Monitoring Stem Cell Research
The President's Council on Bioethics
Washington, D.C.
January 2004
www.bioethics.gov
(The following commissioned paper was prepared
at the request of the President’s Council on Bioethics;
the Council has not itself verified the accuracy of the information
contained therein, nor does it necessarily endorse any of
the author's conclusions or opinions. Additionally, the Council
has not edited this paper either for style or content.)
Appendix M
Potential Use of Cellular Therapy
for Patients with Heart Disease
Silviu Itescu M.D.
Departments of Medicine and Surgery,
Columbia University, New York, NY
Summary
Congestive heart failure remains a major public health problem,
and is frequently the end result of cardiomyocyte apoptosis
and fibrous replacement after myocardial infarction, a process
referred to as left ventricular remodelling. Cardiomyocytes
undergo terminal differentiation soon after birth, and are
generally considered to irreversibly withdraw from the cell
cycle. In response to ischemic insult, adult cardiomyocytes
undergo cellular hypertrophy, nuclear ploidy, and a high degree
of apoptosis. A small number of human cardiomyocytes retain
the capacity to proliferate and regenerate in response to
ischemic injury, however whether these cells are derived from
a resident pool of cardiomyocyte stem cells or from a renewable
source of circulating bone marrow-derived stem cells that
home to the damaged myocardium is at present not known. Replacement
and regeneration of functional cardiac muscle after an ischemic
insult to the heart could be achieved by either stimulating
proliferation of endogenous mature cardiomyocytes or resident
cardiac stem cells, or by implanting exogenous donor-derived
or allogeneic cells such as fetal or embryonic cardiomyocyte
precursors, bone marrow-derived mesenchymal stem cells, or
skeletal myoblasts. The newly formed cardiomyocytes must
integrate precisely into the existing myocardial wall in order
to augment synchronized contractility and avoid potentially
life-threatening alterations in the electrical conduction
of the heart. A major impediment to survival of the implanted
cells is altered immunogenicity by prolonged ex vivo
culture conditions. In addition, concurrent myocardial revascularization
is required to ensure viability of the repaired region and
prevent further scar tissue formation. Human adult bone marrow
contains endothelial precursors which resemble embryonic angioblasts
and can be used to induce infarct bed neovascularization after
experimental myocardial infarction. This results in protection
of cardiomyocytes against apoptosis, induction of cardiomyocyte
proliferation and regeneration, long-term salvage and survival
of viable myocardium, prevention of left ventricular remodelling
and sustained improvement in cardiac function. It is reasonable
to anticipate that cell therapy strategies for ischemic heart
disease will need to incorporate (1) a renewable source of
proliferating, functional cardiomyocytes, and (2) angioblasts
to generate a network of capillaries and larger size blood
vessels for supply of oxygen and nutrients to both the chronically
ischemic endogenous myocardium and to the newly-implanted
cardiomyocytes.
Introduction
Congestive heart failure remains a major public health problem,
with recent estimates indicating that end-stage heart failure
with two-year mortality rates of 70-80% affects over 60,000
patients in the US each year1.
In Western societies heart failure is primarily the consequence
of previous myocardial infarction2.
As new modalities have emerged which have enabled significant
reduction in early mortality from acute myocardial infarction,
affecting over 1 million new patients in the US annually,
there has been a paradoxical increase in the incidence of
post-infarction heart failure among the survivors. Current
therapy of heart failure is limited to the treatment of already
established disease and is predominantly pharmacological in
nature, aiming primarily to inhibit the neurohormonal axis
that results in excessive cardiac activation through angiotensin-
or norepinephrine-dependent pathways. For patients with end-stage
heart failure treatment options are extremely limited, with
less than 3000 being offered cardiac transplants annually
due to the severely limited supply of donor organs3,4,
and implantable left ventricular assist devices (LVADs) being
expensive, not proven for long-term use, and associated with
significant complications5-7.
Clearly, development of approaches that prevent heart failure
after myocardial infarction would be preferable to those that
simply ameliorate or treat already established disease.
Heart Failure After Myocardial Infarction Results From
Progressive Ventricular Remodelling. Heart failure after
myocardial infarction occurs as a result of a process termed
myocardial remodelling. This process is characterized by myocyte
apoptosis, cardiomyocyte replacement by fibrous tissue deposition
in the ventricular wall8-10,
progressive expansion of the initial infarct area and dilation
of the left ventricular lumen11,12.
Another integral component of the remodelling process appears
to be the development of neoangiogenesis within the myocardial
infarct scar13,14,
a process requiring activation of latent collagenase and other
proteinases15.
Under normal circumstances, the contribution of neoangiogenesis
to the infarct bed capillary network is insufficient to keep
pace with the tissue growth required for contractile compensation
and is unable to support the greater demands of the hypertrophied,
but viable, myocardium. The relative lack of oxygen and nutrients
to the hypertrophied myocytes may be an important etiological
factor in the death of otherwise viable myocardium, resulting
in progressive infarct extension and fibrous replacement.
Since late reperfusion of the infarct vascular bed in both
humans and experimental animal models significantly benefits
ventricular remodelling and survival16-18,
we have postulated that methods to successfully augment vascular
bed neovascularization might improve cardiac function by preventing
loss of hypertrophied, but otherwise viable, cardiac myocytes.
Inability Of Damaged Myocardium To Undergo Repair Due
To Cell Cycle Arrest Of Adult Cardiomyocytes. Cardiomyocytes
undergo terminal differentiation soon after birth, and are
thought by most investigators to irreversibly withdraw from
the cell cycle. Analysis of cardiac myocyte growth during
early mammalian development indicates that cardiac myocyte
DNA synthesis occurs primarily in utero, with proliferating
cells decreasing from 33% at mid-gestation to 2% at birth19.
While ventricular karyokinesis and cytokinesis are coupled
during fetal growth, resulting in increases in mononucleated
cardiac myocytes, karyokinesis occurs in the absence of cytokinesis
for a transient period during the post-natal period, resulting
in binucleation of ventricular myocytes without an overall
increase in cell number. A similar dissociation between karyokinesis
and cytokinesis characterizes the primary adult mammalian
cardiac response to ischemia, resulting in myocyte hypertrophy
and increase in nuclear ploidy rather than myocyte hyperplasia20,21.
Moreover, in parallel with an inability to progress through
cell cycle, ischemic adult cardiomyocytes undergo a high degree
of apoptosis.
When cells proliferate, the mitotic cycle progression is
tightly regulated by an intricate network of positive and
negative signals. Progress from one phase of the cell cycle
to the next is controlled by the transduction of mitogenic
signals to cyclically expressed proteins known as cyclins
and subsequent activation or inactivation of several members
of a conserved family of serine/threonine protein kinases
known as the cyclin-dependent kinases (cdks)22.
Growth arrest observed with such diverse processes as DNA
damage, terminal differentiation, and replicative senescence
is due to negative regulation of cell cycle progression by
two functionally distinct families of Cdk inhibitors, the
Ink4 and Cip/Kip families19.
The cell cycle inhibitory activity of p21Cip1/WAF1 is intimately
correlated with its nuclear localization and participation
in quaternary complexes of cell cycle regulators by binding
to G1 cyclin-CDK through its N-terminal domain and to proliferating
cell nuclear antigen (PCNA) through its C-terminal domain
23-26.
The latter interaction blocks the ability of PCNA to activate
DNA polymerase, the principal replicative DNA polymerase27.
For a growth-arrested cell to subsequently enter an apoptotic
pathway requires signals provided by specific apoptotic stimuli
in concert with cell-cycle regulators. For example, caspase-mediated
cleavage of p21, together with upregulation of cyclin A-associated
cdk2 activity, have been shown to be critical steps for induction
of cellular apoptosis by either deprivation of growth factors28
or hypoxia of cardiomyocytes29.
Throughout life, a mixture of young and old cells is present
in the normal myocardium. Although most myocytes seem to be
terminally differentiated, there is a fraction of younger
myocytes (15-20%) that retains the capacity to replicate30.
Moreover, recent observations have suggested that some human
ventricular cardiomyocytes also have the capacity to proliferate
and regenerate in response to ischemic injury31,32.
The dividing myocytes can be identified on the basis of immunohistochemical
staining of proliferating nuclear structures such as Ki67
and cell surface expression of specific surface markers, including
c-kit (CD117). Whether these cells are derived from a resident
pool of cardiomyocyte stem cells or are derived from a renewable
source of circulating bone marrow-derived stem cells that
home to the damaged myocardium remains to be determined.
More importantly, the signals required for homing, in situ
expansion and differentiation of these cells are, at present,
unknown. Gaining an understanding of these issues would open
the possibility of manipulating the biology of endogenous
cardiomyocytes in order to augment the healing process after
myocardial ischemia.
Strategies For The Use Of Cellular Therapy To Improve
Myocardial Function. Replacement and regeneration of functional
cardiac muscle after an ischemic insult to the heart could
be achieved by either stimulating proliferation of endogenous
mature cardiomyocytes or resident cardiac stem cells, or by
implanting exogenous donor-derived or allogeneic cardiomyocytes.
The newly formed cardiomyocytes must integrate precisely into
the existing myocardial wall in order to augment contractile
function of the residual myocardium in a synchronized manner
and avoid alterations in the electrical conduction and syncytial
contraction of the heart, potentially resulting in life-threatening
consequences. In addition, whatever the source of the cells
used, it is likely that concurrent myocardial revascularization
must also occur in order to ensure viability of the repaired
region and prevent further scar tissue formation. The following
section discusses various methods of using cellular therapies
to replace damaged myocardium or re-initiate mitosis in mature
endogenous cardiomyocytes, including transplanted bone marrow-derived
cardiomyocyte or endothelial precursors, fetal cardiomyocytes,
and skeletal myoblasts.
Potential Role For Bone Marrow-Derived Or Embryonic Cardiomyocyte
Lineage Stem Cells In Myocardial Repair/Regeneration.
Over the past several years, a number of studies have suggested
that stem cells can be used to generate cardiomyocytes ex
vivo for potential use in a range of cardiovascular diseases33-37.
Multipotent bone marrow-derived mesenchymal stem cells have
been identified in adult murine and human bone marrow functionally
by their ability to differentiate to lineages of diverse mesenchymal
tissues, including bone, cartilage, fat, tendon, and both
skeletal and cardiac muscle36,
and phenotypically by their expression of specific surface
markers and lack of hematopoietic lineage markers such as
CD34 or CD4535.
It is well established that murine embryonic stem (ES) cells
can give rise to cardiomyocytes in vitro and in vivo38,39.
Recently, Kehat et al. were able to demonstrate that human
embryonic stem cells can also differentiate in vitro
into cells with characteristics of cardiomyocytes37.
However, there are striking differences in the human and murine
stem cell models, and this needs to be taken into account
when extrapolating results of mouse experiments to the human
condition. For example, human ES cells have a very low efficiency
of differentiation to cardiomyocytes compared with murine
ES cells, and a considerably slower time course (a median
of 11 days vs 2 days). Whether these differences reflect
true variations between species, or differences in the experimental
protocols, remains to be determined.
Irrespective whether the cardiomyocyte lineage stem cell
precursors are obtained from adult bone marrow or embryonic
sources, the newly generated cardiomyocytes appear to resemble
normal cardiomyocytes in terms of phenotypic properties, such
as expression of actinin, desmin and troponin I, and function,
including positive and negative chronotropic regulation of
contractility by pharmacological agents and production of
vasoactive factors such as atrial and brain natriuretic peptides.
However, in vivo evidence for functional cardiac improvement
following transplantation of adult bone marrow-derived or
ES-derived cardiomyocytes has been exceedingly difficult to
show to date. In part this may be because the signals required
for cardiomyocyte differentiation and functional regulation
are complex and poorly understood. For example, phenotypic
and functional differentiation of mesenchymal stem cells to
cardiomyocyte lineage cells in vitro requires culture
with exogenously added 5-azacytidine33,34.
Alternatively, the poor functional data obtained to date may
reflect immune-mediated rejection of cells which have been
modified during the ex vivo culture process or poor
viability due to the lack of a sufficient vascular supply
to the engrafted cells (see below).
Potential Role For Autologous Skeletal Myoblasts In Myocardial
Repair. An alternative approach to replacing damaged myocardium
involves the use of autologous skeletal myoblasts40.
The procedure involves harvesting a patient's skeletal
muscle cells, expanding the cells in a laboratory, and re-injecting
the cells into the patient's heart. Perceived advantages
of the approach include ease of access to the cellular source,
the fact that immunosuppression is not needed, and the lack
of ethical dilemmas associated with the use of allogeneic
or embryonic cells. It has also been argued that using relatively
ischemia-resistant skeletal myoblasts rather than cardiomyocytes
might enable higher levels of cell engraftment and survival
in infarcted regions of the heart, where cardiomyocytes would
probably perish41.
Successful engraftment of autologous skeletal myoblasts
into injured myocardium has been reported in multiple animal
models of cardiac injury. These studies have demonstrated
survival and engraftment of myoblasts into infarcted or necrotic
hearts40,
differentiation of the myoblasts into striated cells within
the damaged myocardium40,
and improved myocardial functional performance40,42,43.
Other studies have shown that the survival of transplanted
myoblasts can be improved by heat shock pretreatment44,
and have confirmed that the benefits of skeletal myoblast
transfer are additive with those of conventional therapies,
such as angiotensin-converting enzyme inhibition45.
More recently, the procedure has been reported anecdotally
to result in improved myocardial function in humans46.
On the basis of these preliminary results, clinical trials
have begun both in Europe and in the United States. In addition
to demonstrating functional improvement in large, prospective
series, questions that remain to be addressed include whether
the skeletal myoblasts can make meaningful electromechanical
connections to the surrounding endogenous cardiomyocytes through
gap junctions, whether the cellular mass will contract in
concert, and whether electrical impulses will be transmitted
to the myoblast tissue without inducing significant tachyarrhythmias.
Poor Survival Of Cells Transplanted Into Damaged
Myocardium After Ex Vivo Culture. A major
limitation to successful cellular therapy in animal models
of myocardial damage has been the inability of the introduced
donor cells to survive in their host environment, whether
such transplants have been congenic (analogous to the autologous
scenario in humans) or allogeneic. It has become clear that
a major impediment to survival of the implanted cells is the
alteration of their immunogenic character by prolonged ex
vivo culture conditions. For example, whereas myocardial
implantation of skeletal muscle in the absence of tissue culture
does not induce any adverse immune response and results in
grafts showing excellent survival for up to a year, injection
of cultured isolated (congenic) myoblasts results in a massive
and rapid necrosis of donor myoblasts, with over 90% dead
within the first hour after injection47-50.
This rapid myoblast death appears to be mediated by host natural
killer (NK) cells50
which respond to immunogenic antigens on the transplanted
myoblasts altered by exposure to tissue culture conditions48.
It seems likely that a similar mechanism of host NK cell-mediated
rejection will apply also to transplanted ES-derived, cultured
cardiomyocytes51
since massive death of injected donor cells is recognized
as a major problem with transplanted cardiomyocytes, especially
in the inflammatory conditions that follow infarction52.
In this regard, the report that cultured mesenchymal stem
cells obtained from adult human bone marrow are not rejected
on transfer to other species is intriguing53,
needs confirmation in humans, and requires detailed investigation
into possible tolerogenic mechanisms.
Concomitant Induction Of Vascular Structures Augments
Survival and Function Of Cardiomyocyte Precursors.
An additional explanation for the poor survival of transplanted
cardiomyocytes or skeletal myoblasts may be that viability
and prolonged function of transplanted cells requires an augmented
vascular supply. Whereas many transplanted cardiomyocytes
die by apoptosis, cultured cardiomyocytes that incorporate
more vascular structures in vivo demonstrate significantly
greater survival52.
Moreover, in situations where transplanted cardiomyocyte precursors
contained an admixture of cells also giving rise to vascular
structures, survival and function of the newly formed cardiomyocytes
has been significantly augmented.
In one study, direct injection of whole rat bone marrow
into a cryo-damaged heart resulted in neovascularization,
cardiomyocyte regeneration and functional improvement34.
More recently, systemic delivery of highly purified bone marrow-derived
hematopoietic stem cells in lethally irradiated mice contributed
to the formation of both endothelial cells and long-lived
cardiomyocytes in ischemic hearts 53.
Most strikingly, significant improvement in cardiac function
of mice who had previously undergone LAD ligation was demonstrated
after direct myocardial injection of syngeneic bone marrow-derived
stem cells, defined on the basis of c-kit (CD117) expression54.
This population of cells contains a mixture of cellular elements
in addition to cardiomyocyte precursors, including CD117-positive
endothelial progenitors (see below). These cells were found
to proliferate and differentiate into myocytes, smooth muscle
cells and endothelial cells, resulting in the partial regeneration
of the destroyed myocardium and prevention of ventricular
scarring. Together, these findings raise the intriguing possibility
that for long-term in vivo viability and functional
integrity of stem cell-derived cardiomyocytes it may be necessary
to induce neovascularization by co-administration of endothelial
cell progenitors (see below).
Endothelial Precursors And Formation Of Vascular
Structures During Embryogenesis. In order to develop
successful methods for inducing neovascularization of the
adult heart, one needs to understand the process of definitive
vascular network formation during embryogenesis. In the pre-natal
period, hemangioblasts derived from the human ventral aorta
give rise to cellular elements involved in both vasculogenesis,
or formation of the primitive capillary network, and hematopoiesis
55,56.
In addition to hematopoietic lineage markers, embryonic hemangioblasts
are characterized by expression of the vascular endothelial
cell growth factor receptor-2, VEGFR-2, and have high proliferative
potential with blast colony formation in response to VEGF57-60.
Under the regulatory influence of various transcriptional
and differentiation factors, embryonic hemangioblasts mature,
migrate and differentiate to become endothelial lining cells
and create the primitive vasculogenic network. The differentiation
of embryonic hemangioblasts to pluripotent stem cells and
to endothelial precursors appears to be related to co-expression
of the GATA-2 transcription factor, since GATA-2 knockout
embryonic stem cells have a complete block in definitive hematopoiesis
and seeding of the fetal liver and bone marrow61.
Moreover, the earliest precursor of both hematopoietic and
endothelial cell lineage to have diverged from embryonic ventral
endothelium has been shown to express VEGF receptors as well
as GATA-2 and alpha4-integrins62.
Subsequent to capillary tube formation, the newly-created
vasculogenic vessels undergo sprouting, tapering, remodelling,
and regression under the direction of VEGF, angiopoietins,
and other factors, a process termed angiogenesis. The final
component required for definitive vascular network formation
to sustain embryonic organogenesis is influx of mesenchymal
lineage cells to form the vascular supporting structures such
as smooth muscle cells and pericytes.
Characterization Of Endothelial Progenitors, Or
Angioblasts, In Human Adult Bone Marrow. In studies
using various animal models of peripheral ischemia a number
of groups have shown the potential of adult bone marrow-derived
elements to induce neovascularization of ischemic tissues63-69.
In the most successful of these63,
bone marrow-derived cells injected directly into the thighs
of rats who had undergone ligation of the left femoral artery
and vein induced neovascularization and augmented blood flow
in the ischemic limb as documented by laser doppler and immunohistochemical
analyses. Although the nature of the bone marrow-derived
endothelial progenitors was not precisely identified in these
studies, the cumulative reports indicated that this site may
be an important source of endothelial progenitors which could
be useful for augmenting collateral vessel growth in ischemic
tissues, a process termed therapeutic angiogenesis.
In more recent studies, our group has identified such endothelial
progenitors in human adult bone marrow70.
By employing both in vitro and in vivo experimental
models we have sought to precisely identify the surface characteristics
and biological properties of these bone marrow-derived endothelial
progenitor cells. Following G-CSF treatment, mobilized mononuclear
cells were harvested and CD34+ cells were separated using
anti-CD34 mAb coupled to magnetic beads. 90-95% of CD34+
cells co-expressed the hematopoietic lineage marker CD45,
60-80% co-expressed the stem cell factor receptor CD117, and
<1% co-expressed the monocyte/macrophage lineage marker
CD14. By quadruple parameter analysis, the VEGFR-2 positive
cells within the CD34+CD117bright subset displayed
phenotypic characteristics of endothelial progenitors, including
co-expression of Tie-2, as well as AC133, but not markers
of mature endothelium such as ecNOS, vWF, E-selectin, and
ICAM. Sorting CD34+ cells on the basis of CD117 bright or
dim expression demonstrated that GATA-2 mRNA and protein levels
were significaantly higher in the CD117bright population,
indicating that human adult bone marrow contains cells with
an angioblast-like phenotype.
Since the frequency of circulating endothelial cell precursors
in animal models has been shown to be increased by either
VEGF71
or regional ischemia63-66,
phenotypically-defined angioblasts were examined for proliferative
responses to VEGF and to factors in ischemic serum. CD117brightGATA-2hiangioblasts
demonstrated significantly higher proliferative responses
relative to CD117dimGATA-2lo bone marrow-derived
cells from the same donor following culture for 96 hours with
either VEGF or ischemic serum. The expanded angioblast population
consisted of large blast cells, defined by forward scatter,
which continued to express immature markers, including GATA-2
and CD117bright, but not markers of mature endothelial
cells, including eNOS or E-selectin, indicating blast proliferation
without differentiation under these culture conditions. However,
culture on fibronectin with endothelial growth medium resulted
in outgrowth of monolayers with endothelial morphology and
functional and phenotypic features characteristic of endothelial
cells, including uniform uptake of acetylated LDL, and co-expression
of CD34, factor VIII, and eNOS. Thus, G-CSF treatment of
adult humans mobilizes into the peripheral circulation a bone-marrow
derived population with phenotypic and functional characteristics
of embryonic angioblasts, as defined by specific surface phenotype,
high proliferative responses to VEGF and cytokines in ischemic
serum, and ability differentiate into endothelial cells by
culture in medium enriched with endothelial growth factors.
Human Angioblasts Induce Neovascularization Of The
Myocardial Infarct Zone. Intravenous injection of
freshly-obtained human angioblasts into athymic nude rats
who had undergone ligation of the left anterior descending
(LAD) coronary artery resulted in infarct zone infiltration
within 48 hours. Few human cells were detected in unaffected
areas of hearts with regional infarcts or in myocardium of
sham-operated rats. Histologic examination at two weeks post-infarction
revealed that injection of human angioblasts was accompanied
by a significant increase in infarct zone microvascularity,
cellularity, and numbers of capillaries, and by reduction
in matrix deposition and fibrosis in comparison to controls.
Neovascularization was significantly increased within both
the infarct zone and in the peri-infarct rim in rats receiving
angioblasts compared with controls receiving saline or other
cells which infiltrated the heart (e.g. CD34- cells or saphenous
vein endothelial cells, SVEC). The neovascularization induced
by human angioblasts was due to both an increase in capillaries
of human origin as well as of rat origin, as defined by monoclonal
antibodies with specificity for human or rat CD31 endothelial
markers. Capillaries of human origin, defined by co-expression
of DiI fluorescence and human CD31, but not rat CD31, accounted
for 20-25% of the total myocardial capillary vasculature,
and was located exclusively within the central infarct zone
of collagen deposition. In contrast, capillaries of rat origin,
as determined by expression of rat, but not human, CD31, demonstrated
a distinctively different pattern of localization, being absent
within the central zone of collagen deposition and abundant
both at the peri-infarct rim between the region of collagen
deposition and myocytes, and between myocytes.
Human Angioblasts Protect Hypertrophied Endogenous
Cardiomyocytes Against Apoptosis. By concomitantly
staining rat tissues for the myocyte-specific marker desmin
and performing DNA end-labeling using the TUNEL technique,
temporal examinations demonstrated that the infarct zone neovascularization
induced by injection of human angioblasts prevented an eccentrically-extending
pro-apoptotic process evident in saline controls. Thus, at
two weeks post-infarction, myocardial tissue of LAD-ligated
rats who received saline demonstrated 6-fold higher numbers
of apoptotic myocytes compared with that from rats receiving
intravenous injections of human angioblasts. Moreover, these
myocytes had distorted appearance and irregular shape. In
contrast, myocytes from LAD-ligated rats who received human
angioblasts had regular, oval shape, and were significantly
larger than myocytes from control rats.
Human Angioblasts Induce Sustained Regeneration/
Proliferation Of Endogenous Cardiomyocytes. In addition
to protection of hypertrophied myocytes against apoptosis,
human angioblast-dependent neovascularization resulted in
a striking induction of regeneration/proliferation of endogenous
rat cardiomyocytes at the peri-infarct rim72.
At two weeks after LAD ligation, rats receiving human angioblasts
demonstrated numerous "fingers" of cardiomyocytes of rat origin,
as determined by expression of rat MHC class I molecules,
extending from the peri-infarct region into the infarct zone.
The islands of cardiomyocytes at the peri-infarct rim in animals
receiving human angioblasts contained a high frequency of
rat myocytes with DNA activity, as determined by dual staining
with mAbs reactive against cardiomyocyte-specific troponin
I and rat Ki67. In contrast, in animals receiving saline
there was a high frequency of cells with fibroblast morphology
and reactivity with rat Ki67, but not troponin I, within the
infarct zone. The number of cardiomyocytes progressing through
cell cycle at the peri-infarct region of rats receiving human
angioblasts was 40-fold higher than that at sites distal to
the infarct, 20-fold higher than that found in non-infarcted
hearts, and 5-fold higher than that at the peri-infarct rim
of animals receiving saline72.
Neovascularization Of Acutely Ischemic Myocardium
By Human Angioblasts Prevents Ventricular Remodelling And
Causes Sustained Improvement In Cardiac Function.
By 15 weeks post-infarction, rats receiving human angioblasts
demonstrated markedly smaller scar sizes together with increased
mass of viable myocardium within the anterior free wall.
Whereas collagen deposition and scar formation extended almost
through the entire left ventricular wall thickness in controls,
with aneurysmal dilatation and typical EKG abnormalities,
the infarct scar extended only to 20-50% of the left ventricular
wall thickness in rats receiving CD34+ cells. Moreover, pathological
collagen deposition in the non-infarct zone was markedly reduced
in rats receiving CD34+ cells. At 15 weeks, the mean proportion
of scar/normal left ventricular myocardium was 13% in rats
receiving CD34+ cells compared with 36-45% for each of the
other groups studied (saline, CD34-, SVEC).
Remarkably, by two weeks after injection of human angioblasts,
and in a parallel time-frame with the observed neo-vascularization,
left ventricular ejection fraction (LVEF) recovered by a mean
of 22%. This effect was long-lived, with LVEF recovering
by a mean of 34% at the end of follow-up, 15 weeks post injection.
Neither CD34- cells nor SVEC demonstrated similar effects.
At 15 weeks post-infarction, mean cardiac index in rats injected
with human angioblasts was only reduced by 26% relative to
normal rats, whereas for each of the other groups it was reduced
by 48-59%. Together, these results indicate that the neovascularization,
reduction in peri-infarct myocyte apoptosis and increase in
cardiomyocyte regeneration/proliferation observed at two weeks
prevented myocardial replacement with fibrous tissue and caused
sustained improvement in myocardial function.
Potential Use Of Angioblasts In Combination With
Cardiomyocyte Progenitors For Repair and Regeneration of Ischemic
Myocardium. While increasing capillary density through
angioblast-dependent neovascularization is a promising approach
for preventing apoptotic death and inducing regeneration of
endogenous cardiomyocytes following acute myocardial infarction,
the role of angioblast therapy for the treatment of congestive
heart failure following chronic ischemia is at present unknown.
Nevertheless, it is reasonable to anticipate that cellular
therapies for congestive heart failure due to ischemic cardiomyopathy
will need to address two interdependent processes: (1) a renewable
source of proliferating, functional cardiomyocytes, and (2)
development of a network of capillaries and larger size blood
vessels for supply of oxygen and nutrients to both the chronically
ischemic, endogenous myocardium and to the newly-implanted
cardiomyocytes. To achieve these endpoints it is likely that
co-administration of angioblasts and mesenchymal stem cells
will be needed in order to develop regenerating cardiomyocytes,
vascular structures, and supporting cells such as pericytes
and smooth muscle cells. Future studies will need to address
the timing, relative concentrations, source and route of delivery
of each of these cellular populations in animal models of
acute and chronic myocardial ischemia.
In addition to synergistic cellular therapies, it is likely
that optimal regimens for the treatment of acute and chronically
ischemic hearts will require a combined approach employing
additional pharmacological strategies. For example, augmentation
in myocardial function might be achieved by combining infusion
of human angioblasts and cardiomyocyte progenitors together
with beta blockade, ACE inhibition or AT1-receptor
blockade to reduce angiotensin II-dependent cardiac fibroblast
proliferation and collagen secretion73-76.
Understanding the potential of defined lineages of stem cells
or undifferentiated progenitors, and their interactions with
pharmacological interventions, will lead to better and more
focussed clinical trial designs using each cell type independently
or in combination, depending on which particular clinical
indication is being targeted.
_________________
ENDNOTES/References
- Effects of enalapril on mortality in severe
congestive heart failure. Results of the Cooperative North
Scandinavian Enalapril Survival Study (CONSENSUS) Trial
Study Group. N Engl J Med 1987; 316(23):1429-1435.
- Mahon NG, O'Roke C, Codd MB, et al. Hospital
mortality of acute myocardial infarction in the thrombolytic
era. Heart 1999;81:478-82.
- Hognes JR. In The Artificial Heart: Prototypes,
Policies and Patients. Washingtion, DC: National Academy
Press; 1991:1-312.
- Annual Report of the US Scientific Registry
for Organ Transplantation and the Organ Procurement and
Transplantation Network - 1990. Washingtion, DC: US Department
of Health and Human Services: 1990.
- Frazier OH, Rose EA, Macmanus Q, et al.
Multicenter clinical evaluation of the Heartmate 1000 IP
left ventricular assist device. Ann Thorac Surg 1992; 102:578-587.
- McCarthy PM, Rose EA, Macmanus Q, et al.
Clinical experience with the Novacor ventricular assist
system. J Thorac Cardiovasc Surg. 1991; 102:578-587.
- Oz MC, Argenziano M, Catanese KA, et al.
Bridge experience with long-term implantable left ventricular
assist devices. Are they an alternative to transplantation?
Circulation. 1997; 95:1844-1852.
- Colucci WS. Molecular and cellular mechanisms
of myocardial failure. Am J Cardiol 1997;80:15L-25L.
- Ravichandran LV and Puvanakrishnan R.
In vivo labeling studies on the biosynthesis and degradation
of collagen in experimental myocardial myocardial infarction.
Biochem Intl 1991;24:405-414.
- Agocha A, Lee H-W, Eghali-Webb M. Hypoxia
regulates basal and induced DNA synthesis and collagen type
I production in human cardiac fibroblasts: effects of TGF-beta,
thyroid hormone, angiotensis II and basic fibroblast growth
factor. J Mol Cell Cardiol 1997;29:2233-2244H.
- Pfeffer JM, Pfeffer MA, Fletcher PJ, Braunwald
E. Progressive ventricular remodeling in rat with myocardial
infarction. Am J Physiol 1991; 260:H1406-14.
- White HD, Norris RM, Brown MA, Brandt
PWT, Whitlock RML, Wild CJ. Left ventricular end systolic
volume as the major determinant of survival after recovery
from myocardial infarction. Circulation 1987; 76:44-51.
- Nelissen-Vrancken H, Debets J, Snoeckx
L, Daemen M, Smits J. Time-related normalization of maximal
coronary flow in isolated perfused hearts of rats with myocardial
infarction. Circulation 1996;93:349-355.
- Kalkman EAJ, Bilgin YM, van Haren P, van
Suylen R-J, Saxena PR, Schoemaker RG.Determinants of coronary
reserve in rats subjected to coronary artery ligation or
aortic banding. Cardiovasc Res 1996.
- Heymans S, Luutun A, Nuyens D, et al.
Inhibition of plasminogen activators or matrix metalloproteinases
prevents cardiac rupture but impairs therapeutic angiogenesis
and causes cardiac failure. Nat Med 1999;5:1135-1142.
- Hochman JS and Choo H. Limitation of
myocardial infarct expansion by reperfusion independent
of myocardial salvage. Circulation 1987;75:299-306.
- White HD, Cross DB, Elliot JM, et al.
Long-term prognostic importance of patency of the infarct-related
coronary artery after thrombolytic therapy for myocardial
infarction. Circulation 1994;89:61-67.
- Nidorf SM, Siu SC, Galambos G, Weyman
AE, Picard MH. Benefit of late coronary reperfusion on ventricular
morphology and function after myocardial infarction. J Am
Coll Cardiol 1992;20:307-313.
- MacLellan WR and Schneider MD. Genetic
dissection of cardiac growth control pathways Annu. Rev.
Physiol. 2000. 62:289-320.
- Soonpaa MH, Field LJ. Assessment of cardiomyocyte
DNA synthesis in normal and injured adult mouse hearts.
Am J Physiol 272, H220-6 (1997).
- Kellerman S, Moore JA, Zierhut W, Zimmer
HG, Campbell J, Gerdes AM. Nuclear DNA content and nucleation
patterns in rat cardiac myocytes from different models of
cardiac hypertrophy. J Mol Cell Cardiol 24, 497-505
(1992).
- Hill MF, Singal PK. Right and left myocardial
antioxidant responses during heart failure subsequent to
myocardial infarction. Circulation 1997 96:2414-20.
- Li, Y., Jenkins, C.W. , Nichols, M.A.
and Xiong, Y. (1994) Cell cycle expression and p53 regulation
of the cyclin-dependent kinase inhibitor p21. Oncogene,
9, 2261-2268.
- Steinman, R.A. , Hoffman, B. , Iro, A.
, Guillouf, C. , Liebermann, D.A. and El-Houseini, M.E.
(1994) Induction of p21 (WAF1/CIP1) during differentiation.
Oncogene, 9, 3389-3396.
- Halevy, O. , Novitch, B.G. , Spicer, D.B.
, Skapek, S.X. , Rhee, J. , Hannon, G.J. , Beach, D. and
Lassar, A.B. (1995) Correlation of terminal cell cycle arrest
of skeletal muscle with induction of p21 by MyoD. Science,
267, 1018-1021.
- Andres, V. and Walsh, K. (1996) Myogenin
expression, cell cycle withdrawal and phenotypic differentiation
are temporally separable events that precedes cell fusion
upon myogenesis. J. Cell Biol., 132, 657-666.
- Tsurimoto, T. PCNA Binding Proteins.
Frontiers in Bioscience, 4:849-858, 1999.
- Levkau B, Koyama H, Raines EW, Clurman
BE, Herren B, Orth K, Roberts JM, Ross R. Cleavage of p21cip1/waf1
and p27 kip1 mediates apoptosis in endothelial cells through
activation of cdk2: role of a caspase cascade. Mol Cell.
1998;1:553-563.
- Adachi S, et al. Cyclin A/cdk2 activation
is involved in hypoxia-induced apoptosis in cardiomyocytes
Circ Res. 88:408, 2001.
- Anversa P, and Nadal-Ginard B. Myocyte
renewal and ventricular remodelling Nature 415, 240 - 243,
2002.
- Kajstura J, Leri A, Finato N, di Loreto
N, Beltramo CA, Anversa P. Myocyte proliferation in end-stage
cardiac failure in humans. Proc Natl Acad Sci USA 95, 8801-8805
(1998).
- Beltrami AP, et al. Evidence that human
cardiac myocytes divide after myocardial infarction. N
Engl J Med 344, 1750-7 (2001).
- Makino S, Fukuda K, Miyoshi S, et al.
Cardiomyocytes can be generated from marrow stromal cells
in vitro. J Clin Invest 1999, 103:697-705.
- Tomita S, Li R-K, Weisel RD, et al. Autologous
transplantation of bone marrow cells improves damaged heart
function. Circulation 1999; 100:II-247.
- Pittenger MF, Mackay AM, Beck SC, et al.
Multilineage potential of adult human mesenchymal stem
cells. Science 1999, 284:143-147.
- Liechty KW, MacKenzie TC, Shaaban AF,
et al. Human mesenchymal stem cells engraft and demonstrate
site-specific differentiation after in utero transplantation
in sheep. Nat Med 2000, 6:1282-6.
- Kehat I, Kenyagin-Karsenti D, Snir M,
Segev H, Amit M, Gepstein A, Livne E, Binah O, Itskovitz-Eldor
J, Gepstein L. Human embryonic stem cells can differentiate
into myocytes with structural and functional properties
of cardiomyocytes. J Clin Invest. 2001; 108: 407-14.
- Klug MG, Soonpaa MH, Koh GY, Field LJ
(1996) Genetically selected cardiomyocytes from differentiating
embryonic stem cells form stable intracardiac grafts. J
Clin Invest 98:216-224.
- Hescheler J, Fleischmann BK, Wartenberg
M, Bloch W, Kolossov E, Ji G, Addicks K, Sauer H (1999)
Establishment of ionic channels and signalling cascades
in the embryonic stem cell-derived primitive endoderm and
cardiovascular system. Cells Tissues Organs 165:153-164.
- DA Taylor, BZ Atkins, P Hungspreugs, TR
Jones, MC Reedy, KA Hutcheson, DD Glower, WE. Regenerating
functional myocardium: improved performance after skeletal
myoblast transplantation. Nature Medicine 1998, 4: 929-933.
- L Field: Future therapy for cardiovascular
disease. In Proceedings of the NHLBI Workshop Cell Transplantation:
Future Therapy for Cardiovascular Disease? Columbia, MD;
1998.
- CE Murry, RW Wiseman, SM Schwartz, SD
Hauschka: Skeletal myoblast transplantation for repair of
myocardial necrosis. J Clin Invest 1996, 98: 2512-2523.
- BZ Atkins, MT Hueman, JM Meuchel, MJ Cottman,
KA Hutcheson, DA Taylor: Myogenic cell transplantation improves
in vivo regional performance in infarcted rabbit myocardium.
J Heart Lung Transpl 1999, 18: 1173-1180.
- Suzuki K, Smolenski RT, Jayakumar J, Murtuza
B, Brand NJ, Yacoub MH (2000) Heat shock treatment enhances
graft cell survival in skeletal myoblast transplantation
to the heart. Circulation 102:III216-221.
- Pouzet B, Ghostine S, Vilquin J-T, Garcin
I, Scorsin M, Hagege AA, Duboc D, Schwartz K, Menasche P
(2001) Is skeletal myoblast transplantation clinically relevant
in the era of angiotensin-converting enzyme inhibitors?
Circulation 104:I223-228.
- Menasche P, Hagege AA, Scorsin M, Pouzet
B, Desnos M, Duboc D, Schwartz K, Vilquin J-T, Marolleau
J-P (2001) Myoblast transplantation for heart failure. Lancet
347:279.
- Beauchamp JR, Morgan JE, Pagel CN, Partridge
TA (1999) Dynamics of myoblast transplantation reveal a
discrete minority of precursors with stem cell-like properties
as the myogenic source. J Cell Biol 144:1113-1122.
- Smythe GM, Grounds MD (2000) Exposure
to tissue culture conditions can adversely affect myoblast
behaviour in vivo in whole muscle grafts: implications for
myoblast transfer therapy. Cell Transplant 9:379-393.
- Smythe GM, Hodgetts SI, Grounds MD (2001)
Problems and solutions in myoblast transfer therapy. J Cell
Mol Med 5:33-47.
- Hodgetts SI, Beilharz MW, Scalzo T, Grounds
MD (2000) Why do cultured transplanted myoblasts die in
vivo? DNA quantification shows enhanced survival of donor
male myoblasts in host mice depleted of CD4+ and CD8+ or
NK1.1+ cells. Cell Transplant 9:489-502.
- Maier S, Tertilt C, Chambron N, Gerauer
K, Huser N, Heidecke C-D, Pfeffer K (2001) Inhibition of
natural killer cells results in acceptance of cardiac allografts
in CD28-/- mice. Nature Med 7:557-562.
- Zhang M, Methot D, Poppa V, Fujio Y, Walsh
K, Murry CE (2001) Cardiomyocyte grafting for cardiac repair:
graft cell death and anti-death strategies. J Mol Cell Cardiol
33:907-921.
- Jackson KA, Majka SM, Wang H, Pocius J,
Hartley CJ, Majesky MW, Entman ML, Michael LH, Hirschi KK,
Goodell MA (2001) Regeneration of ischemic cardiac muscle
and vascular endothelium by adult stem cells. J Clin Invest
107:1395-1402.
- Orlic D, Kajstura J, Chimenti S, et al.
Bone marrow cells regenerate infarcted myocardium. Nature
2001, 410:701-705.
- Tavian M, Coulombel L, Luton D, San Clemente
H, Dieterlen-Lievre F, Peault B. Aorta-associated CD34
hematopoietic cells in the early human embryo. Blood 1996;87:67-72.
- Jaffredo T, Gautier R, Eichmann A, Dieterlen-Lievre
F. Intraaortic hemopoietic cells are derived from endothelial
cells during ontogeny. Development 1998;125:4575-4583.
- Kennedy M, Firpo M, Choi K, Wall C, Robertson
S, Kabrun N, Keller G. A common precursor for primitive
erythropoiesis and definitive haematopoiesis. Nature 1997;386:488-493.
- Choi K, Kennedy M, Kazarov A, Papadimitriou,
Keller G. A common precursor for hematopoietic and endothelial
cells. Development 1998;125:725-732.
- Elefanty AG, Robb L, Birner R, Begley
CG. Hematopoietic-specific genes are not induced during
in vitro differentiation of scl-null embryonic stem cells.
Blood 1997;90:1435-1447.
- Labastie M-C, Cortes F, Romeo P-H, Dulac
C, Peault B. Molecular identity of hematopoietic precursor
cells emerging in the human embryo. Blood 1998;92:3624-3635.
- Tsai FY, Keller G, Kuo FC, Weiss M, Chen
J, Rosenblatt M, Alt FA, Orkin SH. An early hematopoietic
defect in mice lacking the transcription factor GATA-2.
Nature 1994;371:221-225.
- Ogawa M, Kizumoto M, Nishikawa S, Fujimoto
T, Kodama H, Nishikawa SI. Blood 1999;93:1168-1177.
- Asahara, T. et al. Isolation of putative
progenitor cells for endothelial angiogenesis. Science 1997;
275:964-967.
- Folkman, J. Therapeutic angiogenesis in
ischemic limbs. Circulation 1998; 97:108-110.
- Takahashi, T. et al. Ischemia- and cytokine-induced
mobilization of bone marrow-derived endothelial progenitor
cells for neovascularization. Nat. Med. 1999; 5:434-438.
- Kalka, C. et al. Transplantation of ex
vivo expanded endothelial progenitor cells for therapeutic
neovascularization. Proc. Natl. Acad. Sci. USA 2000; 97:3422-3427.
- Rafii S, Shapiro F, Rimarachin J, Nachman
R, Ferris B, Weksler B, Moore AS, Asch AS. Isolation and
characterization of human bone marrow microvascular endothelial
cells: hematopoietic progenitor cell adhesion. Blood 1994;84:10-19.
- Shi Q, Rafii S, Wu MH-D, et al. Evidence
for circulating bone marrow-derived endothelial cells. Blood
1998;92:362-367.
- Lin Y, Weisdorf DJ, Solovey A, Hebbel
RP. Origins of circulating endothelial cells and endothelial
outgrowth from blood. J Clin Invest 2000;105:71-77.
- Kocher AA, Schuster MD, Szabolcs MJ, Takuma
S, Burkhoff D, Wang J, Homma S, Edwards NM, Itescu S. Neovascularization
of ischemic myocardium by human bone-marrow-derived angioblasts
prevents cardiomyocyte apoptosis, reduces remodeling and
improves cardiac function. Nature Med. 2001; 7: 430-6.
- Asahara T, Takahashi T, Masuda H, Kalka
C, Chen D, Iwaguro H, Inai Y, Silver M, Isner JM. VEGF contributes
to postnatal neovascularization by mobilizing bone marrow-derived
endothelial progenitor cells. EMBO J 1999; 18:3964-3972.
- Kocher A, Schuster M, Szabolcs M, Itescu
S. Cardiomyocyte regeneration after neovascularization
of ischemic myocardium by human bone marrow-derived angioblasts.
Nature Medicine 2002, in press.
- McEwan PE, Gray GA, Sherry L, Webb DJ,
Kenyon CJ. Differential effects of angiotensin II on cardiac
cell proliferation and intramyocardial perivascular fibrosis
in vivo. Circulation 1998;98:2765-2773.
- Kawano H, Do YS, Kawano Y, Starnes V,
Barr M, Law RE, Hsueh WA. Angiotensin II has multiple profibrotic
effects in human cardiac fibroblasts. Circulation 2000;101:1130-1137.
- Pfeffer MA, Braunwald E, Moye LA, et al.
Effect of captopril on mortality and morbidity in patients
with left ventricular dysfunction after myocardial infarction.
Results of the survival and ventricular enlargement trial.
The SAVE investigators. N Engl J Med 1992;327:669-677.
- Pitt B, Segal R, Martinez FA, et al. Randomised
trial of losartan versus captopril in patients over 65 with
heart failure (Evaluation of Losartan in the Elderly Study,
ELITE). Lancet 1997;349:747-752.