Improving Cardiac Repair by Stem Cell Factor Post-Myocardial Infarction

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Scholarship@Western

Electronic Thesis and Dissertation Repository

4-19-2013 12:00 AM

Improving Cardiac Repair by Stem Cell Factor Post-Myocardial

Infarction

Fuli Xiang

The University of Western Ontario

Supervisor Dr Qingping Feng

The University of Western Ontario Graduate Program in Physiology

A thesis submitted in partial fulfillment of the requirements for the degree in Doctor of Philosophy

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POST-MYOCARDIAL INFARCTION

(Thesis format: Integrated Article)

by

Fu-Li Xiang

Graduate Program in Physiology

A thesis submitted in partial fulfillment of the requirements for the degree of

Doctor of Philosophy

The School of Graduate and Postdoctoral Studies The University of Western Ontario

London, Ontario, Canada

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Abstract

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that overexpression of hSCF increased epicardial activation, enhanced epicardium-derived cell (EPDC) proliferation and migration via SCF/c-kit signaling, and promoted arteriogenesis post-MI. In conclusion, M-SCF improves cardiac repair and function through inhibiting myocardial apoptosis and cardiac remodeling, and by promoting neovascularization via c-kit+ stem cell recruitment and EPDC activation and migration.

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Co-Authorship Statement

The studies outlined in Chapters 2-4 were performed by Fu-Li Xiang in the laboratory of Dr. Qingping Feng, with the assistance of co-authors as listed below.

Dr. Qingping Feng contributed to experimental design, data interpretation, and manuscript preparation for all experiments. Dr. P. Chidiac, Dr P. Zhu and Dr J. Robbins contributed to manuscript preparation for Chapter 2. Additionally, Dr. Sharon Lu designed and created the hSCF/tTA mice and assisted with the troubleshooting in all the experiments. Mrs. Murong Liu coordinated the animal breeding and genotyping.

Chapter 2: Dr. L Hammoud performed the RT-PCR and western blot analysis of hSCF expression (Figure 2.1).

Chapter 3: Yin Liu assisted with the fluorescent imaging (Figure 3.5 and 3.7).

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Acknowledgments

I would like to thank my supervisor and mentor Dr. Qingping Feng for his help, support, and guidance throughout my Ph.D. studies. Dr. Feng is one of the most hard-working people I have ever met in my life and his commitment to excellence has made him an excellent researcher and a highly respected professor. His passion for research and craving for knowledge inspires me all the time. It is one of the best decisions I made in my life to work in his lab. He creates a very positive environment in the lab that fosters academic learning, creativity, and the career development for future cardiovascular investigators.

To the Feng laboratory, I could not have asked for a more stimulating and supportive environment to work. I would like to particularly thank Dr. Sharon Lu. She is a wonderful teacher and a good friend. To all other members of the Feng lab you have enriched my experience as a graduate student and made my stay in Feng lab enjoyable. To my advisory committee Dr. Peter Chidiac, Dr. David Hess and Dr. Douglas Jones: as a group you always provided unique perspectives and valuable suggestions on my studies and helped strengthen them. On an individual level Dr. Chidiac was the most approachable and lovely person in the department, Dr. Hess was a constant source of support and was very helpful in terms of unique suggestions that solidified my studies. Dr. Jones, was a model of professionalism and his guidance with regards to my career will always be appreciated.

To Heart and Stroke Foundation of Canada, the doctoral research award gave me financial support for my PhD studies, and to attend and present in international conferences.

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I also would like to acknowledge the Lawson Health Research Institute, and the Heart Failure Group at Western for providing me with a very stimulating and supportive environment that helped me to converse scientifically and become a skilled presenter. Additionally, I truly enjoyed my experience TAing for Physiology 3130y. Tom, Jamie and Chris were all wonderful people to work with.

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List of Tables

Table 2.1 General information of control and hSCF/tTA mice subjected to sham or MI surgeries ... 79

Table 2.2 Hemodynamic parameters of WT and hSCF/tTA mice 5 days post-MI ... 85

Table 2.3 Hemodynamic parameters of control and hSCF/tTA mice 30 days post-MI ... 86

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List of Figures

Figure 1.1 Cardioprotective PI3K/Akt signaling pathways in ischemia/reperfusion (I/R). ... 8

Figure 1.2 Potential cell sources for cardiogenesis post-MI... 16

Figure 1.3 Illustration of soluble (S-SCF) and membrane-associated SCF (M-SCF). ... 28

Figure 1.4 Proposed cellular mechanisms by which membrane-associated SCF (M-SCF) regulates cardiac function post-MI. ... 34

Figure 2.1 Generation and characterization of the inducible hSCF/tTA transgenic mouse. .. 76

Figure 2.2 Mouse SCF expression in the heart post-MI. ... 80

Figure 2.3 Survival and LV function 30 days post-MI... 83

Figure 2.4 Cardiac function 5 days post-MI. ... 87

Figure 2.5 Myocardial apoptosis in the peri-infarct area 5 days post-MI... 90

Figure 2.6 c-kit+ and c-kit+/VEGFR2+ cells retained in the peri-infarct area 5 days post-MI.92 Figure 2.7 c-kit+ and c-kit+/VEGFR2+ cells retained in the peri-infarct area 30 days post-MI. ... 94

Figure 2.8 Mast cell staining using toluidine blue in heart tissue sections from WT and hSCF/tTA mice. ... 98

Figure 2.9 Growth factor expression in the peri-infarct area 5 days post-MI... 100

Figure 2.10 LV hypertrophy 30 days post-MI or sham operations... 102

Figure 2.11 Myocardial capillary density 5 days post-MI. ... 105

Figure 2.12 Myocardial capillary density 30 days post-MI. ... 107

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Figure 3.1 Effects of hSCF overexpression on infarct size after I/R. ... 130

Figure 3.2 Myocardial apoptosis in peri-infarct area after I/R. ... 132

Figure 3.3 Myocardial Akt phosphorylation in WT and hSCF/tTA mice after I/R... 135

Figure 3.4 Myocardial eNOS phosphorylation in WT and hSCF/tTA mice after I/R. ... 137

Figure 3.5 c-kit+ cells in WT and hSCF/tTA hearts. ... 139

Figure 3.6 Cardioprotective cytokine expression in WT and hSCF/tTA mice after I/R. ... 142

Figure 3.7 Sub-population of c-kit+ cells in the myocardium after I/R. ... 145

Figure 3.8 Overexpression of hSCF in c-kit+ CSCs in vitro. ... 148

Figure 4.1 Epicardial Wt1 expression 3 days post-MI. ... 169

Figure 4.2 Arteriolar density in the peri-infarct area 5 days post-MI. ... 172

Figure 4.3 In vitro culture of EPDCs. ... 175

Figure 4.4 Outgrowth, proliferation and growth factor expression in cultured EPDCs. ... 177

Figure 4.5 Expression of hSCF protein in adenovirus treated EPDCs. ... 179

Figure 4.6 Transwell EPDC migration. ... 182

Figure 4.7 Lineage tracing of Wt1+ cell derived EPDCs 5 days post-MI... 185

Figure 4.8 Expression of hSCF protein in adenovirus treated hearts. ... 187

Figure 4.9 In vitro lineage tracing of Wt1+ cell derived EPDCs. ... 189

Figure 4.10 Fate-mapping of Wt1+ cell derived EPDCs post-MI... 192

Figure 5.1 Summarized cellular mechanisms by which M-SCF improves cardiac function and animal survival post-MI... 207

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Figure 5.3 Expression of soluble and membrane-associated hSCF in the serum of WT and hSCF/tTA mice. ... 214

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List of Appendices

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List of Abbreviations, Symbols and Nomenclature

ACE: angiotensin-converting-enzyme

Ad-GFP: adenoviral green fluorescent protein construct

Ad-hSCF: adenoviral membrane-associated human stem cell factor construct Ad-LacZ: adenoviral LacZ construct

BAD: B cell lymphoma (Bcl)-xl/Bcl-2–associated death promoter bFGF: basic fibroblast growth factor

BMP2: bone morphogenetic protein-2 BMSCs: Bone marrow-derived stem cells CDCs: cardiosphere-derived cells

CSCs: cardiac stem cells

CSPCs: cardiac side population cells DAB: diaminobenzidine tetrahydrochloride DOX: doxycycline

E: embryonic day

ELISA: enzyme-linked immunosorbent assay EMT: epithelial-mesenchymal transition eNOS: endothelial nitric oxide synthase EPCs: endothelial progenitor cells EPDCs: epicardium-derived cells EPO: erythropoietin

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xviii GFP: green fluorescent protein

GSK-3β: glycogen synthase kinase 3β HF: heart failure

HGF: hepatocyte growth factor

hPGK: human phosphoglycerate kinase

hSCF: membrane-associated human stem cell factor HSCs: hematopoietic stem cells

I/R: ischemia and reperfusion IGF-1: insulin-like growth factor 1 IHD: ischemic heart disease IP: intraperitoneal

JAK/STAT: Janus kinase/signal transducers and activators of transcription LV: left ventricle

LVEF: left ventricle ejection fraction

LVW/BW: left ventricle to body weight ratio MAP: mean artery pressure

MHC: myosin heavy chain MI: myocardial infarction

MMLV: moloney murine leukemia virus MNCs: mononuclear cells

MOI: multiplicity of infection

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xix mTOR: mammalian target of rapamycin

NF-κB: nuclear factor kappa B P0: postnatal day 0

PAMP: preload adjusted maximum power PDK1: phosphoinositide-dependent kinase 1 PI3K: phosphoinositide-3-kinase

PKC: protein kinase C PLC-γ: phospholipase-γ

PMA: phorbol 12-myristate 13-acetate RISK: reperfusion injury salvage kinase SCF: stem cell factor

SH2: Src homology 2

S-SCF: soluble stem cell factor SW: stroke work

TGF β: transforming growth factor β tTA: tetracycline activator

TTC: triphenyltetrazolium chloride VEGF: vascular endothelial growth factor

VEGFR2: vascular endothelial growth factor receptor 2 vWF: von Willebrand factor

WT: wild type Wt 1: Wilms tumor 1

α-SMA: α-smooth muscle actin

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xx *, † and ‡are used for denoting statistical significance.

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1

CHAPTER 1

Literature Review

1.1 ISCHEMIC HEART DISEASE (IHD)

IHD is a condition in which the heart muscle is damaged or works inefficiently due to an absence or relative deficiency of its blood supply. Most often caused by atherosclerosis, the clinical manifestation of IHD includes angina pectoris, acute myocardial infarction, chronic ischemic heart disease, and sudden death. IHD is the leading cause of death and a financial burden worldwide.1 For example, in the United States, IHD resulted in 445,687 deaths (about one of every five deaths) in 2005 and was reported to place a direct and indirect cost of $165.4 billion in 2009.1 Despite optimal treatment with existing drugs, the prognosis of IHD remains poor. Heart transplantation is the last but an effective option for end-stage IHD patients. The five-year survival rate of heart transplantation has achieved 70% in the United States.1 However, the limited donors for heart transplantation are not meeting the need of IHD patients. Thus, novel and effective treatments for IHD are in high demand.

1.1.1 Physiology of the Heart

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capillaries that supply the heart with oxygen. The outer layer of the heart wall is the epicardium, consisting of mesothelium and connective tissues.

The heart pumps blood through two series circulations by coordinated actions of the atria and ventricles.3 Oxygenated blood from the lungs is received by the left atrium and is subsequently pumped into the aorta (systemic circulation) by the left ventricle (LV). The blood flows through arteries and capillaries to all tissues in the body to supply them with oxygen and nutrients. The deoxygenated blood returns to the right atrium of the heart from veins and is then pumped into the pulmonary circulation via the right ventricle to be re-oxygenated in the lungs. The heart generates continuous and sufficient blood circulation and can, by demand, increases its output. The coronary circulation is critical to maintain heart muscle function. Insufficient coronary perfusion caused by coronary artery narrowing or constriction results in cardiac ischemia leading to impaired cardiac function.4

1.1.2 Myocardial Infarction (MI)

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and recruited to the infarcted region.6 Following acute MI, re-establishing coronary blood flow by thrombolysis or primary angioplasty is widely used to salvage viable myocardium. However, the oxidative stress and abrupt metabolic changes immediately after reperfusion have been shown to initiate further cellular injury.7,8 Healing of the infarcted myocardium leads to scar formation and ventricular remodeling, which is characterized by ventricular enlargement, fibrosis and hypertrophic growth of the peri-infarct and non-peri-infarct myocardium. The remodeled LV causes cardiac dysfunction, clinical heart failure and increased mortality.5,9

1.1.3 Heart Failure (HF)

HF is a clinical syndrome characterized by impaired cardiac function and altered

structure, leading to dyspnea and fatigue at rest or with exertion.10 Patients who survive a

MI are at an increased risk of developing HF. The extent of LV systolic and diastolic

dysfunction is associated with clinical symptoms and cardiovascular mortality. HF can be

caused by the continuous abnormal loading conditions post-MI as a result of the infarct

scar formation. The LV remodeling is a dynamic process that is driven by a number of

factors.11 During cardiac remodeling post-MI, the LV becomes dilated, hypertrophic, and

undergoes morphological and pathophysiological alterations. Pathologically, there is

extensive fibrosis as well as disarray of abnormally structured myocardial cells.

Furthermore, apoptosis becomes more active, resulting in additional loss of myocardial

cells.12 It is the combination of these pathological changes that alters the structure of the

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1.1.4 Current Treatment for MI and HF

After an acute MI, patients often undergo revascularization in order to regain adequate blood supply to the myocardium. The two most widely employed treatments are angioplasty, in which the blocked artery is mechanically opened, and bypass surgery, in which vessels from elsewhere in the body are attached to bypass the blocked artery and re-establish blood flow. Also, MI patients receive pharmacological therapy to relieve angina, prevent thrombus propagation and to stabilize the “vulnerable” plaque.13 Anti-anginal drugs, which affect the balance between oxygen supply and demand, include nitroglycerin, β-adrenergic blockers, and calcium channel blockers. To reduce thrombus formation, anti-platelet and antithrombotic treatments are also recommended, unless contraindicated. Antiplatelet agents include aspirin, thienopyridines, and glycoprotein IIb/IIIa inhibitors. Commonly used antithrombotic agents include unfractionated heparin, low-molecular-weight heparin, fondaparinux, and bivalirudin. Furthermore, statin therapy promotes plaque stabilization and restores endothelial function, and has been shown to reduce the incidence of myocardial infarction, recurrent ischemia and mortality.

HF is a chronic disease that requires lifelong management including pharmacological therapy and lifestyle modification.14 Angiotensin-converting-enzyme (ACE) inhibitors are now considered a first-choice in HF drug therapy. ACE inhibitors limit the formation of angiotensin II resulting in vessel dilation as well as the prevention

of cardiac hypertrophy, fibrosis and decreased afterload and preload of the heart.

Angiotensin II receptor blockers, which have similar effects, can be used as an alternative

for patient who are unable to tolerant ACE inhibitors. Diuretics are prescribed for patients

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force of cardiac contractions and relieves heart failure symptoms, especially when the

patient is not responding to ACE inhibitors and diuretics. Beta-blockers are prescribed to

reduce heart rate and lower blood pressure. They also inhibit ventricular remodeling and

reduce the risk of arrhythmias and sudden death.15 Lifestyle modifications include losing

weight, quitting smoking, avoiding alcohol and caffeine, eating a saturated-fat

low-sodium diet, exercising and reducing stress, all of which have been shown to alleviate

symptoms, slow disease progression and improve quality of life.

In spite of significant progress in clinical treatment, the prognosis of HF remains

poor. At present there is no cure for patients with end-stage HF except heart transplantation. Drug treatment can attenuate myocardial remodeling and improve

survival, but does not reduce the size of the scar. Unfortunately, angioplasty or bypass

surgery does not reverse the damage induced by MI. LV assist devices can prolong

patient lives only for a few years.16,17 On the other hand, although the survival rate after

heart transplantation is excellent, the source of transplantable donor hearts is scarce.

Therefore, there is a need for developing new and effective therapies to treat patients with

HF post-MI. Approaches to limit cardiomyocyte loss, rebuild the coronary microvascular

network and impede cardiac remodeling have been widely investigated to improve

cardiac function post-MI.

1.2 NEW APPROACHES TO TREAT IHD

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Approaches to decrease myocardial cell death, enhance cardiac regeneration and impair cardiac remodeling have shown promising beneficial outcomes in animal models.

1.2.1 Inhibition of Myocardial Apoptosis

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apoptosis during I/R injury have been demonstrated to decrease infarct size and improve cardiac function.28-30 Additionally, activation of pro-survival kinases including Akt has been shown to provide significant anti-apoptotic effects in MI and I/R.

1.2.2 Activation of Akt Signaling

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Figure 1.1 Cardioprotective PI3K/Akt signaling pathways in ischemia/reperfusion

(I/R).

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size and protects against myocardial apoptosis after I/R by phosphorylation of eNOS via the PI3K/Akt signaling pathway.34 Akt can also directly inhibit caspase-mediated cell death through the phosphorylation of BAD, releasing Bcl-2 family members to maintain mitochondrial permeability 43 and the phosphorylation of caspase-9.44 Additionally, Akt can protect against cardiomyocyte apoptosis by regulating gene expression of Glut-1, vascular endothelial growth factor (VEGF), Bcl-2 and forkhead transcription factors.45 Furthermore, Akt plays a critical role in endothelial homeostasis and angiogenesis by maintaining endothelial cell viability, regulating migration and promoting the homing of endothelial progenitor cells (EPCs) in the ischemic heart after I/R.46,47

1.2.3 Improvement of Cardiac Repair post-MI

The endogenous healing process post-MI is manifested by increased matrix synthesis, fibroblast proliferation and the release of pro-fibrotic cytokines, resulting in scar formation. The infarct size, which is critical to disease progression and prognosis, is determined by the area of coronary artery occlusion and dynamic changes during the remodeling process.48 The ideal cardiac repair would include the complete regeneration of the myocardium and neovascularization, which has been observed in Zebra fish,49 the adult newt,50 and the neonatal mouse.51 However, the ability to regenerate myocardial tissue follow injury is diminished in the adult mammals.51

1.2.3.1Neovascularization post-MI

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1.2.3.2Endogenous Cardiomyocyte Regeneration post-MI

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1.3 STEM CELLS IN CARDIAC REPAIR POST-MI

Stem cells are defined by their unique properties: self-renewal capacity and the potential to differentiate into one or more specialized cell types.86 Stem cells have the ability to undergo asymmetric and symmetric cell division. Asymmetric cell division gives rise to two daughter cells that have different fates, one maintaining the stem cell population and the other generating differentiated cells.87 Stem cells can be classified into 3 major categories: embryonic, induced pluripotent and adult stem cells.74,88,89 Both embryonic and induced pluripotent stem cells are pluripotent and can generate any cell type in the body. However, they present a high risk of teratoma formation, which makes them unacceptable for clinical treatment at this time. Interestingly, small populations of clonogenic, self-renewing, and multipotent cells have been found in different adult tissues including bone marrow, vascular walls, adipose tissues, skeletal muscles, heart and brain as well as the epithelium of the lung, liver, pancreas, digestive tract, skin,

limbus, retina, breast, ovaries, prostate and testis.90,91 These multipotent stem cells, referred to as adult stem cells, have the potential to differentiate into mature cell lineages in tissues from which they originate and contribute to postnatal organ homeostasis. Furthermore, bone marrow-derived stem/progenitor cells may also migrate to distant peripheral sites after intense injury and contribute to tissue repair.91,92 Adult stem cells reside in a specific cellular niche, which ensures a balance between the maintenance of the stem cell pool and the production of daughter progenitor cells engaged in lineage differentiation.93 The in vivo stimulation of adult stem cells or the use of their ex vivo

expanded and differentiated progenies is emerging as a promising approach for cell

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bone marrow-derived stem cells, resident cardiac stem cells and epicardium-derived cells

have shown cardiac myogenic and vasculogenic potential, which make them favorable

candidates for cardiac repair post-MI.77,94 (Figure 1.2)

1.3.1 Bone Marrow-derived Stem Cells (BMSC)

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Figure 1.2 Potential cell sources for cardiogenesis post-MI.

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trophic factors released from the HSCs, which are referred to as paracrine effects.104

MSCs were originally identified as a small subpopulation of bone marrow cells which have the ability to differentiate into tissue types of mesodermal origin, such as chondrocytes, adipocytes and osteocytes.105 MSCs have been successfully isolated from various non-marrow tissues such as umbilical cord blood 106 and adipose tissue.107 DNA demethylating agents, such as 5-azacytidine, have been shown to induce MSC transdifferentiation into cardiomyocytes in vitro.108,109 Cell therapy using MSCs has been well-documented to improve cardiac performance and promote neovascularization, which are now considered mostly due to the paracrine effects instead of cardiomyocyte regeneration.110,111 There are several advantages of using MSCs in cell therapy in IHD compared to HSCs. First, they can be easily cultured and expanded in vitro to the large population needed for transplantation. Second, they can be engineered in vitro to achieve specific characteristics. Third, they have been shown to contribute to both myocardial and vascular repair post-MI. Last, they have immunological privilege by regulating immune cell activity, which makes MSCs a potential candidate for allogenetic treatment.112

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be needed in order to achieve the best outcome for BMSCs stem cell therapy in IHD patients. The lack of benefit of bone marrow-derived MNCs therapy suggests that it is not an optimal stem cell type for IHD treatment.

Endothelial progenitor cells (EPCs) are a rare population of cells that circulate in the blood and have the ability to differentiate into endothelial cells and promote neovascularization. Bone marrow is the major source of adult EPCs. EPCs share many markers with HSCs, which makes it difficult to identify EPCs strictly by specific marker expression. The most widely used markers for EPCs include c-kit, CD34, CD133 and vascular endothelial growth factor receptor 2 (VEGFR2). In vitro culture of mononuclear bone marrow cells yields 2 types of EPCs: early outgrowth and late outgrowth EPCs.115 The early outgrowth EPCs have been shown to be of hematopoietic origin and potently induce angiogenesis without incorporating in blood vessels, while the late outgrowth EPCs are considered real EPCs which can be clonally expanded and give rise to endothelial cells. During neovascularization, EPCs are released from bone marrow to the peripheral circulation and mobilize to the target tissue. Mobilization of EPCs after cardiac ischemia is also observed in patients after acute MI.116 Animal studies have demonstrated that EPC transplantation or treatment with agents that enhance EPC mobilization and recruitment post-MI improves cardiac function and reduces cardiac remodeling via angiogenesis and vasculogenesis.52,117-120

1.3.2 Cardiac Stem Cells (CSCs)

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low in the adult heart, they are committed to generate cardiomyocytes and their numbers can be expanded ex vivo for therapeutic purposes.66 Different approaches are employed to identify the resident CSCs, including detection of specific surface markers such as c-kit, and assessment of their unique physiological properties such as the ability to form cardiospheres in culture or to efflux Hoechst 33342 dye (side population progenitors).

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Cardiosphere-derived cells (CDCs) are also CSCs identified through their ability to migrate from cultured cardiac tissues and form floating spheres. CDCs are isolated from minimally invasive percutaneous endomyocardial biopsies from the adult human heart 131 and the myocardium from other species (mice, rats and pigs).132,133 The CDCs are a mixture of c-kit+ cells and supporting endothelial and mesenchymal cells that can be amplified in culture and grown to millions in a few weeks.133 Intracoronary delivery of CDCs has been shown to improve cardiac repair and function in animal MI models.131,134 A phase I clinical trial was carried out to study the safety and efficacy of CDCs in patients with a recent MI (ClinicalTrials.gov Identifier: NCT00893360). Reduction in scar mass, increased viable heart mass and regional contractility was observed in 17 patients with MI, 6 months after CDCs treatment, while no significant changes in ejection fraction, end-diastolic and end-systolic volume were found.135 Cell therapies with autologous c-kit+ CSCs or CDCs after MI have been shown to be safe and beneficial, and warrant phase 2 studies to test their efficacy.

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multipotent CSCs marked by the LIM-homeodomain transcription factor islet-1 (Isl-1) has been shown to give rise to cardiomyocytes, endothelial and smooth muscle cells both in vitro and in vivo. The Isl-1+ CSCs are mainly located in the outflow tract and right atrium of fetal or very young postnatal hearts and are negative for c-kit and Sca-1.139 The fact that Isl-1+ CSCs exist in very low numbers in the adult heart limits their therapeutic potential. Furthermore, the property of cardiac progenitor cells to expel toxic molecules through an ATP-binding cassette transporter 140 is also employed to define a pool of CSCs. They are termed cardiac side population cells (CSPs) because after exposure to the DNA-binding dye, Hoechst 33342, they can clear the fluorochrome and appear on the side of the flow cytometry profile.141 CSPs are positive for Sca-1 and negative for c-kit.142 CD31-Sca-1+ CSPs form colonies in vitro and differentiate into cardiomyocytes.142,143 When transplanted into animals with MI, CSPs can migrate to the injured myocardium and differentiate into cardiomyocyte- and endothelial-like cells.144,145

To date, whether the resident CSCs identified by different methods represent distinct populations of stem/progenitor cells or they are actually the same line of stem cells at different stage of maturation remains unknown. Exploitation of CSCs in stem cell therapy for IHD is certainly an exciting and promising strategy. However, further investigations of the endogenous regulatory mechanisms of CSCs survival, proliferation and differentiation under physiological or pathological conditions are definitely needed before stem cell therapy can be safely and effectively applied to IHD patients.

1.3.3 Epicardium-derived Cells (EPDCs)

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1.4 STEM CELL FACTOR (SCF)

SCF, also known as Steel factor, mast cell growth factor, or c-kit ligand, binds to c-kit and promotes survival, proliferation, mobilization, and adhesion of all c-kit -expressing cells. c-kit is a tyrosine kinase receptor expressed in stem cells and progenitors including BMSCs, CSCs and some EPDCs 122,163,164 while SCF is ubiquitously expressed as a glycosylated transmembrane protein widely throughout the body by stromal cells,165 fibroblasts, endothelial cells and the myocardium. Alternative mRNA splicing leads to two isoforms of SCF: soluble (S-SCF) and membrane-associated (M-SCF), which differ in the absence or presence of a particular proteolytic cleavage site encoded by exon 6.166 S- and M-SCF have distinct but overlapping roles. M-SCF is the dominant isoform in vivo and induces more persistence and long-term support of target cell survival via juxtacrine, but may be less effective in stimulating growth.166-168

1.4.1 SCF Biology

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melanogenesis, and works in synergy with other growth factors. In vitro, SCF treatment induces survival, proliferation and migration of HSCs, melanoblasts and primordial germ cells.173 In vivo, recombinant S-SCF elicits a multilineage response in peripheral counts and increases bone marrow cell mass, which protects against lethal irradiation by accelerating hematopoietic recovery.174-176

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13-acetate (PMA) is able to increase the shedding in COS-1 cells transfected with mouse M-SCF.168 The biological activity of the S-SCF released from M-SCF is similar to that of the proteolytic product of the full length SCF.

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Figure 1.3 Illustration of soluble (S-SCF) and membrane-associated SCF (M-SCF).

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Thus, it is not surprising that M-SCF provides greater survival and proliferation signals compared to the soluble isoform.181,186 It also appears that the 2 isoforms of SCF have different signaling pathways for their biological activities. For example, although both S-SCF and M-S-SCF can induce proliferation of erythroid progenitors via activation of p38 and ERK pathways, M-SCF-induced signaling is more sensitive to ERK inhibition while p38 inhibition works better on S-SCF.186 Thus, through expression of different SCF isoforms, stromal cells regulate the activity, survival, proliferation, migration and adhesion of local c-kit expressing cells as well as those in remote organs and tissues.

1.4.2 SCF receptor c-kit

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kinase, PI3 kinase, phospholipase-γ (PLC-γ), Src and Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathways, all of which contribute to the biological effects of SCF.197 SCF/c-kit signaling can be inhibited by blockage of SCF/ c-kit binding via neutralizing antibody, ACK2,198 or inhibition of c-kit receptor activation by AG 1296.199

1.4.3 Potential Clinical Applications of SCF

Recombinant human SCF is currently used to mobilize BMSCs for collection of HSCs for hematopoietic reconstitution200 and to prevent chemotherapy-induced anemia and thrombocytopenia.201 SCF has also been indicated as a drug target for cancer treatment.202,203 Analysis of serum SCF levels is recommended to aid the diagnosis of many diseases including pancreatic cancer,184 gastrointerstitial stromal tumor,204 primary uveal melanoma,205 acute renal allograft rejection,206 delayed and slow graft function207 and Alzheimer’s disease.185

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1.4.4 SCF and IHD

The role of granulocyte colony-stimulating factor (G-CSF) and S-SCF in MI was first studied by Orlic et al, showing enhanced BMSCs mobilization and recruitment to the infarcted heart and improved cardiac function in mice post-MI.68 Those beneficial effects resulted from decreased myocardial apoptosis,218 reduced arrhythmia,219 increased neovascularization 220 and impaired cardiac remodeling.221 However, Ohtsuka et al found SCF treatment alone failed to induce cardiac protective effects post-MI.220 Myocardial expression of SCF is decreased post-MI. Augmentation of SCF expression through transplanted MSCs has been shown to increase angiogenesis and cardiac function.222 Moreover, cardiac transplantation of MSCs transfected with a full length mouse SCF cDNA plasmid, which express both S- and M-SCF, enhanced EPC recruitment and myocardial angiogenesis for about 2 weeks post-MI. However, there was no change in animal survival among all treatment groups.222 This is not surprising as more than 95% of the transplanted MSCs are cleared within 7 days following transplantation into the myocardium.223 Intra-myocardial administration of SCF sustainably mobilized exogenously administered Lin-c-kit+ bone marrow-derived cells from the circulation homing to the infarcted myocardium up to 72 hours post-MI, however, cardiac function was not investigated in this study.224 The molecular and cellular mechanisms by which SCF regulates cardiac function post-MI are not fully understood.

1.5 OVERALL HYPOTHESIS AND AIMS

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injections of SCF increased recruitment of BMSCs to the infarcted heart.224 Although numerous animal studies using stem cell therapy showed significant protection post-MI, only minor improvements have been observed in most clinical trials,113,114,225,226 while two recent trials showed no significant benefit at all.75,76,226 Thus, a better understanding of the pathophysiology of different types of stem cells in cardiac repair post-MI is needed.

SCF is expressed as a glycosylated trans-membrane protein by many cells and tissues, including the myocardium.227 SCF binds to its receptor c-kit and plays a critical role in cell survival, proliferation, mobilization, and adhesion. c-kit positive adult stem cells including HSCs, EPCs and CSCs122,163,164 have been demonstrated to be potential candidates for cardiac repair post-MI, which implies a possible regulatory role of SCF in MI. M-SCF is more potent than S-SCF in supporting cell survival and proliferation.19 In adult mice lacking M-SCF, cardiac morphology and function at basal conditions were normal, but significantly higher mortality and worse cardiac function were observed post-MI.228 To study the role of M-SCF in cardiac repair post-MI, we generated a novel mouse model of inducible cardiac specific over-expression of hSCF. The aim of this thesis was to study the effects of M-SCF on cardiac function post-MI and to investigate the underlying cellular mechanisms especially the role of M-SCF in regulation of c-kit+ stem/progenitor cell activity in the ischemic heart (Figure 1.4). Our overall hypothesis

was that cardiac-specific overexpression of hSCF protects the heart via inhibition of

myocardial apoptosis and enhancement of neovascularization and stem cell activity

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Figure 1.4 Proposed cellular mechanisms by which membrane-associated SCF

(M-SCF) regulates cardiac function post-MI.

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In order to induce cardiac-specific hSCF overexpression, a hSCF transgenic mouse was generated with the hSCF transgene expression under the control of a tetracycline-responsive element containing the α-MHC promoter. The hSCF mice were then crossed

with cardiac-specific tetracycline activator (tTA) mice to generate hSCF/tTA double transgenic mice. The hSCF/tTA transgenic mice specifically express the hSCF in the heart. These transgenic mice were healthy and fertile with no cardiac abnormalities.229 Furthermore, myocardial expression of hSCF in hSCF/tTA mice can be abrogated by treatment with doxycycline (DOX). Thus, the specific effects of hSCF can be tested in the absence and presence of DOX to turn on and off hSCF expression in the heart.

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CSCs, recent studies have shown that EPDCs are another potential resource of cardiovascular progenitors.230 Thus, the third aim of my thesis was to investigate the role of hSCF in EPDC activation, migration into the sub-epicardium and contribution to cardiac repair post-MI.161 So I hypothesized in Chapter 4 that cardiac specific over-expression of hSCF promotes epicardial activation. We found that hSCF overover-expression increased EPDC activation, proliferation and mobilization which contribute to myocardial neovascularization post-MI.

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