Cardiovascular Research 65 (2005) 656 – 664
www.elsevier.com/locate/cardiores
Review
Growth factor-induced therapeutic angiogenesis and arteriogenesis in the
heart—gene therapy
Johanna E. Markkanena, Tuomas T. Rissanena, Antti Kivel7a, Seppo Yl7-Herttualaa,b,c,*
a
b
Department of Biotechnology and Molecular Medicine, A. I. Virtanen Institute, Kuopio, Finland
Department of Medicine, University of Kuopio and Kuopio University Hospital, Kuopio, Finland
c
Gene Therapy Unit, University of Kuopio and Kuopio University Hospital, Kuopio, Finland
Received 22 July 2004; received in revised form 24 September 2004; accepted 11 October 2004
Available online 11 November 2004
Time for primary review 34 days
Abstract
Myocardial ischemia is one of the most promising targets of gene therapy. Although several growth factors and delivery approaches have
yielded positive results in preclinical studies, first clinical studies have shown little or no real clinical benefit to the patients. It is likely that
less than optimal gene therapy approaches have been used so far, and more thorough preclinical studies are needed in order to establish safe,
efficient pro-angiogenic therapy. Growth factor, gene transfer vector, delivery method and target microenvironment need to be chosen based
on the therapeutic target. It has become apparent that induction of large collateral arteries in the myocardium may need a different approach
than rapid growth of neovasculature around infarction scar. Large animal models are necessary in the determination of optimal therapeutic
agent, dose and clinically relevant delivery strategy.
D 2004 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.
Keywords: Angiogenesis; Collateral arteries; Capillaries; Gene delivery; VEGF
1. Introduction
Collateral vessel growth by the induction of the
expression of growth factors, such as vascular endothelial
growth factor (VEGF), by shear stress is a natural process
occurring in human myocardium undergoing vessel occlusion [1]. Gradual occlusion of the vessel redirects blood
flow into quiescent collateral vessels and increased shear
stress induces enlargement of these vessels with thickening
of the smooth muscle layer surrounding the vessels [2].
Individual capacity to grow these channels is highly
variable, and requires a slow course of disease progression.
Impaired blood flow in the tissue downstream from the
occlusion creates a hypoxic environment, which induces
expression of several growth factors, such as VEGF and
* Corresponding author. Department of Biotechnology and Molecular
Medicine, A. I. Virtanen Institute, University of Kuopio, P.O. Box 1627,
FIN-70211 Kuopio, Finland. Tel.: +358 17 162075; fax: +358 17 163751.
E-mail address: [email protected] (S. Yl7-Herttuala).
fibroblast growth factors (FGFs). Expression of these
growth factors in turn triggers a process termed angiogenesis, growth of capillary level vessels. The processes of
naturally occurring angiogenesis and arteriogenesis are,
however, impaired by old age [3], high cholesterol levels
[4], diabetes [5] and endothelial dysfunction, i.e., the same
attributes often associated with patients who are no longer
suitable candidates for conventional revascularization techniques. Angiogenesis in response to exogenous angiogenic
cytokines and growth factors is, however, at least partially
able to override these phenomena, thus supporting the
usefulness of gene therapy approach [6]. The goal of growth
factor gene therapy is to induce local growth of blood
vessels with techniques suitable for most patients [7].
Both collateral growth and angiogenesis are targets for
gene therapy in the myocardium. Induction of collateral
growth can be beneficial both in severe, multivessel arterial
occlusive disease as a preventive treatment and after acute
occlusion of the vessel to improve the function of the
conducting vessel [8]. Angiogenesis increases local tissue
0008-6363/$ - see front matter D 2004 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.cardiores.2004.10.030
J.E. Markkanen et al. / Cardiovascular Research 65 (2005) 656–664
perfusion and would be a preferred response after acute
myocardial infarction to increase perfusion in the ischemic
area and to salvage hibernating myocardium.
2. Mechanisms of blood vessel growth
Growth of blood vessels has different mechanisms
depending on the goal, initial stimulus and tissue. In
developing embryo, coronary vessels are formed by
vasculogenesis, a process in which the course and even
the caliber of proximal and distal arteries are governed in the
absence of blood flow [9]. Although growth of the blood
vessels in adults mainly follows the same basic mechanisms,
data obtained in different tissue environments should be
applied to myocardium with caution [10].
In adult myocardium, arterial remodelling occurs mainly
in response to alterations in blood flow and shear stress
against the vascular wall. Diameter of an artery determines
the velocity of blood flow in it, and any deviation from that
relationship results either in growth or regression of the
vessel. Collateral vessels form thin-walled, microvascular
anastomoses that connect coronary arteries at all levels.
Once a stenosis forms in the conducting coronary artery, or a
major artery is acutely occluded, blood flow takes the path
of the lowest resistance via the collateral vessels into the
periphery. Blood flow, hydrostatic pressure and shear stress
against the wall of the pre-existing collateral vessels
increase rapidly leading to activation of the endothelium
and up-regulation of vascular growth factors [11]. When
vessels enlarge and develop a fully formed media by
proliferation and remodelling in response to these growth
factors, a biological bypass is formed to compensate for the
decreased flow in the conducting vessel [12]. Collateralization takes from weeks to months to be completed, and
despite the rapid growth and increase in the diameter,
collaterals never reach the conductance of the coronary
vessel they replace.
Myocardium extracts normally 70% of oxygen from the
circulating blood leaving little capacity to increase oxygen
uptake. Myocardium is also highly dependent on aerobic
metabolism. Immediate dilatation of the quiescent, preexisting collaterals can normally replace up to one half of
the flow required to support the tissue downstream. Areas
completely deprived of blood flow will undergo necrosis
and form fibrous scar tissue in the healing process, whereas
tissue receiving insufficient blood flow turns into a power
save mode of hibernating, viable myocardium. Extent of
tissue damage and, thus, restoration of heart function and
even survival of the patient are therefore dependent on the
rapid restoration of blood flow. Gene therapy approaches
aim to overexpress factors naturally up-regulated during
growth of collaterals and thereby speed up the arteriogenesis
process. Another, less explored possibility is to prepare the
collateral network for ischemic insult by enhancing collateral vessel growth in myocardium prior to complete
657
occlusion of coronaries. Such approach may be beneficial
for patients with severe, wide-spread coronary artery
disease. Long-term, low-level expression of growth factors
would probably produce an optimal, sustained response
(Fig. 1).
Collateral vessel growth takes place proximal to the
occlusion site in the normoxic myocardium. Prevailing
theory suggests that normoxic tissues are largely unresponsive to angiogenic stimuli. When the goal is to induce
arteriogenesis in the myocardium, it is therefore essential to
use growth factors that are also potent in normoxic tissues
and gene therapy vectors that produce a sufficient gene
transfer efficiency in healthy tissue. As growth of collateral
vessels is a combination of endothelial changes, smooth
muscle cell proliferation and remodelling of connective
tissue, growth factors used should be able to affect, either
directly or indirectly, a variety of cell types from endothelial
cells to smooth muscle cells and maybe also fibroblasts.
Angiogenesis, growth and enlargement of capillary
vessels occur naturally in response to ischemia. Scar formed
after myocardial infarction is surrounded by hibernating but
viable myocardium, and a network of neovessels is formed
within days to nourish the endangered tissue. Angiogenesis
starts with proliferation and migration of endothelial cells
that spread out from the pre-existing capillaries to form new
vascular sprouts. Newly formed vessels are susceptible to
regression until pericyte coverage is obtained. Such
intermediate stage, a plasticity window, is required for the
vascular network to be trimmed to respond to the metabolic
Fig. 1. Therapeutic time window for gene therapy in the myocardium.
Therapeutic target determines the time course and optimal vector for gene
therapy. Prior to development of myocardial ischemia stimulation of
collateral growth with low level, long-term expression of growth factors
may prevent myocardial ischemia. After acute myocardial infarction (AMI),
rescue of hibernating myocardium requires efficient short-term expression
of growth factor and induction of both angiogenesis and arteriogenesis.
Growth of large-scale vessels takes months to complete, and long-term
expression of growth factors may precipitate the process.
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J.E. Markkanen et al. / Cardiovascular Research 65 (2005) 656–664
needs of the tissue [13]. Growth of excessive capillary
network increases local tissue perfusion, but in order to
compensate the flow in the occluded vessel territory and
sustain cardiac function, larger vessels need to be grown
from upstream of the ischemic area. Angiogenesis in the
ischemic zone lowers the local resistance of the capillary
bed and results in increased flow in the arteries upstream
with the same pressure [14].
Rapid, efficient overexpression of growth factors in the
peri-infarct zone is a promising gene therapy approach.
Patients that have undergone acute myocardial infarction
often require invasive treatment, and gene transfer could be
performed without additional interventions either independently or in combination with intravascular revascularization
strategies or with bypass surgery.
Capillary network in the infarct zone requires flow from
the larger arteries upstream from the ischemic area, and
large collateral arteries require a functional capillary network for effective exchange of oxygen and nutrients.
Although angiogenesis and arteriogenesis can exist as
independent processes and have several distinct features,
both processes of vessel growth are connected also in
natural response to decreased flow in the myocardium.
Several growth factors, including VEGFs, FGFs, PDGFs,
MCP-1 and GM-CSF, have been shown to induce both
angiogenesis and arteriogenesis [7]. In addition, both
processes require proliferation and migration of the same
cell types, mainly endothelial cells and smooth muscle cells.
As both processes are required simultaneously for optimal
clinical outcome, similarities are important clues in search
for optimal therapeutic genes.
3. What to deliver?
Gene therapy approaches usually aim to locally overexpress one of the growth factors naturally participating
in the process of vascular growth and repair [7,15] (Fig.
2). Whereas growth of collateral vessels is likely
governed mostly by hemodynamic factors, angiogenesis
process in the heart is initiated by hypoxia and induced
by growth factors secreted by the surrounding cells. One
therapeutic strategy is to overexpress hypoxia inducible
regulators of gene expression like hypoxia inducible
factor-1alpha (HIF-1a). The aim of such approaches is
to mimic natural responses to ischemic stress, and to
produce a natural cascade of growth factors downstream
from the hypoxic switch. One of the main downstream
targets of HIF1-a, VEGF was found in infracted human
myocardium years before its characterization and naming
[16]. VEGF is the most potent angiogenic growth factor
known, capable of producing vasodilation, increased
vascular permeability, proliferation and migration of
endothelial and likely also smooth muscle cells [17].
Endothelial cells proliferate and migrate to form vascular
sprouts, connect with other blood vessels and eventually
form a lumen [18]. Thin-walled endothelial tubes are at
this point dependent on continuous growth factor expression and prone to regression [19,20]. Newly formed
vascular network is trimmed to respond to needs of
surrounding tissue probably by means of oxygen tension,
negative regulators of angiogenesis and by blood flow
[13]. Sustained expression of growth factors such as
VEGF at this stage is alone sufficient to prevent vessel
regression and promote stabilization of the vessels [21].
The next step of vessel growth is maturation of
capillaries, acquisition of smooth muscle cell coat and
deposition of extracellular matrix. Angiopoietins display a
consistent expression pattern in angiogenic processes
throughout the body. Angiopoietin-2 (Ang-2) destabilizes
vessels by promoting extracellular matrix degradation and
by loosening connections between endothelial cells and is
expressed at the sites of angiogenesis and vessel regression
[22]. The function of Ang-2 seems to be dependent on the
presence or absence of VEGF. When expressed alone,
loosening of endothelial cell connections induces apoptosis,
but the presence of VEGF provides required survival signals
for endothelial cells, and disattachment leads to cell
proliferation and migration [23]. Overexpression of Ang-2
alone has been inefficient to trigger angiogenesis in in vivo
experiments [24,25]. In contrast, angiopoietin-1 (Ang-1) is
expressed in the quiescent state and has been suggested to
reduce vascular permeability and stabilize vessels both by
establishing endothelial cell connections and by recruiting
periendothelial cells [26–29]. Surprisingly, although the role
of Ang-1 is suggested to be more stabilizing and related to
the quiescent state, several studies have shown that Ang-1
has also both angiogenic and arteriogenic properties when
overexpressed alone or in combination with VEGF [30,31].
Hallmark of vessel maturation, recruitment of perivascular
cells, has been shown to be largely dependent on yet another
family of growth factors, platelet-derived growth factors
(PDGFs) [32]. Although overexpression of PDGFs alone
may not be an optimal stimulus for vessel growth,
successful combination approaches with VEGF have been
published [33] (Fig. 3).
Although the initiator of collateral growth seems to be
increased flow and shear stress, the result is activation of
the endothelium and production of several growth factors
[11,12]. One of the most widely studied molecules,
monocyte chemoattractant protein-1 (MCP-1), exerts its
functions by increasing monocyte infiltration into the
growing vessels. After they have reached the site of
collateral growth, macrophages start to release a variety of
growth factors to simulate cell proliferation [34]. Increased
shear stress induces also directly expression of several
growth factors via shear stress responsive element, for
example VEGFs, FGFs and PDGFs. Several angiogenic
proteins have also shown to be potent inducers of
arteriogenesis although their role in natural process of
collateral growth is unclear. Ang-1, FGF-4, placental
growth factor (PlGF), hepatocyte growth factor (HGF)
J.E. Markkanen et al. / Cardiovascular Research 65 (2005) 656–664
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Fig. 2. Variety of growth factors participating in the angiogenesis process. Most gene therapy approaches aim to overexpress one of the growth factors which
are naturally up-regulated during the angiogenesis process. While normal human myocardium does not produce VEGF (A), cells surrounding a myocardial
infarct zone stain positive for VEGF (B). Capillaries in normal human myocardium (C) and angiogenesis in the edge of infarct scar (D). VEGF overexpression
in the pig myocardium by adenoviral gene transfer (F) induces dramatic capillary enlargement in the pig myocardium 6 days after gene transfer (H) while
VEGF expression is weak (E) and capillaries in the control gene (AdLacZ)-transduced myocardium remain unchanged (G).
and a large number of other growth factors have improved
collateral vessel growth in animal models of hindlimb and
myocardial ischemia [25,35,36].
A wide variety of growth factors participate in the growth
of blood vessels in the heart and overexpression of one
factor may not produce an optimal response. It is therefore
likely that a combination of growth factors or a factor
capable of up-regulating other factors should be used.
Recently, adenoviral overexpression of Hif1-a was shown
to up-regulate the expression of multiple angiogenic growth
factors in vivo [37]. Some growth factors have been shown
to display species-specific actions and should therefore be
tested in more than one species before use in human
applications.
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J.E. Markkanen et al. / Cardiovascular Research 65 (2005) 656–664
Fig. 3. Growth factors for gene therapy from nature’s own angiogenesis tool
box. Angiogenesis is a complex, multi-step process in which every step has
its own mixture of growth factors. Almost every single one of them has
been studied for its potency to orchestrate the whole process of vessel
growth. The natural trigger for angiogenesis is hypoxia. Hypoxia induces
expression of variety of growth factors including VEGF and Ang-2.
Pericytes detach from the capillaries, basement membrane is degraded by
matrix metalloproteineses (MMPs) and plasma proteins extravasate to form
a protein-rich matrix in the interstitial space. Growth factors up-regulated
by hypoxia and released from the extracellular matrix induce proliferation
and migration of endothelial cells and pericytes. Excessive neovessel
network is then trimmed to respond to the metabolic needs of the tissue.
Vessels that receive insufficient blood flow regress, while others with
sufficient flow mature, require pericyte coverage and deposit a basement
membrane.
tissue extending from the patent coronary vessel to the
hibernating, jeopardized myocardium (Fig. 4).
Also, the microenvironment for transduction and the
target cells for the produced growth factor should be
considered [40]. Growth factors exert their functions via
specific receptors expressed on the target cells, and
therapeutic effect can only be achieved when both ligands
and receptors are able to interact. Neither vectors nor
produced proteins will efficiently penetrate intact basement
membrane, much less will they penetrate larger caliber
vessel wall with multiple smooth muscle cell (SMC) layers.
Growth factors intended to act on arterial endothelium
should therefore be available in the vessel lumen, as well as
factors acting on mural cells in the tissue surrounding the
vasculature. Also the solubility of the growth factor
determines the bioavailability of the ligand. A growth factor
that binds efficiently to heparin proteoglycans on cell
surface will stay in the vicinity of the producing cells
whereas a soluble growth factor will diffuse further and
cover a larger tissue area [41].
Whereas local delivery of a growth factor in a wrong
microenvironment is inefficient, too widespread overexpression of the growth factor may have serious side effects.
Expression can be directed to the right tissue compartment
by using a proper delivery method but localized gene
transfer gives only a rough estimate of the transduced area
and it is difficult to restrict expression to a specific cell type.
Tissue- or cell-specific targeting of gene delivery vectors
has been developed to improve myocardial specificity of the
gene transfer [42].
Another possibility to improve both angiogenesis and
arteriogenesis in the ischemic tissue is to inhibit endogenous
anti-angiogenic processes and negative feedback loops such
as up-regulation of Thrombospondin-1 by VEGF [38].
Inhibition of endogenous Hif1-a inactivation improved
angiogenesis in the ischemic limb [39].
4. Where to deliver?
Where and how the gene therapy should be delivered
depends on the therapeutic goal and on the properties of the
growth factor itself. Growth of capillary vessels is desired in
the infarct edge area and collateral growth in the healthy
Fig. 4. Therapeutic goal determines gene delivery strategy and microenvironment. Collateral growth is needed upstream and around the
occlusion site in the normoxic myocardium. Process requires proliferation
of smooth muscle cells around pre-existing collaterals while effects on the
endothelium are secondary to the increase in the blood flow. Angiogenesis,
on the other hand, is needed in the periphery around the infarction scar and
growth factors are mainly required to stimulate proliferation and migration
of endothelial cells.
J.E. Markkanen et al. / Cardiovascular Research 65 (2005) 656–664
5. How to deliver?
A wide variety of delivery methods have been
introduced to achieve therapeutic angiogenesis in the
myocardium ranging from systemic delivery of growth
factors via mouse tail vein to highly sophisticated, local
delivery methods in large animal models. Majority of the
models require invasive procedures, such as thoracotomy,
which themselves cause damage and therefore start repair
processes and induce expression of endogenous growth
factors in young, healthy animals. More importantly,
highly invasive, stressful gene delivery methods are not
suitable for very sick patients most urgently in need for
alternative therapies.
Growth factors or gene transfer vectors can be injected
into coronary arteries via regular angiography catheters.
Intracoronary (IC) approach is relatively simple, does not
require specialized equipment or specially trained physicians and does not require additional invasive procedures
when done in combination with angiography. Usually the
aim of IC approaches is transduction of endothelial cells.
Although beneficial therapeutic effects have been reported
in animal models, several studies have been published
showing the low clinical efficacy of this method [7].
Intravenous and intra-arterial approaches are possible without the need for specialized equipment. As intravenous
delivery of VEGF recombinant protein has been shown to
be ineffective [43], efficacy of the intracoronary delivery
depends on the first-pass effect [44]. Endothelium is an
effective barrier for both proteins and viral particles. When
tissue distribution of bFGF was studied after IC and i.v.
administrations, 0.88% and 0.26% of 125I-labelled bFGF
were found in the myocardium, respectively [45].
Therapeutic agents can also be delivered to the tissue
surrounding the vessels. In the intrapericardial approach,
vector is delivered into the pericardium surrounding the
heart. Although good transduction efficiency can be
achieved in the peri- and epicardium, no therapeutic effect
on collateral perfusion has been observed [46]. Although
high concentration of therapeutic protein is produced in the
pericardial fluid, proteins may not penetrate into the
myocardium and reach the growing collateral vessels. More
localized delivery can be achieved with a collar placed
around the treated vessel or with direct injection with a
needle catheter [47]. Even high local protein concentration
around the vessel leads mainly to angiogenic response in the
adventitia of the vessels and does not penetrate deeper into
the vessel wall [48].
Intramyocardial gene transfer allows vector delivery
directly to the target area. Direct intramyocardial injections
can be combined with traditional surgical procedures.
Percutaneous techniques such as NOGA and Stiletto
injection catheters for direct, intramyocardial injections are
suitable for patients too compromised to undergo invasive
surgical procedures and allow local delivery of the growth
factors [49,50]. Furthermore, techniques for the evaluation
661
of therapeutic effects and potential side effects essential for
clinical trials are readily available in large animal models.
In large animal models, techniques developed for human
use are applicable and can therefore be more directly
transferred to clinical trials.
6. How to deliver? Growing selection of gene transfer
vectors
One therapeutic goal should be to determine the vector
used for gene transfer. Treatment after acute myocardial
infarction probably requires a different time course of gene
expression than treatment of chronic myocardial ischemia.
Also, damaged myocardium after infarction forms a different microenvironment than relatively healthy myocardium
preconditioned for decreasing oxygen tension during slow
progression of arterial occlusive disease in chronic ischemia.
Growth factors can be delivered into vascular tissues as
recombinant proteins either directly or using substances that
slowly release protein into the tissue. The main limitation of
the protein approach is short half-life of growth factors in
the tissue, as growth of blood vessels requires the presence
of angiogenic stimulus until a steady state of the vessel
structure is achieved [51] or flow is increased to a level
sufficient to prevent vessel regression. Genes encoding
growth factors can be delivered either nonvirally using
plasmid constructs or using viral vectors. Plasmids are easy
to produce and safe, but their main drawback in myocardium is a very low transduction efficiency [52]. Transduction efficiency of plasmid constructs can be significantly
improved with liposomes [53].
Availability of viral vectors is increasing as new viruses
are developed for gene therapy, and already established
vectors are modified to improve their properties. An ideal
viral vector for gene transfer would have high transduction
efficiency, low toxicity, no immunogenicity, capacity to
carry large gene constructs, selective gene expression in
desired cell type and possibility to regulate gene expression.
Such vector has not yet been created, and gene transfer
vector has to be chosen based on what are the most
important qualities for the therapeutic application.
Salvage of local tissue perfusion after myocardial
infarction requires quick, high transduction efficiency.
Adenovirus is one of the most widely used and studied
gene transfer vectors. It has high transduction efficiency,
relatively high transgene capacity and it can be produced
with high titers. Transgene expression is transient and lasts
2–3 weeks in large mammals with normal immune system,
time frame sufficient to build capillary network and increase
tissue perfusion.
Growth of collateral vessels and arterialization of
neovessels require longer transgene expression, from
several weeks up to months. This can be achieved using
episomal vectors or vectors that integrate into the host cell
genome. Adeno-associated viruses (AAV) have lower
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J.E. Markkanen et al. / Cardiovascular Research 65 (2005) 656–664
transduction efficiency than adenoviruses but have natural
tropism to muscle tissue and transgene expression lasting
up to a year after gene transfer [54]. DNA-capacity of
AAVs is sufficient for most growth factors, but the
maximal capacity (b5 kb) limits the possibility to develop
regulated gene expression systems. Lentiviruses have
higher capacity for transgenes and they are also capable
of transducing both quiescent and proliferating cells. To
overcome the safety issues arising from integration into the
host cell genome, several regulated expression systems
have been developed, but the main limitations for their use
in cardiovascular approaches are low transduction efficiency and low titers of virus preparations.
7. How much to deliver?
As with all drugs, gene therapy approaches also have a
therapeutic window, a concentration at which they produce
an optimal therapeutic response and minimal side effects. In
addition to variation in response to a given concentration of
a growth factor, patients may also display considerable
variation in the transduction efficiency and level of protein
production. The challenge with viral delivery approaches is
thus to determine the correct viral dose that produces an
optimal amount and the target tissue distribution of the
growth factor in a given tissue environment.
In conventional pharmacological approaches, drugs are
usually distributed systemically. One of the main advantages
of gene therapy is the possibility to deliver therapeutic
proteins locally. On the other hand, it makes determination
of correct dose and delivery more challenging. Whereas
concentration of a drug in the blood stream can easily be
determined, it takes far more preclinical experience in large
animal models to be able to determine correct viral dose to
achieve an optimal local protein concentration [40]. Spreading of the virus solution in the target tissue, transduction
efficiency, level of protein production by the transduced
cells and the solubility of the growth factor are all equally
important determinants of the resulting therapeutic effect.
A vast majority of preclinical gene therapy experiments
have been done in mouse models. Although mouse models
are invaluable for transgenic and gene knock-out experiments and in understanding the biological function of
growth factors, information derived from small animal
models should be applied to human therapies with caution
[7]. Viral loads and resulting protein concentrations
achieved in mouse experiments are not necessarily applicable for human use. For example, injection of 1.01011
viral particles [55] or a volume of 3 ml [56] into tail vein of
a mouse would respond to 2.51014 viral particles and a
volume of 7 l in man. Also, the small tissue mass may
complicate the determination of both efficacy and correct
dose of a growth factor. It is easy to cover the entire
myocardium of a mouse with a few injections, while
covering the hundreds of times larger heart of a pig (or a
Fig. 5. Relative sizes of pig, rabbit and mouse hearts. Large animal models
are required in the preclinical studies of growth factor-induced angiogenesis
in the myocardium to develop clinically relevant strategies. Mouse heart
weighs approximately 2000 times less than pig or human heart. Delivery
strategies, virus titers and volumes and side effects like needle track in the
myocardium are in a whole different scale than in the pig or human heart.
man) is a much harder task. Also as trivial a detail as the
needle track caused by the intramuscular injection has a
whole different proportions in mouse than in man. For
example, a 28-G needle causes approximately a 500-Amwide, necrotic needle track in the muscle tissue in which
inflammatory cells and damaged myocytes act as sources of
endogenous growth factors. That needle track is 1/8 of the
width of the left ventricle muscle of a mouse, but only 1/200
of the width of the left ventricle in man (Fig. 5).
8. What are the risks?
Side effects of gene therapy may be caused by the
delivery process, the vector carrying the transgene or the
gene product itself. Since one of the main goals of gene
therapy is to find treatment strategies feasible for no option
patients, delivery of the gene therapy vector itself should be
as a noninvasive and low risk procedure as possible.
Surgical approaches and long-term or repeated infusions
are therefore less likely to be relevant clinically feasible
delivery strategies for myocardial angiogenesis.
Both viral and nonviral gene delivery vectors can cause
adverse effects in vivo. High doses of naked plasmids have
been reported to cause necrosis and inflammation [57].
Most viruses produce an immune reaction which may lead
to a range of clinical symptoms, from transient rise in body
temperature to septic shock. Vectors integrating into the
host genome may activate oncogenes, interfere with
J.E. Markkanen et al. / Cardiovascular Research 65 (2005) 656–664
normal gene transcription or even promote mutations in the
host genome [7].
Side effects of angiogenic growth factors in large animal
models and man are poorly documented, as invasive
approaches in small animal models have masked side
effects specific for angiogenic molecules in the heart. Also,
follow-up times of these studies have been quite short. One
of the main concerns in angiogenic gene therapy is
excessive vessel growth in the transduced tissue, so-called
hemangioma or glomeruloid body formation [58]. Such
structures will, however, usually undergo blood flowdependent remodeling and normalization of the vascular
architecture [59]. Systemic expression of the growth factors
may produce harmful side effects in the distant organs, i.e.,
promote tumor growth, retinopathy or arthritis. Regulation
of gene expression by tissue specific promoters or vectors
regulated by conditions like hypoxia may help to limit
angiogenic effects to target tissues [60]. Although vascular
permeability and subsequent edema are known side effects
of VEGFs, only recently its manifestation in the heart,
pericardial effusion, was documented as a dose-limiting side
effect [52]. Thus, comprehensive evaluation of potential
side effects is needed in large animal models to determine
the optimal dose and delivery strategy for each vector and
gene combination.
Acknowledgements
This study was supported by grants from Finnish
Academy, Sigrid Juselius Foundation, Maud Kuistila
Foundation and Finnish Foundation for Cardiovacular
Research.
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