and Regulation of Conjunctival Epithelial Cell Replacement. 17th Singapore General Hospital Scientific Congress, 2008. (Young Investigator Award, Merit). 135 2. Zhu Hong-Yuan, RW Beuerman. Integrin signaling pathways involved in the conjunctiva stem cell. 2009, 18th Singapore General Hospital scientific congress. (Young Investigator Award, Merit). 3. Zhu Hong-Yuan, Riau AK, RW Beuerman. Epithelial microfilament regulators show regional distribution in mouse conjunctiva. 2009, USA ARVO. Florida. 4. Zhu Hong-Yuan, RW Beuerman. Betamethasone and Ketorolac used to control postsurgical intraocular inflammation in the eye have different molecular target in the inflammatory pathway. 2010, International Singapore Symposium of Immunology. 5. Zhu Hong-Yuan, RW Beuerman. Mouse Model of Postsurgical Intraocular Inflammation. 2010, USA ARVO. Florida. (Travel grant) 6. Zhu Hong-Yuan, RW Beuerman. Betamethasone and Ketorolac used to control postsurgical intraocular inflammation in the eye have different molecular target in the inflammatory pathway. 2010, Singhealth Duke-NUS scientific congress, Singapore. (Allied Health Best Paper Award, Merit) 7. Zhu Hong-Yuan, SW Yeo, RW beuerman. Therapeutic Targets for PostOperative Intra-Ocular Inflammation. 2011, Asia Arvo, Singapore 8. Zhu Hong-Yuan, SW Yeo, RW beuerman. Cytoskeleton Regulators in Corneal Fibrosis. 19th Singapore General Hospital Scientific Congress, 2011 (Young Investigator Award, Merit). 136 D. Publications 1. Moesin as a key regulator in corneal fibrosis. Zhu Hong Yuan, SW Yeo, Beuerman RW. The Ocular Surface. 2012. In press. 2. Epithelial microfilament regulators show regional distribution in mouse conjunctiva Zhu Hong-Yuan, Riau AK, Beuerman RW. Mol Vis. 2010 ;16:2215-24. 3. A polycationic antimicrobial and biocompatible hydrogel with microbe membrane suctioning Li P, Poon YF, Li W, Zhu Hong-Yuan, Yeap SH, Cao Y, Qi X, Zhou C, Lamrani M, Beuerman RW, Kang ET, Mu Y, Li CM, Chang MW, Leong SS, Chan-Park MB. Nature Material. 2011;10:149-56. 4. Expression of neural receptors in mouse meibomian gland. Zhu Hong-Yuan, Riau AK, Barathi VA, Chew J, Beuerman RW. Cornea. 2010 Jul;29(7):794-801. 5. Stimulation of specific cytokines in human conjunctival epithelial cells by defensins HNP1, HBD2, and HBD3 Li J, Zhu Hong-Yuan, Beuerman RW. Invest Ophthalmol Vis Sci. 2009;50(2):644-53. [...]... sections in hematoxylin for 2-3 minutes (staining time can be modified depending on desired staining density) 4 Wash the slides in running tap water for 2 minutes 5 Identify the nucleus staining with Scott’s tap water for 2 minutes 6 Wash the slides in tap water for 2 minutes 7 Counterstain in eosin for 3 minutes (staining time can be modified depending on desired staining intensity 8 Rinse in 95% ethanol... involvement of specific members of this class of proteins in the development of corneal fibrosis has not been developed 13 1.8.1 Specific aim The specific aims in this study were: 1 To develop a mouse model with corneal fibrosis 2 To dissect specific cytoskeleton regulators active in corneal fibrosis 3 To evaluate the in vivo role of specific cytoskeleton regulators in the regulation of corneal fibrosis. .. cytoskeletal regulators involved in the control of fibrosis in the cornea” 1.8 Hypothesis Based on the strong associations of fibrosis with cytoskeleton regulators , my hypothesis is that this class of proteins which links the intracellular cytoskeleton with the extracellular environment may be a target for therapeutic development for corneal scar; however, as this is a large group of proteins, evidence for involvement... deficient in gelsolin are protected from bleomycininduced fibrosis and gelsolin expression is crucial for the pulmonary fibrosis (Oikonomou N et al 2009) 10 Profilin participates in the reorganization of the cytoskeleton (Hinz B et al 2001b) Profilin mRNA was observed to be up-regulated by using in situ hybridization in hypertrophic fibrosis (Wu J et al 2004) Vinculin, moesin, and ezrin, were elevated in. .. TGF-β1 up-regulated the expression of a variety of cytoskeleton regulators (cofilin, profilin etc.) The details about several cytoskeleton regulators which are implicated to be involved in the fibrosis are shown below Mechanical stimulation is transduced from extracellular environment into intracellular cytoskeleton of myofibroblasts through transmembrane receptorsintegrins that link to intracellular stress... 2003) Cytoskeleton contains three main components: microfilaments, intermediate filaments, and microtubules (Heuser JE et al 1980; Fey EG et al 1984) 1.1.4 Cytoskeleton regulators in biology Rearrangements of cytoskeleton are central to the aforementioned function of cytoskeleton Several studies have highlighted the importance of cytoskeleton regulators in the control of dynamic rearrangements of cytoskeleton. .. fibers through a variety of cytoskeleton regulators (vinculin, talin, paxillin, gelsolin, FAK, ezrin–radixin–moesin proteins) (Geiger B et al 2001) Phenotypic transition of human lung fibroblasts-tomyofibroblasts induced by TGF-β1 depends on integrin signaling via focal adhesion kinase (FAK) (Thannickal VJ et al 2003) FAK, a nonreceptor protein tyrosine kinase, is central for the mechanoresponses, and... rearrangement of cytoskeleton architecture in the corneal stroma keratocytes results in increased expression of α-SMA after rabbit corneal injury Myofibroblast differentiation in mesangial cells is modulated by structure and composition of the cytoskeleton (Patel K et al 2003) Fibroblasts-tomyofibroblasts differentiation in the lung fibrosis is controlled by cytoskeleton 8 tension (Blaauboer ME et al 2011) Cytoskeleton. .. et al 2006) Paxillin has a pivotal role in transducing signals from extracellular environment into intracellular cytoskeleton (Turner CE et al 2000) and mediates the rearrangement of actin cytoskeleton into contractile elements (Cowin AJ et al 2003; O’Kane S et al 1997) The ability of paxillin to modulate fibronectin activities in wound healing may be important in the mechanism of fibrosis- free fetal... consequence of multiple stimuli, but focus is put on TGF-β1 and cytoskeleton regulators in this thesis since the cytoskeletal tension and TGF-β1 play a main role in regulating the acquisition and maintenance of the myofibroblast phenotype (Hinz B et al 2001b; Arora PD et al 1999a; Blaauboer ME et al 2011; Hinz B et al 2003) 1.4.1 TGF-β1 induced fibrosis TGF-β1 is a multifunctional regulatory cytokine and . group of proteins, evidence for involvement of specific members of this class of proteins in the corneal fibrosis has not been developed. Recently, interest in the ERM (ezrin/radixin/moesin). within corneal stroma keratocytes in corneal fibrosis ……………………………………………………………… 47 4.4 Characterization of moesin expression in the corneal fibrosis ……………… 49 4.5 Role of moesin in the corneal. transformation of corneal stroma keratocytes-to-myofibroblasts is predominently responsible for the corneal fibrosis. vii To define the in vivo role of specific cytoskeleton regulators in mediating the