The Role of Mitochondrial Reactive Oxygen Species in the Development of Hypoxia-induced Pulmonary Hypertension Pubblico
Adesina, Sherry (2015)
Published
Abstract
Pulmonary hypertension (PH) is characterized by increased pulmonary vascular resistance, pulmonary vascular remodeling, and increased pulmonary pressures that result in right ventricular hypertrophy and if untreated, right heart failure and death. Pathogenic derangements in PH include an imbalance in the production of vasodilating and vasoconstricting mediators and enhanced proliferation of pulmonary vascular wall cells. Numerous studies have noted increased reactive oxygen species (ROS) in patients and models of PH. ROS are produced as intermediates in the redox reactions leading from O2 to H2O and comprise both free radicals (superoxide, O2•-) and non-radical derivatives of oxygen (e.g. hydrogen peroxide, H2O2). ROS in the form of O2•- and H2O2 play a vital role in vascular cell signaling, and regulate cellular proliferation, differentiation, and apoptosis. Current evidence suggests that ROS generated by both mitochondrial respiration and NADPH oxidases (Noxes) may contribute to PH pathogenesis by altering vascular cell proliferation and apoptotic signaling pathways. If hypoxia increases mitochondrial ROS generation (mtROS) to stimulate Nox expression, then targeted reduction of mtROS will prevent pulmonary vascular wall proliferation, remodeling, and PH pathogenesis. The overall goal of this project was to assess if reducing mtROS levels by increasing mitochondrial antioxidants could mitigate hypoxia-induced aberrations in Nox expression, vascular remodeling, and molecular signaling. To our knowledge, the direct assessment of the contribution of mtROS to Nox expression and activity in hypoxia-induced PH has not been previously reported. Using three transgenic models (MCAT, TghSOD2, and TghTrx2), studies emphasize: A) the importance of mtROS in hypoxia-induced PH pathogenesis, and B) that targeted therapies directed at lowering mtH2O2 may be uniquely effective in reducing pulmonary vascular cell proliferation, remodeling, and PH.
Table of Contents
Index of Tables i
Index of Figures ii
List of Abbreviations iv
Chapter 1: Introduction 1
PAH Etiology 4
PH Pathobiology 8
Current PH Therapy 11
Experimental Animal Models of PH 13
Monocrotaline 14
Hypoxia 15
Hypoxia + Sugen 18
Model Differences 19
Hypoxia-induced ROS 21
Superoxide 25
Hydrogen peroxide 26
Antioxidants 27
Roles and Sources of Oxidative Stress in PH 29
NADPH Oxidases 30
Background and Isoforms 30
Nox2 and Nox4 32 Nox2 and Nox4 in PH 34
Mitochondria 36
Mitochondrial-Derived ROS Production 37
Possible Roles of Mitochondrial-Derived ROS in PH 39
Summary 41
Proposed Research 43
CHAPTER 2: Materials and Methods 47
Littermate Control and Transgenic Mice 48
Mitochondrial Catalase (MCAT) 48
Transgenic SOD2 Overexpression (TghSOD2) 51
Thioredoxin 2 Overexpression(TghTrx2) 54
Hypoxia Exposure 56
Right Ventricular Systolic Pressure Assessment 56
Right Ventricular Hypertrophy Measurement 57
Echocardiography 58
Assessment of Pulmonary Arteriolar Muscularization 58
Mitochondria Isolation 60
Amplex Red Detection of H2O2 in Pulmonary Tissue 61
Real-time qRT-PCR mRNA Analysis 62
Western Blot Analysis 65
Confocal Microscopy of Mitochondrial ROS 69
MitoPy1 69
MitoSOX 70
Statistical Analysis 70
CHAPTER 3: Mitochondrial Catalase Expression Prevents Hypoxia-induced PH 72
INTRODUCTION 73
RESULTS 77
DISCUSSION 98
CHAPTER 4: Overexpression of SOD2 Exacerbates Hypoxia-induced PH 106
INTRODUCTION 107
RESULTS 110
DISCUSSION 127
CHAPTER 5: Overexpression of Thioredoxin2 in Hypoxia-induced PH 133
INTRODUCTION 134
RESULTS 138
DISCUSSION 153
CHAPTER 6: Discussion 157
Mitochondrial ROS Regulation of Noxes 158
Mitochondrial H2O2 Regulation of PH 163
Future Studies 166
Conclusions 169
References 173
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