# Cellular Therapy for Repair of Cardiac Damage after Acute Myocardial Infarction

Authors: Matthew M. Cook; Katarina Kollar; Gary P. Brooke; Kerry Atkinson
Journal: International Journal of Cell Biology
 (2009)

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## Abstract

Cardiovascular diseases, particularly acute myocardial infarction, are the leading causes of death worldwide. Important advances have been made in the secondary treatment for cardiovascular diseases such as heart transplantation and medical and surgical therapies.  Although these therapies alleviate symptoms, and may even improve survival, none can reverse the disease process and directly repair the lasting damage. Thus, the cure of cardiovascular diseases remains a major unmet medical need. Recently, cellular therapy has been proposed as a candidate treatment for this. Many stem and progenitor cell populations have each been suggested as a potential basis for such therapy. This review assesses some of the more notable exogenous adult cell candidates and provides insights into the mechanisms by which they may mediate improvement in cardiac function following acute myocardial infarction. Research into the cellular therapy field is of great importance for the further planning of clinical trials for cardiac cellular myoplasty.


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## Body

## 1. Introduction

Cardiovascular
disease (CVD) is a broad term
referring to all diseases that involve the heart and/or blood vessels. CVD is the leading cause of death worldwide,
estimated at causing 17.5 million deaths in 2005. Of these an estimated 7.6
million were due to ischaemic heart disease (IHD) which is a subset of CVD and is characterised by occlusion of a coronary
artery causing decreased blood flow and deprivation of oxygen and nutrients to
the high energy-requiring cardiomyocytes in the myocardium [1]. This situation
is also known as acute myocardial infarction (AMI)
or more commonly a heart attack. While this may be an acute or transient
condition (i.e., the heart is reperfused after a temporary occlusion), the ischemic
damage due to loss of blood flow causes significant cardiomyocyte death and the
subsequent irreversible formation of a fibrotic scar [2]. This, in
turn, leads to dyskinesis of the ventricular wall, diminished heart function,
and chronic heart failure (CHF).Within the last 10–15 years,
pharmaceutical therapies (aspirin, angiotensin converting enzyme-inhibitors,β-blockers), percutaneous coronary interventions, coronary artery bypass graft
surgery, left ventricular assist devices and biventricular pacing have made a
significant impact on the cardiovascular disease burden. Heart transplantation
is also well established, but the need for long-term immune suppression and the
chronic donor organ shortages suggest that it is unlikely to evolve into a
viable definitive treatment for the majority of persons with cardiac disease.
Although these therapies ameliorate symptoms, and may even improve survival,
none can claim to directly reverse the disease process itself.With
recent advances in medicine and associated technologies, a number of novel
therapies for the repair of the myocardium following ischaemia have been
suggested. The most prominent of these
is the use of cells to repair existing cardiac damage with bone marrow being
the commonest source due to its readily accessible nature and autologous
sourcing. Bone marrow mononuclear cells (BMCs), peripheral blood stem cells (PBSCs), mesenchymal stem
cells (MSCs), and endothelial
progenitor cells (EPCs)
have been investigated in animal and human studies. However, many of these cell populations have
been incompletely characterised and are thought to be a mixture of several
related subpopulations. At present, BMCs are the most common source for cell-based therapy and, due
to the 40 years of experience with bone marrow transplants used for treating
haematological diseases, they have rapidly been applied to clinical trials in
severe cardiac disease. Although the first phase I/II controlled clinical trials using BMCs to improve cardiac function after AMI were recently published (with variable results)  [3, 4], it is still unclear as to which bone marrow
cell population contributed to the repair of cardiac muscle damaged by
ischaemia in these trials. This review will clarify the potential of purified
adult cell populations to repair the damaged myocardium and outline several
possible mechanisms of repair.


## 2. Stem/Progenitor Cell Populations

Stem cells are defined by two key characteristics. Firstly, they must be able to
self-renew in that they can go through many cell division cycles whilst
remaining in an unchanged and undifferentiated state. Their second property is
the capacity to differentiate into multiple specialised cell types [5],
and the potency of stem cells is often defined by their potential to
differentiate. At one end of the spectrum stem cells are able to differentiate
into specialised mature cells from all three germ layers. These are termed
pluripotent stem cells with embryonic stem cells (derived from blastocyst of a
developing embryo) as the prime example. Alternately, multipotent stem cells
are restricted to differentiating into certain closely related lineages often
of the same germ layer [5, 6].
Adult (or somatic) stem cells are derived from postnatal or mature tissues and
are primarily multipotent. However, some subsets have shown plasticity across
multiple germ layers [7, 8]. Some
adult stem cells have a limited capacity for self-renewal and may thus be
better classified as adult progenitor cells. Although progenitor cells are also
defined by the properties of self-renewal and multipotency, these are observed
to a lesser extent compared to pluripotent stem cells. Hence, progenitor cells
are more committed to specific lineage differentiation than stem cells (Figure 1).Figure 1
Differentiation and self-renewal potential of embryonic stem cells, adult stem
cells, and progenitor cells.


### 2.1. Mesenchymal Stem Cells (MSCs)

MSCs were first recognised by Friedenstein et al., who identified a
plastic adherent, fibroblast-like population (Figure2) that could regenerate
rudiments of normal bone in vivo
[9–11]. MSCs were initially identified within the stroma of the bone
marrow and were subsequently found to provide support for haematopoiesis by secreting many
colony-stimulating factors and growth factors important in the proliferation,
differentiation and survival of haematopoietic cells [12–15]. Although a majority of the literature is
concerned with bone marrow derived MSCs, they have also, in more recent times, been isolated from
other organs including placenta, adipose, cord-blood, and liver [8, 16–21]. Recently, they have been shown to be
ubiquitous since they have been shown to be an integral part of the composition
of endothelium (perivascular cells or pericytes) [22, 23].Mesenchymal stem cell morphology by light microscopy. (a) MSCs culture (×100), (b)
Cytospin and Giemsa stained (×1000).
(a)(b)Although
no single marker specific for MSCs has yet been described, they are characteristically
negative for typical
haematopoietic lineage antigens such
as CD45, CD34, and CD14 and positive for CD44, CD73, CD90, CD105, CD166, and
Stro-1 [24–29]. MSCs also produce a large
number of growth factors and cytokines, including vascular endothelial growth
factor (VEGF)  [13]. This suggests
MSCs involvement in paracrine
mechanisms. However, the current hallmark for MSCs is their ability to differentiate into mature
cell populations of the mesodermal lineage such as bone, cartilage, tendon,
muscle, and adipose tissue [25, 28, 30, 31]. Interestingly, some groups have also shown MSCs to have plasticity beyond the mesodermal
lineage with the ability to differentiate into neurons and astrocytes
(ectodermal) as well as hepatocytes (endodermal)  [7, 8].MSCs also appear to have a significant advantage for cell therapy in that
they are immunologically privileged and even in large outbred animals can be transplanted
across major histocompatibility (MHC)
barriers without the need for immune suppression [32–34]. This has important implications in that MSCs can be taken from a healthy unrelated donor and
cryopreserved ready for use in patients with a wide variety of pathologies [35]. In addition to this relative lack of
immunogenicity, MSCs
are actively suppressive of T cell function [36, 37]. This immune suppressive capability has been
successfully exploited in the clinic where unrelated, MHC-unmatched
or mismatched MSCs
have been used to treat patients with acute graft-versus-host disease (GVHD)  [33, 38, 39].MSCs show promise as a cellular therapeutic agent due to their ease of
expansion, immuno-privileged status, and ability to self-renew and to differentiate
across multiple mature cell lineages. Because of their multipotency, they have
been investigated in cardiovascular disease [40, 41], neurological disease [42], osteogenesis imperfecta [43–45], osteoarthritis [46], GVHD [33, 39], and
liver fibrosis [47]. Controversially, MSCs have been induced to differentiate into
cardiomyocytes in mice [48] and humans [41], and several studies have shown improved
myocardial function after myocardial ischaemia in rodents and pigs [40, 41]. This indicates their potential for
treatment of damage caused by ischemic myocardial infarction in human. Finally,
there are some reports that MSCs
migrate preferentially to sites of inflammation as opposed to the bone marrow,
as has been reported in some studies to be the case in unperturbed animals [26, 28].


### 2.2. Haematopoietic Stem/Progenitor Cells (HSCs/HPCs)

HSCs/HPCs
are the best characterised adult stem cell and are the only stem or progenitor cells that are in
routine clinical use today. HSCs
normally reside in the bone marrow and are responsible for making all blood
cell types and thus continually reconstituting the haematopoietic and immune systems [49, 50]. These cells are used clinically in bone
marrow transplants to treat a number of blood disorders including
leukaemia, aplastic anaemia, and severe combined immunodeficiency [51, 52]. The functional hallmark of a true HSCs is the in
vivo ability to reconstitute all blood lineages (from a single cell)
following otherwise lethal total body irradiation (TBI).In the human, HSCs/HPCs
are identified by cell surface expression of CD34, a cell surface glycoprotein [49, 53, 54]. However, isolation of CD34+ cells from umbilical cord blood, bone marrow, or peripheral blood aphaeresis
product leads to a relatively heterogeneous population, while true HSCs, as defined by
single-cell repopulation capacity, represent less then 0.1% of CD34+ enriched cells [55]. Thus, a CD34 enriched population is often
referred to as an HPC population
containing lineage-committed cells as well as HSCs.
Although CD34 is also expressed in the mouse, the regulation of the CD34 gene
is different to that in the human [53]. Thus, it is not a reliable HPC marker in the mouse. Furthermore, it has been
shown that CD34−/low murine HSCs can reconstitute the haematopoietic system following TBI [54].Murine studies of HSCs often use the
following markers: c-kit+, CD45+, and lineage−/low.
Lineage−/low status is commonly defined as negativity for CD5 (T cells),
CD11b (myeloid cells), CD45R (B cells), Gr-1 (granulocytes), and Ter119
(erythrocytes). This population can be further purified with the addition of
the Sca-1+ marker to the panel and these HSCs are termed LSK cells (Lineage/Sca-1/c-kit) (Figure 3).
However, these cells still represent a heterogeneous population with
approximately one in ten having the ability to repopulate the haematopoietic
system [56]. More recently, the signal lymphocyte
activating molecule (SLAM)
receptors (CD48, CD150, and CD244) have been used to further enrich the LSK
population to derive a more primitive HSC population [57–59]. In humans, earlier HSCs are identified by the phenotypes CD34+ CD90+ or CD34+ CD38− [60].Morphology of (a) haematopoietic progenitor cells displaying a heterogeneous
morphology and (b) the more purified LSK showing a more homogeneous morphology
(light microscopy×1000).
(a)(b)HPCs migrate preferentially to the bone marrow in healthy animals and the
molecular mechanisms of this migration are well described, including rolling
and tethering of HPCs
on bone marrow endothelium, followed by their arrest and firm adhesion to the
endothelium [61–63]. It is not known if intravenously injected HPCs migrate preferentially
to acutely inflamed tissue.It is debated as to whether bone marrow cells enriched for HPCs participate in the repair of cardiomyocytes following infarction. Orlic et al. (2001) and Rota et al. (2007) have shown that bone
marrow cells enriched for c-kit are capable of differentiating into cardiomyocytes
[64, 65]. However, these c-kit+ enriched
populations were heterogeneous and may have contained stromal cell populations.
Furthermore, HPCs
have been observed to differentiate into skeletal muscle fibres during muscle
regeneration [66]. Conversely, it has also been shown that HPCs do not differentiate
into cardiomyocytes following infarction and rather take on a mature
haematopoietic fate which may, in turn, give rise to differentiated
haematopoietic cells, responsible for the inflammatory wound healing process [67, 68]. Various other actions of HPCs on injured tissue have been proposed including
secretion of cytokines and chemokines, inhibition of apoptosis, and suppression
of immune reactions [69, 70].


### 2.3. Endothelial Progenitor Cells (EPCs)

Historically, neoangiogenesis (the formation of new blood vessels) was
thought to occur by the proliferation of existing endothelial cells. However,
in 1997 Asahara et al. discovered “putative progenitor endothelial
cells” in adult peripheral blood [71]. In the last 10 years, this discovery has
been confirmed and it is now commonly accepted that EPCs play an essential role in adult blood
vessel formation, endothelial repair, and endothelial homeostasis [72, 73]. These postnatal cells have been extensively
explored for their innate capacity to contribute to angiogenesis in both
pathological and unperturbed states. Like MSCs and HPCs, EPCs can also be
derived from the bone marrow. They circulate in peripheral blood in a very low
numbers, but can be mobilised into the blood using molecules such as
granulocyte colony-stimulating factor (G-CSF) [74].EPCs derived from adult human peripheral blood
were initially characterised by expression of both CD34+ and Flk-1+ [71]. Flk-1 is also known as vascular endothelial
growth factor receptor (VEGFR)-2 in mice and kinase insert domain receptor
(KDR) in humans. Along with vascular endothelial growth factor receptor-1
(VEGFR-1, also known as Flt-1), VEGFR-2 serves to mediate the actions of VEGF,
which is recognised as an essential regulator of angiogenesis [74, 75]. VEGFR-2 expression is considered to be a
marker of progenitor cell commitment to the endothelial lineage and is now
typically used in conjunction with other antigens, such as vascular-endothelium
(VE)-cadherin (CD144) and CD31 (also known as platelet/endothelial cell
adhesion molecule-1 or PECAM-1) to identify putative angioblasts or EPCs [76, 77]. Another key marker is CD133 (prominin-1)
and, like CD34, it is present on both HPCs and cells that exhibit a potent blood-vessel forming capacity [78, 79]. In the developing embryo, EPCs and HSCs arise from a common precursor called the haemangioblast [80, 81]. Hence, a majority of markers used to
identify EPCs are also prevalent on
haematopoietic progenitors. Although these markers prove useful in identifying
populations enriched for the angioblast or EPCs,
it should be recognised that defining hierarchical relationships is far from currently
agreed upon [77, 82].It is now more common for EPCs to be isolated from peripheral blood, bone marrow, or
umbilical cord blood (UCB) based on their morphologic and adherent
characteristics when cultured on fibronectin or collagen-coated plates in the
presence of appropriate growth media and supplemental angiogenic
differentiation factors. These factors
include VEGF, fibroblast growth factor-2 (FGF-2), insulin-like growth factor-1
(IGF-1), and epidermal growth factor (EGF)  [75, 76, 83, 84]. The majority of cells that appear in early
stages of culture (within the first 15 days) are thought to have originated
from a CD14+ (macrophage/monocyte) subpopulation of mononuclear
cells. These cells are often referred to as early-outgrowth endothelial
progenitor cells (EO-EPCs)  [75, 83, 85]. Conversely, late-outgrowth EPCs (LO-EPCs) do not
appear in culture for 2-3 weeks and
exhibit the “classic endothelial” phenotype with contact inhibition and
cobblestone monolayer morphology and the ability to form in vitro tube-like structures when seeded on Matrigel (Figure 4). These LO-EPCs also show exponential growth
kinetics and a capacity for ex vivo expansion [75, 77, 83, 85, 86].Endothelial progenitor cells derived from
human umbilical cord blood (a) exhibit classic cobble-stone morphology (light
microscopy×40)
and (b) form tube-like structures when seeded on Matrigel
basement membrane matrix (light microscopy ×100).
(a)(b)It has been extensively shown that EPCs are associated with neoangiogenesis following
tissue injury in animal models of hind-limb ischaemia and myocardial infarction
[4, 84, 87, 88]. Conversely, however, administration of EPCs into human patients with CVD is yet to show efficacy with regard to vessel
formation, even though these patients had a better clinical outcome [89]. This observation suggests that improvement
may have been due to, at least in part, a paracrine function.


### 2.4. Non-Stem/Progenitor Cell Populations

It is well established that monocytes/macrophages play an important role in
angiogenesis, first shown in 1977 [90]. This occurs by the release of tumour
necrosis factor-α (TNF-α), thrombospondin, and angiogenic factors such as VEGF,
angiopoietin, and matrix metalloproteinases (MMPs)  [91–93]. Cells of the macrophage lineage play a major
role in the innate immune response and contribute to wound healing, tissue
repair, and bone remodeling [94]. Any disturbance of tissue normality such as
infection, aberrant cell turnover, or tissue damage leads to an inflammatory
response and a rapid recruitment of macrophages. During inflammation monocytes
and macrophages phagocytose foreign particles (cellular debris or pathogens)
and stimulate lymphocytes and other immune cells to respond to the pathogen by
release of a variety of cytokines and chemoattractants that can modulate the migration
of circulating cells and their adhesion to local endothelial cells [2, 91, 93, 95].A novel lineage of monocytes has recently been isolated that is thought to play a
key role in the revascularisation process. These cells are isolated from mice
as CD11b+ monocytes (or from human as CD14+ and CD16−)
and express the angiogenic marker Tie-2 (tunica internal endothelial cell
kinase-2)  [96–99]. Tie-2 is a receptor tyrosine kinase
expressed principally on vascular endothelium and is also expressed on HPCs and EPCs.
Its ligands are the angiopoietin growth factors that promote the growth of new
blood vessels and are involved with migration of Tie-2-expressing cells to
sites of inflammation [96–98]. Tie-2 has also been hypothesised to be
involved in the attenuation of proinflammatory mediators.Tie-2 expressing monocytes (TEMs)
have been isolated from the bone marrow of mice by plastic adherence and stimulation
with macrophage colony-stimulating factor (M-CSF or CSF-1). These cells have a large
nucleus surrounded by a cytoplasm with an abundance of vacuoles (Figure5).
They have also been identified in the peripheral blood of mice [96] and from human PBMCs [98, 99]. However, the literature on this subject has been focused on how TEMs may
contribute to tumour-associated angiogenesis and thus these cells have often been
identified and isolated from various mouse and human tumours. It is thought
that TEMs may contribute to tumour angiogenesis by providing paracrine support
to nascent blood vessels and by the sequestering of endothelial cells. TEMs produce
proangiogenic factors and, when injected in Matrigel matrix plugs implanted
under the skin of rodents, promote robust angiogenesis whilst not forming new
blood vessels themselves. These data suggest that their recruitment to the site
of ischaemic injury is sufficient to support revascularisation [96, 98, 100].Analysis of Tie-2 expressing monocytes by (a) culture
morphology (light microscopy×100) and (b) Giemsa staining (light microscopy ×1000).
(a)(b)Monocytes are attracted to the myocardium by
overexpression of monocyte chemoattractant protein-1 (MCP-1), where there is
evidence that they form CD31-negative (PECAM) tunnels. Monocytes and
macrophages “drill tunnels” using matrix metalloproteinases (MMPs). However it is yet to be demonstrated whether
these become new vessels [100–102]. It has
been proposed that neoangiogenesis occurs via the contribution of monocytes,
macrophages, and circulating EPCs.


## 2.1. Mesenchymal Stem Cells (MSCs)

MSCs were first recognised by Friedenstein et al., who identified a
plastic adherent, fibroblast-like population (Figure2) that could regenerate
rudiments of normal bone in vivo
[9–11]. MSCs were initially identified within the stroma of the bone
marrow and were subsequently found to provide support for haematopoiesis by secreting many
colony-stimulating factors and growth factors important in the proliferation,
differentiation and survival of haematopoietic cells [12–15]. Although a majority of the literature is
concerned with bone marrow derived MSCs, they have also, in more recent times, been isolated from
other organs including placenta, adipose, cord-blood, and liver [8, 16–21]. Recently, they have been shown to be
ubiquitous since they have been shown to be an integral part of the composition
of endothelium (perivascular cells or pericytes) [22, 23].Mesenchymal stem cell morphology by light microscopy. (a) MSCs culture (×100), (b)
Cytospin and Giemsa stained (×1000).
(a)(b)Although
no single marker specific for MSCs has yet been described, they are characteristically
negative for typical
haematopoietic lineage antigens such
as CD45, CD34, and CD14 and positive for CD44, CD73, CD90, CD105, CD166, and
Stro-1 [24–29]. MSCs also produce a large
number of growth factors and cytokines, including vascular endothelial growth
factor (VEGF)  [13]. This suggests
MSCs involvement in paracrine
mechanisms. However, the current hallmark for MSCs is their ability to differentiate into mature
cell populations of the mesodermal lineage such as bone, cartilage, tendon,
muscle, and adipose tissue [25, 28, 30, 31]. Interestingly, some groups have also shown MSCs to have plasticity beyond the mesodermal
lineage with the ability to differentiate into neurons and astrocytes
(ectodermal) as well as hepatocytes (endodermal)  [7, 8].MSCs also appear to have a significant advantage for cell therapy in that
they are immunologically privileged and even in large outbred animals can be transplanted
across major histocompatibility (MHC)
barriers without the need for immune suppression [32–34]. This has important implications in that MSCs can be taken from a healthy unrelated donor and
cryopreserved ready for use in patients with a wide variety of pathologies [35]. In addition to this relative lack of
immunogenicity, MSCs
are actively suppressive of T cell function [36, 37]. This immune suppressive capability has been
successfully exploited in the clinic where unrelated, MHC-unmatched
or mismatched MSCs
have been used to treat patients with acute graft-versus-host disease (GVHD)  [33, 38, 39].MSCs show promise as a cellular therapeutic agent due to their ease of
expansion, immuno-privileged status, and ability to self-renew and to differentiate
across multiple mature cell lineages. Because of their multipotency, they have
been investigated in cardiovascular disease [40, 41], neurological disease [42], osteogenesis imperfecta [43–45], osteoarthritis [46], GVHD [33, 39], and
liver fibrosis [47]. Controversially, MSCs have been induced to differentiate into
cardiomyocytes in mice [48] and humans [41], and several studies have shown improved
myocardial function after myocardial ischaemia in rodents and pigs [40, 41]. This indicates their potential for
treatment of damage caused by ischemic myocardial infarction in human. Finally,
there are some reports that MSCs
migrate preferentially to sites of inflammation as opposed to the bone marrow,
as has been reported in some studies to be the case in unperturbed animals [26, 28].


## 2.2. Haematopoietic Stem/Progenitor Cells (HSCs/HPCs)

HSCs/HPCs
are the best characterised adult stem cell and are the only stem or progenitor cells that are in
routine clinical use today. HSCs
normally reside in the bone marrow and are responsible for making all blood
cell types and thus continually reconstituting the haematopoietic and immune systems [49, 50]. These cells are used clinically in bone
marrow transplants to treat a number of blood disorders including
leukaemia, aplastic anaemia, and severe combined immunodeficiency [51, 52]. The functional hallmark of a true HSCs is the in
vivo ability to reconstitute all blood lineages (from a single cell)
following otherwise lethal total body irradiation (TBI).In the human, HSCs/HPCs
are identified by cell surface expression of CD34, a cell surface glycoprotein [49, 53, 54]. However, isolation of CD34+ cells from umbilical cord blood, bone marrow, or peripheral blood aphaeresis
product leads to a relatively heterogeneous population, while true HSCs, as defined by
single-cell repopulation capacity, represent less then 0.1% of CD34+ enriched cells [55]. Thus, a CD34 enriched population is often
referred to as an HPC population
containing lineage-committed cells as well as HSCs.
Although CD34 is also expressed in the mouse, the regulation of the CD34 gene
is different to that in the human [53]. Thus, it is not a reliable HPC marker in the mouse. Furthermore, it has been
shown that CD34−/low murine HSCs can reconstitute the haematopoietic system following TBI [54].Murine studies of HSCs often use the
following markers: c-kit+, CD45+, and lineage−/low.
Lineage−/low status is commonly defined as negativity for CD5 (T cells),
CD11b (myeloid cells), CD45R (B cells), Gr-1 (granulocytes), and Ter119
(erythrocytes). This population can be further purified with the addition of
the Sca-1+ marker to the panel and these HSCs are termed LSK cells (Lineage/Sca-1/c-kit) (Figure 3).
However, these cells still represent a heterogeneous population with
approximately one in ten having the ability to repopulate the haematopoietic
system [56]. More recently, the signal lymphocyte
activating molecule (SLAM)
receptors (CD48, CD150, and CD244) have been used to further enrich the LSK
population to derive a more primitive HSC population [57–59]. In humans, earlier HSCs are identified by the phenotypes CD34+ CD90+ or CD34+ CD38− [60].Morphology of (a) haematopoietic progenitor cells displaying a heterogeneous
morphology and (b) the more purified LSK showing a more homogeneous morphology
(light microscopy×1000).
(a)(b)HPCs migrate preferentially to the bone marrow in healthy animals and the
molecular mechanisms of this migration are well described, including rolling
and tethering of HPCs
on bone marrow endothelium, followed by their arrest and firm adhesion to the
endothelium [61–63]. It is not known if intravenously injected HPCs migrate preferentially
to acutely inflamed tissue.It is debated as to whether bone marrow cells enriched for HPCs participate in the repair of cardiomyocytes following infarction. Orlic et al. (2001) and Rota et al. (2007) have shown that bone
marrow cells enriched for c-kit are capable of differentiating into cardiomyocytes
[64, 65]. However, these c-kit+ enriched
populations were heterogeneous and may have contained stromal cell populations.
Furthermore, HPCs
have been observed to differentiate into skeletal muscle fibres during muscle
regeneration [66]. Conversely, it has also been shown that HPCs do not differentiate
into cardiomyocytes following infarction and rather take on a mature
haematopoietic fate which may, in turn, give rise to differentiated
haematopoietic cells, responsible for the inflammatory wound healing process [67, 68]. Various other actions of HPCs on injured tissue have been proposed including
secretion of cytokines and chemokines, inhibition of apoptosis, and suppression
of immune reactions [69, 70].


## 2.3. Endothelial Progenitor Cells (EPCs)

Historically, neoangiogenesis (the formation of new blood vessels) was
thought to occur by the proliferation of existing endothelial cells. However,
in 1997 Asahara et al. discovered “putative progenitor endothelial
cells” in adult peripheral blood [71]. In the last 10 years, this discovery has
been confirmed and it is now commonly accepted that EPCs play an essential role in adult blood
vessel formation, endothelial repair, and endothelial homeostasis [72, 73]. These postnatal cells have been extensively
explored for their innate capacity to contribute to angiogenesis in both
pathological and unperturbed states. Like MSCs and HPCs, EPCs can also be
derived from the bone marrow. They circulate in peripheral blood in a very low
numbers, but can be mobilised into the blood using molecules such as
granulocyte colony-stimulating factor (G-CSF) [74].EPCs derived from adult human peripheral blood
were initially characterised by expression of both CD34+ and Flk-1+ [71]. Flk-1 is also known as vascular endothelial
growth factor receptor (VEGFR)-2 in mice and kinase insert domain receptor
(KDR) in humans. Along with vascular endothelial growth factor receptor-1
(VEGFR-1, also known as Flt-1), VEGFR-2 serves to mediate the actions of VEGF,
which is recognised as an essential regulator of angiogenesis [74, 75]. VEGFR-2 expression is considered to be a
marker of progenitor cell commitment to the endothelial lineage and is now
typically used in conjunction with other antigens, such as vascular-endothelium
(VE)-cadherin (CD144) and CD31 (also known as platelet/endothelial cell
adhesion molecule-1 or PECAM-1) to identify putative angioblasts or EPCs [76, 77]. Another key marker is CD133 (prominin-1)
and, like CD34, it is present on both HPCs and cells that exhibit a potent blood-vessel forming capacity [78, 79]. In the developing embryo, EPCs and HSCs arise from a common precursor called the haemangioblast [80, 81]. Hence, a majority of markers used to
identify EPCs are also prevalent on
haematopoietic progenitors. Although these markers prove useful in identifying
populations enriched for the angioblast or EPCs,
it should be recognised that defining hierarchical relationships is far from currently
agreed upon [77, 82].It is now more common for EPCs to be isolated from peripheral blood, bone marrow, or
umbilical cord blood (UCB) based on their morphologic and adherent
characteristics when cultured on fibronectin or collagen-coated plates in the
presence of appropriate growth media and supplemental angiogenic
differentiation factors. These factors
include VEGF, fibroblast growth factor-2 (FGF-2), insulin-like growth factor-1
(IGF-1), and epidermal growth factor (EGF)  [75, 76, 83, 84]. The majority of cells that appear in early
stages of culture (within the first 15 days) are thought to have originated
from a CD14+ (macrophage/monocyte) subpopulation of mononuclear
cells. These cells are often referred to as early-outgrowth endothelial
progenitor cells (EO-EPCs)  [75, 83, 85]. Conversely, late-outgrowth EPCs (LO-EPCs) do not
appear in culture for 2-3 weeks and
exhibit the “classic endothelial” phenotype with contact inhibition and
cobblestone monolayer morphology and the ability to form in vitro tube-like structures when seeded on Matrigel (Figure 4). These LO-EPCs also show exponential growth
kinetics and a capacity for ex vivo expansion [75, 77, 83, 85, 86].Endothelial progenitor cells derived from
human umbilical cord blood (a) exhibit classic cobble-stone morphology (light
microscopy×40)
and (b) form tube-like structures when seeded on Matrigel
basement membrane matrix (light microscopy ×100).
(a)(b)It has been extensively shown that EPCs are associated with neoangiogenesis following
tissue injury in animal models of hind-limb ischaemia and myocardial infarction
[4, 84, 87, 88]. Conversely, however, administration of EPCs into human patients with CVD is yet to show efficacy with regard to vessel
formation, even though these patients had a better clinical outcome [89]. This observation suggests that improvement
may have been due to, at least in part, a paracrine function.


## 2.4. Non-Stem/Progenitor Cell Populations

It is well established that monocytes/macrophages play an important role in
angiogenesis, first shown in 1977 [90]. This occurs by the release of tumour
necrosis factor-α (TNF-α), thrombospondin, and angiogenic factors such as VEGF,
angiopoietin, and matrix metalloproteinases (MMPs)  [91–93]. Cells of the macrophage lineage play a major
role in the innate immune response and contribute to wound healing, tissue
repair, and bone remodeling [94]. Any disturbance of tissue normality such as
infection, aberrant cell turnover, or tissue damage leads to an inflammatory
response and a rapid recruitment of macrophages. During inflammation monocytes
and macrophages phagocytose foreign particles (cellular debris or pathogens)
and stimulate lymphocytes and other immune cells to respond to the pathogen by
release of a variety of cytokines and chemoattractants that can modulate the migration
of circulating cells and their adhesion to local endothelial cells [2, 91, 93, 95].A novel lineage of monocytes has recently been isolated that is thought to play a
key role in the revascularisation process. These cells are isolated from mice
as CD11b+ monocytes (or from human as CD14+ and CD16−)
and express the angiogenic marker Tie-2 (tunica internal endothelial cell
kinase-2)  [96–99]. Tie-2 is a receptor tyrosine kinase
expressed principally on vascular endothelium and is also expressed on HPCs and EPCs.
Its ligands are the angiopoietin growth factors that promote the growth of new
blood vessels and are involved with migration of Tie-2-expressing cells to
sites of inflammation [96–98]. Tie-2 has also been hypothesised to be
involved in the attenuation of proinflammatory mediators.Tie-2 expressing monocytes (TEMs)
have been isolated from the bone marrow of mice by plastic adherence and stimulation
with macrophage colony-stimulating factor (M-CSF or CSF-1). These cells have a large
nucleus surrounded by a cytoplasm with an abundance of vacuoles (Figure5).
They have also been identified in the peripheral blood of mice [96] and from human PBMCs [98, 99]. However, the literature on this subject has been focused on how TEMs may
contribute to tumour-associated angiogenesis and thus these cells have often been
identified and isolated from various mouse and human tumours. It is thought
that TEMs may contribute to tumour angiogenesis by providing paracrine support
to nascent blood vessels and by the sequestering of endothelial cells. TEMs produce
proangiogenic factors and, when injected in Matrigel matrix plugs implanted
under the skin of rodents, promote robust angiogenesis whilst not forming new
blood vessels themselves. These data suggest that their recruitment to the site
of ischaemic injury is sufficient to support revascularisation [96, 98, 100].Analysis of Tie-2 expressing monocytes by (a) culture
morphology (light microscopy×100) and (b) Giemsa staining (light microscopy ×1000).
(a)(b)Monocytes are attracted to the myocardium by
overexpression of monocyte chemoattractant protein-1 (MCP-1), where there is
evidence that they form CD31-negative (PECAM) tunnels. Monocytes and
macrophages “drill tunnels” using matrix metalloproteinases (MMPs). However it is yet to be demonstrated whether
these become new vessels [100–102]. It has
been proposed that neoangiogenesis occurs via the contribution of monocytes,
macrophages, and circulating EPCs.


## 3. Possible Mechanisms of Tissue Repair by Cells

### 3.1. Differentiation into Cardiomyocytes and Cell Fusion

Stem cells may have
the potential to replace necrotic myocardium, form new cardiomyocytes, and
restore cardiac function after AMI [5, 103]. Cardiomyocyte differentiation in vitro
has been shown with embryonic stem cells [104], HPCs [64, 65], MSCs [41], and EPCs [105]. Few studies, however, have been able
to replicate this phenomenon in vivo
and significant cell numbers have failed to differentiate in the myocardium [67, 106]. Although these studies showed a lack
of physical repair of the myocardium, there was still, in most cases, an
improvement in cardiac function. These data suggest that there may be other mechanisms
mediating functional improvement. One alternative to differentiation is the
occurrence of cell fusion to support cardiomyogenesis. However, the occurrence
of this is also rare [107–109].


### 3.2. Paracrine Function

Stem cells secrete
many soluble factors that may directly or indirectly have reparative qualities.
In cardiac ischaemia, these factors may signal through pathways that act to
promote angiogenesis, decrease apoptosis, increase the efficiency of cardiomyocyte
metabolism, or modulate inotrophy (formation of fibrous scar) [2]. There
is evidence of bone marrow mononuclear cell-conditioned media showing
reparative features in preclinical models of AMI [110–113]. This conditioned media contained a
variety of cytokines including VEGF, interleukin-1 (IL-1), placental-derived
growth factor (PDGF), IGF-1 and MCP-1.
It has also been shown that these factors were significantly upregulated
when the cells are cultured in a hypoxic state [114]. Transfusion of this media led to
increased capillary density, decreased infarct size, and improvement of cardiac
function following myocardial infarction [110, 112]. Whilst transfusion of the conditioned
media is potentially seen as a useful therapeutic agent, the need for the donor
cells to be transfused was still apparent as it was shown that some functional
reparative mechanisms only occurred when the bone marrow derived cells were
present. There is also a need for such a pool of cytokines to be constantly
replenished, a function that can be performed by constant transfusion of
conditioned media or a single transfusion of a cellular “cytokine factory”.


### 3.3. Vascular Remodeling

Interventions that
increase the perfusion to areas of restricted blood flow may cause salvage of
border-zone cardiomyocytes as well as partial reversal of pathological
myocardial remodeling. Increased vascularisation may occur via two pathways.
Firstly, it may occur by angiogenesis which is the formation of new branches of
blood vessels from pre-existing vessels and thus an increase in perfusion.
Secondly, it may occur by an increase in diameter of existing vessels causing
an increase in local perfusion [104]. The success of both
these events is determined by the pre-existence of a vascular network,
activation of the endothelium by fluid pressure stress, invasion of bone marrow
derived cells, and proliferation of endothelial and smooth muscle cells [115]. Cell therapy may
act in a paracrine mechanism to increase the production of proangiogenic
cytokines (as mentioned above) and stimulate host cells to remodel the existing
vasculature by either forming new branches or converting an arteriole into an
artery. Alternately, donor cells may physically embed in the ischaemic tissue
and become new vessel branches.Various studies have
shown revascularisation in animal models of hind-limb ischaemia and myocardial
infarction with EPCs [87, 116–118], HPCs [64, 66], BMCs [112, 119], and MSCs [120]. One such study by Kocher et al. [81]
showed that human mobilised CD34+ cells homed to the infarcted
myocardium where they were incorporated into newly developed coronary
circulation. However, similar to the differentiation process above, there is
limited evidence showing that the cells themselves are directly or physically
participating in this event [121]. This, once again, reinforces the
potential value of the paracrine function of infused cells as they are often
associated with an increase in capillary density in the infarcted region.


### 3.4. Cardiac Protection

Donor cell secretion
of numerous cytokines is believed to attenuate the apoptotic state. For
example, IGF-1 [122], hepatocyte growth
factor (HGF)  [123], and FGF [124] have all been shown to be
cardioprotective molecules by attenuation of cardiomyocyte apoptosis.Another form of cardiac
protection is by modification of the immune response and this has subsequently
been acknowledged as a potential therapeutic target [104]. This has been shown
with MSCs, which suppress T-cell responses and thus have local immunosuppressive
functionalities [32, 39]. However, cardiac protection through
these mechanisms must be viewed with caution as many proinflammatory and
proapoptotic molecules are also involved in angiogenic responses.


## 3.1. Differentiation into Cardiomyocytes and Cell Fusion

Stem cells may have
the potential to replace necrotic myocardium, form new cardiomyocytes, and
restore cardiac function after AMI [5, 103]. Cardiomyocyte differentiation in vitro
has been shown with embryonic stem cells [104], HPCs [64, 65], MSCs [41], and EPCs [105]. Few studies, however, have been able
to replicate this phenomenon in vivo
and significant cell numbers have failed to differentiate in the myocardium [67, 106]. Although these studies showed a lack
of physical repair of the myocardium, there was still, in most cases, an
improvement in cardiac function. These data suggest that there may be other mechanisms
mediating functional improvement. One alternative to differentiation is the
occurrence of cell fusion to support cardiomyogenesis. However, the occurrence
of this is also rare [107–109].


## 3.2. Paracrine Function

Stem cells secrete
many soluble factors that may directly or indirectly have reparative qualities.
In cardiac ischaemia, these factors may signal through pathways that act to
promote angiogenesis, decrease apoptosis, increase the efficiency of cardiomyocyte
metabolism, or modulate inotrophy (formation of fibrous scar) [2]. There
is evidence of bone marrow mononuclear cell-conditioned media showing
reparative features in preclinical models of AMI [110–113]. This conditioned media contained a
variety of cytokines including VEGF, interleukin-1 (IL-1), placental-derived
growth factor (PDGF), IGF-1 and MCP-1.
It has also been shown that these factors were significantly upregulated
when the cells are cultured in a hypoxic state [114]. Transfusion of this media led to
increased capillary density, decreased infarct size, and improvement of cardiac
function following myocardial infarction [110, 112]. Whilst transfusion of the conditioned
media is potentially seen as a useful therapeutic agent, the need for the donor
cells to be transfused was still apparent as it was shown that some functional
reparative mechanisms only occurred when the bone marrow derived cells were
present. There is also a need for such a pool of cytokines to be constantly
replenished, a function that can be performed by constant transfusion of
conditioned media or a single transfusion of a cellular “cytokine factory”.


## 3.3. Vascular Remodeling

Interventions that
increase the perfusion to areas of restricted blood flow may cause salvage of
border-zone cardiomyocytes as well as partial reversal of pathological
myocardial remodeling. Increased vascularisation may occur via two pathways.
Firstly, it may occur by angiogenesis which is the formation of new branches of
blood vessels from pre-existing vessels and thus an increase in perfusion.
Secondly, it may occur by an increase in diameter of existing vessels causing
an increase in local perfusion [104]. The success of both
these events is determined by the pre-existence of a vascular network,
activation of the endothelium by fluid pressure stress, invasion of bone marrow
derived cells, and proliferation of endothelial and smooth muscle cells [115]. Cell therapy may
act in a paracrine mechanism to increase the production of proangiogenic
cytokines (as mentioned above) and stimulate host cells to remodel the existing
vasculature by either forming new branches or converting an arteriole into an
artery. Alternately, donor cells may physically embed in the ischaemic tissue
and become new vessel branches.Various studies have
shown revascularisation in animal models of hind-limb ischaemia and myocardial
infarction with EPCs [87, 116–118], HPCs [64, 66], BMCs [112, 119], and MSCs [120]. One such study by Kocher et al. [81]
showed that human mobilised CD34+ cells homed to the infarcted
myocardium where they were incorporated into newly developed coronary
circulation. However, similar to the differentiation process above, there is
limited evidence showing that the cells themselves are directly or physically
participating in this event [121]. This, once again, reinforces the
potential value of the paracrine function of infused cells as they are often
associated with an increase in capillary density in the infarcted region.


## 3.4. Cardiac Protection

Donor cell secretion
of numerous cytokines is believed to attenuate the apoptotic state. For
example, IGF-1 [122], hepatocyte growth
factor (HGF)  [123], and FGF [124] have all been shown to be
cardioprotective molecules by attenuation of cardiomyocyte apoptosis.Another form of cardiac
protection is by modification of the immune response and this has subsequently
been acknowledged as a potential therapeutic target [104]. This has been shown
with MSCs, which suppress T-cell responses and thus have local immunosuppressive
functionalities [32, 39]. However, cardiac protection through
these mechanisms must be viewed with caution as many proinflammatory and
proapoptotic molecules are also involved in angiogenic responses.


## 4. Conclusions

The field of
regenerative medicine is quickly evolving and has been applied to many aspects
of medicine including the treatment of CVD.
Cellular therapy has been proposed as a candidate treatment for CVD. However, a
majority of the clinical studies have used mixed populations of cells. Thus, it
is still unclear as to which cell population contributed to the repair of
cardiac muscle damaged by ischaemia in these trials. This review has outlined
the characteristics of some notable purified cell populations that can be used
to elucidate the possible roles and contributions of each cell type. Each cell
type has its own advantages and limitations towards application for the treatment
of CVD. This review has also
described the potential mechanisms by which these cells mediate improvement in
cardiac function following myocardial infarction. Possible mechanisms of cardiac
cellular myoplasty include the generation of proangiogenic proteins,
stimulation of new blood vessel formation, decrease in apoptosis of cardiac
cells, decrease in pathological cardiac remodeling, differentiation into
cardiomyocytes and cell fusion. Taking into consideration the various
mechanisms of repair and the characteristics of each cell population, a
cocktail of specific cell types may need to be considered for successful CVD cellular
therapy.



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*Source: 906507-2009-03-29.xml*