Download Cardiac reparation: fixing the heart with cells, new vessels and genes

European Heart Journal Supplements (2002) 4 (Supplement D), D73-D81
Cardiac reparation: fixing the heart with cells,
new vessels and genes
P. M e n a s c h e 1 and M. D e s n o s 2
1Department of Cardiovascular Surgery, H@ital Bichat Claude Bernard, and 2Department of Cardiology, H6pital
Europden Georges Pompidou and Facultd de Mddecine Necker-Enfants Malades, Paris, France
Cell-based interventions, angiogenesis and gene therapy are
among the newest treatment modalities that have been
proposed to improve outcomes in patients with ischaemic heart
disease. Experimental data have established that implantation
of contractile cells into fibrous post-infarction scars can allow
those tissues to regain some functionality. Although clinical
data are still preliminary, they support the concept of cell
transplantation and raise realistic hopes that it will find a place
among strategies to ameliorate heart failure in the future.
Therapeutic angiogenesis is another promising means of
ameliorating ischaemic symptoms as there is already
experimental evidence that angiogenic growth factors can
stimulate the development of functionally significant new
blood supply. In addition, successful correction of major
abnormalities of calcium cycling by adenoviral gene transfer
represents an encouraging finding in gene therapy. However,
the complexity" of gene dysregulation that is involved in heart
failure complicates identification of the culprit genes, and
important safety issues remain to be addressed before such
therapies may proceed to clinical trials.
Introduction
This increase will add to the already enormous financial
burden of heart failure, associated with the costs of lifetime
treatment, nursing home care and repeated hospitalizations.
Heart failure is already estimated to consume 1-2% of the
total health care budget of Western countries[ll, and this
figure is likely to continue to rise.
Improvements in medical therapy, primarily betablockers, angiotensin-converting enzyme inhibitors and
aldosterone antagonists, have dramatically improved the
prognosis of ischaemic heart failure. However, the overall
outcome in heart failure patients remains dismal. A recent
survey using the database of the U.K. National Health
Service in Scotland[2] indicated that, with the exception of
lung cancer, heart failure has a worse 5-year survival than
many common cancers. In the most severe, drug-refractory
forms of cardiac failure, more aggressive approaches such
as ventricular resynchronization and cardiac transplantation
may be indicated. The role of conservative surgical
strategies (correction of mitral valve incompetence or
restoration of left ventricular geometry) remains limited,
Cell-based interventions, angiogenesis and gene therapy are
among the newest treatment modalities that have been
proposed to improve outcomes in patients with ischaemic
heart disease and, more specifically, those with cardiac
failure. It is well documented that infarcted areas of the
myocardium evolve toward fibrous scars that, if extensive,
can lead to heart failure through excessive remodelling and
reduced pump function. The clinical relevance of this
problem is reflected in the high incidence of heart failure
(approximately 120,000 new cases each year in France and
500,000 in the U.S.A.). The incidence of heart failure is
expected to increase because of ageing of the population
and improved survival after acute myocardial infarction.
Correspondence: Dr Philippe Menasch6, Department of
Cardiovascular Surgery, H6pita[ Bichat Claude Bernard, 46, rue Henri
Huchard, 75018 Paris, France.
1520-765X/02/0D0073 + 09 $35.00/0
(Eur Heart J Supplements 2002; 4 (Suppl D): D73-D81)
© 2002 The European Society of Cardiology
Key Words: Angiogenesis, cellular transplantation, gene
therapy, heart failure.
© 2002 The European Society of Cardiology
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P. MenaschO and M. Desnos
and implantation of left ventricular assist devices is still at a
developmental stage. The limitations of all of these
approaches mandate the search for alternate therapeutic
options, such as cell-based interventions, angiogenesis and
gene therapy, which are the focus of the present review.
Cell-based interventions
The objective of cellular therapy is to repopulate fibrous
scars with new contractile cells, with the aim of restoring
some functionality to these akinetic areas. This objective
could theoretically be achieved through three distinct
approaches: multiplication of residual myocytes, transforming fibroblasts in the scar, and implanting exogenous
contractile cells.
been extensively investigated in the laboratory and is now
being tested in the first clinical trials in humans. It should be
noted that most experiments to date have focused on
ischaemic, segmental cardiomyopathies (as does the present
review). However, preliminary studies suggest that the
putative benefits of cellular transplantation might extend to
globally dilated cardiomyopathies, whether idiopathic[8,91 or
following doxorubicin therapy[l°l.
Myocyte transplantation:
proof of concept
Transplantation of contractile cells (i.e. foetal cardiomyocytes or skeletal myoblasts [satellite cells]) has been
tested in small and large animal models of myocardial
infarction generated by coronary artery ligation, endovascular
coronary artery embolization, or cryoinjury. In those animal
models myocyte transplantation has been shown to result in
Multiplication of residual myocytes
successful cell engraftment and improved function[ 111.
Evidence for cell engraftment has been obtained with
This approach would involve forcing residual cardiomyocytes various techniques, depending on the type of implanted
to re-enter the mitotic cycle, thus expanding the number of cells. Foetal cardiomyocytes may be identified by pre-transcontractile elements. Such a strategy has previously been plantation cell transfection with genes encoding betaconsidered unrealistic because adult cardiac cells were galactosidase (which gives cells a blue colour after
considered to be terminally differentiated and therefore unable appropriate histological processing). Alternatively,
to multiply. This assumption has now been challenged by explanted specimens can be subjected to immunoexperimental and clinico-pathological studies[3 51that suggest histochemical analysis for the alpha-actin smooth muscle
that cardiomyocytes in infarcted or failing human hearts do isoform, which is normally present in foetal but not in adult
retain the capacity to re-enter the cell cycle. However, the cardiomyocytes[121. A further approach (used in our own
number of 'new' cells that can be generated appears far too studies) is to use the Y-chromosome as a genetic marker for
low to compensate for the loss of cardiomyocytes that results male foetal cells implanted into female myocardium[131.
from an infarct large enough to cause heart failure. An
Skeletal myoblasts are easier to identify than are foetal
altemative approach involving residual myocytes might be to cardiomyocytes because they differentiate into typical
stimulate cardiomyocyte DNA replication by expression of multinucleated myotubes, which can be identified
transgenes that encode viral oncoproteins or endogenous histologically. In addition, skeletal myoblasts can also be
cellular proteins that are involved in cell cycle control. identified by pre-implantation labelling with a fluorescent
However, such a strategy raises major safety issues that cast dye (e.g. 4'6'-diamidino-2-phenylindole, which binds to the
doubt on its clinical applicability[6].
nucleus), or by staining with antibodies that are specific for
skeletal muscle myosin.
Interestingly, engrafted myotubes display a composite
phenotypic pattern, co-expressing embryonic, fast and slow
Transforming fibroblasts in the scar
myosins[14]. By analogy to the events that occur following
dynamic cardiomyoplasty, it may be speculated that
This approach would involve transforming the fibroblasts stretching and/or repeated electro-mechanical stimulation
that constitute the post-infarction scar into contractile cells. triggers 'reprogramming' of engrafted myoblasts toward the
Theoretically, this could be achieved by transfection with expression of the slow fibre phenotype. A study reported by
the MyoD master gene, which controls the skeletal muscle Chin et al.[ Is] supports the concept of 'milieu-induced
differentiation programme. Although there have been some differentiation'. However, our own studies[141 and those of
successful experimental results[V], this approach is fraught others do not support this paradigm, in that intrawith technical problems that render its clinical application myocardially implanted myoblasts do not turn into cardiounlikely in the forseeable future.
myocytes (as demonstrated by their failure to stain positively
for the cardiac-specific alpha-myosin heavy chain), but
remain committed to a skeletal muscle phenotype.
Interestingly, transplanted foetal cells establish gap
Implanting exogenous contractile cells
junctions with neighbouring host cardiomyocytes[161. This
is not the case with skeletal myoblasts, however. Once
To date, the most promising approach consists of implanting skeletal myoblasts have differentiated into myotubes, they
the scar with exogenous contractile cells. This strategy has downregulate N-cadherin and connexin-43 (the major
Era"HeartJ Supplements,Vol. 4 (Suppl D) April 2002
Cardiac reparation
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proteins responsible for mechanical and electrical coupling, in function. Fibroblasts, for example, improve post-infarct
respectively), and thus appear to remain insulated from the diastolic performance, but are unable to augment contractile
surrounding myocardial tissue[171. However, it is possible function[25,261.
that myotubes may be indirectly connected with cardiomyocytes tba'ough the extracellular matrix (see below).
Finally, engrafted myofibres have been reported to align
Foetal cardiomyocytes
with the transverse axis of the heart[14]. However, the extent
to which this phenomenon influences their ability to restore
mechanical function is unknown.
Foetal cardiomyocytes were the first cell type to be
Transplantation of either foetal cardiomyocytesEl3 18] or investigated for transplantation, and their successful
skeletal myoblasts[19-221 into post-infarct scars has clearly experimental use has been pivotal to a convincing 'proof of
been shown to improve global and regional contractile concept'. However, their clinical usefulness is undermined by
performance, Evidence for improved function has been problems related to ethics, availability and immunogenicity.
provided by ex vivo (Langendorff-type isolated heart The encouraging results obtained with intra-cerebral
perfusion) and in vivo (echocardiography, sonomicrometry) transplantation of brain tissue in patients with Parkinson's
techniques. Animal hearts that have received such cell disease cannot readily be extrapolated to the heart, which
transplants demonstrate higher developed pressures, ejection tolerates allografts much less well than does the brain.
fi'actions and pre-load recruitable stroke work indices. They
also show decreased ventricular remodelling, evidenced by
smaller left ventricular end-diastolic dimensions/volumes and
Skeletal myoblasts
limitation of scar expansion. Our experiments in a sheep
model suggest that limitation of scar expansion could be
related to regression of post-infarct fibrosis in favour of newly The limitations of foetal cardiomyocytes account for the
formed myotubes (paper submitted). In the same model current interest in skeletal myoblasts (satellite cells). These
Doppler tissue imaging shows that, in the myoblast-grafted myogenic stem cells normally lie in a quiescent state under
segments of the infarct area, transmyocardial velocity the basal membrane of skeletal muscular fibres. In case of
gradients are increased during both systole and diastole. This injury, they are rapidly mobilized, proliferate and fuse to
finding provides direct evidence for the contribution of regenerate the damaged fibres. From the clinical point of
transplanted cells to the overall improvement in function.
view, skeletal myoblasts offer several advantages. First, their
A causal relationship between the presence of engrafted autologous origin enables large-scale clinical applicability.
cells and functional outcome was also suggested by the data Second, it is possible to grow a large number of cells from a
reported by Taylor et a/.[201. In their rabbit model of small biopsy. Third, they have a well-differentiated myogenic
cryoinjury implanted with autologous myoblasts, in hearts lineage, which minimizes the risk of tumourigenicity.
where incorporation of donor cells was unsuccessful, there Reinecke and Murry[27] reported cases of graft overgrowth
was no improvement in mechanical function. In rat that distorted left ventricular endocardial contours, but this
experiments[23], the post-transplantation improvement in effect was probably due to the culture conditions used, and
fimction was additive to that obtained with angiotensin- was not observed in our studies. Finally, skeletal myoblasts
converting enzyme inhibitors, a finding that clearly have a high resistance to ischaemia, which should enhance
strengthens the clinical rationale for the procedure. Finally, their survival after implantation into the hostile environment
the functional outcome appears to be tightly linked to the of the post-infarction scar.
number of cells injected[24], which has important implications
Our findings show that, in spite of the lack of gap junctions
for clinical trials.
with host cardiomyocytes, skeletal myoblasts improve
The question of the duration of functional benefit of fnnction to a similar extent to that with foetal cellsI281,
cellular transplantation has yet to be addressed in clinical justifying our choice of skeletal myoblasts for clinical trials.
trials, but there are encouraging data from animal studies. In However, because engrafted myoblasts remain committed to
our studies in rats and sheep, we found that the improvement their myogenic programming, it is conceivable that their
in fimction observed in myoblast-injected hearts early after contractile performance might be improved by pretransplantation remains stable over 1 year (paper submitted).
implantation engineering with genes encoding critical cardiacMyotubes were also evident in the grafted myocardium after specific proteins. Possible candidates include connexin-43
1 year. These fibres express a slow myosin, a finding that is (which is a major constituent of gap junctions[ag]) and cardiacconsistent with the observed resistance of intra-myocardial type dihydropyridine membrane receptors (which are required
skeletal grafts to fatigue[14], which suggests their ability to for triggering a calcium-induced calcium release pattern of
sustain a cardiac-type workload in the long term.
excitation~ontraction coupling[30]).
Which cell type?
S t e m cells
As mentioned above, implanted cells have to possess
contractile properties if they are to enable an improvement
Myoblasts are not the only option for cell transplantation.
Stem cells are currently the focus of growing interest (even
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P. Menaschd and M. Desnos
in the lay press). The term 'stem cells' actually encompasses
two completely different types of cells: bone marrow and
embryonic stem cells.
cells have been reported to be functionally less effective
than similarly cryopreserved skeletal myoblasts[9l.
Embryonic stem cells
Bone marrow stem cells
Bone marrow stem cells have 'pluripotentiality', which
should allow them to differentiate into various cell types,
including cardiomyocytes. In a clinical situation, these cells
would have the advantages of autologous origin and easy
retrieval via bone marrow aspiration (or even simple blood
collection after pharmacological stimulation of the bone
marrow).
It is unclear whether, before intra-myocardial implantation, bone marrow stem cells should first be cultured under
conditions that promote differentiation into 'cardiac' cells or
whether they should be transplanted unmodified, relying on
local signals to drive them toward the cardiac phenotype. All
experiments based on the former approach have used the
compound 5-azacytidine. However, this 'de-represses' a
wide array of genes, raising important safety issues should a
human heart be injected with cells modified in this manner.
A second major question is whether bone marrow stem
cells should be implanted regardless of type, or whether a
particular subpopulation should be selected. At first sight, it
seems attractive to use the CD34 + progenitors. However,
because these only represent a small percentage of the total
bone marrow cell population, the restricted number of cells
available might not be enough for any significant
improvement in cardiac fimction. It might be possible to
expand them in vitro but this, in turn, might compromise
their pluripotentiality. Pretransplantation mobilization of
progenitor cells from the bone marrow by cytokines would
then represent a more clinically relevant alternative. Another
subpopulation of interest is the bone marrow stromal
(mesenchymal) cells. Once implanted into a myocardial
environment, these cells have been reported to receive
signals that drive them toward cardiomyogenic
differentiation[311. However, the true 'cardiac' transformation
of any type of bone marrow-derived cells must be carefully
verified. If they only become 'muscular-type' cells, then
native myoblasts would offer an easier option.
Evidence for cardiac differentiation has been provided by
a recent study conducted by Orlic et al.[321. Injection of a
selected subpopulation of bone marrow cells (Lin- c-kitPos)
into an infarcted mouse myocardium resulted in their
transformation into cardiac, smooth muscle and endothelial
cells, and was associated with an improvement in function.
This finding is theoretically important, but should be
interpreted cautiously for two reasons. First, the small
number of progenitors made it necessary to use cells from
several mice to inject a single individual, thereby raising all
the issues associated with allografting. Second, injections
were made in a fresh infarct (3-5 h after coronary artery
ligation). The microenvironmental signals that drove the
cells toward the various reported lineages might be different
from those that are present in the clinically relevant setting
of an old post-infarction scar, as encountered in patients
with heart failure.
Finally, it is interesting to note that, in the hamster model
of dilated cardiomyopathy, cryopreserved bone marrow
Eur Heart J Supplements,Vol. 4 (Suppl D) April 2002
The challenges posed by embryonic stem cells are even
greater than with bone marrow stem cells. Aside from the
ethical and regulatory problems, therapeutic cloning of
mammalian cells is still fraught with major technical
difficulties. The supply of human eggs is limited, and it is
difficult for cloned eggs to reach the blastocyst stage. These
problems make it unlikely that these 'personalized' cell
lineages will be available in the near future. One alternative
to the use of embryonic stem cells is to 'reprogramme' the
patient's own cells, rather than cloning them. They could be
'rewound' back to an embryonic stem cell-like phenotype,
which could then be orientated toward the desired cell
lineage. Another option might be to genetically engineer
allogenic embryonic stem cells to make them match the
intended graft recipient (and thus overcome an immune
response from the host). These approaches are still at a very
early experimental stage, however, and it is not possible to
predict whether they will ever become clinically useful.
Stem cells versus myoblasts
This discussion of the problems associated with stem cell
transplantation does not imply that unmodified, native
skeletal myoblasts are necessarily the most appropriate
choice for cell transplantation; they are simply the most
practical choice at the moment. It is quite likely that skeletal
myoblasts represent just one early step on the long journey
of cellular therapy for heart failure. However, it appears that
early optimism regarding stem cell transplantation is
premature, because so many issues remain unresolved.
Although an attractive concept, stem cell transplantation
may take a considerable time to become a clinical reality. In
contrast, skeletal muscle cell transplantation has already
reached the stage of clinical trials.
Where should cells be injected?
Thus far, most experimental studies of any kind of cell
transplantation in cardiac failure have used cell injection
through multiple epicardial puncture sites. For example, this
direct vision approach has been adopted in early clinical
trials of skeletal myoblast transplantation (see below).
However, the attractive possibility of delivering cells
percutaneously through an endoventricular catheter is
generating increasing interest among interventional
cardiologists. This mode of delivery is made possible by
recent improvements in catheter design and navigation
systems. Its tecbmical feasibility has now been established in
animals and in humans. However, no data are yet available
for cell survival following passage through these catheters,
or for the functional efficacy of this 'blind' approach. In
addition, the potential problem of cells being 'squeezed' by
the heartbeat into the left ventricular cavity and
subsequently migrating into the systemic circulation has not
been thoroughly addressed.
Cardiac reparation
The epicardial and endocardial routes are not mutually
exclusive. A further mode ofmyoblast administration is via
the intra-coronary route. This has been used successfully in
situ in mouse[33] and in explanted rat hearts[341, but its
clinical practicality remains debatable.
Regardless of the route chosen in future, it should be
recognized that the physical process of injection still leads
to a high cell death rate. Optimization of the procedure of
cell transplantation will require improved delivery devices
that enhance early post-injection cell survival.
Alternatively, cell viability might be enhanced by blockade
of apoptosis, promotion of angiogenesis, inhibition of
matrix proteases, or even pre-implantation heat-shock
treatment[351. Finally, major advances in tissue engineering
also raise the possibility that cell engr~iftment could be
achieved by seeding donor cells on biodegradable
scaffolds[36]. One of the most attractive applications of
these 'cellularized' grafts is the repair of congenital heart
defects[37].
How does cellular transplantation
improve cardiac function?
The mechanisms by which cellular transplantation improves
heart function still remain largely unknown. At least three
hypotheses exist, which are not mutually exclusive, and are
currently topics of intensive research.
Limitation of infarct expansion
It is conceivable that, through their elastic properties,
implanted cells provide a 'scaffold' that limits post-infarct
expansion, thus preserving the left ventricle from excessive
remodelling. This hypothesis is strongly supported by the
finding of reduced end-diastolic volumes in celltransplanted hearts.
Intrinsic contractile properties
The observation that cell contractility is required for
maximal functional benefit suggests that the intrinsic
contractile properties of implanted ceils are also important.
In the case of foetal cardiomyocytes, the presence of gap
junctions increases the likelihood of synchronous
propagation of electrical impulses between host and grafted
cardiac cells. The mechanism is more difficult to understand
in the case of skeletal myoblasts, which lack these
junctions. Conceivably, myoblasts could contract in
response to the mechanical stress exerted by the surrounding cardiomyocytes, although this implies that both cell
types are connected to the extracellular matrix through
which the mechanical impulses would propagate. Myoblast
'tethering' to this matrix remains to be confirmed.
D77
Release of growth and~or angiogenic factors
It is possible that transplanted cells might release growth
and/or angiogenic factors that could enhance graft survival
and stimulate contractile fimction in hibernating cardiac
cells. This hypothesis is speculative and is not supported by
experimental findings. Our studies show that myoblast
transplantation fails to increase angiogenesis beyond that
observed in control individuals receiving an equivalent
volume of cell-free culture medium alone, through similar
epicardial punctures. (Epicardial puncture alone can trigger
angiogenesis[381, but the response is too small to be
functionally relevant.) Attempts have been made to
genetically engineer myoblasts to express the vascular
endothelial growth factor (VEGF). This has been reported
to result in increased angiogenesis and better haemodynamics as compared with unmodified myoblasts[391.
However, another study[ 401 found that injection of VEGFtransfected myoblasts into immunodeficient mice results in
the formation of vascular mmours, raising doubts regarding
the clinical applicability of this technique.
An early clinical trial of myoblast
transplantation
The weight of experimental data on cellular and, more
specifically, myoblast transplantation accumulated over the
past decade led us to initiate the first phase ] human trial.
This was approved by the French Regulatory Health
Authorities and our Institutional Ethics Committee in the
Spring of 2000. The first patient received intra-myocardial
injections of his own cultured skeletal myoblasts on 15 June
2000E41]. The trial is completed and full results will be
published separately. However, it is possible to make some
general comments.
The primary objectives of the trial were to assess the
feasibility and safety of the procedure. Efficacy was only a
secondary end-point, given the lack of a control group and
the potentially confounding effect of the concomitant
bypass. Therefore, the study does not allow us to draw any
definite conclusions regarding the specific effects of
myoblast transplantation on functional outcome. However,
functional outcome will be a primary end-point of a
multicentre prospective placebo-controlled randomized
phase II trial planned for 2002.
Eligibility for inclusion in the phase I trial was based on
the following: impairment of left ventricular function, as
reflected by an echocardiographically determined ejection
fraction of 0.35 or less; history of myocardial infarction
with a residual discrete, akinetic and non-viable scar
(assessed by dobutamine echocardiography and fluorodeoxyglucose positron emission tomography); and an
indication for concomitant coronary artery bypass grafting
in remote (i.e. different from the transplanted area) and
viable, but ischaemic myocardium.
The trial followed a straightforward three-stage protocol.
First, a muscle biopsy was taken from the thigh under local
anaesthesia. Second, the minced muscle was grown for
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P. Menaschd and M. Desnos
2-3 weeks in the cell culture laboratory using customized
techniques in order to obtain a pure (at least 50%
myoblasts) and high (at least 400 x 106) cell yield.
Microbiological controls were performed throughout this
expansion phase. Third, the cells were reimplantated into
the post-infarct scar, while the chest was open for coronary
artery bypass grafting. On completion of the bypass grafts,
the cells (which had been concentrated into 4-6 ml fluid)
were injected at 25-50 sites in the post-infarction scar. A
pre-bent microneedle was used to create subepicardial
'pockets', thus avoiding inadvertent intra-cavitary cell
delivery. The transepicardial injections were done according
to a 'virtual grid', coveting the entire area of scar tissue. It
is evident that the procedure is feasible (assuming the
availability of adequately equipped Good Manufacturing
Practices facilities and expertise in large-scale cell
expansion for clinical purposes). The key to technical
success is the quality of cell cultures.
With regard to safety, bleeding from the multiple puncture
sites does not appear to be a problem, either intra- or postoperatively. However, ventricular arrhythmias are a potential
concern. Although they have not been observed in our
animal experiments with skeletal myoblasts or with AT-1
cardiomyocytes derived from a differentiated tumour line[421,
they have occurred in some of our patients. The mechanism
of these arrhythmias (circus rhythm or ectopic pacemaker)
has not yet been elucidated. Post-infarction scars in patients
represent a potentially arrhythmogenic substrate, which can
never exactly be duplicated in animal models. The potential
risk for ventricular arrhythmias has led us to implement
some safety measures. We now start amiodarone prophylaxis
at the time of biopsy and continue until 3 months postoperatively. Patients also receive close in-hospital
monitoring for at least 2 weeks (to cover the period during
which most of these arrhythmias are likely to occur).
A further issue that is relevant to arrhythmias is the
avoidance of intra-myocardial 'over-cellularization'.
Optimizing the number of cells to be delivered is difficult.
On the one hand, it is known that up to 90% of injected cells
die within a few hours after implantation. Hence, we have
transplanted a large number of cells (up to one billion) in
order to compensate for this high attrition rate and to
maximize the chance of an improvement in function. On the
other hand, increasing the number of cells increases the
volume of injection and the number of puncture sites. This
may amplify the inflammatory response to needle punctures
and the subsequent clearance of dead cells. Compounds
released by inflammatory cells that invade the transplantation areas (particularly cytokines and nitric oxide)
could increase the vulnerability of the myocardium to
arrhythmias. Future dose-ranging studies should help in
'fine-tuning' the number of cells required to obtain the
optimal risk-benefit ratio. In the meantime, ongoing
laboratory experiments should help in gaining an understanding of the mechanisms of arrhythmias and should
assist in developing appropriate preventive strategies.
Any efficacy data obtained from this phase I study will be
difficult to interpret because of the methodological
limitations outlined above. A confounding effect of the
concomitant revascularization can never be completely
Eur Heart J Supplements,Vol. 4 (Suppl D) April 2002
ruled out. However, preliminary findings do appear to show
that scar tissue implanted with myoblasts can regain
functionality, supporting the concept of cellular
transplantation as a means of augmenting heart function.
Angiogenesis
Angiogenesis, which provides new blood supply to the
diseased heart, is not designed primarily to improve the
function of the failing myocardium, but to relieve ischaemic
symptoms in patients who are unsuitable for more
conventional forms of revascularization (angioplasty or
bypass surgery). Proof of concept for angiogenesis has been
obtained from animal models of myocardial ischaemia.
Compelling data show that administration of angiogenic
growth factors such as VEGF and basic fibroblast growth
factor (either as recombinant proteins or by gene transfer)
can increase myocardial blood supply through neovascularization.
The initial clinical trials with these factors[ 43] yielded
mixed, although generally encouraging, results. A recent
placebo-controlled study[44], in which naked plasmid DNA
encoding VEGF-2 was injected through an endoventricular
catheter, reported improved myocardial perfusion,
paralleled by a reduction in angina in the treated group.
However, several issues remain to be addressed, including
the nature of compounds to be administered (gene or
protein), the optimal dose schedule (single or repeated
administration) and the route of delivery. Percutaneous
catheter-based
intra-coronary
or
endoventricular
administration is one delivery option. Another is a direct
surgical approach using direct intra-myocardial injections
or subepicardial encapsulation of sustained-release
polymers[45]. These various approaches must be evaluated in
terms of maximizing drug distribution and retention in the
target myocardial tissue. However, they must also be
evaluated with reference to adverse events, in particular
atherosclerotic
plaque
expansion/destabilization,
development of functionally abnormal blood vessels,
proliferative retinopathy, the risks associated with viral
vectors, and (albeit less likely) acceleration of occult
malignancies[46?. These issues were comprehensively
reviewed by an Expert Panel Report of the Angiogenesis
Foundation and the Angiogenesis Research CenterI471.
At the basic science level it is interesting to note that,
when naked plasmid DNA encoding the 165-amino-acid
isoform of human VEGF was injected into the myocardium
of patients with chronic myocardial ischaemia, there was a
rise in plasma levels of VEGF. This resulted in mobilization
of endothelial progenitor cells. These could home in foci of
neovascularization and differentiate into endothelial
cells[48]. This finding raises the hypothesis that the
therapeutic development of new blood vessels may not be
restricted to 'classical' angiogenesis (defined as the
proliferation and migration of fully differentiated
endothelial cells). It could also occur through enhanced
vasculogenesis, which is the primary process responsible
for the growth of vasculature in the embryo.
Cardiac reparation
Gene therapy
Several experimental studies provide convincing evidence
that gene therapy can be an effective means of improving
the function of the failing heart. The complexity of the
problem differs markedly, however, depending on the
specific type of heart failure. Gene therapy may be
predicted to be most successful in monogenic disorders
such as familial dilated or hypertrophic cardiomyopathy.
The validity of this approach is demonstrated by the
efficacy of a recombinant adeno-associated virus-mediated
transfection of the gene encoding delta-sarcoglycan in
reversing morphological and functional alterations in the
hamster model of dilated cardiomyopathy[491.
The situation is far more complex in the case of ischaemic
heart failure, which results from the dysregulation of several
signalling pathways, complicating the identification of
candidate genes. Nevertheless, genetic manipulation of three
major areas has been investigated in ischaemic heart
failureESO]: calcium handling, beta-adrenergic signalling and
apoptosis. Abnormalities of calcium homeostasis associated
with heart failure were addressed by del Monte et al.[511. In
cardiomyocytes from failing human hearts, those investigators
showed that adenoviral gene transfer resulted in overexpression of SERCA2a (the pump that contributes to calcium
removal from the cytosol by re-accumulating it in the
sarcoplasmic reticulum). This led to an increase in both
protein expression and pump activity, accompanied by
normalization of the maj or abnormalities of calcium handling.
It remains uncertain whether such improvements in
contractile parameters at the cellular level can affect left
ventricular function and ultimately survival of patients with
advanced heart failure. However, encouragingly, the same
group[521 also found that over-expression of SERCA2a
induced by gene transfer in vivo was effective in
normalizing systolic and diastolic function in a rat model of
pressure-overload hypertrophy in transition to failure. Other
studies have shown that adenoviral gene transfer of beta-2adrenoreceptors or an inhibitor of beta-adrenoreceptor
kinase 1 restores beta-adrenergic signalling. However, the
clinical utility of this approach might be hampered by
possible adverse effects associated with a sustained increase
in cytosolic cyclic adenosine monophosphate (cAMP)J50].
Apoptosis represents another potential target for gene
therapy. Programmed cell death pathways can be blocked
by adenoviral gene-transfer-induced over-expression of
antiapoptotic agents such as Bcl-2, or compounds that
potentiate the antiapoptotic effect of some growth factors
(e.g. phosphatidylinositol-3-kinase and insulin-like growth
factor-I).
All of the strategies described thus far target genes that
are functionally impaired or defective. However, inducing
expression of genes in tissues where they are normally
silent may offer an alternative approach. For example, a
recent study[ 531 has reported the recruitment of cAMPdependent contractility in response to transfection of the
myocardium with the V2 vasopressin receptor genes (which
are normally expressed only in the kidney).
In spite of all these encouraging results, there is still a
wide gap between laboratory findings and the clinical
D79
application of gene therapy in heart failure. Expected
improvements in vector technology and gene delivery
systems will no doubt help to fill this gap. However, studies
in vitro or in rodents still need to be extended to large
animal models before proceeding toward clinical trials of
gene therapy[54]. Although the message conveyed by a
recent enthusiastic review[ sS] is that many of the concerns
raised by cardiac gene therapy are unfounded, most
clinicians and regulatory authorities would still consider
that important safety and efficacy issues remain to be
addressed. Such issues include control of the targeted
protein expression as well as anticipation of potential
adverse outcomes , particularly inflammation, autoimmunity
and oncogenesis. Clarification of these questions is of
crucial importance; in contrast to pharmacological
interventions, which can be terminated if untoward effects
become evident, gene therapy is basically irreversible once
implemented. Whatever the eventual place of gene therapy
in heart failure, however, studies in this area are already
contributing to a better understanding of the
pathophysiology of the disease and to the identification of
new molecular targets for classical drug therapies.
Conclusion
In conclusion, skeletal myoblast transplantation is the cellbased intervention that is most likely to become applicable
in clinical practice in the immediate future. As outlined in
the report of the workshop on cellular transplantation held
under the auspices of the U.S. National Heart, Lung and
Blood Institute[561, several key questions still need to be
addressed. These include the advantages and disadvantages
of different donor cells; the extent to which cell engraftment
affects cardiac function actively (by increasing contractility)
or passively (by limiting infarct expansion and
remodelling); the development of strategies to enhance cell
survival; and the identification of cardiac diseases for which
cell engraftment may be beneficial. The huge amount of
research in this area should provide answers to these key
questions in the near future.
As research progresses, it is likely that cellular therapy
will also take advantage of advances in gene discovery and
developmental biology. Genetic manipulation of donor cells
to make them express cardioprotective recombinant proteins,
or the delivery of genes that encode proteins that are
involved in angiogenesis are just two examples of such an
interplay. Thus, cellular transplantation, angiogenesis and
gene therapy must not be viewed as separate entities. In fact,
they are likely to be mutually beneficial and complementary.
By taking advantage of all three approaches, we may
develop combined strategies that will significantly improve
outcomes in patients with heart failure.
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