The Role of Mitochondrial Reactive Oxygen Species in the Development of Hypoxia-induced Pulmonary Hypertension Open Access

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, Tg^hSOD2, and Tg^hTrx2), 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

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Molecular & Systems Pharmacology
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Biology, Physiology Biology, Molecular
Keyword	reactive oxygen species mitochondria transgenic model hydrogen peroxide superoxide pulmonary hypertension hypoxia NADPH oxidase
Committee Chair / Thesis Advisor	Sutliff, Roy, Emory University Hart, Michael, Emory University
Committee Members	Brown, Lou Ann, Emory University Griendling, Kathy, Emory University

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