Understanding the Functional Contribution of Heme to the Progression of Atherosclerosis in Beta Thalassemia Restricted; Files Only

Abstract

 

Background

Children with Beta Thalassemia (BT) present with an increase in carotid intima-medial thickness, an early sign suggestive of premature atherosclerosis. However, it is unknown if there is a direct relationship between BT and atherosclerotic disease. BT is a hemolytic anemia which has increased free heme in the vasculature. We hypothesize that heme-mediated oxidative stress creates a proatherogenic environment.

Methods

Both male and female, WT (littermates) and BT (Hbbth3/+) mice were placed on a 3-month high fat diet with LDL receptor suppression via proprotein convertase subtilisin/kexin type 9 (PCSK9) gain-of-function mutation (D377Y). In addition, the effect of hemopexin (HPX) on the progression of atherosclerosis was evaluated by overexpressing HPX with an adeno-associated virus. Atherosclerosis was evaluated in the descending aorta via en face analysis and histology of the aortic roots. In parallel, we evaluated atherosclerotic plaque accumulation in a drug-induced model of hemolysis using phenylhydrazine (PHZ). We also evaluated the effect of defiriprone (DFP)-mediated iron chelation in the progression of atherosclerosis in BT mice.

Results

Aortic en face analysis revealed elevated plaque accumulation in both male and female BT mice compared to WT mice (WT female: 10.6 ± 2.9 % vs. BT female: 23.3 ± 1.5 % and WT male: 28.1 ± 2.4 % vs. BT male: 45.5 ± 2.4 %, total percentage plaque area relative to luminal area, mean ± SEM). Hemopexin therapy was able to decrease plaque accumulation in BT mice measured via aortic en face (BT: 45.5 ±. 2.4 % vs. BT + HPX: 31.7 ± 2.5 %) and aortic root lesion area analysis (BT: 444.7 ± 38.1 µm² x 10³ vs BT + HPX: 318.8 ± 28.6 µm² x 10³).

Conclusion

Our data demonstrate for the first time that the underlying pathophysiology of BT leads to accelerated atherosclerosis and shows that heme contributes to atherosclerotic plaque development in BT.  

Table of Contents

 

CHAPTER 1: INTRODUCTION 1

1.1 Introduction 2

1.1.1 Underlying Pathophysiology in Beta Thalassemia 2

1.2 Clinical Manifestation and Disease Management in Beta Thalassemia 7

1.3 Clinical observations suggest that Beta Thalassemia is associated with Accelerated Atherosclerosis 12

1.4 Intravascular hemolysis in Beta Thalassemia 13

1.4.1 Pathophysiology of Hemolysis 16

1.4.2 Contribution intravascular hemolysis to oxidative stress 16

1.4.3 Evidence of Intravascular hemolysis in Beta Thalassemia 28

1.5. Summary and Objectives 34

1.5.1 Summary 34

1.5.2 Objectives 34

CHAPTER 2: ACCELERATED ATHEROSCLEROSIS IN BETA THALASSEMIA 36

2.1 Introduction to Atherosclerosis 37

2.1.1 Blood Vessel Structure 37

2.1.2 Pathogenesis of Atherosclerosis 38

2.1.3 Models of Atherosclerosis 46

2.2 Methods 50

2.3. Results 53

2.3.1 Beta Thalassemia’s Underlying Pathophysiology leads to Accelerated Atherosclerosis 53

2.3.2 Secondary Model Confirms Accelerated Atherosclerosis in Beta Thalassemia 55

2.3.3 Beta Thalassemia is a Hemolytic Anemia with Intravascular Hemolysis 59

2.4 Discussion 59

CHAPTER 3: OVEREXPRESSING HEMOPEXIN IN HEMOLYTIC MICE ON A LONG-TERM MODEL OF ATHEROSCLEROSIS 65

3.1 Introduction to HPX experiments 66

3.1.1 Introduction 66

3.1.2 Heme and Atherosclerosis 70

3.1.3 Model of hemolysis 72

3.2 Methods 73

3.2.1 Phenylhydrazine 74

3.2.2 Beta Thalassemia 74

3.2.3 Hemopexin therapy 74

3.2.4 Assays 74

3.2.5 Statistical Analysis 75

3.3 Results 75

3.3.1 Phenylhydrazin-mediated Hemolysis leads to Accelerated Atherosclerosis that can be Reduced with Hemopexin Therapy 76

3.3.2 Beta Thalassemia leads to Accelerated Atherosclerosis that can be Reduced with Hemopexin Therapy 79

3.4 Discussion 97

CHAPTER 4: DEFERIPRONE DECREASES ATHEROSCLEROSIS IN BETA THALASSEMIC MICE,BUT DOES NOT PROVIDE AN ADDITIVE BENEFIT WITH HEMOPEXIN THERAPY 102

4.1 Introduction 103

4.1.1 Key Proteins and Pathways 103

4.1.2 Iron and Atherosclerosis 107

4.1.3 Iron Chelators 109

4.2. Methods 111

4.3 Results 112

4.3.1 Deferiprone reduces atherosclerosis in Beta Thalassemia 112

4.4 Discussion 115

CHAPTER 5: DISCUSSION/FUTURE DIRECTIONS 118

5.1 Overview 119

5.2 Does Beta Thalassemia underlying pathophysiology lead to accelerated atherosclerosis? 119

5.3 Is intravascular hemolysis a prominent feature of Beta Thalassemia? 123

5.4 Is hemopexin therapy a promising therapeutic in Beta Thalassemia for atherosclerosis? 123

5.5 Implications for Accelerated Atherosclerosis in Beta Thalassemia and Future directions 125

5.5.1 Changing Landscape in Beta Thalassemia 125

5.5.2 Are other hemolytic anemias associated with an increased risk for accelerated atherosclerosis? 126

5.5.3. What are the contributions of the other adaptive and pathological responses to hemolysis? 128

5.6. Summary 131

REFERENCES 132

 

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Molecular & Systems Pharmacology
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, Physiology Engineering, Biomedical
Keyword	Hemolytic anemia Atherosclerosis Sickle cell disease Beta Thalassemia
Committee Chair / Thesis Advisor	W. Robert Taylor, Emory University
Committee Members	Roy Sutliff, Emory University David Archer, Emory University Lou Ann Brown, Emory University

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