MOLECULAR AND COMPUTATIONAL APPROACHES TO UNDERSTANDING KELOID SCARRING OOI NICK SERN, BRANDON (B. Eng. (Hons.), NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY GRADUATE PROGRAMME IN BIOENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2010 ACKNOWLEDGEMENTS First and foremost, I would like to express my sincerest gratitude to the Graduate Programme in Bioengineering for giving me the opportunity to pursue my PhD studies. By having access to the wonderful resources at the National University of Singapore, I have been greatly facilitated in my quest for knowledge and learning. I would also like to thank my supervisors, Prof Phan Toan Thang and Prof Thiagarajan for their invaluable advice and enthusiastic support throughout my candidature. I have learnt a great deal from my interactions with them and I believe that this will stand me in good stead for my future endeavors. A big thank you also goes out to my Thesis Steering Committee comprising of Prof Bay Boon Huat and Dr Martin Lindsay Buist for their constructive advice and helpful suggestions. Their comments have helped shape this thesis in more ways than one. In all areas of work, colleagues play an immense role in the learning and development of any project. Here I am indebted to Dr Anandaroop Mukhopadhyay, Dr Masilamani Jeyakumar, Ms Audrey Khoo, Mr Ong Chee Tian, Ms Zhou Yue and Mr Do Dang Vinh from the Wound Healing and Stem Cell Research Group, and to Dr Geoffrey Koh and Mr Liu Bing from the Computational Systems Biology Group. It is through them that I have learnt the in vitro and in silico techniques that were essential to my project. Their companionship has also been most welcome during the long years of my candidature. Special thanks also go to Dr Lim Cheh Peng from the Institute of Molecular and Cell Biology for checking my paper manuscripts, and also for the helpful support in microarray work and analysis. i Last but definitely not least, I would like to thank my family and countless friends who have supported and encouraged me throughout my PhD years. It is with their unwavering support and with God’s grace that I now stand at the brink of completion of this project. ii TABLE OF CONTENTS Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix List of Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi List of Figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii List of Symbols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xv List of Presentations and Publications . . . . . . . . . . . . . . . . . . . . . . . . xx Chapter One: Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1 Backround and motivations for the study . . . . . . . . . . . . . . . . 1.2 Approach and methodology . . . . . . . . . . . . . . . . . . . . . . . . 1.3 Contributions of the thesis . . . . . . . . . . . . . . . . . . . . . . . . . 1.4 Organization of the thesis . . . . . . . . . . . . . . . . . . . . . . . . . Chapter Two: Literature Review . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Wound healing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 2.1.1 Hemostasis and inflammation . . . . . . . . . . . . . . . . . . 2.1.2 Proliferation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.3 Remodeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Keloid scarring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 2.2.1 Keloid versus hypertrophic scar . . . . . . . . . . . . . . . . 10 2.2.2 Epidemiology . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 2.2.3 Clinical presentation . . . . . . . . . . . . . . . . . . . . . . 11 2.2.4 Histopathology . . . . . . . . . . . . . . . . . . . . . . . . . . 12 iii 2.2.5 Etiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 2.2.6 Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Chapter Three: Materials and Methods . . . . . . . . . . . . . . . . . . . . . 17 3.1 Media and chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 3.2 Cell isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 3.2.1 Keloid keratinocyte and fibroblast database . . . . . . . . . . 18 3.2.2 Keratinocyte culture from keloid scar and normal skin . . . 18 3.2.3 Fibroblast culture from keloid scar and normal skin . . . 19 3.2.4 Cell counting . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 3.3 HDGF experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 3.3.1 Immunohistochemistry . . . . . . . . . . . . . . . . . . . . . . 20 3.3.2 Serum stimulation of fibroblasts . . . . . . . . . . . . . . . . 21 3.3.3 Keratinocyte-fibroblast co-culture . . . . . . . . . . . . . . . 21 3.3.4 Treatment of fibroblasts with HDGF . . . . . . . . . . . . . 22 3.3.5 Treatment of keloid co-cultures with inhibitors . . . . . . . 22 3.3.6 Smad-null and Smad-overexpression cell assay . . . . . . . . 23 3.3.7 MTT assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 3.3.8 Western blotting . . . . . . . . . . . . . . . . . . . . . . . . . 24 3.3.9 Quantification and statistical analysis . . . . . . . . . . . . . 25 3.4 Microarray experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 3.4.1 Cell culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 3.4.2 RNA extraction . . . . . . . . . . . . . . . . . . . . . . . . . 26 3.4.3 cRNA preparation and labeling . . . . . . . . . . . . . . . . . 27 iv 3.4.4 Affymetrix chip hybridization and scanning . . . . . . . . . . 27 3.4.5 Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 3.5 Reverse engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 3.5.1 Preparation of additional microarray samples . . . . . . . . 29 3.5.2 Data preprocessing . . . . . . . . . . . . . . . . . . . . . . . . 29 3.5.3 Application of the fREDUCE algorithm . . . . . . . . . . . . 30 3.5.4 Pathways selected for influence approach . . . . . . . . . . 31 3.5.5 Application of the ARACNE and BANJO algorithms . . . 33 3.5.6 Estimation of the performance of the algorithms . . . . . . 34 Chapter Four: The Role of Hepatoma-derived Growth Factor in Keloid Pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 4.2 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 4.2.1 HDGF expression is increased in keloid scar dermis. . . . . 39 4.2.2 Serum stimulation and epithelial-mesenchymal interactions had no effect on intracellular HDGF expression . . . . . . . . . . . . 41 4.2.3 Epithelial-mesenchymal interactions in keloid co-culture increased secretion of HDGF . . . . . . . . . . . . . . . . . . . . . . . . . 42 4.2.4 Increased keloid fibroblast proliferation upon stimulation with HDGF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 4.2.5 Treatment of fibroblasts with HDGF activated the ERK pathway, increased the secretion of VEGF, and decreased the secretion of collagen I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 v 4.2.6 Treatment with mTOR and Sp1 inhibitors did not significantly affect the production of HDGF . . . . . . . . . . . . . . . . . . . . . 49 4.2.7 Knockout of Smad 2/3 signaling increases intracellular HDGF expression while knockout of Smad signaling increases extracellular HDGF expression . . . . . . . . . . . . . . . . . . 50 4.3 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 Chapter Five: Genome Wide Transcriptional Profiling of Serum Starved Keloid and Normal Fibroblasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 5.2 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66 5.2.1 The time factor did not result in any systematic differences in the transcriptional profile of the fibroblast cells . . . . . . . . . 66 5.2.2 Genes significantly upregulated in keloid compared to normal fibroblasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 5.2.3 Genes significantly downregulated in keloid compared to normal fibroblasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 5.2.4 Hierarchical clustering and principal components analysis revealed that genes chosen were capable of distinguishing between keloid and normal samples . . . . . . . . . . . . . . . . . . . . . . . . . . 72 5.2.5 DAVID analysis suggests a role for immunological factors and ribosomal proteins in keloid pathogenesis . . . . . . . . . . . 74 5.3 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 vi Chapter Six: Reverse Engineering Gene Networks in Keloid and Normal Fibroblasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 6.2 Algorithms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 6.2.1 fREDUCE . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 6.2.2 ARACNE . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96 6.2.3 BANJO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98 6.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98 6.3.1 Binding motifs found from fREDUCE for keloid versus normal fibroblasts under serum starvation condition . . . . . . . . . 100 6.3.2 Binding motifs found from fREDUCE for keloid versus normal fibroblasts under serum induced condition . . . . . . . . . . 101 6.3.3 Binding motifs found from fREDUCE for sets C and D suggest consistent effects from steroid induction for both keloid and normal fibroblasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102 6.3.4 Not many binding motifs found from fREDUCE for sets E and F. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 6.3.5 Mean sensitivity performance of BANJO in recovering influence networks was significantly better than that of ARACNE . . . 106 6.3.6 Transcriptional networks were better suited for reverse engineering compared to cytokine receptor interactions and intracellular signaling networks. . . . . . . . . . . . . . . . . . . . . . . . . 107 6.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108 vii Chapter Seven: Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114 Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117 Appendices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132 A.1 Full list of 181 genes upregulated in keloid compared to normal fibroblasts using the MAS 5.0 summarization algorithm (P < 0.05) . . . . . . . . 132 A.2 Full list of 290 genes downregulated in keloid compared to normal fibroblasts using the MAS 5.0 summarization algorithm (P < 0.05) . . . . . . . . 137 A.3 Full list of 86 genes upregulated in keloid compared to normal fibroblasts using the RMA summarization algorithm (P < 0.05) . . . . . . . . . . 145 A.4 Full list of 258 genes downregulated in keloid compared to normal fibroblasts using the RMA summarization algorithm (P < 0.05) . . . . . . . . . . 147 A.5 List of genes differentially expressed using both the RMA and MAS 5.0 summarization algorithm (P < 0.05) . . . . . . . . . . . . . . . . . . . . 154 A.6 Cytokine-cytokine receptor interaction from the KEGG database (Benjamini corrected P-value = 0.094) . . . . . . . . . . . . . . . . . . . . . . . . . 160 A.7 Toll-like receptor signaling pathway from the KEGG database (Benjamini corrected P-value = 0.246) . . . . . . . . . . . . . . . . . . . . . . . . . 161 viii SUMMARY Keloid scars are aberrations in the wound healing process, resulting in the appearance of protrusive crab like extensions growing into normal tissue. They not subside with time, and may develop over the most minor of skin wounds, such as insect bites or acne. Aside from being an aesthetic impediment, keloids are frequently associated with itchiness, pain and, when involving the skin overlying a joint, restricted range of motion. To date, none of the known treatment modalities have proven optimal. In recent years, a systems approach to understanding biology has gained eminence, in part due to the limitations of a purely reductionist approach in explaining biological phenomena. However, there are merits to the reductionist approach; much of what we know of biology today can be attributed to the work of molecular biologists of the past. In this dissertation, we will adopt both these approaches to tackling the keloid problem. In the first part of this thesis, we examined the role played by a novel growth factor, the hepatoma-derived growth factor (HDGF), in keloid pathogenesis. Using a combination of immunohistochemical staining and Western blots, we found that secreted HDGF is increased in the keloid condition and its secretion is modulated by epithelial– mesenchymal interactions. Furthermore, exogenous HDGF exerts a proliferative effect on keloid fibroblasts and increases the production of the angiogenic factor VEGF, indicating that it plays some role in the process of angiogenesis. With the advent of high throughput technology, researchers are no longer confined to the study of individual molecules. In the second part of this dissertation, we utilized the microarray platform to assess the global transcriptional differences between ix 1.747925 1.730545 1.719725 1.686518 1.676297 1.667339 HDLBP COL5A3 PTK7 NONO NT5DC2 ATF4 1.664031 1.634808 1.627513 1.615317 1.612627 1.597447 1.58056 1.568977 1.562242 1.544172 1.544152 1.531647 1.521308 1.51899 1.513227 1.499035 1.469517 1.468484 1.459514 1.456908 1.443996 1.441773 1.436187 1.43166 1.425044 1.417303 LOC100130624 FAM155A PQBP1 RPL13 RPS8 HOXA11 MGC87895 /// RPS14 RPL3 EIF1 RPL13 RPL8 LOC642741 RPS5 C20orf149 EPHB3 RPL13 RPL10L RPL3 DBN1 RPL3 RPL10 PARVB RPS16 SHMT2 RPLP2 SLC1A4 1.415545 1.413611 1.408501 1.405121 1.403469 RPL3 YBX1 RPL13 RPS6 PPP2R3A 1.386019 1.379153 1.360253 1.34878 EIF3B RPS17L4 RPS2 TREX2 /// UCHL5IP RPS13 GRB10 RPL9 1.33581 1.32547 1.303224 high density lipoprotein binding protein collagen, type V, alpha PTK7 protein tyrosine kinase non-POU domain containing, octamer-binding 5'-nucleotidase domain containing activating transcription factor (tax-responsive enhancer element B67) hypothetical LOC100130624 family with sequence similarity 155, member A polyglutamine binding protein ribosomal protein L13 ribosomal protein S8 homeobox A11 similar to ribosomal protein S14 /// ribosomal protein S14 ribosomal protein L3 eukaryotic translation initiation factor ribosomal protein L13 ribosomal protein L8 similar to ribosomal protein L3 ribosomal protein S5 chromosome 20 open reading frame 149 EPH receptor B3 ribosomal protein L13 ribosomal protein L10-like ribosomal protein L3 drebrin ribosomal protein L3 ribosomal protein L10 parvin, beta ribosomal protein S16 serine hydroxymethyltransferase (mitochondrial) ribosomal protein, large, P2 solute carrier family (glutamate/neutral amino acid transporter), member ribosomal protein L3 Y box binding protein ribosomal protein L13 ribosomal protein S6 protein phosphatase (formerly 2A), regulatory subunit B'', alpha eukaryotic translation initiation factor 3, subunit B ribosomal protein S17-like ribosomal protein S2 three prime repair exonuclease /// UCHL5 interacting protein ribosomal protein S13 growth factor receptor-bound protein 10 ribosomal protein L9 0.037741 0.042118 0.041423 0.017365 0.044871 0.004086 0.039839 0.027042 0.003057 1.97E-04 0.023227 0.037859 0.026445 0.024636 0.038827 7.69E-04 0.001713 0.001728 0.010015 0.047073 0.037648 0.002784 0.030904 0.015644 0.021666 0.025091 0.018933 1.48E-04 0.004886 0.002008 0.008827 0.046909 0.016536 0.040024 7.69E-04 0.043219 0.011736 0.020335 0.04918 0.029039 0.014841 0.00654 0.023425 0.046875 146 1.300171 1.276438 1.274737 RPL38 NUDT3 PARD3 1.27258 1.264076 1.261194 1.255499 1.245831 1.23863 RPL27 RPL17 CDYL RPS9 RP5-1077B9.4 hCG_21078 /// RPL27A EIF1 RPS24 LOC100130553 /// RPS18 INPPL1 RPL28 1.22714 1.216908 1.170586 1.161676 1.146715 ribosomal protein L38 nudix-type motif par-3 partitioning defective homolog (C. elegans) ribosomal protein L27 ribosomal protein L17 chromodomain protein, Y-like ribosomal protein S9 invasion inhibitory protein 45 hCG21078 /// ribosomal protein L27a 0.021022 0.04918 0.047977 eukaryotic translation initiation factor ribosomal protein S24 hypothetical protein LOC100130553 /// ribosomal protein S18 inositol polyphosphate phosphatase-like ribosomal protein L28 0.033374 0.026217 0.02368 0.040303 0.018933 0.049917 0.007091 0.017708 0.002151 0.022554 0.037601 A.4 Full list of 258 genes downregulated in keloid compared to normal fibroblasts using the RMA summarization algorithm (P < 0.05) Fold change 44.34992 Gene Symbol Gene Title CXCL6 41.64695 CXCL1 37.03845 C2 /// CFB 33.61232 29.71772 21.50683 20.34249 19.02187 18.80414 HSD11B1 TNFAIP6 CXCL2 TNFAIP6 IL8 SLC39A8 16.76682 SLC39A8 14.08095 13.78165 C3 RSAD2 12.01816 11.75815 11.65778 10.88791 9.990352 9.679501 SOD2 IL8 CCL2 SFRP1 G0S2 IFI44L chemokine (C-X-C motif) ligand (granulocyte chemotactic protein 2) chemokine (C-X-C motif) ligand (melanoma growth stimulating activity, alpha) complement component /// complement factor B hydroxysteroid (11-beta) dehydrogenase tumor necrosis factor, alpha-induced protein chemokine (C-X-C motif) ligand tumor necrosis factor, alpha-induced protein interleukin solute carrier family 39 (zinc transporter), member solute carrier family 39 (zinc transporter), member complement component radical S-adenosyl methionine domain containing superoxide dismutase 2, mitochondrial interleukin chemokine (C-C motif) ligand secreted frizzled-related protein G0/G1switch interferon-induced protein 44-like Corrected p-value 9.29E-10 1.89E-05 1.75E-08 1.50E-06 0.008827 5.89E-05 0.011388 0.003057 1.22E-05 7.69E-04 2.94E-04 0.013835 0.002706 0.001761 9.46E-07 0.026217 0.001019 0.04903 147 8.604651 8.378408 8.213031 8.204023 8.195308 8.068323 7.890376 7.244113 CHI3L2 IL6 CA12 OAS1 GCH1 CA12 SOD2 IFIT1 7.188362 6.98734 TNFAIP3 IFIT3 6.850272 TNFSF10 6.773449 6.696858 6.448252 6.327625 6.259846 6.225736 6.107744 OAS1 NAMPT IFIH1 CA12 HERC6 CTSS GBP1 5.973147 IFIT2 5.971855 5.964412 5.887205 5.848494 5.825303 5.720924 5.663569 5.519223 5.507072 5.327916 5.289413 CA12 CA12 HERC5 CXCL3 SOD2 NAMPT BTN3A2 WTAP WTAP CCL5 ABCA8 5.146521 SLC39A14 4.979725 RARRES3 4.976672 4.80152 IFI44 AKR1B1 4.67249 4.650329 4.624918 4.616492 4.304027 4.30355 LAP3 PTGES TNFAIP2 CCL5 MT1X NFKBIA chitinase 3-like interleukin (interferon, beta 2) carbonic anhydrase XII 2',5'-oligoadenylate synthetase 1, 40/46kDa GTP cyclohydrolase carbonic anhydrase XII superoxide dismutase 2, mitochondrial interferon-induced protein with tetratricopeptide repeats tumor necrosis factor, alpha-induced protein interferon-induced protein with tetratricopeptide repeats tumor necrosis factor (ligand) superfamily, member 10 2',5'-oligoadenylate synthetase 1, 40/46kDa nicotinamide phosphoribosyltransferase interferon induced with helicase C domain carbonic anhydrase XII hect domain and RLD cathepsin S guanylate binding protein 1, interferon-inducible, 67kDa interferon-induced protein with tetratricopeptide repeats carbonic anhydrase XII carbonic anhydrase XII hect domain and RLD chemokine (C-X-C motif) ligand superoxide dismutase 2, mitochondrial nicotinamide phosphoribosyltransferase butyrophilin, subfamily 3, member A2 Wilms tumor associated protein Wilms tumor associated protein chemokine (C-C motif) ligand ATP-binding cassette, sub-family A (ABC1), member solute carrier family 39 (zinc transporter), member 14 retinoic acid receptor responder (tazarotene induced) interferon-induced protein 44 aldo-keto reductase family 1, member B1 (aldose reductase) leucine aminopeptidase prostaglandin E synthase tumor necrosis factor, alpha-induced protein chemokine (C-C motif) ligand metallothionein 1X nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha 0.019936 4.59E-04 2.40E-04 0.015085 2.29E-04 0.00631 0.001861 0.035976 8.07E-05 0.014427 0.03935 0.044028 0.001998 0.016536 0.006223 0.009306 8.67E-05 0.003902 0.049917 0.004125 4.59E-04 0.012534 5.04E-04 7.69E-04 0.023627 0.042157 9.55E-05 0.009169 0.002448 0.006956 0.024324 0.003712 0.037647 3.95E-04 0.024205 0.012578 7.11E-04 0.010015 0.020822 4.59E-04 148 4.096058 4.09106 4.075102 4.064827 4.041319 PLSCR1 CLU MT1E TNFAIP3 GBP1 3.909078 3.828279 CLU STEAP1 3.82739 3.806854 3.680742 3.657572 MT2A IFI35 XAF1 MT1E /// MT1H /// MT1M /// MT1P2 3.653427 3.645952 CXCL5 PSMB9 3.586836 3.463399 3.378218 IL15RA ICAM1 UCHL1 3.338111 3.33326 3.289887 3.285261 3.285236 3.279497 MT1X NFIB TMEM100 NRCAM TLR3 SLC1A1 3.275539 SLC11A2 3.262109 3.245122 3.224767 3.219482 3.194404 IL32 LRRN3 MT1F PTGFR PARP12 3.130064 3.075876 3.055434 3.049139 2.993775 2.925094 ICAM1 MID1 MT1P2 VCAM1 CP AMPD3 2.918264 2.903807 2.896697 IFI30 APOL3 SLC11A2 2.892534 STAT1 phospholipid scramblase clusterin metallothionein 1E tumor necrosis factor, alpha-induced protein guanylate binding protein 1, interferon-inducible, 67kDa clusterin six transmembrane epithelial antigen of the prostate metallothionein 2A interferon-induced protein 35 XIAP associated factor metallothionein 1E /// metallothionein 1H /// metallothionein 1M /// metallothionein pseudogene chemokine (C-X-C motif) ligand proteasome (prosome, macropain) subunit, beta type, (large multifunctional peptidase 2) interleukin 15 receptor, alpha intercellular adhesion molecule ubiquitin carboxyl-terminal esterase L1 (ubiquitin thiolesterase) metallothionein 1X nuclear factor I/B transmembrane protein 100 neuronal cell adhesion molecule toll-like receptor solute carrier family (neuronal/epithelial high affinity glutamate transporter, system Xag), member solute carrier family 11 (proton-coupled divalent metal ion transporters), member interleukin 32 leucine rich repeat neuronal metallothionein 1F prostaglandin F receptor (FP) poly (ADP-ribose) polymerase family, member 12 intercellular adhesion molecule midline (Opitz/BBB syndrome) metallothionein pseudogene vascular cell adhesion molecule ceruloplasmin (ferroxidase) adenosine monophosphate deaminase (isoform E) interferon, gamma-inducible protein 30 apolipoprotein L, solute carrier family 11 (proton-coupled divalent metal ion transporters), member signal transducer and activator of transcription 1, 0.011388 0.026445 0.047073 0.001023 0.021022 0.033121 0.014024 0.029119 0.025091 0.023346 0.021022 0.004989 0.03417 0.011711 1.10E-04 0.004886 0.026959 0.026219 0.04263 7.94E-04 0.039578 7.69E-04 3.06E-04 4.59E-04 0.00287 0.023227 0.027574 0.048335 3.40E-05 0.033374 0.035137 0.047073 8.67E-05 0.010801 0.023627 0.042118 2.81E-04 0.032961 149 2.862865 MMD 2.810585 2.798357 2.72018 2.711745 ABLIM1 DDX60 FILIP1L GPRC5B 2.703468 2.693878 HIST1H2BD PSME2 2.689429 2.688505 2.687159 BTN3A2 CFLAR LGALS3BP 2.650053 2.647512 2.634461 2.629179 2.62574 2.595849 2.594678 2.593336 2.569516 2.54052 2.528263 2.522403 2.520541 2.514324 CXCL5 DRAM MT1M UBE2L6 MT1F CHEK2 CFLAR NMI CFLAR SIRPA CEBPD BTN3A3 CYB5A SLC11A2 2.498402 2.490228 2.454265 2.411988 2.410709 2.408566 2.390128 PALM RNF114 TRIM38 SLC25A28 STAT4 CYB5A KIAA0391 /// PSMA6 SLC15A3 GHR C1QTNF1 NFIB PANX1 LRRN3 RGS3 NT5E MARCH3 CYB5A TRIM38 2.366543 2.361601 2.354626 2.351021 2.347035 2.336691 2.333014 2.319342 2.311114 2.27852 2.251881 91kDa monocyte to macrophage differentiationassociated actin binding LIM protein DEAD (Asp-Glu-Ala-Asp) box polypeptide 60 filamin A interacting protein 1-like G protein-coupled receptor, family C, group 5, member B histone cluster 1, H2bd proteasome (prosome, macropain) activator subunit (PA28 beta) butyrophilin, subfamily 3, member A2 CASP8 and FADD-like apoptosis regulator lectin, galactoside-binding, soluble, binding protein chemokine (C-X-C motif) ligand damage-regulated autophagy modulator metallothionein 1M ubiquitin-conjugating enzyme E2L metallothionein 1F CHK2 checkpoint homolog (S. pombe) CASP8 and FADD-like apoptosis regulator N-myc (and STAT) interactor CASP8 and FADD-like apoptosis regulator signal-regulatory protein alpha CCAAT/enhancer binding protein (C/EBP), delta butyrophilin, subfamily 3, member A3 cytochrome b5 type A (microsomal) solute carrier family 11 (proton-coupled divalent metal ion transporters), member Paralemmin ring finger protein 114 tripartite motif-containing 38 solute carrier family 25, member 28 signal transducer and activator of transcription cytochrome b5 type A (microsomal) KIAA0391 /// proteasome (prosome, macropain) subunit, alpha type, solute carrier family 15, member growth hormone receptor C1q and tumor necrosis factor related protein nuclear factor I/B pannexin leucine rich repeat neuronal regulator of G-protein signaling 5'-nucleotidase, ecto (CD73) membrane-associated ring finger (C3HC4) cytochrome b5 type A (microsomal) tripartite motif-containing 38 0.006815 0.026332 0.043219 0.004067 0.016484 0.015237 0.001077 0.025091 0.015237 0.013645 0.015587 0.003902 0.008827 0.03412 9.22E-04 5.84E-05 0.00493 0.044028 5.48E-04 1.38E-05 0.019171 0.048335 0.011736 0.006815 0.009971 0.047073 0.021022 0.001713 3.39E-04 0.015085 0.027574 0.027138 0.001713 0.048335 0.040444 0.03639 0.016484 0.026787 0.002448 0.001642 0.023425 0.032743 150 2.23989 2.235737 CFLAR PPFIBP2 2.224926 2.22473 2.215581 2.210316 HLA-F TAPBP HLA-C AK3L1 /// AK3L2 2.20811 2.17376 2.169356 2.167533 2.146688 2.13148 2.124391 2.12098 HLA-F SMC2 CYLD NOVA1 C10orf26 HLA-B HTATIP2 SLC39A8 2.108726 2.097694 2.097592 2.092583 2.089171 2.072831 2.0599 TNIP1 SAMHD1 PON2 CFLAR HLA-B CFLAR DHRS3 2.055893 2.048616 2.023844 DKFZP586H2123 CFLAR HLA-B /// MICA 2.021447 2.007685 2.001211 1.998355 1.981507 BIRC3 PDPN LY6E ZC3H12A HLA-A /// HLAA29.1 /// HLA-B /// HLA-G /// HLAH /// HLA-J 1.96592 1.942084 1.935575 1.927046 1.924471 1.911548 1.909791 1.896043 1.877315 1.876992 CSF1 HTATIP2 ACP2 PON2 IFNGR1 FAM117A C14orf159 HLA-G MT1F HLA-C CASP8 and FADD-like apoptosis regulator PTPRF interacting protein, binding protein (liprin beta 2) major histocompatibility complex, class I, F TAP binding protein (tapasin) major histocompatibility complex, class I, C adenylate kinase 3-like /// adenylate kinase 3like major histocompatibility complex, class I, F structural maintenance of chromosomes cylindromatosis (turban tumor syndrome) neuro-oncological ventral antigen chromosome 10 open reading frame 26 major histocompatibility complex, class I, B HIV-1 Tat interactive protein 2, 30kDa solute carrier family 39 (zinc transporter), member TNFAIP3 interacting protein SAM domain and HD domain paraoxonase CASP8 and FADD-like apoptosis regulator major histocompatibility complex, class I, B CASP8 and FADD-like apoptosis regulator dehydrogenase/reductase (SDR family) member regeneration associated muscle protease CASP8 and FADD-like apoptosis regulator major histocompatibility complex, class I, B /// MHC class I polypeptide-related sequence A baculoviral IAP repeat-containing podoplanin lymphocyte antigen complex, locus E zinc finger CCCH-type containing 12A major histocompatibility complex, class I, A /// major histocompatibility complex class I HLAA29.1 /// major histocompatibility complex, class I, B /// major histocompatibility complex, class I, G /// major histocompatibility complex, class I, H (pseudogene) /// major histocompatibility complex, class I, J (pseudogene) colony stimulating factor (macrophage) HIV-1 Tat interactive protein 2, 30kDa acid phosphatase 2, lysosomal paraoxonase interferon gamma receptor family with sequence similarity 117, member A chromosome 14 open reading frame 159 major histocompatibility complex, class I, G metallothionein 1F major histocompatibility complex, class I, C 0.023911 0.003755 0.009477 0.002016 0.006519 0.003202 0.00287 0.002998 0.046985 0.04244 0.025091 0.006956 0.026762 0.004086 0.027814 0.047977 0.025091 0.003645 0.002024 0.00197 0.001019 0.037791 8.55E-04 0.018933 0.001713 0.033129 0.027138 5.99E-05 0.006852 0.015085 0.003902 0.006566 0.014803 0.018356 0.001713 0.010796 0.008626 1.10E-04 7.69E-04 151 1.87264 1.87205 1.85887 1.857911 MEIS3P1 TAPBPL HLA-G BASP1 1.847917 1.844846 1.843371 1.837641 1.822478 1.811002 1.805608 1.801476 1.785207 1.763702 1.752336 1.748331 1.718806 1.700887 1.683461 1.668382 1.667567 1.651713 MICALL2 SIRPA GFRA1 HLA-E DENND2D TRIM38 NNMT FTH1 PION NNMT HLA-C NR4A3 PROCR PDCD5 HSPB8 HLA-C PDCD1LG2 SLCO3A1 1.641853 SLC1A1 1.629699 1.626722 1.625006 1.61725 1.617092 HLA-G BTN3A1 HLA-A LARGE AK3L1 /// AK3L2 1.592655 1.589381 PION NFKBIE 1.584692 1.576378 1.571031 RNF8 CD59 LYN 1.563836 MMP2 1.556934 1.533629 NRP2 P2RX4 1.523406 NFKB1 1.522656 SVEP1 1.518637 WWC1 Meis homeobox pseudogene TAP binding protein-like major histocompatibility complex, class I, G brain abundant, membrane attached signal protein MICAL-like signal-regulatory protein alpha GDNF family receptor alpha major histocompatibility complex, class I, E DENN/MADD domain containing 2D tripartite motif-containing 38 nicotinamide N-methyltransferase ferritin, heavy polypeptide pigeon homolog (Drosophila) nicotinamide N-methyltransferase major histocompatibility complex, class I, C nuclear receptor subfamily 4, group A, member protein C receptor, endothelial (EPCR) programmed cell death heat shock 22kDa protein major histocompatibility complex, class I, C programmed cell death ligand solute carrier organic anion transporter family, member 3A1 solute carrier family (neuronal/epithelial high affinity glutamate transporter, system Xag), member major histocompatibility complex, class I, G butyrophilin, subfamily 3, member A1 major histocompatibility complex, class I, A like-glycosyltransferase adenylate kinase 3-like /// adenylate kinase 3like pigeon homolog (Drosophila) nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, epsilon ring finger protein CD59 molecule, complement regulatory protein v-yes-1 Yamaguchi sarcoma viral related oncogene homolog matrix metallopeptidase (gelatinase A, 72kDa gelatinase, 72kDa type IV collagenase) neuropilin purinergic receptor P2X, ligand-gated ion channel, nuclear factor of kappa light polypeptide gene enhancer in B-cells sushi, von Willebrand factor type A, EGF and pentraxin domain containing WW and C2 domain containing 0.007091 0.001292 0.003202 0.026445 7.91E-04 0.002908 0.031214 0.042803 0.009234 0.014427 0.033657 0.032417 0.026445 0.015237 0.001075 0.010796 0.027362 0.039578 0.04761 0.016484 0.004125 0.00287 0.014024 0.006852 0.0442 0.002743 0.013456 0.033481 0.003955 0.019276 0.016484 4.56E-04 0.029499 5.99E-05 0.02645 0.021666 0.042118 0.011711 0.045278 152 1.499967 1.497518 1.493991 1.489115 LSAMP APOL2 PGK1 GBA /// GBAP 1.488427 KHDC1 /// SPA17 1.482473 PSTPIP2 1.466329 POLD3 1.449279 1.446802 DTNA RELB 1.445625 ELF3 1.440517 CYP27A1 1.439601 1.429019 1.406325 HYPK ACP6 TFDP2 1.397198 1.388266 1.38517 1.382151 CTSS B2M CSTB SNCA 1.368093 1.367311 1.349224 1.321152 PDLIM4 NFE2L1 FTH1 ARHGEF10L 1.320407 1.313343 1.308686 1.307239 1.272 1.266423 LGALS8 FTHP1 EDNRB HLA-F SLC19A3 SLC11A2 1.25115 1.249708 RBKS CAND2 1.246255 1.238541 1.232883 1.225532 1.196416 1.174331 SNX11 C6orf64 ZMIZ2 CSF1 NFIB TFDP2 1.155878 WDR48 limbic system-associated membrane protein apolipoprotein L, phosphoglycerate kinase glucosidase, beta; acid (includes glucosylceramidase) /// glucosidase, beta; acid, pseudogene KH homology domain containing /// sperm autoantigenic protein 17 proline-serine-threonine phosphatase interacting protein polymerase (DNA-directed), delta 3, accessory subunit dystrobrevin, alpha v-rel reticuloendotheliosis viral oncogene homolog B E74-like factor (ets domain transcription factor, epithelial-specific ) cytochrome P450, family 27, subfamily A, polypeptide Huntingtin interacting protein K acid phosphatase 6, lysophosphatidic transcription factor Dp-2 (E2F dimerization partner 2) cathepsin S beta-2-microglobulin cystatin B (stefin B) synuclein, alpha (non A4 component of amyloid precursor) PDZ and LIM domain nuclear factor (erythroid-derived 2)-like ferritin, heavy polypeptide Rho guanine nucleotide exchange factor (GEF) 10-like lectin, galactoside-binding, soluble, ferritin, heavy polypeptide pseudogene endothelin receptor type B major histocompatibility complex, class I, F solute carrier family 19, member solute carrier family 11 (proton-coupled divalent metal ion transporters), member ribokinase cullin-associated and neddylation-dissociated (putative) sorting nexin 11 chromosome open reading frame 64 zinc finger, MIZ-type containing colony stimulating factor (macrophage) nuclear factor I/B transcription factor Dp-2 (E2F dimerization partner 2) WD repeat domain 48 0.009696 0.047977 0.042095 0.036143 0.004067 0.008121 0.025058 0.037974 0.009818 0.02046 0.04263 0.019813 0.039972 0.006852 0.046985 0.024654 0.017681 0.006815 0.041423 0.032417 0.013128 0.030511 0.044328 0.03731 0.046273 0.039578 0.031469 0.018933 0.018933 0.020736 0.020092 0.044823 0.033374 0.013754 0.047977 0.029499 0.006116 153 1.146091 1.131624 1.106074 1.104509 SP100 TLR1 CXCL5 TAF1B 1.101118 BRCA2 SP100 nuclear antigen toll-like receptor chemokine (C-X-C motif) ligand TATA box binding protein (TBP)-associated factor, RNA polymerase I, B, 63kDa breast cancer 2, early onset 0.015085 0.033657 0.044823 0.037317 0.027138 A.5 List of genes differentially expressed using both the RMA and MAS 5.0 summarization algorithm (P < 0.05) Regulation (Keloid vs Normal) down Gene Symbol Gene Title CXCL6 down CXCL1 down down down down down down down down down down down down down down down down down down down down down down down down down down down down down IL8 HSD11B1 IL8 CCL5 CXCL2 RSAD2 C2 /// CFB CXCL5 TNFAIP6 CXCL3 IL32 CP CHI3L2 TNFAIP6 C3 SLC39A8 CXCL5 G0S2 OAS1 TNFSF10 SLC39A8 GCH1 CCL5 SLC19A3 HERC5 IL6 NRCAM CCL2 ABCA8 chemokine (C-X-C motif) ligand (granulocyte chemotactic protein 2) chemokine (C-X-C motif) ligand (melanoma growth stimulating activity, alpha) interleukin hydroxysteroid (11-beta) dehydrogenase interleukin chemokine (C-C motif) ligand chemokine (C-X-C motif) ligand radical S-adenosyl methionine domain containing complement component /// complement factor B chemokine (C-X-C motif) ligand tumor necrosis factor, alpha-induced protein chemokine (C-X-C motif) ligand interleukin 32 ceruloplasmin (ferroxidase) chitinase 3-like tumor necrosis factor, alpha-induced protein complement component solute carrier family 39 (zinc transporter), member chemokine (C-X-C motif) ligand G0/G1switch 2',5'-oligoadenylate synthetase 1, 40/46kDa tumor necrosis factor (ligand) superfamily, member 10 solute carrier family 39 (zinc transporter), member GTP cyclohydrolase chemokine (C-C motif) ligand solute carrier family 19, member hect domain and RLD interleukin (interferon, beta 2) neuronal cell adhesion molecule chemokine (C-C motif) ligand ATP-binding cassette, sub-family A (ABC1), member 154 down down down down down down down down down down down down down down down down down down down down down down down down down down down down down down down down down down SFRP1 TMEM100 SOD2 CTSS IFI30 HERC6 TLR3 RARRES3 SOD2 TNFAIP3 CA12 CTSS IFIT2 LRRN3 IFIT3 BIRC3 CA12 GBP1 TNFAIP2 CA12 PTGES GBP1 NAMPT SLC39A8 CA12 STAT4 CA12 LRRN3 MT1X NAMPT MT1M WTAP MT1P2 ELF3 down down down down down down down down down down down down down TNFAIP3 SOD2 MT1E ZC3H12A ICAM1 SLC39A14 MT1F AKR1B1 SNCA WWC1 ICAM1 WTAP SLC15A3 secreted frizzled-related protein transmembrane protein 100 superoxide dismutase 2, mitochondrial cathepsin S interferon, gamma-inducible protein 30 hect domain and RLD toll-like receptor retinoic acid receptor responder (tazarotene induced) superoxide dismutase 2, mitochondrial tumor necrosis factor, alpha-induced protein carbonic anhydrase XII cathepsin S interferon-induced protein with tetratricopeptide repeats leucine rich repeat neuronal interferon-induced protein with tetratricopeptide repeats baculoviral IAP repeat-containing carbonic anhydrase XII guanylate binding protein 1, interferon-inducible, 67kDa tumor necrosis factor, alpha-induced protein carbonic anhydrase XII prostaglandin E synthase guanylate binding protein 1, interferon-inducible, 67kDa nicotinamide phosphoribosyltransferase solute carrier family 39 (zinc transporter), member carbonic anhydrase XII signal transducer and activator of transcription carbonic anhydrase XII leucine rich repeat neuronal metallothionein 1X nicotinamide phosphoribosyltransferase metallothionein 1M Wilms tumor associated protein metallothionein pseudogene E74-like factor (ets domain transcription factor, epithelialspecific ) tumor necrosis factor, alpha-induced protein superoxide dismutase 2, mitochondrial metallothionein 1E zinc finger CCCH-type containing 12A intercellular adhesion molecule solute carrier family 39 (zinc transporter), member 14 metallothionein 1F aldo-keto reductase family 1, member B1 (aldose reductase) synuclein, alpha (non A4 component of amyloid precursor) WW and C2 domain containing intercellular adhesion molecule Wilms tumor associated protein solute carrier family 15, member 155 down down down down down down IFI35 MT1E /// MT1H /// MT1M /// MT1P2 AMPD3 MT2A LAP3 SLC11A2 down down down down down MARCH3 CLU STEAP1 GFRA1 NFKBIA down down down MMD MT1X SLC11A2 down down down down down down down down down down down down down down down down down down down down down down down down down PTGFR NR4A3 PLSCR1 UCHL1 CHEK2 MT1F EDNRB IL15RA STAT1 SMC2 SAMHD1 ABLIM1 MT1F HLA-F CFLAR SIRPA HLA-C SLC25A28 AK3L1 /// AK3L2 TAPBPL NOVA1 DTNA PDPN LGALS3BP PSME2 down SLC1A1 interferon-induced protein 35 metallothionein 1E /// metallothionein 1H /// metallothionein 1M /// metallothionein pseudogene adenosine monophosphate deaminase (isoform E) metallothionein 2A leucine aminopeptidase solute carrier family 11 (proton-coupled divalent metal ion transporters), member membrane-associated ring finger (C3HC4) Clusterin six transmembrane epithelial antigen of the prostate GDNF family receptor alpha nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha monocyte to macrophage differentiation-associated metallothionein 1X solute carrier family 11 (proton-coupled divalent metal ion transporters), member prostaglandin F receptor (FP) nuclear receptor subfamily 4, group A, member phospholipid scramblase ubiquitin carboxyl-terminal esterase L1 (ubiquitin thiolesterase) CHK2 checkpoint homolog (S. pombe) metallothionein 1F endothelin receptor type B interleukin 15 receptor, alpha signal transducer and activator of transcription 1, 91kDa structural maintenance of chromosomes SAM domain and HD domain actin binding LIM protein metallothionein-1F major histocompatibility complex, class I, F CASP8 and FADD-like apoptosis regulator signal-regulatory protein alpha major histocompatibility complex, class I, C solute carrier family 25, member 28 adenylate kinase 3-like /// adenylate kinase 3-like TAP binding protein-like neuro-oncological ventral antigen dystrobrevin, alpha Podoplanin lectin, galactoside-binding, soluble, binding protein proteasome (prosome, macropain) activator subunit (PA28 beta) solute carrier family (neuronal/epithelial high affinity glutamate transporter, system Xag), member 156 down down down down down down down down down down down down CFLAR HIST1H2BD PROCR CFLAR CYB5A FILIP1L PSTPIP2 HLA-F CFLAR PPFIBP2 RNF114 SLC11A2 down down down down down down down down down down down down down down down down down down CFLAR DRAM CFLAR HLA-G LYN HLA-B HTATIP2 MICALL2 SIRPA HLA-G CYB5A TAPBP PON2 NT5E TRIM38 HLA-B CFLAR KIAA0391 /// PSMA6 TRIM38 NFKBIE down down down down down down down down down down down down down C10orf26 LY6E PANX1 CSF1 TNIP1 RELB ACP2 PON2 TRIM38 HLA-C HLA-A /// HLA-A29.1 /// HLA-B /// HLA-G /// CASP8 and FADD-like apoptosis regulator histone cluster 1, H2bd protein C receptor, endothelial (EPCR) CASP8 and FADD-like apoptosis regulator cytochrome b5 type A (microsomal) filamin A interacting protein 1-like proline-serine-threonine phosphatase interacting protein major histocompatibility complex, class I, F CASP8 and FADD-like apoptosis regulator PTPRF interacting protein, binding protein (liprin beta 2) ring finger protein 114 solute carrier family 11 (proton-coupled divalent metal ion transporters), member CASP8 and FADD-like apoptosis regulator damage-regulated autophagy modulator CASP8 and FADD-like apoptosis regulator major histocompatibility complex, class I, G v-yes-1 Yamaguchi sarcoma viral related oncogene homolog major histocompatibility complex, class I, B HIV-1 Tat interactive protein 2, 30kDa MICAL-like signal-regulatory protein alpha major histocompatibility complex, class I, G cytochrome b5 type A (microsomal) TAP binding protein (tapasin) paraoxonase 5'-nucleotidase, ecto (CD73) tripartite motif-containing 38 major histocompatibility complex, class I, B CASP8 and FADD-like apoptosis regulator KIAA0391 /// proteasome (prosome, macropain) subunit, alpha type, tripartite motif-containing 38 nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, epsilon chromosome 10 open reading frame 26 lymphocyte antigen complex, locus E pannexin colony stimulating factor (macrophage) TNFAIP3 interacting protein v-rel reticuloendotheliosis viral oncogene homolog B acid phosphatase 2, lysosomal paraoxonase tripartite motif-containing 38 major histocompatibility complex, class I, C major histocompatibility complex, class I, A /// major histocompatibility complex class I HLA-A29.1 /// major histocompatibility complex, class I, B /// major histocompatibility complex, class I, G /// major histocompatibility complex, class I, H 157 down down down down down down down down down down down down down down up up up up up HLA-H /// HLA-J NNMT HLA-C BASP1 IFNGR1 FTH1 HLA-C HLA-B /// MICA CD59 NNMT HLA-A MMP2 up up up up CSTB FTH1 B2M POSTN COL15A1 HOXA11 KCNJ6 JUP /// KRT19 IGFBP3 ATXN1 CADM1 SEMA5A up up up GPSM2 FAM155A MICAL2 up up CADM1 SEMA5A up up SHMT2 MICAL2 up FARP1 up up up up MGC87895 /// RPS14 TBC1D2 ECM1 SLC25A6 up PDGFRB (pseudogene) /// major histocompatibility complex, class I, J (pseudogene) nicotinamide N-methyltransferase major histocompatibility complex, class I, C brain abundant, membrane attached signal protein interferon gamma receptor ferritin, heavy polypeptide major histocompatibility complex, class I, C major histocompatibility complex, class I, B /// MHC class I polypeptide-related sequence A CD59 molecule, complement regulatory protein nicotinamide N-methyltransferase major histocompatibility complex, class I, A matrix metallopeptidase (gelatinase A, 72kDa gelatinase, 72kDa type IV collagenase) cystatin B (stefin B) ferritin, heavy polypeptide beta-2-microglobulin periostin, osteoblast specific factor collagen, type XV, alpha homeobox A11 potassium inwardly-rectifying channel, subfamily J, member junction plakoglobin /// keratin 19 insulin-like growth factor binding protein ataxin cell adhesion molecule sema domain, seven thrombospondin repeats (type and type 1like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5A G-protein signaling modulator (AGS3-like, C. elegans) family with sequence similarity 155, member A microtubule associated monoxygenase, calponin and LIM domain containing cell adhesion molecule sema domain, seven thrombospondin repeats (type and type 1like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5A serine hydroxymethyltransferase (mitochondrial) microtubule associated monoxygenase, calponin and LIM domain containing FERM, RhoGEF (ARHGEF) and pleckstrin domain protein (chondrocyte-derived) similar to ribosomal protein S14 /// ribosomal protein S14 TBC1 domain family, member extracellular matrix protein solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member platelet-derived growth factor receptor, beta polypeptide 158 up up up CTSB FHOD1 SLC25A6 up up up up up up up up up NXN PTK7 LAMA2 LOC644191 /// LOC728937 /// RPS26 PARVB COL1A1 SHMT2 NONO ATF4 up up up up up up up up RPS9 RPL13 RPS8 RPL13 RPS6 RPL8 PPP2R3A RPL13 cathepsin B formin homology domain containing solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member Nucleoredoxin PTK7 protein tyrosine kinase laminin, alpha similar to hCG15685 /// similar to 40S ribosomal protein S26 /// ribosomal protein S26 parvin, beta collagen, type I, alpha serine hydroxymethyltransferase (mitochondrial) non-POU domain containing, octamer-binding activating transcription factor (tax-responsive enhancer element B67) ribosomal protein S9 ribosomal protein L13 ribosomal protein S8 ribosomal protein L13 ribosomal protein S6 ribosomal protein L8 protein phosphatase (formerly 2A), regulatory subunit B'', alpha ribosomal protein L13 159 A.6 Cytokine-cytokine receptor interaction from the KEGG database (Benjamini corrected P-value = 0.094) 160 A.7 Toll-like receptor signaling pathway from the KEGG database (Benjamini corrected P-value = 0.246) 161 [...]... scars To this end, an understanding of the molecular etiology of keloids would be useful Furthermore, since keloid formation is generally considered to be a form of abnormal wound healing, any insights gained from this endeavour would also increase our understanding of the wound healing process 1.2 Approach and methodology We have decided to adopt both top down as well as reductionist approaches to understanding. .. possible factor underlying the growth and formation of keloids is their resistance to apoptosis Keloid fibroblasts was found to be more resistant to Fas mediated apoptosis (Chodon et al 2000) and the overexpression of insulin-like growth factor-1 (IGF-1) receptor inhibited ceramid-induced apoptosis (Ishihara et al 2000) Furthermore, decreased expression of proapoptotic genes (Sayah et al 1999) and increased... compared to that inferred from keloid data, suggesting a more robust network in the keloid condition This would mean that targeting NFKB alone may not be sufficient to reduce its transcriptional products in keloid fibroblasts The work done in this thesis, utilizing both molecular and computational approaches, has advanced our understanding by shedding light on some of the important players and key networks... 1999) and increased expression of apoptotic inhibitors (Messadi et al 2004) have also been observed in keloid fibroblasts Tissue hypoxia could be another contributory factor to pathogenesis An increased level of hypoxia marker, hypoxia induced factor-1α (HIF-1α) was detected in keloid tissues and hypoxia appears to elevate the expression of plasminogen activator inhibitor-1 (PAI-1) (Zhang et al 2003) Increased... this study, motivating a more global approach to understanding keloid scarring through the use of microarray technology Results from the microarray experiments were then analyzed computationally to provide further insights into the molecular mechanisms underpinning this condition 5 Single factor Microarray Gene networks Figure 1.1: Summary of the three main approaches used in this study An in depth analysis... factors and cytokines are released from the serum of the disrupted blood vessels and degranulating platelets (Werner & Grose 2003) Following hemostasis, neutrophils infiltrate into the wound site and monocytes are activated to become wound macrophages These inflammatory cells serve two purposes: firstly as a means of removing foreign material, bacteria and damaged matrix components by phagocytosis, and. .. proliferate and migrate from the wound edge leading to the process of reepithelialization (Santoro & Gaudino 2005) In addition, local factors in the wound microenvironment such as low pH and reduced oxygen tension initiate the release of angiogenic factors leading to the migration and proliferation of endothelial cells (Knighton et al 1983) Massive angiogenesis leads to the formation of new blood vessels, and. .. bundles are thick and are randomly oriented as swirls and whorls (Rockwell, Cohen & Ehrlich 1989) Keloid formations are characterized by active angiogenesis and hypoxia (Appleton, Brown & Willoughby 1996) Occlusion of some microvessels by excessive endothelial cells may lead to local hypoxic conditions and apoptosis (Kischer 1992) 12 2.2.5 Etiology Several etiological factors for keloids have been... type-B receptor 4 fREDUCE fast-Regulatory Element Detection Using Correlation with Expression GEO Gene Expression Omnibus G0S2 G0/G1switch 2 GO Gene Ontology GPR137B G-protein-coupled receptor 137B GPR153 G-protein-coupled receptor 153 GPSM2 G-protein signaling modulator 2 GRB10 Growth factor receptor-bound protein 10 HATH Homologous to amino terminus of HDGF HDGF Hepatoma-derived growth factor HIF-1α... found to increase the proliferation of keloid fibroblasts and was also found to increase the production of the vascular endothelial growth factor (VEGF) However, one of the hallmarks of keloids is an increased extracellular matrix production, and HDGF did not seem to contribute to this aspect of keloid formation In the second part of this dissertation, we used the microarray platform in an attempt to . MOLECULAR AND COMPUTATIONAL APPROACHES TO UNDERSTANDING KELOID SCARRING OOI NICK SERN, BRANDON (B. Eng. (Hons.), NUS) . transcriptional products in keloid fibroblasts. The work done in this thesis, utilizing both molecular and computational approaches, has advanced our understanding by shedding light on some. players and key networks in keloid scarring. In addition, the results from this study has generated new and promising future areas of research, and is a small step forward to finding a solution to