Investigations on keloid pathogenesis and therapy

INVESTIGATIONS ON KELOID PATHOGENESIS AND THERAPY Anandaroop Mukhopadhyay (B.Pharm), The Tamilnadu Dr M.G.R Medical University, INDIA) THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHARMACY NATIONAL UNIVERSITY OF SINGAPORE 2007 i ACKNOWLEDGEMENT It is my pleasure to thank the many people who made this thesis possible. To start with, I would like to thank my Ph.D. supervisor Associate Professor Chan Sui Yung for her excellent guidance and support. Had it not been for her, I wouldn’t have had the courage to explore new horizons. Her critical evaluation of my research and constant encouragement invigorated me to push the limits. It is difficult to overstate my gratitude to my co-supervisor Assistant Professor Phan Toan Thang. He took me in his research team knowing fully well that I had no experience in skin biology. I thank him for having confidence in me and providing me the opportunity to be a part of the cutting edge research in skin pathogenesis in his laboratory. With his enthusiasm, his inspiration, and his great efforts to explain things clearly and simply, he helped to make research fun for me. Throughout the period of writing my thesis, he provided encouragement, sound advice, good teaching, good company, and lots of good ideas. I would have been lost without him. I am deeply indebted and thankful to my parents Subhash Chandra Mukhopadhyay and Gopa Mukhopadhyay for instilling in me, values, that helped me comprehend the importance of knowledge and pursue it. My brothers Abhiroop Mukhopadhyay and Ritobrata Banerjee have always been my mentors and I am thankful to them for their invaluable advice. I would also like to thank my uncle Dr M.G Banerjee for always believing in me, and the rest of my family members for their support. Finally I am grateful to all my friends and lab mates for making my graduate life enjoyable. ii TABLE OF CONTENTS Acknowledgement List of Publications List of Figures Abbreviations Summary BACKGROUND AND INTRODUCTION 1.1 OVERVIEW OF THE WOUND HEALING PROCESS 1.2 KELOID SCAR 1.2.1 Clinical Characteristics of Keloid .7 1.2.2 Epidemiology .9 1.2.3 Histopathology 1.2.4 Molecular Pathogenesis 11 1.2.5 Current Treatment .16 1.3 OBJECTIVES OF THE PRESENT STUDY 19 MATERIALS AND METHODS 21 2.1 MEDIA AND CHEMICALS .22 2.2 RECOMBINANT GROWTH FACTORS AND ANTIBODIES 22 2.3 PREPARATION OF NORMAL AND KELOID TISSUE 2.4 IMMUNOHISTOCHEMISTRY 24 EXTRACTS 23 2.4.1 Preparation of Paraffin Sections of Normal and Keloid Tissues .24 2.4.2 Pretreatment of Paraffin Sections for Immunohistochemistry .24 2.4.3 Probing with Antibody and Developing 25 2.5 CELL CULTURE .25 2.5.1 Keloid Keratinocyte and Fibroblast Database 25 2.5.2 Keloid and Normal Keratinocytes from Keloid scar and Normal Skin .26 2.5.3 Keloid and Normal Fibroblast from Keloid Scar and Normal Skin 26 2.5.4 Keratinocyte-fibroblast Coculture .27 2.6 CELL COUNTING 29 2.7 TREATMENT OF FIBROBLASTS WITH GROWTH FACTOR .29 2.8 TREATMENT OF CELLS WITH GLEEVEC .29 2.9 SERUM STIMULATION 30 2.10 MTT ASSAY .30 2.11 FIBROBLAST-POPULATED COLLAGEN LATTICE (FPCL) 31 2.11.1 Preparation of Fibroblast-Populated Collagen Lattices 31 2.11.2 Macroscopic Evaluation of FPCL Contraction 32 2.12 WESTERN BLOTTING 32 2.13 IMMUNOASSAY 33 iii 2.14 FLOW CYTOMETRY .33 2.15 RNASE PROTECTION ASSAY .34 2.16 RATE OF ATP SYNTHESIS .34 2.17 MEASUREMENT OF INTRACELLULAR ATP 35 2.18 STATISTICAL ANALYSIS 35 ROLE OF EPITHELIAL-MESENCHYMAL INTERACTIONS IN WOUND CONTRACTION AND SCAR CONTRACTURE 36 3.1 INTRODUCTION 37 3.2 RESULTS 39 3.3 DISCUSSION 46 ROLE OF ACTIVIN-FOLLISTATIN SYSTEM IN KELOID PATHOGENESIS .50 4.1 INTRODUCTION 51 4.2 RESULTS 53 4.3 DISCUSSION 67 ROLE OF PROTEOGLYCANS IN KELOID PATHOGENESIS 73 5.1 INTRODUCTION 74 5.2 RESULTS 77 5.3 DISCUSSION 94 ROLE OF STEM CELL FACTOR AND C-KIT SYSTEM IN KELOID PATHOGENESIS 101 6.1 INTRODUCTION 102 6.2 RESULTS 104 6.3 DISCUSSION 117 INVESTIGATION OF GLEEVEC AS A THERAPEUTIC AGENT FOR KELOID SCARS 124 7.1 INTRODUCTION 125 7.2 RESULTS 127 7.3 DISCUSSION 134 General Conclusion Appendix iv LIST OF PUBLICATIONS Publication Papers 1. Mukhopadhyay A, Tan EK, Khoo YT, Chan SY, Lim IJ, Phan TT. Conditioned medium from keloid keratinocyte/keloid fibroblast coculture induces contraction of fibroblastpopulated collagen lattices. Br J Dermatol 2005 Apr; 152(4):639-45. 2. Phan TT, Lim IJ, Aalami O, Lorget F, Khoo A, Tan EK, Mukhopadhyay A, Longaker MT Smad3 signaling plays an important role in keloid pathogenesis via epithelialmesenchymal interactions. J Pathol 2005 Oct; 207(2):232-42. 3. Khoo A, Ong CT, Tan EK, Mukhopadhyay A, IJ Lim, TT Phan. The role of Connective Tissue Growth Factor (CTGF) in the biology of epithelial-mesenchymal interactions of keloid pathogenesis. J Cell Physiol 2006 Aug; 208(2):336-43 4. Mukhopadhyay A, Chan SY, Lim IJ, Phillips DJ, Phan TT. The role of Activin System in Keloid Pathogenesis. Am J Physiol-Cell Physiol 2006 Sep 13(in press) 5. Ong CT, Khoo A, Tan EK, Mukhopadhyay A, Do DV, Han CH, IJ Lim, TT Phan. Epithelial-mesenchymal interactions in keloid pathogenesis modulate vascular endothelial growth factor expression and secretion. J Pathol 2007; 211:95-108 6. Do DV, Mukhopadhyay A, Lim IJ, Phan TT. Roles of epithelial-mesenchymal interactions in keloid scar pathogenesis and carcinogenesis. A review. Current Signal Transduction Therapy (invited review paper, in press). 7. Mukhopadhyay A, Fan S, Do DV, Khoo A, Ong CT, Lim IJ, Phan TT. Role of HGF /cMet system in keloid pathogenesis. Br J Dermatol, (in press). v Submitted manuscripts 1. Mukhopadhyay A, Khoo A, Chan SY, Aalami O, Lim IJ, Phan TT. Specific Target of Sp1 Transcription Factor: A Novel Therapeutic Approach for Keloids. 2. Mukhopadhyay A, Wong MY, Chan SY , Do DV , Khoo A , Ong CT , Cheong HH , Lim IJ , Phan TT. Syndecan-2 and Decorin - Proteoglycans with a difference: Implications in keloid pathogenesis. Presentations 1. NHG Annual Scientific Congress, Singapore, October 2004 2. AAPS (American Association of Pharmaceutical Scientists) Annual Meeting and Exposition, Baltimore, USA, November 2004 3. 17th Pharmacy Congress and Intervarsity Symposium, Singapore, July 2005 4. 3rd Meeting of the WHSS (Wound Healing Society of Singapore), Singapore, August 2005 5. Inaugural AAPS-NUS Student Chapter Symposium, Singapore, September 2005 6. Controlled Release Society Conference, Vienna, Austria, July 2006 7. Biostar Congress, Stuttgart, Germany, October 2006 vi LIST OF FIGURES FIGURE 1: SCHEMATIC REPRESENTATION OF DIFFERENT STAGES OF WOUND REPAIR (ADAPTED FROM WERNER ET AL., 2003). .6 FIGURE 2: KELOID FORMATION IN DIFFERENT PATIENTS (ADAPTED FROM MARNEROS ET AL., 2004) .8 FIGURE 3: H&E STAINING OF NORMAL SKIN AND KELOID SCAR TISSUE .10 FIGURE 4: SCHEMATIC REPRESENTATION OF PATHWAYS POTENTIALLY RESULTING IN ACCUMULATION OF COLLAGEN IN FIBROTIC SKIN DISEASES (ADAPTED FROM UITTO ET AL., 2007) 16 FIGURE 5: COCULTURE OF EPIDERMAL KERATINOCYTES AND DERMAL FIBROBLASTS AS AN IN VITRO MODEL TO STUDY EPITHELIAL-MESENCHYMAL INTERACTION .ERROR! BOOKMARK NOT DEFINED. FIGURE 6: EFFECT OF CONDITIONED MEDIA COLLECTED FROM KELOID KERATINOCYTE (KK)/KELOID FIBROBLAST (KF) CO CULTURES ON COLLAGEN CONTRACTION BY KFS .42 FIGURE 7: COMPARISON OF CONTRACTION OF COLLAGEN GEL LATTICE INCORPORATED WITH KELOID FIBROBLASTS (KFS) AND NORMAL FIBROBLASTS (NFS). 43 FIGURE 8: Α-SMOOTH MUSCLE ACTIN (Α-SMA) EXPRESSION BY KELOID FIBROBLASTS (KFS) (A) OR NORMAL DERMAL FIBROBLASTS (NFS) (B). .44 FIGURE 9: EFFECT OF ANTI TRANSFORMING GROWTH FACTOR (TGF)-Β1 NEUTRALIZING ANTIBODY ON COLLAGEN CONTRACTION BY KELOID FIBROBLAST (KF) STRAIN 48 (KF48) INDUCED BY CONDITIONED MEDIA COLLECTED FROM KELOID KERATINOCYTE (KK) STRAIN 48 (KK48)/KF48 COCULTURE. .45 FIGURE 10: INCREASED LOCALIZATION OF ACTIVIN-A AND FOLLISTATIN IN THE BASAL LAYER OF EPIDERMIS IN NORMAL AND KELOID TISSUES 57 FIGURE 11: EXPRESSION OF ENDOGENOUS ACTIVIN-ΒA MRNA DERIVED FROM NORMAL AND KELOID TISSUES. .58 FIGURE 12: ELEVATED LEVELS OF ACTIVIN-A OBTAINED FROM KELOID FIBROBLAST CULTURES .59 FIGURE 13: INCREASED PROLIFERATION OF NORMAL FIBROBLASTS WHEN COCULTURED WITH ACTIVIN-A OVEREXPRESSING HΒAHACAT CELLS. .60 vii FIGURE 14: INCREASED PROLIFERATION OF NORMAL AND KELOID FIBROBLASTS TREATED WITH RHACTIVINA. .61 FIGURE 15: EFFECT OF ACTIVIN-A, FOLLISTATIN OR TGF-Β1 ON COLLAGEN, FIBRONECTIN AND Α-SMA EXPRESSION 63 FIGURE 16: ACTIVIN-A EXPRESSION INCREASED BY EPITHELIAL-MESENCHYMAL INTERACTIONS RESULTING IN A FIBROTIC PHENOTYPE. .72 FIGURE 17: ELEVATED LEVELS OF SYNDECAN-2 IN TISSUE EXTRACTS OBTAINED FROM KELOID TISSUE. .81 FIGURE 18: SERUM GROWTH FACTORS UPREGULATED SYNDECAN-2 EXPRESSION IN NORMAL AND KELOID FIBROBLASTS. .82 FIGURE 19: INCREASED ECTODOMAIN SHEDDING OF SYNDECAN-2 IS OBSERVED IN KELOID COCULTURES AS COMPARED TO MONOCULTURES 83 FIGURE 20: EXOGENOUS RHFGF-2 STIMULATES SHEDDING OF SYNDECAN-2 FROM FIBROBLAST CELL SURFACE INTO THE CONDITIONED MEDIA 84 FIGURE 21: INCREASED LOCALIZATION OF FGF-2 IN THE BASAL LAYER OF EPIDERMIS AND DERMIS IN KELOID TISSUE. .85 FIGURE 22: ELEVATED LEVELS OF FGF-2 IN TISSUE EXTRACTS OBTAINED FROM KELOID TISSUE. .86 FIGURE 23: COCULTURED NORMAL AND KELOID KERATINOCYTES EXPRESS INCREASED LEVELS OF FGF-2 AS COMPARED TO MONOCULTURED KERATINOCYTES. .87 FIGURE 24: COCULTURED NORMAL AND KELOID FIBROBLASTS EXPRESS INCREASED LEVELS OF FGF-2 AS COMPARED TO CELLS IN MONOCULTURE .88 FIGURE 25: DOWNREGULATION OF DECORIN IN TISSUE EXTRACTS OBTAINED FROM KELOID TISSUE 89 FIGURE 26: SERUM GROWTH FACTORS DOWNREGULATE DECORIN EXPRESSION IN NORMAL AND KELOID FIBROBLASTS. .90 FIGURE 27: INCREASED SECRETORY DECORIN IN COCULTURED NORMAL FIBROBLASTS AND NOT KELOID FIBROBLASTS. .91 FIGURE 28: EFFECT OF DECORIN ON COLLAGEN, FIBRONECTIN AND Α-SMA EXPRESSION 92 FIGURE 29: ECTODOMAIN SHEDDING OF SYNDECAN-2 BY FGF-2 STIMULATED BY EPITHELIAL-MESENCHYMAL INTERACTIONS RESULTING IN A FIBROTIC PHENOTYPE 100 viii FIGURE 30: ELEVATED LEVELS OF SCF IN TISSUE EXTRACTS OBTAINED FROM KELOID TISSUE 107 FIGURE 31: SERUM GROWTH FACTORS UPREGULATED SCF EXPRESSION IN NORMAL AND KELOID FIBROBLASTS. .108 FIGURE 32: INCREASED LEVELS OF SCF IN CONDITIONED MEDIA OBTAINED FROM KELOID COCULTURES AS COMPARED TO MONOCULTURES 109 FIGURE 33: ELEVATED LEVELS OF C-KIT AND PHOSPHO C-KIT (TYR 703, TYR 721) IN TISSUE EXTRACTS OBTAINED FROM KELOID TISSUE. 110 FIGURE 34: INCREASED LOCALIZATION OF C-KIT IN THE BASAL LAYER OF EPIDERMIS OF KELOID TISSUE. 111 FIGURE 35: C-KIT UPREGULATED IN KELOID KERATINOCYTES AS COMPARED TO NORMAL KERATINOCYTES. .112 FIGURE 36: INCREASED ECTODOMAIN SHEDDING OF C-KIT IN KELOID KERATINOCYTE/KELOID FIBROBLAST COCULTURES. .112 FIGURE 37: TACE OVEREXPRESSED IN KELOID SCAR AS COMPARED TO NORMAL SKIN 115 FIGURE 38: UPREGULATION OF TACE IN KELOID FIBROBLAST COCULTURES AS COMPARED TO NORMAL COCULTURES. .116 FIGURE 39: C-KIT SYSTEM IN KELOID PATHOGENESIS 122 FIGURE 40: NO SIGNIFICANT DIFFERENCE IN THE PROLIFERATIVE POTENTIAL OF KELOID KERATINOCYTES AND KELOID FIBROBLASTS 129 FIGURE 41: GLEEVEC DOWNREGULATES EXPRESSION OF PHOSPHO AKT, PHOSPHO MTOR, PHOSPHO C-KIT (TYR 721). 130 FIGURE 42: GLEEVEC DOWNREGULATES EXPRESSION OF COLLAGEN, FIBRONECTIN, Α-SMA, VEGF, HDGF, TGF-Β1, SCF, FGF-2. 131 FIGURE 43: GLEEVEC SIGNIFICANTLY REDUCES THE CONTRACTION OF COLLAGEN GEL LATTICE INCORPORATED WITH KELOID FIBROBLASTS. 132 FIGURE 44: GLEEVEC SIGNIFICANTLY REDUCES THE INTRACELLULAR ATP AND THE RATE OF ATP SYNTHESIS IN KELOID FIBROBLASTS. 133 FIGURE 45: SUMMARY OF VARIOUS GROWTH FACTORS INVOLVED IN KELOID PATHOGENESIS AND THE POSSIBLE THERAPEUTIC TARGETS. 139 ix ABBREVIATIONS ALK Activin receptor – like kinase BMP Bone morphogenetic protein CTGF Connective tissue growth factor DMEM Dulbecco’s modified Eagle medium ECM Extracellular matrix EGF Epidermal growth factor ELISA Enzyme-Linked Immunosorbent Assay ERK Extraregulated Kinase FCS Fetal calf serum FGF-2 Basic fibroblast growth factor FPCL Fibroblast populated collagen lattice FPD Fibroproliferative disorder GADPH glyceraldehyde-3-phosphate dehydrogenase HBSS Hank’s balanced salt solution HDGF 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XVI Appendix List of patients NORMAL NF/NK1 NF2 NF/NK4 NF8 NF/NK9 NF/NK5 NF/NK3 NS31 NS32 NS33 NS34 NS35 KELOID KF6 KF46 KF3 KS/KF/KK48 KF/KK31 KF/KK32 KF/KK43 KF/KK16 KS/KF/KK 25 KF/KK30 KF/KK45 KF/KK24 KF/KK29 KS50 KS51 KS52 KS53 KS54 KS55 KS56 KS57 F M M F F F F F M M F M Chinese Indian Chinese Malay Chinese Chinese Chinese Malay Chinese Chinese Chinese Malay 27 years 32 years years 28 years 24 years 35 years 12 years 13 years 20 years 29 years 35 years 33 years Breast Abdominal Earlobe Breast Breast Breast Groin Groin Forearm Thigh Breast Forearm - F M F F F M M F M F F M F M F M M F M F Chinese Chinese Malay Indian Chinese Indian Chinese Chinese Indian Chinese Malay Malay Indian Malay Chinese Indian Malay Malay Chinese Chinese 16 years 36 years 20 years 23 years 17 years 28 years 34 years 19 years 25 years 30 years 23 years 21 years 18 years 10 years 18 years 21 years 28 years 16 years 25 years 22 years Earlobe Forearm Earlobe Earlobe Earlobe Chest Elbow Earlobe Forearm Face Forearm Chest Earlobe Face Earlobe Elbow Face Forearm Earlobe Earlobe 1 1.5 1.5 0.5 1.5 1 1.5 1 1.5 1.5 XVII XVIII [...]... all been demonstrated to be elevated in keloids However their role in keloid pathogenesis is not well understood At higher magnification using scanning electron microscopy, the random nature of fiber orientation, varying fiber length and poor bundle formation in keloids are observed Figure 3: H&E staining of normal skin and keloid scar tissues Normal Skin Keloid Scar 10 1.2.4 Molecular Pathogenesis. .. cells, mediators, growth factors, and cytokines, leading to inflammation, cell proliferation, ECM deposition, contraction and remodeling Thus a disruption in the cellular harmony leads to either a delayed healing response or execessive healing The over healing response is marked by an over exuberant deposition of collagen and other extracellular matrix components like fibronectin by fibroblasts resulting... interactions have been demonstrated to influence the expression of various transcription factors, growth factors and cytokines in vitro Phan and his group (2005) demonstrated that epidermal-dermal interactions in keloids resulted in activation of the TGFβ-Smad axis which in turn played a crucial profibrotic role in keloid pathogenesis Lim and his co-workers (2006) further demonstrated the modulation of... appreciation about the effect of silicone gel sheeting among the physicians, the mode of action of silicone is still unknown Although silicone gel is comfortable, it requires active patient compliance and long-term application which is challenging on mobile and angled anatomical sites (Sproat et al., 1992) Corticosteroid Injection: Injection of triamcinolone is efficacious for the treatment of keloid. .. epithelialmesenchymal interaction towards a contractile phenotype It was observed that epithelial-mesenchymal interactions increased the contractile response of both normal and Keloid fibroblasts in vitro In addition KF’s were shown to have an elevated contracile response to signals from the extracellular envirionment than normal fibroblasts, due to the increased expression of α-SMA, a contractile protein 1... wound, does not regress spontaneously, grows in pseudotumor fashion with distortion of the lesion and tends to recur after excision They are notoriously resistant to therapy Numerous treatment modalities are available, none of which are consistently effective (Poochareon et al., 2003) Fibroproliferative disorders (FPD) involve various pathologic fibrotic conditions which constitute a leading cause... effects for keloids but its practical use is limited to ear lobes It is generally used as a post operative adjunct for earlobe keloids (Brent et al., 1978; Russell et al., 2001) Radiotherapy: Radiotherapy has been used as monotherapy, and in combination with surgery, for hypertrophic scars and keloids It is effective in reducing the recurrence rate of scars However, monotherapy remains controversial... follistatin, in keloid pathogenesis The findings strongly suggest that activin-A is a potent inducer of fibroblast activation and involved in the pathogenesis of keloids They also emphasize the importance of follistatin in regulating activin-A bioactivity and suggest a possible therapeutic potential of follistatin in the treatment and prevention of keloids Most of the growth factors and cytokines involved... Week 6 1.2 KELOID SCAR 1.2.1 Clinical Characteristics of Keloid Keloid scars represent a pathological response to cutaneous injury and occur only in humans The term keloid is derived from a Greek word “khele” meaning crab claw (Ladin et al., 1995) Keloids usually appear as firm broad nodules, often erythematous and with a shiny surface (Marneros et al., 2004) A keloid scar extends beyond the confines... –dimethylformamide (DMF) and paraformaldehyde were all purchased from Sigma Chemical Co (USA) Methanol and acetic acid were purchased from Lab-Scan 2.2 RECOMBINANT GROWTH FACTORS AND ANTIBODIES Mouse anti-α- SMA monoclonal antibody (Sigma), mouse anti-TGF- β1 neutralizing antibody (R&D systems), Mouse anti-Collagen I,III monoclonal antibody (Monosan®antibodies-The Netherlands), rabbit anti-fibronectin monoclonal antibody . 25 2.5.1 Keloid Keratinocyte and Fibroblast Database 25 2.5.2 Keloid and Normal Keratinocytes from Keloid scar and Normal Skin 26 2.5.3 Keloid and Normal Fibroblast from Keloid Scar and Normal. INTERACTIONS IN WOUND CONTRACTION AND SCAR CONTRACTURE 36 3.1 INTRODUCTION 37 3.2 RESULTS 39 3.3 DISCUSSION 46 4 ROLE OF ACTIVIN-FOLLISTATIN SYSTEM IN KELOID PATHOGENESIS 50 4.1 INTRODUCTION. KERATINOCYTE (KK) /KELOID FIBROBLAST (KF) CO CULTURES ON COLLAGEN CONTRACTION BY KFS 42 FIGURE 7: COMPARISON OF CONTRACTION OF COLLAGEN GEL LATTICE INCORPORATED WITH KELOID FIBROBLASTS (KFS) AND NORMAL