Effects of hydrogen peroxide on different models of wound healing

EFFECTS OF HYDROGEN PEROXIDE ON DIFFERENT MODELS OF WOUND HEALING LOO ENG KIAT, ALVIN NATIONAL UNIVERSITY OF SINGAPORE 2011 Acknowledgements I started this project knowing next to nothing about wound healing so this thesis would not have been possible without the guidance and discussions from the well-experienced (past and present) members of the lab including (alphabetical order) Aina Hoi, Ho Rongjian, Irwin Cheah, Jan Gruber, Jetty Lee, Long Lee Hua, Sebastian Schafer, Sherry Huang, Ryan Hartwell, Tang Soon Yew and Wong Yee Ting. I would also like to thank John Common and Ng Kee Woei from Prof. Birgit Lane’s lab in Institute of Medical Biology for helping me get started with the scratch wound assay. I would like to give thanks to Pan Ning, Mary Ng and A/P Ong Wei Yi for helping me get started with the histology work. My thesis advisory committee members, A/P Phan Toan Thang and Prof Sit Kim Ping have also been especially helpful and supportive throughout my PhD studies. Most importantly, I would like to thank my supervisor, Prof. Barry Halliwell, for being extremely patient with me, nudging me towards the right direction without ever telling me what (or what not) to do, giving me more than my fair share of opportunities and believing in me even when I have doubts about myself. Finally, I would also like to thank all the administrative staff (past and present) in NGS office, particularly Wee An-Hway Ivy, Chuan Irene Christina and Elissa Horn, all NGS Scholars’ Alliance members and all the staff and students of neurobiology program for making the past four years memorable and interesting. i Contributors to the thesis Animal handling, surgery and tissue collection were performed by Ho Rongjian (HR), Wong Yee Ting (WYT) and myself, Loo Eng Kiat Alvin (LEKA). Experiments for figure 3.4 were performed by HR. Experiments for figures 4.7, 4.8, 4.9 and 4.10 were performed by HR and analyzed by HR and LEKA. Experiments for figures 4.14 and 4.15 were performed and analyzed by WYT. All other experiments were performed by LEKA. I would like to thank all the contributors to the thesis. Journal publications and international conference attended Published Loo, A. E.; Ho, R.; Halliwell, B. Mechanism of hydrogen peroxide-induced keratinocyte migration in a scratch-wound model. Free Radic Biol Med 51:884-892; 2011. In preparation Loo, A. E. & Halliwell, B. Keratinocytes and fibroblasts display differential sensitivity to H2O2. Loo, A. E.; Wong, Y.T.; Halliwell, B. Effects of H2O2 on wound healing and oxidative damage in an excision wound model. Conference poster presented at Society for Free Radical Biology and Medicine 17th Annual Meeting, Nov 17th – 21 2010. Loo, A.E.; Ho, R.; Wong, Y.T.; Halliwell, B. Mechanism of hydrogen peroxide induced keratinocyte cell sheet migration. ii Acknowledgements i Contributors to thesis ii Journal publications and international conference attended ii Table of Content iii Summary vi List of table vii List of figures vii List of abbreviations and keywords ix Chapter A review of the role of H2O2 in wound healing 1.1 1.2 The wound healing process 1.1.1 Inflammatory phase 1.1.2 Is inflammation beneficial in wound healing 1.1.3 Proliferation and Remodelling Phase Evidence for increased ROS and oxidative damage in wounds 1.2.1 Superoxide anion (O2•-) 10 1.2.2 Hydrogen Peroxide (H2O2) 11 1.2.3 Evidence for increased ROS in wounds 12 1.2.4 Evidence for increased oxidative damage in 14 wounds 1.3 1.4 H2O2 as a signaling molecule 16 1.3.1 Principles of H2O2 signaling 16 1.3.2 H2O2, re-epithelialization and MAPK 18 1.3.3 H2O2 as a regulator of cytokine expression 22 Hypothesis and objectives 29 Chapter Materials and methods 2.1 Materials 31 2.2 Culturing of cells 34 2.3 Monolayer scratch wound migration assay 34 2.4 Multiple scratch wound assay 35 iii 2.5 Effect of conditioned medium on ERK and p38 activation 36 2.6 Western blot analysis 37 2.7 Quantification of Cytokines in conditioned Medium 39 2.8 Lactate dehydrogenase activity assay 40 2.9 RNA extraction and semi-quantitative RT-PCR 41 2.10 Effect of H2O2 conditioned medium on cell migration 43 2.11 Cell viability assay 43 2.12 Degradation of H2O2 by HaCaT keratinocytes 44 2.13 Immunofluorescence staining of pERK and pp38 in HaCaT 45 keratinocytes 2.14 Co-culture model of cell migration 46 2.15 Animal handling and excision wound model 48 2.16 Preparation of histological sections 49 2.17 Immunohistochemistry 49 2.18 Connective tissue stain 51 2.19 Immunofluorescence staining of macrophages and 52 neutrophils in animal tissues 2.20 F2-Isoprostanes extraction and analysis 52 Chapter 3, Results I Effects of H2O2 on in vitro models of wound healing 3.1 Effect of H2O2 on HaCaT keratinocytes migration 56 3.1.1 56 Low concentrations of H2O2 increases keratinocyte migration and induce phosphorylation of ERK1/2 and p38 MAPK 3.1.2 Persistent ERK1/2 phosphorylation is needed for 65 H2O2-induced cell migration 3.1.3 EGF receptor phosphorylation is upstream of 67 ERK1/2 but not p38 MAPK phosphorylation 3.1.4 EGF receptor phosphorylation induced by scratch 69 wounding is ligand-dependent but that induced by iv H2O2 is ligand-independent 3.1.5 Role of p38 in H2O2-induced cell migration, a 71 cautionary tale 3.1.6 Persistent ERK1/2 signaling is needed for H2O2- 74 induced proliferation 3.2 Effects of H2O2 on cytokine secretion in keratinocytes 77 3.2.1 77 Conditioned media from H2O2 treated keratinocytes can activate ERK 3.2.2 H2O2 induces expression of VEGF mRNA 80 3.2.3 H2O2 induces expression of pro-inflammatory and 81 pro-angiogenic cytokines 3.2.4 Conditioned medium alone does not induce cell 86 migration 3.3 Effects of H2O2 on a keratinocyte-fibroblast co-culture 87 model of wound healing 3.3.1 3.4 Co-culture model of re-epithelialization Brief discussion 88 92 Chapter 4, Results II Effects of H2O2 on an in vitro models of wound healing 4.1 Biphasic effects of H2O2 on wound closure 93 4.2 Effects of H2O2 on connective tissue formation and MMP 94 production 4.3 Effects of H2O2 on angiogenesis 101 4.4 Effects of H2O2 on leukocyte recruitment 102 4.5 Effects of H2O2 on ERK1/2 and p38 phosphorylation 107 4.6 Effects of wounding and H2O2 on lipid oxidation 110 4.7 Brief discussion 113 Effects of H2O2 on ERK1/2 phosphorylation and HaCaT 115 Chapter Discussion 5.1 keratinocyte migration 5.2 Effects of H2O2 on p38 phosphorylation and HaCaT 118 v keratinocyte migration 5.3 Effects of H2O2 on re-epithelialization in the co-culture model 5.4 Effects of antioxidants on re-epithelialization in the monolayer scratch wound model and co-culture model 120 122 123 128 5.5 Effects of H2O2 on cytokine secretion 132 5.6 Effects of H2O2 on wound closure and connective tissue 134 formation in the excision wound model 5.7 Effects of H2O2 on angiogenesis in the excision wound 134 136 model 5.8 Effects of H2O2 on ERK1/2 and p38 phosphorylation in the excision wound model 5. Effects of H2O2 on inflammatory cell infiltration in an in vivo model of wound healing 5.10 Effects of H2O2 on oxidative damage in the excision wound model Conclusion 140 vi Summary It has been established that low concentrations of H2O2 are produced in wounds. Yet at the same time, there is evidence that excessive oxidative damage is correlated with chronic wounds. In this thesis we explored the effects of H2O2 in keratinocyte cell culture models and an in vivo excision wound model of wound healing. H2O2 stimulates a persistent ERK phosphorylation in HaCaT keratinocytes which was found to be important in cell proliferation and migration. H2O2 also increases the production of proinflammatory and pro-angiogenic cytokines such as Vascular endothelial growth factor, Interleukin-8, Granulocyte-macrophage colony-stimulating factor, Tumor necrosis factor-α, interleukin-6 and Interferon gamma-induced protein 10, in HaCaT keratinocytes. H2O2 was found to increase re-epithelialization in a primary fibroblast-keratinocyte co-culture model as well. In a C57BL/6 mice excision wound model, low concentrations of H2O2 (10 mM) were found to enhance angiogenesis while high concentrations of H2O2 (166 mM) retarded wound closure and connective tissue formation. High concentrations of H2O2 also increased the levels of MMP-8 in the wounds, which could be the cause of reduced connective tissue formation. Wounding was found to increase oxidative lipid damage, as measured by F2isoprostanes, but H2O2 treatment does not significantly increase it even at concentrations that delay wound healing. This challenges the putative claim that oxidative damage contributes to the pathology of poor healing wounds. vii List of Tables Table Table Table Table Standard reduction potential of some common radical species Effects of selected cytokines on wound healing Cytokines that are measured in the custom bead-based multiplex ELISA Comparison of cytokine concentrations h after H2O2 stimulation and with literature values of cytokine concentration required to stimulate an observable phenotype 18 21-22 75 115 List of Figures Fig. 1.1 Fig. 1.2 Fig. 2.1 Fig. 2.2 Fig. 2.3 Fig. 2.4 Fig. 2.5 Fig. 3.1 Fig. 3.2 Fig. 3.3 Fig. 3.4 Fig. 3.5 Fig. 3.6 Fig. 3.7 Fig. 3.8 Fig. 3.9 Fig. 3.10 Fig. 3.11 Fig. 3.12 Fig. 3.13 Fig. 3.14 Fig. 3.15 Fig. 3.16 Fig. 3.17 Fig. 4.1 Fig. 4.2 Fig. 4.3 Schematic diagram illustrating the multiple oxidation states of thiols. Schematic diagram of the mitogen-activated protein kinase signaling cascade Representative photos of the multiple scratch wound assay A representative calibration curve for protein quantification by DC protein assay Standard curve for cell number determination by crystal violet staining A representative calibration curve for H2O2 quantification by FOX2 assay Schematic diagram of the layout of the keratinocyte-fibroblast co-culture re-epithelialization assay Low concentrations of H2O2 stimulate cell proliferation and migration Both scratch wounding and H2O2 activate ERK1/2 Both scratch wounding and H2O2 activate p38 Cells show localization of phosphorylated ERK1/2 and p38 at the wound edge when scratch wounded Sustained ERK1/2 activity is needed for cell migration Scratch wounding and H2O2-induce EGFR phosphorylation is associated with ERK1/2 but not p38 phosphorylation. EGFR phosphorylation induced by H2O2 is ligand-independent but the EGFR phosphorylation induced by scratch wounding is ligand-dependent p38 is not needed for H2O2-induced migration but high concentrations of p38 inhibitor can inhibit keratinocyte migration A crosstalk exists between ERK and p38 where inhibition of one pathway leads to activation of the other Sustained ERK1/2 activation is needed for H2O2-induced proliferation Conditioned media induce ERK1/2 but not p38 phosphorylation in quiescent cells H2O2 induce the expression of VEGF-A 121 and 165 H2O2-induced cytokine secretion and their dependence on the ERK and p38 pathway The LDH activity of conditioned medium from cells treated with U0126, SB203580 and/or H2O2 Conditioned medium from cells treated with 500 µM H2O2 is not sufficient to induce cell migration H2O2 promotes keratinocyte migration in a co-culture model of reepithelialization and NAC retards it Cell viability of fibroblast-keratinocyte co-culture after treatment with H2O2 or NAC Biphasic effect of H2O2 on wound closure Validation of Masson-Goldner trichrome stain quantification 166 mM H2O2 retards connective tissue formation but 10 mM H2O2 does not affect connective tissue formation 17 20 36 38 40 45 47 58 - 60 61 - 62 63 - 64 64 66 68 – 69 70 72 - 73 75 76 - 77 78 - 80 81 84 -85 86 87 90 - 91 92 94 96 97 viii Fig. 4.4 Fig. 4.5 Fig. 4.6 Fig. 4.7 Fig. 4.8 Fig. 4.9 Fig. 4.10 Fig. 4.11 Fig. 4.12 Fig. 4.13 Fig. 4.14 Fig. 4.15 166 mM H H2O2 treatment increases MMP-8 Effect of H2O2 on MMP-9 levels Effect of H2O2 on TIMP-1 levels 10 mM H2O2 induce angiogenesis but 166 mM H2O2 has no effect on angiogenesis Time course of neutrophil infiltration 166 mM H2O2 causes persistent neutrophil infiltration on day postwounding Time course of macrophage infiltration H2O2 does not affect macrophage infiltration on day post-wound Wounding increases ERK1/2 phosphorylation which can be further increased by 166 mM H2O2 treatment Wounding increases p38 phosphorylation which can be further increased by 166 mM H2O2 treatment Wounding increases F2-isoprostanes levels but 166 mM H2O2 does not increase it further Comparison of F2-isoprostanes levels per unit fresh tissue weight and changes in arachidonic acid over time 99 100 101 102 104 105 106 107 109 110 112 113 ix [41] Soininen, R.; Haka-Risku, T.; Prockop, D. 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Myofibroblasts are aligned along the lines of contraction which in turn are aligned in the direction of skin tension [46] In full thickness wounds, contraction is an important part of healing In animals with loose skin, such as the dorsal area of rodents, it is the predominant mechanism for wound closure Myofibroblasts have increased expression of smooth muscle differentiation markers such as α-smooth... application of a 0.15% solution of H2O2 did not affect wound closure in mice but promoted angiogenesis On the other hand a 3% solution delayed wound healing and even higher concentrations killed the mice [58] Similarly, knockout of the antioxidant enzyme peroxiredoxin 6 results in severe hemorrhages during wound healing [66] From these observations, we hypothesized that low levels of ROS may contribute to wound. .. acute and chronic wounds compared to intact skin but chronic wounds have higher levels of MDA than acute wounds [67-69] F2-isoprostanes, another marker of lipid peroxidation, have also been shown to be higher in chronic wound fluids than acute wound fluids [70] However studies on wound fluid fails to answer the fundamental question of whether wounding increases oxidative damage Using 4-Hydroxynonenal as... healing This assumption is wrong on several accounts Firstly, while neutrophils and macrophages are important sources of ROS, they are not the only source of ROS in wounds Resident cells are also important sources of ROS and will be discussed in section 1.3 Secondly, inflammation need not be associated with increased ROS Upon phagocytosis of a foreign body, the oxygen consumption rate of phagocytes increase... infection [23-25] It is clear that ROS are not the only way that inflammatory cells kill bacteria and therefore the presence of inflammatory cells in wounds cannot be taken as unequivocal proof of increased ROS in wounds Having established that the investigation of the role of ROS in wounds is a problem distinct from the role of inflammation in wounds, what is our current knowledge of the role of inflammation... current knowledge of the role of inflammation in wound healing? Removal of macrophages from wounds with the use of macrophage antisera retards wound healing [26] while the removal of neutrophils with antisera results in faster healing [27] PU.1 is a key transcription factor for the differentiation of common myeloid progenitor cells to the granulocytes and monocytes lineage PU.1 knockout mice lack macrophages,... after wounding [3] The concentrations of H2O2 found in wounds range from 50 [58] – 250 [3] µM, depending on the model and method used It was further found that DUOX (there is only 1 isoform of DUOX in zebrafish) is the cellular source of ROS in this model The production of H2O2 precedes the infiltration of leukocytes and is shown to serve as a leukocyte chemoattractant In vitro studies have also demonstrated... is complex and there is no one straightforward answer to whether inflammation is beneficial in wound healing Only one fact is certain; none of the inflammatory cells are absolutely required for healing [30] 1.1.3 Proliferation and Remodelling Phase The two main activities of the proliferation phase are the restoration of the epidermis and the dermis After these, the healed wound is remodeled into a... the role of ROS in wounds, one would need to measure the amount of oxidative damage in wounds 1.2.4 Evidence for increased oxidative damage in wounds Previous studies have attempted to measure oxidative damage in wounds However these are not without limitations The most commonly used oxidative damage marker studied in wound healing is malondialdehyde (MDA), a product of lipid peroxidation Among the . H 2 O 2 on an in vitro models of wound healing 4.1 Biphasic effects of H 2 O 2 on wound closure 4.2 Effects of H 2 O 2 on connective tissue formation and MMP production 4.3 Effects of H 2 O 2 . monolayer scratch wound model and co-culture model 5.5 Effects of H 2 O 2 on cytokine secretion 5.6 Effects of H 2 O 2 on wound closure and connective tissue formation in the excision wound. model 5.7 Effects of H 2 O 2 on angiogenesis in the excision wound model 5.8 Effects of H 2 O 2 on ERK1/2 and p38 phosphorylation in the excision wound model 5. 9 Effects of H 2 O 2 on inflammatory