Bone marrow mononuclear cells and acute myocardial infarction

Stem cell transplantation is emerging as a potential therapy to treat heart diseases. Promising results from early animal studies led to an explosion of small, non-controlled clinical trials that created even further excitement by showing that stem cell transplantation improved left ventricular systolic function and enhanced remodelling. However, the specific mechanisms by which these cells improve heart function remain largely unknown. A large variety of cell types have been considered to possess the regenerative ability needed to repair the damaged heart. One of the most studied cell types is the bone marrow-derived mononuclear cells and these form the focus of this review. This review article aims to provide an overview of their use in the setting of acute myocardial infarction, the challenges it faces and the future of stem cell therapy in heart disease.


Introduction
Despite the recent advances in percutaneous inter vention, drug and device therapy, patients with acute myocardial infarction (AMI) and resulting left ventricular impairment have 13% mortality at 1 year [1]. Following the loss of over one billion cardiomyocytes in a functionally signifi cant MI, the overloaded surviving cardiomyocytes undergo abnormal remodelling, eventually leading to heart failure. Th is condition, a leading cause of death and disability in the developed world, is associated with 5-year mortality rates of up to 70% in symptomatic patients [2]. Current conventional therapies do not correct underlying defects in cardiac muscle cell number [3].
Th e only therapeutic option that currently addresses cardiomyocyte loss is heart transplantation. However, due to stringent selection criteria and chronic shortage of donor hearts, the vast majority of patients are deemed unsuitable or never receive a transplant. Th erefore, preventing this progression post-MI is a major challenge requiring novel therapeutic strategies such as stem cell transplantation to improve the prognosis and quality of life for these patients.
Th e traditional view that the heart is a terminally diff erentiated organ has been challenged by the discovery of diff erentiation of stem cells into cardiomyocytes in animal and human hearts [4][5][6][7]. Th is in turn has led to the exciting possibility for regenerative therapy for cardio myocyte loss after a MI. Th e demonstration of functional recovery of myocardium through cardiomyogenesis and neoangiogenesis in AMI in murine models by Orlic and colleagues [8] generated tremendous interest in the potential of bone marrow-derived stem cells. Since then, the cardiomyogenic ability of these cells has been challenged. However, studies continue to demon strate improvement in cardiac function and reduction in infarct size. It should be noted that progenitor cells also contribute to cardiac repair by mechanisms beyond the growth of new cardiomyocytes and as such may off er an 'indirect' benefi t.

Animal and human trials
Th e most promising and obvious cell type for the growth of new cardiomyocytes is the embryonic stem cell; however, considerable technical and ethical issues exist with these cells, which must be overcome before their successful use in humans. Adult stem cells are an attractive option to explore for transplantation as they are autologous, but their diff erentiation potential is more restricted than embryonic stem cells. Currently, the major sources of adult cells used for basic research and in clinical trials originate from the bone marrow. Th e bone marrow mononuclear subset is heterogeneous and comprises mesenchymal stem cells, haematopoietic progenitor cells and endothelial progenitor cells. Th e diff erentiation capacity of diff erent populations of bone marrow-derived stem cells into cardiomyocytes has been studied intensively. Th e results are rather confusing and diffi cult to compare, since diff erent isolation and identifi cation

Abstract
Stem cell transplantation is emerging as a potential therapy to treat heart diseases. Promising results from early animal studies led to an explosion of small, non-controlled clinical trials that created even further excitement by showing that stem cell transplantation improved left ventricular systolic function and enhanced remodelling. However, the specifi c mechanisms by which these cells improve heart function remain largely unknown. A large variety of cell types have been considered to possess the regenerative ability needed to repair the damaged heart. One of the most studied cell types is the bone marrow-derived mononuclear cells and these form the focus of this review. This review article aims to provide an overview of their use in the setting of acute myocardial infarction, the challenges it faces and the future of stem cell therapy in heart disease. methods have been used to determine the cell population studied. To date, only mesenchymal stem cells seem to form cardiomyocytes, and only a small percentage of this population will do so in vitro or in vivo. Pragmatically, the translation of the basic science into clinical research has followed a common pathway: injection of bone marrow-derived mononuclear cells (BMMNCs) as a source of stem cells into the heart. Table 1 provides a summary of clinical trials using BMMNCs in patients with acute MI.

Trials with no sham bone marrow harvest or intracoronary re-infusion in the control group
In the fi rst human trial, Strauer and colleagues [9] reinfused intracoronary BMMNCs 7 days after myocardial infarction (MI). Th e mean number of mononuclear cells was 2.8 × 10 7 . Th ere was a signifi cant improvement in myocardial perfusion and a reduction in the infarct region in the cell therapy group. Th e Transplantation of Progenitor Cells and Regeneration Enhancement in Acute Myocardial Infarction (TOPCARE-AMI) investigators randomised patients into intracoronary infusion of BMMNCs or ex vivo expanded circulating progenitor cells 4 days after MI [10]. Th ere was a signifi cant improve ment in global and regional left ventricular (LV) function in both groups and a benefi cial eff ect on the post-infarction remodelling process manifest by a profound improvement in wall motion abnormalities in the infarct area and a signifi cant reduction in end-systolic LV volume at 4 months post-MI. Th e LV ejection fraction (LVEF) further improved at 12 months, resulting in a total increase of 9.3% at 1 year [11]. Of interest, there was no diff erence between the two active treatment groups. Th e mean number of infused cells was 245 × 10 6 , which contained haematopoietic progenitor, mesenchymal and stromal cells. However, a major limitation of both of these trials was the lack of a control group receiving sham bone marrow harvest or intracoronary re-infusion.
Another trial in which there was no sham procedure is the Autologous Stem-Cell Transplantation in Acute Myocardial Infarction (ASTAMI) trial, which included only patients with acute anterior MI. Th e intracoronary re-infusion of BMMNCs 4 to 8 days after infarction did not have a benefi cial eff ect on LVEF compared to percutaneous coronary intervention (PCI) alone at 6 months [12]. Th is lack of benefi cial eff ect may be explained by the diff erent cell processing protocols used in this trial. Cell processing protocols may have a signifi cant impact on the functional capacity of bone marrow-derived stem cells [13]. Comparison of diff erent isolation protocols revealed a vastly reduced recovery of mononuclear cells and nullifi cation of the neovascularisation capacity when the ASTAMI cell isolation and storage protocol was used [13].
Th e Bone Marrow Transfer to Enhance ST-Elevation Infarct Regeneration (BOOST) trial, a slightly larger trial, included 60 patients that were randomised to receive intra coronary BMMNCs or standard therapy 4.8 days after successful PCI following AMI. Th ere was a signifi cant improvement in global LVEF in the cell treatment group at 6 months without an eff ect on LV remodelling [14]. However, this improvement was not maintained at 18 months. Th e mean number of bone marrow cells that were infused contained 9.5 × 10 6 CD34 + and 3.6 × 10 6 haematopoietic colony-forming cells. Th e improvement in LVEF did not correlate with the number of CD34 + cells or haematopoietic colony forming cells. Again, a major limitation of the BOOST trial is that the control group did not undergo a sham bone marrow harvest or intracoronary infusion.
Th e fi rst long-term study involving 62 patients who underwent intracoronary BMMNC transplantation 7 days post-AMI not only resulted in an early signifi cant improvement in ejection fraction (EF) and infarct size, but there was also a signifi cant reduction in mortality and improvement in exercise capacity compared to controls at 5 years [15].

Randomised controlled trials
Th e Transcatheter Transplantation of Stem Cells for Treat ment of Acute Myocardial Infarction (TCT-STAMI) trial, which included a control group receiving a placebo infusion, showed a signifi cant (approximately 5%) improvement in LVEF of patients receiving intracoronary BMMNCs at 6 months [16].
Intracoronary bone marrow derived progenitor cells in acute infarction (REPAIR-AMI), a large random ized double-blind controlled trial that included over 200 patients, showed an improvement in the primary endpoint in the treatment group that was an absolute change in global LVEF from baseline to 4 months, as measured by quantitative left ventricular angiography [17]. Furthermore, the pre-specifi ed cumulative endpoint of death, MI, or revascularisation was signifi cantly reduced, and this benefi t was maintained at one year follow-up [18]. Th e mean increase in LVEF in the BMMNC group was 2.5% and there was an inverse relationship between the baseline EF and the degree of improvement. For example, patients with a baseline EF below the median value (48.9%) had an absolute increase in global EF that was three times higher than that in the placebo group. In contrast, the improvement in LVEF in patients with a baseline EF that was above the median value was non-signifi cant (0.3%). Th e timing of cell infusion post-PCI also had an eff ect on the primary endpoint. Patients in whom the cells were infused ≥5 days post-PCI were the only ones who derived benefi t.
By contrast, the LEUVEN-AMI study by Janssens and colleagues [19] showed that intracoronary re-infusion of BMMNCs within 24 hours of reperfusion was associated with a greater reduction in infarct size and improved regional systolic function, but no overall improve ment in global left ventricular function compared to controls.

Trials that used two diff erent cell populations
More recently, the Myocardial Regeneration by Intracoronary Infusion of Selected Population of Stem Cells in Acute Myocardial Infarction (REGENT) trial, which included patients with anterior MI, uniquely compared two cell types. Patients were random ized to receive intracoronary infusion of unselected (n = 80) or selected CD34 + CXCR4 + (n = 80) BMMNCs, or to the control group (n = 40) [20]. Although patients in the treatment group had a 3% improvement in LVEF, this did not reach statistical signifi cance. However, the primary endpoint analysis included <60% of the total population of patients, which is likely to be responsible for the failure in the improvement in LVEF to achieve statistical signifi cance. Subgroup analysis showed that baseline EF below the median value (37%) was an indepen dent predictor of signifi cant (≥5%) increase in LVEF after treatment with BMMNCs. Th e HEBE trial compared the intracoronary infusion of BMMNCs or mononuclear peripheral blood cells to standard therapy alone following an AMI [21]. Th e intracoronary BMMNCs were delivered between 3 to 8 days after AMI. Th ey showed no eff ect of either treatment on regional or global left ventricular function.

Benefi ts beyond ejection fraction
A recent comprehensive systematic review that included 13 trials with a total of 811 patients showed an improvement in LVEF by 2.99% in the BMMNC group compared to standard reperfusion therapy [22].
A previous meta-analysis by Lipinski and colleagues [23] that included 10 trials with AMI showed that intracoronary stem cell therapy (within the fi rst 14 days after infarction) was associated with a small but signifi cant (3.0%) improvement in LV systolic function compared to standard medical therapy. It was also associated with a non-signifi cant reduction in death and rehospitalisation from heart failure. Although they found no signifi cant association between the benefi ts of intracoronary cell injection and the number of injected cells, there was a trend toward a statistically signifi cant association with the injected volume, suggesting the possible presence of a dose-response relationship. Th e improvement in LVEF was observed in both bone marrow and peripheral mononuclear cells. Similar conclusions were reached in the meta-analysis by Abdel-Latif and colleagues [24], which included 18 studies and showed that stem cell therapy signifi cantly increased LVEF by 3.66%.
In contrast to animal models, the improvement in LV function in most clinical trials is at best modest. However, it should be noted that several of our established therapies that have an impact on prognosis in patients with MI and a reduced LV function, such as angiotensin-converting enzyme inhibitors, β-blockers [25], thrombolytic therapy and percutaneous coronary intervention [26,27], are associated with similar improve ments in LVEF. It is likely that adult stem cells exert their benefi t on cardiac remodelling through an 'indirect' para crine eff ect, and that the small functional benefi t seen with this therapy may translate into significant long-term improvement in exercise tolerance and survival [15].
Th e main surrogate markers used as an end-point have been EF and perfusion defects, which correlate poorly with prognosis and quality of life [28,29]. Th erefore, in the future, the validation of progenitor cell therapy for clinical use may depend on the demonstration of a benefi t with regard to clinical outcomes such as improvement in prognosis, quality of life [30], New York Heart Association functional classifi cation and exercise capacity.

The debated hypothesis
Th e divergent fi ndings from current trials may be due to several factors. Th ere appears to be an inverse relationship between the benefi t seen with stem/progenitor cell therapy and the baseline LV function, with cell therapy being most eff ective in patients with a lower LVEF [17,20]. Furthermore, patients with longer ischaemic time (>5 hours) may be more likely to have signifi cant improve ment of LVEF following the BMMNC infusion [20].
Th e timing of cell infusion may also play a role on the derived benefi t. Although the REPAIR-AMI trial suggests that the enhanced improvement of the LVEF was confi ned to patients who were treated ≥5 days after primary PCI, the investigators of the HEBE and REGENT trials showed no interaction between the timing of cell infusion and derived benefi t. Th e meta-analysis by Martin-Rendon and colleagues [22], however, showed that the benefi t of stem cell therapy was even greater when the BMMNCs were infused >7 days after MI. Th e eff ect of timing on the benefi cial eff ects of BMMNC adminis tration is further supported by the study by Lai and colleagues [31] that showed that intracoronary BMMNC administration provided cardio-protection in a fashion similar to ishaemic preconditioning. Th is benefi t was only seen when the myocardium had not been preconditioned by other means. An ongoing study at our centre, the REGENERATE-AMI (ClinicalTrial.gov NCT00765453), is designed to study the delivery of BMMNCs at very early time points (within 6 hours of PCI). Th e purpose of this design is to replicate the animal models where very early interventions lead to a significant (40%) improvement in cardiac function [8].
Th e dose of infused BMMNCs has varied between diff erent trials with variable results. Th ere appears to be a dose-dependent improvement in EF, with the benefi t of BMMNCs only seen when doses higher than 10 8 are administered [22].

Direct (transdiff erentiation) and indirect (paracrine and angiogenesis) eff ects of stem cells
To date, there is no direct clinical evidence that cellular cardiomyogenesis in fact occurs in the human heart after transplantation of progenitor cells, and over the past few years, various experiments using diff erent types of stem cells have shown that <2% of the transplanted cells transdiff erentiate into cardiomyocytes [32]. Th erefore, the number of cardiac cells produced by cardiac re genera tion alone is unlikely to explain the eff ects seen. In experi ments using a mouse model of MI, bone marrow-derived cells were shown to undergo a very low level of transdiff erentiation into cardiomyocytes and most of these cells continued to diff erentiate along the haematopoietic lineage [33,34]. However, engraftment of these haemato poietic cells at the infarct site led to an improvement in myocardial function that is likely attributed to vasculogenesis, angiogenesis and a paracrine eff ect. Adult stem cells secrete a variety of cytokines, chemokines, and growth factors that are involved in cardiac repair [35] and the production of these factors is increased in response to the hypoxic stress associated with AMI [36]. Takahashi and colleagues [37] showed that BMMNCs in rats produce and release various cytoprotective factors, including vascular endothelial growth factor, platelet-derived growth factor, interleukin-1β, and insulin-like growth factor-1, some of which are signifi cantly up-regulated by hypoxia. Th ese paracrine factors may infl uence adjacent cells and exert their actions via several mechanisms, including myocardial protection and neovascularisation. Furthermore, in humans, direct injection of BMMNCs during acute ischaemia results in a direct cardioprotective eff ect, by abolishing the process of apoptosis and necrosis [38], which in part explains the clinical benefi t seen in clinical trials. Th is cardioprotective mechanism appears to be dose related with the benefi t only seen with injected doses that are ≥5 × 10 6 .
Neo-angiogenesis in the peri-infarct zone is an integral part of the cardiac remodelling process [39]. Under normal circumstances, however, this is seldom suffi cient to meet the demands of the hypertrophied myocardium, and the compensatory tissue growth required for myocardial contractility. One of the therapeutic advantages of bone marrow-derived cells is to induce therapeutic angiogenesis in ischaemic tissues, which in turn would augment oxygen supply [40][41][42][43], and help rescue cells from critical ischaemia [44]. Dowell and colleagues [45] have shown with histological examination at 2 weeks postinfarction that injection of CD34 + cells was accompanied by a signifi cant increase in infarct zone microvascularity, cellularity and fi brosis in comparison to controls. Th ey also showed that neoangiogenesis was increased within both the infarct zone and the peri-infarct rim in rats receiving CD34 + cells compared with saline controls [45].
Paracrine factors released by transplanted stem cells may alter the extracellular matrix, resulting in more favourable post-infarction remodelling and strengthening of the infarct scar. In animal models of MI, the injection of endothelial progenitor cells or bone marrow-derived stem cells signifi cantly improved blood fl ow and cardiac function and reduced left ventricular scarring [46,47]. After an ischaemic event, the effi ciency of engrafment diff ers between diff erent progenitor subpopulations [48,49]. Th e formation of new blood vessels occurs as a result of the interaction of diff erent types of stem cells with cardiomyocytes [46,[50][51][52][53]. Neovascularisation is mediated by the physical integration of progenitor cells into new capillaries [48,54], or through a paracrine eff ect by releasing growth factors that promote angiogenesis [55], depending on the cell type and the circumstances of the cardiac injury.

Route of cell administration
Th e three routes of stem cell delivery that have been used so far in clinical trials are through intracoronary or intramyocardial injection or peripherally through the systemic circulation. It is not yet possible on the basis of existing clinical studies to assert a 'best' mode of delivery. However, it is likely that patients' individual pathobiology as well as the aetiology of their cardiac dysfunction will ultimately dictate the route chosen among potential progenitor cell therapies. Th e advantage of intracoronary delivery is that cells are directly injected into areas of good blood supply rich in nutrients and oxygen, which is essential for cell survival. Myocardial ischemia is a major stimulus for incorporation of circulating progenitor cells, and potently up-regulates the chemo-attractants for neoangiogenesis. Even after infarction, however, the absolute number of progenitor cells detected in the heart is very low [40,41,56,57], but intracoronary infusion of progenitor cells may enhance local accumulation and homing compared to intravenous injection.
By contrast, the benefi t of direct intramyocardial cell delivery into hibernating myocardium is that it negates the need for the uptake of progenitor cells from the circulation. Electromechanical (NOGA) mapping is essential to ensure that the cells are injected in areas of hibernating myocardium [58], as necrotic areas of myocardium and scar tissue lack the necessary cues for cells to engraft and diff erentiate, and cells injected in these areas die immediately [59].

Homing
While homing of haematopoietic progenitor cells to bone marrow has been widely studied [60], the mechanisms of homing of progenitor cells to areas of tissue injury remain poorly understood. Homing is a complex process involving integrins and chemokine receptors, which is greatly enhanced after myocardial ischaemia and hypoxia. It includes adhesion to and transmigration through the endothelium followed by migration and invasion of the target tissue. Homing of cells is dependent on migration out of the vessel into the surrounding myocardium; therefore, underperfused regions of the myocardium are targeted in a less effi cient manner [61]. Th e two key factors that play an important role in homing after a MI are the release of stromal-cell-derived factor (SDF)-1 and a chromatin binding protein (HMGB1). SDF-1 regulates homing of stem cells to ischaemic tissue through integrin-dependent adhesion [62][63][64], and local delivery of SDF-1 can enhance progenitor cell recruitment and neovasculari sation [65,66]. Th e release of HMGB1 may act as a danger signal and stimulate the homing of stem cells to ischaemic tissue [67].
Furthermore, endothelial progenitor cells express a number of chemokine receptors, such as CXCR2, CXCR4 and CXCL12. Th ese chemokines play an important role in the homing and mobilisation of endothelial progenitor cells and their recruitment to the site of ischaemic injury for endothelial recovery [68][69][70][71].
Where is research in this area heading in the next few years?
Th e need for new therapies to treat patients with AMI has led to a swift transition from bench to bedside and a number of clinical trials showing promising potential for stem cell therapy in heart disease. Th e ultimate aim of this research is to develop a technique that grows new functioning heart muscle. However, many obstacles still lie ahead. One of the many remaining unanswered questions is which type of stem/progenitor cell is the best candidate for cardiac regeneration. Th e bone marrow is an attractive source because it is easily accessible and contains a number of stem cells, including haematopoietic and mesenchymal cells. It is likely, however, that bone marrow cells in humans work through an indirect paracrine mechanism. Th e safety and feasibility of bone marrow cells in AMI have been well established in clinical trials. Th is, therefore, supports the need for robust evidence from large double-blind randomised controlled trials to assess their eff ect on clinical endpoints such as mortality and symptoms.
Th e European Society of Cardiology established a task force to investigate the role of stem cells in cardiac repair and published its consensus in a 2006 report [72]. Th e future focus in stem cell therapy should be to provide a better understanding of the mechanism of functional improvements observed and the development of safe and eff ective cell tracking modalities. Th ese areas of research would aid the identifi cation of the best cell candidate for therapeutic use as well as better understanding of myocardial homing and cell survival post-transplantation. It is important to note, however, that the clinical experience has provided a lot of valuable information regarding the approaches to cell therapy in humans, which will of course provide a platform for future trials in this fi eld.
One of the challenges in the future is improving the durability and survival of stem cells in the adverse environ ment they are engrafted into. One of the properties of stem cells is stress resistance [73], although several studies have shown that most stem cells die within a few weeks of delivery into the myocardium [34,[74][75][76]. Th is is probably due to the lack of nutrients and oxygen within the ischaemic environment. Furthermore, heart failure [77], atherosclerosis [78,79] and advanced age [80,81] correlate inversely with the number and function of circulating endothelial progenitor cells. Allogenic cells from young and healthy donors may represent a good solution, but cell rejection requiring immunosuppressive therapies would pose a new problem. Th ere is some evidence that statins improve the survival of the circulating endothelial progenitor cells [82,83]. Furthermore, higher doses of statin therapy are associated with a greater increase in circulating CD34 + and CXCR4 + from the bone marrow, resulting in an increase in coronary fl ow reserve at 8 months [84]. Future trials are on the horizon assessing the role of statin therapy on enhancing the number of endothelial cells in patients with coronary artery disease (Clinicaltrials.gov CT01096875). Endothelial progenitor cells are a subset of haematopoietic cells that have an important role to play in ischaemia by promoting angiogenesis, preventing cardiomyocyte apoptosis and reducing adverse remodelling. It may be that future potential remedies, such as statins, that enhance the function of endothelial progenitor cells may play an important role in improving stem cell survival and function. One of the ways of improving cell survival may be achieved by using viral vectors encoding multiple cytoprotective genes that act on diff erent cell death and apoptotic pathways, or by preconditioning the stem cells with cytokines that result in improved cell engraftment.
Another important issue is the timing of cell administration post-MI. Although animal studies have supported early administration of stem cells post-infarction, in humans the benefi ts of this therapy were greater when administered >4 days after reperfusion (based on avail able evidence). Furthermore, given the seemingly small improvements that these trials have shown, the costeff ective ness of cell therapy will also need to be addressed.
Two ongoing randomised controlled trials (TIME and late TIME studies) may help us understand whether the timing of cell administration plays an important role. Th e TIME study (Clinicaltrials.gov NCT00684021) is a trial designed to assess the eff ect of timing (3 versus 7 days) of BMMNC administration versus placebo in patients with acute MI. Th e LATE TIME study (Clinicaltrials.gov NCT00684060) will assess the eff ect of BMMNC administra tion 2 to 3 weeks after a MI.

Future cells
Animal and human studies have clearly shown that stem cell engraftment into the myocardium is associated with improvement in cardiac function; however, the quest for the optimal population of cells remains a challenge [85,86]. Embryonic stem cells are able to transform into cardiomyocytes and can replicate indefi nitely, although ethical issues -their potential to form teratomas and the need for immunosuppressive therapy -have hindered their use in clinical trials. Furthermore, one of the major limitations of adult stem cells, including skeletal myoblasts and bone marrow-derived stem cells, is their limited ability to cross their lineage boundaries.
Ultimately, cells that more closely resemble embryonic stem cells in their regenerative potential without the ethical issues provide an important future direction. A cell type that comes close, and is on the horizon of being tested for potential clinical application, is the inducible pluripotent stem cell (iPSC). iPSCs can be generated from adult human somatic cells by retroviral transduction [92], have similar diff erentiation potential and may provide an alternative to pluripotent embryonic stem cells.

The future of bone marrow stem cells
For the time being, it is important to establish whether the simple unfractionated bone marrow cell approach has clinical benefi t, given the large number of studies that have been performed using this cell type without providing a clear answer. Meta-analysis suggests a positive eff ect on surrogate cardiac end-points in studies using BMMNCs to treat AMI. Th ere is now a need to perform a large scale clinical trial using clinical hard end-points such as mortality to establish whether the positive eff ects seen on surrogate end-points can indeed translate to meaningful clinical benefi ts.