The Cardiovascular Reparative Properties of Bone Marrow ...

The Cardiovascular Reparative Properties of Bone Marrow Mesenchymal Precursor Cells

Peter James Psaltis Department of Medicine Faculty of Health Sciences The University of Adelaide, South Australia & Cardiovascular Research Centre The Royal Adelaide Hospital, South Australia & Bone and Cancer Research Laboratories Division of Haematology Institute of Medical and Veterinary Science

A thesis submitted to the University of Adelaide in candidature for the degree of Doctor of Philosophy October 2009

Chapter 1 1 General Introduction 0B

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Chapter 1: Introduction

1.1 Central Hypotheses 8B

This thesis explores the biologic and cardiovascular reparative properties of immunoselected bone marrow (BM)-derived mesenchymal precursor cells (MPC).

Specifically it addresses the following central hypotheses: 1. Prospective antibody-based isolation enhances the stem-like characteristics and trophic activity of human BM mesenchymal cell populations, by enriching for the presence of immature MPC. 2. The pleiotropic properties of BM MPC confer reparative benefit to nonischemic cardiac injury, as assessed in an experimental, large animal model of anthracycline-induced cardiotoxicity.

1.2 Cardiomyopathy and Cardiac Failure 9B

1.2.1

Burden of disease 59B

Cardiovascular disease remains the leading cause of morbidity and mortality in Western communities and represents a growing economic burden to most health systems [1]. Congestive cardiac failure (CCF) describes the clinical syndrome whereby the structure or function of the heart impairs its ability to supply sufficient blood flow to meet the body's needs. Common causes H

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of CCF include myocardial infarction (MI) and other forms of ischaemic heart disease (IHD), H

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hypertension, valvular heart disease and nonischaemic cardiomyopathy (NICM) [2]. H

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Cardiac failure is a common and costly condition, afflicting 2.5% of Australians aged 55-64 years and 8.2% of those aged 75 years or over (statistics obtained from Australian Institute of Health and Welfare website). The lifetime risk of developing CCF has been estimated at one in five for both men and women [3]. Although its age-adjusted incidence has remained relatively stable over 2

Chapter 1: Introduction

the past 20 years, its prevalence appears to be increasing and will continue to do so with ageing populations [4]. Indeed, it remains the single most common cause of hospital admissions in people aged over 65 years. It follows, that CCF is associated with high health expenditure, with estimates that it accounts for over $400 million of the total direct health costs attributed to cardiovascular disease in Australia.

With respect to individual patient suffering, CCF impairs quality of life more than other chronic diseases and carries poor prognosis [5]. Mortality rates have been reported at 30-40% one year after diagnosis and 60-70% within five years [6], with death most commonly caused by worsening of cardiac failure or sudden ventricular arrhythmia. The clinical burden of CCF is therefore profound and presents an increasing public health concern, worthy of considerable attention.

1.2.2

Aetiology: ischaemic versus nonischaemic 60B

Classifications of cardiomyopathy are typically based on myocardial structural changes (e.g. dilated, restrictive or hypertrophic) or on underlying aetiology (e.g. ischaemic or nonischaemic) (Table 1.1). Myocardial ischaemia is the commonest cause of cardiomyopathy, weakening and dilating the left ventricle (LV) due to inadequate oxygen delivery, most commonly from obstructive coronary artery disease. In ischaemic cardiomyopathy, there may be permanently damaged, scarred myocardium caused by infarction, or “hibernating” tissue due to prolonged and severe oxygen deprivation from chronic coronary ischaemia.

Nonischaemic cardiomyopathy contributes to approximately one third of clinical CCF and comprises numerous different aetiologies [7]. Although most studies have demonstrated better 3

Table 1.1 Aetiological classification of cardiomyopathy A. Ischaemic Post-myocardial infarction Hibernating myocardium Small vessel ischaemia B. Nonischaemic Idiopathic Familial or Genetic Peripartum Hypertension Tachyarrhythmia Valvular heart disease Infective - Viral myocarditis (e.g. coxsackie) - Human immunodeficiency virus - Trypanosoma cruzi (Chagas disease) - Rheumatic carditis Infiltrative - Amyloidosis - Sarcoidosis - Haemochromatosis Connective tissue disease - Scleroderma - Systemic lupus erythematosus - Marfan’s syndrome - Polyarteritis nodosum - Dermatomyositis or polymyositis - Ankylosing spondylitis - Rheumatoid arthritis Endocrine - Diabetes mellitus - Thyroid disease - Acromegaly - Phaeochromocytoma Substance abuse (e.g. alcohol, cocaine) Iatrogenic (e.g. anthracyclines, steroids) Neoplastic heart disease Congenital heart disease Miscellaneous - Critical illness - Neuromuscular disease - Radiation

Chapter 1: Introduction

prognosis for patients with NICM than for those with ischaemic cardiomyopathy [7-10], this finding has not been universal [6]. The time course of progression of LV dysfunction is variable for the different aetiologies of NICM, as is the risk of sudden and non-sudden death [11]. For example, better long-term prognosis has been observed in patients with peripartum cardiomyopathy, while worse survival rates accompany NICM due to myocardial infiltrative diseases (e.g. amyloidosis, haemochromatosis), human immunodeficiency virus (HIV), anthracycline chemotherapy and connective-tissue disease [7].

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Chapter 1: Introduction

1.2.3

Pathogenesis: cardiac remodelling and cardiomyocyte loss 61B

The manifestations of both ischaemic and nonischaemic cardiomyopathy arise from a complex, multi-step progression of changes within the myocardium referred to as cardiac remodelling. The remodelling process comprises adaptations at genomic, molecular, cellular and interstitial levels within the heart, ultimately resulting in disturbances to cardiac size, shape, systolic and/or diastolic function (Figure 1.1) [12]. Central to the development of cardiomyopathy, is the progressive loss of cardiomyocytes and contractile cardiac tissue [13]. Following severe MI, large-scale death of cardiomyocytes (approximately 1-2x109 cells) occurs via a process of ischaemic necrosis, followed by apoptosis of vulnerable cardiomyocytes in peri-infarct territories. Although more sporadic and insidious, substantial loss of cardiac cell mass is also associated with remodelling in other disease states including chronic IHD, hypertension, valvular disease and NICM.

1.3 Emergence of Cell-based Therapies for Cardiovascular Disease 10B

Conventional management of cardiovascular disease in general and CCF specifically, has evolved from numerous seminal advances [14]. While improvements in pharmacotherapy, addressing the neurohormonal changes associated with CCF (e.g. angiotensin inhibition, betaadrenergic blockade), have provided the mainstay of its treatment, great progress has also been made in the areas of home-care management, treatment and prevention of underlying diseases (e.g. coronary revascularisation techniques for MI/IHD), device therapy (e.g. cardiac resynchronisation pacing, implantable defibrillators, mechanical assist devices) and cardiac transplantation. With the obvious exception of cardiac transplantation, these treatment options are limited by their failure to replace lost cardiomyocytes and myocardial scar tissue with new, 5

Figure 1.1 Cardiac remodelling This schematic depicts the progression of left ventricular remodelling following antero-apical myocardial infarction. Initial cicatrisation and thinning of the myocardial wall occur at the local site of injury, but ultimately the adaptive changes in the LV progress to cause marked global dilatation of the chamber.

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Chapter 1: Introduction

functioning, contractile tissue. This is compounded by the heart’s own inability to replace its lost cell mass by self-regeneration. Consequently, despite improvements in its prognosis over recent decades [15, 16], the morbidity and mortality of CCF remain substantial, and new approaches to treatment have been sought. This has led to the emergence of novel regenerative therapies, including gene transfer, growth factor/cytokine administration and stem cell transplantation.

Cellular therapy has evolved quickly over the last decade, both at the level of in vitro and in vivo preclinical research and more recently, clinical trials of MI, IHD and CCF. Following initial proof-of-concept studies using foetal cardiomyocytes transplanted into fibrotic myocardial scars [17], various other cell types have been examined for their capacity to mediate cardiac and vascular repair [18].

Stem cells are defined by their ability to achieve self-renewal, their high replicative potential and their capacity to differentiate into multiple mature cell types. They can be broadly classified into embryonic stem cells (ESC) and adult-derived stem cells (ASC). Embryonic stem cells are totipotent with the ability to develop all the tissue-forming cells that constitute an entire organism, while ASC reside in the postnatal state in tissue niches and function to replenish cell loss as a consequence of tissue damage and death. The differentiation potential of some ASC populations is not only confined to the cells of their specific tissue of origin, but to other cell lineages as well. As detailed below, not all of the cell types applied to cardiac therapy fulfil these criteria for stem cells.

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Chapter 1: Introduction

1.4 Endogenous Myocardial Repair 1B

The long-standing dogma has been that adult cardiomyocytes undergo terminal differentiation soon after birth, irreversibly withdrawing from the cell cycle. In the postnatal state, uncoupling of cardiomyocyte karyokinesis and cytokinesis helps to explain the characteristic cell hypertrophy and nuclear ploidy, rather than hyperplasia, that occur following cardiac injury [19]. Furthermore, the inability of cardiomyocytes to progress through the cell cycle contributes to their apoptosis. In recent years, however, this long-held depiction of the adult mammalian heart as a post-mitotic organ has been severely challenged. It is now apparent that not all cardiomyocytes in the adult heart lose their ability to replicate. Initially this was inferred from observations that there is an increase in the number of proliferating cardiac cells after MI, especially in the peri-infarct territories, but also in areas of remote, non-infarcted myocardium [20]. Similar evidence of cell division is also evident in cardiomyopathy [21]. These findings prompted investigations into the properties and the functional role of these dividing cells, under both normal tissue homeostasis and pathological conditions.

1.4.1

Circulating non-cardiac cells 62B

Evidence that extrinsic (non-cardiac) cells may contribute to endogenous cardiac repair processes emerged from transplantation studies in which donor and recipient organs were sex-mismatched and small numbers of Y-chromosome-containing cells were identified in the female donor hearts of male transplant recipients [22]. This implied the migration of a non-resident cell type(s) to the heart which contributed to resident endothelial and possibly cardiac cell populations. It is now well-established that in response to myocardial insult, a variety of cell types are mobilised from non-cardiac organs, such as BM, into peripheral blood to assist with cardiac and vascular repair.

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Chapter 1: Introduction

These cells include lymphocytes, monocytes/macrophages, granulocytes and mature endothelial cells, along with immature cells, such as endothelial progenitor cells (EPC), haematopoietic stem cells (HSC) and mesenchymal stromal/stem cells (MSC) [23, 24].

By comparison to healthy individuals and those with chronic angina, patients presenting with acute MI have marked increases in circulating levels of different progenitor cell subtypes (e.g. CD34+/CD117+, CD34+/CXCR4+, CD34+/CD38+, CD34+/CD45+ cells, c-met+) [24, 25]. The mobilisation of these cells in the early stages after MI appears to correlate positively with LV ejection fraction (EF) and negatively with markers of myocardial necrosis (e.g. troponin-T) [26]. Cytokines and chemokines that have been strongly implicated in this process include vascular endothelial growth factor (VEGF) [27, 28], hepatocyte growth factor (HGF) and stromal cellderived factor-1 (CXCL12, SDF-1) and its receptor, CXCR4 [25, 29].

1.4.2

Resident cardiac stem/progenitor cells 63B

In addition to the recruitment of non-cardiac stem cells, there is now compelling evidence demonstrating the existence of resident cardiac stem/progenitor cells in the hearts of adult mammalian species, including humans [30-33]. These lineage negative cells, identified by various techniques including the expression of different antigenic markers (e.g. c-kit, SCA-1, MDR1, ABCG2, Tbx5), possess the capacity for multilineage differentiation into mature cardiomyocytes, endothelial and smooth muscle cells [30, 34, 35]. The Islet-1 transcription factor has also been associated with cardiac stem cells [32], but unlike c-kit or Tbx5, is exclusively expressed by cells from the second heart field, indicating that it is not involved in cardiomyocyte development in the LV. The frequency of c-kit+ cardiac stem cells has been shown to

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Chapter 1: Introduction

approximate 1 per 3-4x104 cardiomyocytes in different species [36, 37], whereas Islet-1+ cells are virtually undetectable after the early post-natal period [34]. Cardiac stem cell niches have been localised to the atria, atrioventricular groove and LV apex, where they form structural and functional connections with mature cardiomyocytes and cardiac fibroblasts [38, 39]. Cytokine/chemokine mediators, such as insulin-derived growth factor-1 (IGF-1) and HGF, appear capable of stimulating the migration of cardiac stem cells out of their niches and promoting their proliferation and differentiation into mature cardiac and vascularspecific cells. Unfortunately, these reparative responses are too limited to achieve endogenous replacement of lost cardiomyocytes in the setting of acute MI or cardiomyopathy. However, paracrine stimulation of resident stem cells may be an important mechanism by which exogenous cell therapy assists cardiac repair. There may also be the therapeutic potential of transplanting harvested, autologous cardiac stem cells to regenerate injured myocardium, as has been successfully applied in a growing number of preclinical studies [30, 31, 33, 38, 40, 41].

1.5 Exogenous Cell Therapy - Embryonic Stem Cells 12B

Embryonic stem cells are derived from the inner cell mass of the blastocyst. They display promising preclinical potential for achieving myocardial regeneration, largely as a result of their enormous proliferative capacity and their toti-differentiation potential. There are numerous reports in the literature describing the cardiogenic potential of murine ESC (mESC) lines and human ESC (hESC), which were first successfully isolated from human blastocysts in 1998 [4245]. Human ESC-derived cardiomyocytes behave structurally and functionally like early-stage cardiomyocytes, expressing characteristic early cell markers and possessing electrophysiological and ultrastructural features that are similar to foetal ventricular cardiomyocytes. Cardiac-

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Chapter 1: Introduction

committed cells can be enriched from hESC culture by using Percoll gradient centrifugation. Initial studies, in which these cells were transplanted into animal models of heart disease, demonstrated their durable engraftment, proliferation and differentiation in vivo and importantly, their integration with host cardiomyocytes [46, 47].

Unfortunately, current techniques for isolating and generating differentiated hESC-derived cardiomyocytes are limited by issues of yield and failure to exclude undifferentiated ESC. The latter issue poses significant risk of teratoma formation at the sites of cell transplantation and in other tissues, following migration of immature ESC to secondary sites. Along with tumour risk, immunorejection and ethico-legal considerations are currently major obstacles hindering the clinical application of hESC [48]. While these problems are not insurmountable, the emerging fields of nuclear transfer technology [49] and induced pluripotent stem cells (iPS) [50, 51] may provide future alternatives to hESC therapy.

1.6 Exogenous Cell Therapy – Adult-derived Cells 13B

Various adult tissues contain resident cell populations that have been evaluated in cardiac research. Most experience has involved cells from skeletal muscle, BM and blood, however, other tissue sites, such as adipose, dental pulp and periodontal ligament, placental and umbilical cord blood and the heart itself, also harbour cell types that are being intensively investigated.

1.6.1

Skeletal myoblasts 64B

Skeletal myoblasts (SkM) are committed skeletal muscle precursor cells that normally lie in a quiescent state under the basal lamina of skeletal muscle fibres. Their normal role is to regenerate 10

Chapter 1: Introduction

skeletal muscle following injury, both by fusion with the surrounding muscle fibre and by differentiation into contractile, mature skeletal myocytes. Autologous SkM are easily accessible through muscle biopsy and can be rapidly expanded in vitro to high cell numbers for therapeutic application. In vitro isolation can be assisted through their expression of the cell surface marker, CD56.

The contractile and ischaemia-resistant properties of SkM have led to their evaluation in both preclinical and clinical studies of cardiomyopathy [52-58]. They have been shown to engraft, proliferate and survive in the presence of myocardial injury, ultimately improving systolic and diastolic cardiac function. However, the mechanisms responsible for these benefits are not related to myoblasts undergoing transdifferentiation into cardiomyocytes [59]. Instead, they remain committed to their skeletal muscle origin in vivo, but may adapt over time to become more fatigue-resistant, by acquiring a greater component of slow-fibre myosin. They also fail to electromechanically couple with host cardiomyocytes through the production of gap junctions and intercalated disc proteins (e.g. N-cadherin and connexin-43) [60]. This may in part explain the incidence of ventricular tachyarrhythmia that has complicated transplantation of SkM in clinical studies [61]. In the absence of cardiac transdifferentiaton and electromechanical coupling, SkM appear to mediate cardiac repair via mechanisms that include autonomous contraction, infarct size reduction and stabilisation of scar tissue [62]. They may also influence cardiac remodelling and angiogenesis in a paracrine fashion by the release of cytokines and growth factors, such as VEGF and HGF [63].

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Chapter 1: Introduction

1.6.2

Bone marrow and blood-derived cells 65B

Bone marrow and peripheral blood contain various types of stem/progenitor cells, with the best defined being HSC, angioblasts/EPC and MSC. Numerous subpopulations of these cell types have been identified through techniques such as flow cytometric analysis, although in many cases they are still incompletely characterised.

1.6.2.1 Unfractionated bone marrow 16B

A paucity of comparative research between the different types of BM-derived stem cells has created uncertainty as to which cell type is optimal for cardiac repair. Many studies, especially those in humans, have avoided the use of specific stem cell fractions, preferring to adopt a “blanket” treatment approach by administering unfractionated bone marrow cells (BMC) or bone marrow mononuclear cells (BM MNC). These preparations are heterogeneous in their cellular composition and include monocytes, lymphocytes, nucleated red cells, immature B- and T-cells and a minor proportion of HSC (