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Functions of Extracellular Pyruvate Kinase M2 in Tissue Repair and Regeneration Georgia State University ScholarWorks @ Georgia State University Biology Dissertations Department of Biology 5-9-2016 Functions of Extracellular Pyruvate Kinase M2 in Tissue Repair and Regeneration Yinwei Zhang Follow this and additional works at: https://scholarworks.gsu.edu/biology_diss Recommended Citation Zhang, Yinwei, "Functions of Extracellular Pyruvate Kinase M2 in Tissue Repair and Regeneration." Dissertation, Georgia State University, 2016. https://scholarworks.gsu.edu/biology_diss/166 This Dissertation is brought to you for free and open access by the Department of Biology at ScholarWorks @ Georgia State University. It has been accepted for inclusion in Biology Dissertations by an authorized administrator of ScholarWorks @ Georgia State University. For more information, please contact [email protected]. FUNCTIONS OF EXTRACELLULAR PYTUVATE KINASE M2 IN TISSUE REPAIR AND REGENERATION by YINWEI ZHANG Under the Direction of Zhi-Ren Liu, PhD ABSTRACT Pyruvate kinase M2 (PKM2) is a glycolytic enzyme expressed in highly proliferating cells. Studies of PKM2 have been focused on its function of promoting cell proliferation in cancer cells. Our laboratory previously discovered that extracellular PKM2 released from cancer cells promoted angiogenesis by activating endothelial cell proliferation and migration. PKM2 activated endothelial cells through integrin αvβ3. Angiogenesis and myofibroblast differentiation are key processes during wound healing. In this dissertation, I demonstrate that extracellular PKM2 released from activated neutrophils promotes angiogenesis and myofibroblast differentiation during wound healing. PKM2 activates dermal fibroblasts through integrin αvβ3 and PI3K signaling pathway. I also claim that extracellular PKM2 plays a role during liver fibrosis. PKM2 protects hepatic stellate cells from apoptosis by activating the survival signaling pathway. INDEX WORDS: Pyruvate kinase M2, Wound healing, Angiogenesis, Myofibroblast differentiation, Liver fibrosis, Apoptosis FUNCTIONS OF EXTRACELLULAR PYRUVATE KINASE M2 IN TISSUE REPAIR AND REGENERATION by YINWEI ZHANG A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the College of Arts and Sciences Georgia State University 2016 Copyright by Yinwei Zhang 2016 FUNCTIONS OF EXTRACELLULAR PYRUVATE KINASE M2 IN TISSUE REPAIR AND REGENERATION by YINWEI ZHANG Committee Chair: Zhi-Ren Liu Committee: Yuan Liu Susanna Greer Electronic Version Approved: Office of Graduate Studies College of Arts and Sciences Georgia State University May 2016 iv DEDICATION I dedicate my dissertation to my father, Suming Zhang, and my mother, Yuan Yuan. Thanks for your unconditional support in my life. I love you. I also dedicate my dissertation to my grandma, my uncle and other family members, for the continued support and encouragement. At last, I dedicate this work to my dear cousin Mujia Erica Yuan. I will always miss you. v ACKNOWLEDGEMENTS I would like to thank my advisor Dr. Zhi-Ren Liu for having me in his lab. He always supports my research work and encourages me to generate novel ideas. I would like express my gratitude to my committee members Dr. Yuan Liu and Dr. Susanna Greer, for the great suggestions and supports on this research project, and also my presentation. I would like to thank Dr. Jenny Yang for letting me use the equipment in her lab and also giving me lots of good ideas. I would like to thank Dr. Siming Wang and Dr. Cheng Ma for the assistance in mass spectrometry experiment. I thank all my previous and current lab members for the help in the lab, especially Dr. Liangwei Li. I thank the biology core facility for the technical support and LaTesha Warren for the help during my PhD years. I would also like to give my appreciation to everyone in the joint group meeting. vi TABLE OF CONTENTS DEDICATION.................................................................................................................. iv ACKNOWLEDGEMENTS ............................................................................................. v TABLE OF CONTENTS ................................................................................................ vi LIST OF FIGURES ........................................................................................................ xii CHAPTER 1 GENERAL BACKGROUND ............................................................... 1 1.1 Glycolysis and Pyruvate Kinase ......................................................................... 1 1.2 Pyruvate Kinase M2 ............................................................................................ 3 1.2.1 Pyruvate kinase M2 structure and regulation ............................................. 3 1.2.2 Location of pyruvate kinase M2 ................................................................... 4 1.3 Functions of PKM2 ............................................................................................. 6 1.4 Wound Healing .................................................................................................... 8 1.4.1 Wound healing process ................................................................................. 8 1.4.2 Neutrophil infiltration................................................................................. 10 1.4.3 Angiogenesis................................................................................................ 12 1.4.4 Myofibroblast differentiation ...................................................................... 14 1.4.5 From immune response to proliferation .................................................... 16 1.4.6 Treatment of wound .................................................................................... 17 1.5 Liver fibrosis and cirrhosis .............................................................................. 17 1.5.1 Introduction of liver fibrosis and cirrhosis ................................................ 17 vii 1.5.2 Pathogenesis of liver fibrosis ...................................................................... 18 1.5.3 Reverse of liver fibrosis ............................................................................... 20 1.6 Rational and Aims ............................................................................................. 21 CHAPTER 2 EXTRACELLULAR PYRUVATE KINASE M2 RELEASED FROM NEUTROPHILS FACILITATES EARLY WOUND HEALING BY PROMOTING ANGIOGENESIS1………………………………………………………………………...……27 2.1 Abstract .............................................................................................................. 27 2.2 Introduction ....................................................................................................... 27 2.3 Results ................................................................................................................ 28 2.3.1 rPKM2 facilitates wound healing by activating angiogenesis. ................. 28 2.3.2 rPKM2 promotes granulation tissue growth during wound healing ........ 29 2.3.3 Application of PKM2 antibody slows wound healing process .................. 30 2.3.4 PKM2 was released from neutrophils in wound healing .......................... 31 2.3.5 Inhibition of neutrophil infiltration reduces extracellular PKM2 ............ 32 2.3.6 Identification of PKM2 release from neutrophil degranulation ............... 33 2.4 Discussion ........................................................................................................... 33 2.5 Materials and Methods ..................................................................................... 34 2.5.1 Mouse Wound Healing Model .................................................................... 34 2.5.2 Tissue Sample Preparation ......................................................................... 35 2.5.3 H&E Staining .............................................................................................. 35 viii 2.5.4 Measurement of Granulation Thickness ................................................... 36 2.5.5 Neutrophil Isolation .................................................................................... 36 2.5.6 Activation of Neutrophils ............................................................................ 36 2.5.7 Beige Mice Wound Healing Protocol ......................................................... 37 2.5.8 Sucrose Gradient Centrifugation ............................................................... 37 CHAPTER 3 EXTRACELLULAR PYRUVATE KINASE M2 ACTIVATES DERMAL MYOFIBROBLAST DIFFERENTIATION ......................................................... 56 3.1 Abstract .............................................................................................................. 56 3.2 Introduction ....................................................................................................... 56 3.3 Results ................................................................................................................ 57 3.3.1 Myofibroblast differentiation is activated in rPKM2 treated wound ........ 57 3.3.2 Effect of extracellular PKM2 on HDFa..................................................... 58 3.3.3 Extracellular PKM2 activates HDFa cells through integrin αvβ3 ........... 58 3.4 Discussion ........................................................................................................... 60 3.5 Materials and Methods ..................................................................................... 61 3.5.1 Induction of HDFa Differentiation
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