Taming of the wild vessel: Promoting vessel stabilization for safe therapeutic angiogenesis

Abstract

VEGF (vascular endothelial growth factor) is the master regulator of blood vessel growth. However, it displayed substantial limitations when delivered as a single gene to restore blood flow in ischaemic conditions. Indeed, uncontrolled VEGF expression can easily induce aberrant vascular structures, and short-term expression leads to unstable vessels. Targeting the second stage of the angiogenic process, i.e. vascular maturation, is an attractive strategy to induce stable and functional vessels for therapeutic angiogenesis. The present review discusses the limitations of VEGF-based gene therapy, briefly summarizes the current knowledge of the molecular and cellular regulation of vascular maturation, and describes recent pre-clinical evidence on how the maturation stage could be targeted to achieve therapeutic angiogenesis.

Full text

1654 Biochemical Society Transactions (2011) Volume 39, part 6
Taming of the wild vessel: promoting vessel
stabilization for safe therapeutic angiogenesis
Silvia Reginato, Roberto Gianni-Barrera and Andrea Banfi1
Cell and Gene Therapy, Department of Biomedicine and Department of Surgery, Basel University Hospital, Hebelstrasse 20, Basel, CH-4031, Switzerland
Abstract
VEGF (vascular endothelial growth factor) is the master regulator of blood vessel growth. However, it
displayed substantial limitations when delivered as a single gene to restore blood flow in ischaemic
conditions. Indeed, uncontrolled VEGF expression can easily induce aberrant vascular structures, and short-
term expression leads to unstable vessels. Targeting the second stage of the angiogenic process, i.e. vascular
maturation, is an attractive strategy to induce stable and functional vessels for therapeutic angiogenesis.
The present review discusses the limitations of VEGF-based gene therapy, briefly summarizes the current
knowledge of the molecular and cellular regulation of vascular maturation, and describes recent pre-clinical
evidence on how the maturation stage could be targeted to achieve therapeutic angiogenesis.
Introduction
The growth of new blood vessels (angiogenesis) is a
fundamental process to allow tissue repair and regeneration
and plays also a crucial role in many pathological conditions,
such as cancer, inflammation and ischaemia. In particular,
therapeutic angiogenesis, i.e. the stimulation of vascular
growth by the delivery of specific growth factors, is
an attractive strategy to restore blood flow in chronic-
ally ischaemic tissue. VEGF (vascular endothelial growth
factor) is the master regulator of angiogenesis in both
development and postnatal life. However, angiogenesis is
a complex multi-step process that requires the temporal
and spatial co-ordination of different factors and cell types.
Endothelial cells proliferate and migrate in response to VEGF
gradients to form new tubular structures. Subsequently, the
maturation of new blood vessels requires the recruitment
of mural cells (pericytes or smooth muscle cells) and the
deposition of a basal lamina [1]. The maturation stage is
crucial to ensure the return to quiescence of the activated
endothelium, as well as the persistence and the functionality
of the new vascular structures. Therefore an understanding
of the cellular and molecular cross-talk during the different
stages of vascular growth is crucial to design therapeutic
strategies that are both safe and effective.
Limitations of VEGF gene delivery for
therapeutic angiogenesis
The results of placebo-controlled clinical trials of VEGF gene
therapy for both peripheral and coronary artery disease were
Key words: angiopoietin, neuropilin-1, platelet-derived growth factor BB (PDGF-BB),
transforming growth factor β(TGFβ), vascular endothelial growth factor (VEGF), vascular
maturation.
Abbreviations used: AAV, adeno-associated vector; Ang1/2, angiopoietin 1/2; CXCR4, CXC
chemokine receptor 4; NEM cell, neuropilin-1-expressing mononuclear cell; NRP1, neuropilin-1;
PDGF-BB, platelet-derived growth factor BB; TGFβ, transforming growth factor β;VEGF,vascular
endothelial growth factor.
1To whom correspondence should be addressed (email abanfi@uhbs.ch).
disappointing [2], and, although VEGF gene delivery was
safe, it did not generate sufficient angiogenesis to correct the
underlying ischaemia. Retrospective analyses have uncovered
several factors that had not been adequately taken into
consideration in designing the first clinical trials and that
could account for the therapeutic results [3]. Of these, we
focus on two limitations of VEGF-based gene therapy that
arise from biological properties of VEGF itself, namely
the need to control its dose distribution in vivo and the
requirement for prolonged expression.
Several lines of evidence suggest that VEGF gene delivery
has a very narrow therapeutic window in vivo: low doses have
no or little angiogenic effects, whereas higher doses rapidly
become unsafe, inducing vessels that frequently display mor-
phological and functional abnormalities. Exogenous VEGF
administration during embryonic vasculogenesis results in
profoundly altered development of vessels with large lumens
[4]. Furthermore, transgenic mice overexpressing VEGF in
the skin, as well as in the heart and liver, display malformed
leaky vessels with unusually large and irregular lumens
[5,6]. The induction of vascular tumours (haemangiomas)
as a consequence of uncontrolled VEGF expression was
also reported in skeletal muscle [7] and in the myocardium
[8] after the delivery of retrovirally transduced myoblasts.
Adenoviral overexpression of VEGF in the skin, fat, heart
and skeletal muscle of mice caused the formation of enlarged,
thin-walled, pericyte-poor vessels, as well as multi-lumenized
glomeruloid structures that resembled tumour-associated
vascular malformations [9,10]. Finally, angioma growth was
observed in infarcted rat hearts after the injection of a VEGF-
encoding plasmid [11].
Previous work from our group has carefully investigated
the dose-dependent effects of VEGF in both normal and
ischaemic skeletal muscle [12,13]. These results indicate that
VEGF does not have an intrinsically narrow dose–response
curve. However, VEGF induces normal or aberrant an-
giogenesis depending on its amount in the microenvironment
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Advances in the Cellular and Molecular Biology of Angiogenesis 1655
around each producing cell, and not on its total dose, as it
remains localized in the extracellular matrix [14]. Therefore
direct in vivo gene therapy approaches, which generate
heterogeneous expression levels around each transduced cell,
lead to a waste of the therapeutic window [14].
Duration of expression is another crucial point to
be considered when delivering VEGF for therapeutic
angiogenesis. On one hand, it is desirable to avoid long-
term expression of a potent growth factor such as VEGF,
but on the other, too brief expression is ineffective. In
a transgenic system of conditional switching of VEGF
expression, it has been shown that VEGF production
for approximately 2 weeks leads to unstable vessels that
promptly regress as soon as the angiogenic stimulus is
stopped. However, if expression was prolonged for at least
4 weeks, the new vessels matured and persisted indefinitely
after VEGF withdrawal [5]. VEGF-dependence of newly
induced vessels has been demonstrated also by injecting
inducible AAVs (adeno-associated vectors) in skeletal muscle,
confirming that sustained VEGF expression for at least 1
month is needed to induce the formation of stable vessels
[15]. Furthermore, abrogation of VEGF signalling with
a recombinant receptor-body (VEGF-Trap) did not cause
regression of new vessels only 4 weeks after implantation of
VEGF-expressing myoblasts in skeletal muscle. Interestingly,
aberrant vessels induced by high VEGF levels never became
VEGF-independent [12].
The need for at least 4 weeks of sustained VEGF expression
in order to generate stable vessels is a challenge for the use
of short-term gene-delivery systems, such as plasmids and
adenoviral vectors, for therapeutic angiogenesis.
Mechanisms of vascular maturation
Vascular maturation, which involves the induction of
endothelial quiescence and protection against VEGF with-
drawal, requires a tight co-ordination of different cell types,
including endothelial cells, smooth muscle cells/pericytes
and recently described populations of myeloid cells [16]
(Figure 1).
Nascent endothelial tubes are coated by pericytes.
During vessel sprouting, pericytes are recruited by PDGF-
BB (platelet-derived growth factor BB) produced by the
migrating tip cell. Genetic studies revealed that mice lacking
PDGF-BB or PDGFRβ(platelet-derived growth factor
receptor β) genes display vascular abnormalities, formation
of microaneurysms and bleeding [17,18]. Pericyte deficiency
in mice lacking the NG2 (nerve/glial antigen-2) proteoglycan
results in immature tumour vessels and impaired basal lamina
assembly [19].
The association between endothelium and pericytes
renders new vessels independent of continued VEGF expres-
sion, as demonstrated in the vascularization of the neonatal
retina as well as in tumours [20,21]. In diabetic retinopathy,
excessive VEGF production leads to the formation of
pericyte-poor and leaky vessels, resulting in blindness [22].
Continuous uncontrolled VEGF overexpression in skeletal
Figure 1 Cellular and molecular cross-talk during vascular
maturation
(A) VEGF drives the release of Ang2, which inhibits Tie2 signalling and
causes the activation of quiescent endothelial cells. Tip cells (yellow)
initiate sprouting and produce PDGF-BB. Trailing endothelial cells (green)
proliferate to form the stalk of the new vessels, which, at this stage, are
not yet invested by pericytes (orange). (B) During vascular maturation,
pericytes, recruited by PDGF-BB, make contact with the new endothelial
structures and exchange signals through different pathways, foremost
among which are the Ang1/Tie2 and TGFβ1/TGFβR1 (TGFβreceptor 1)
axes. VEGF also induces the recruitment of CXCR4+myeloid cells (pink)
that exert a pro-angiogenic activity through paracrine signals. Another
specific subset of myeloid cells (CD11b +/Nrp-1 +, blue) express factors
directly responsible for vascular maturation.
muscle, by retrovirally transduced myoblasts, also induced
aberrant vessels that were not covered by pericytes and failed
to stabilize, remaining dependent on VEGF signalling for
survival [12].
Pericyte–endothelium cross-talk
Pericytes exert their regulatory function on endothelial
cells through both cell–cell contact and secreted signals.
In particular, the role of the TGFβ(transforming growth
factor β) and angiopoietin signalling pathways are the best
understood.
TGFβ1 regulates endothelial cell proliferation and differ-
entiation through the ALK1 and ALK5 (activin receptor-like
kinase 1 and 5) receptors [23]. Both endothelial cells and
pericytes produce TGFβ1 in an inactive form. Only when
cell–cell contact is established does the protein undergo the
cleavage of the latency-associated peptide by plasmin and
become activated [24]. TGFβ1 promotes vessel stabilization
by inhibiting the proliferation and migration of endothelial
cells and by stimulating mural cell differentiation [25].
Furthermore, it has a direct stimulatory effect on the synthesis
and deposition of extracellular matrix components [23].
Angiopoietins are the ligands for the endothelium-specific
tyrosine kinase receptor Tie2 and exert fundamental functions
in both the induction and the maturation stages of vascular
growth. Ang1 (angiopoietin 1) is expressed by pericytes and
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1656 Biochemical Society Transactions (2011) Volume 39, part 6
has been shown to inhibit vascular permeability in the skin, in
tumours and in an in vitro model of the blood–brain barrier by
acting as an agonist for the Tie2 receptor [23]. Furthermore,
Ang1 acts as a survival signal for endothelial cells [26] and
also promotes vascular stabilization by further facilitating
pericyte recruitment [27].
Ang2 (angiopoietin 2) is secreted mainly by endothelial
cells at sites of active angiogenesis and is a partial agonist of
the Tie2 receptor, which makes its actions context-dependent.
In the presence of VEGF, Ang2 promotes sprouting of new
blood vessels and remodelling of the vasculature, since it
induces the dissociation of pericytes from endothelial cells.
In the quiescent vasculature, where Tie2 is constitutively
activated by a basal Ang1 expression, Ang2 acts as a functional
antagonist of Ang1, binding preferentially to Tie2 without
inducing signal transduction and therefore destabilizing
mature vessels [28]. The vessel-destabilizing effect of Ang2
has been also demonstrated after transgenic overexpression
of Ang2 in a normal retina [29].
Myeloid accessory cells
Previous studies have revealed an intriguing role for recruited
bone-marrow-derived circulating cells in promoting both
endothelial sprouting and the stabilization of newly formed
vessels [30] (Figure 1B). In sites of active angiogenesis, VEGF
expression drives the recruitment of a population of bone-
marrow-derived CXCR4 (CXC chemokine receptor 4)-
positive myeloid cells, which are not incorporated into newly
formed vessels, but rather acquire a perivascular position
and are retained through SDF-1 (stromal-cell-derived factor
1)/CXCR4 signalling. These cells synergistically contribute
to the formation of new blood vessels through the secretion
of pro-angiogenic factors and the prevention of their
recruitment significantly inhibits neovascularization [31,32].
Further investigations demonstrated a role for another
population of bone-marrow-recruited cells in the process of
vascular maturation. Zacchigna et al. [33] demonstrated that
cells infiltrating the site of VEGF165-induced angiogenesis
in skeletal muscle are mainly CD11b+cells co-expressing
the VEGF and semaphorin-3A receptor NRP1 (neuropilin-
1), and named them NEM (NRP1-expressing mononuclear)
cells. NEM cells were shown to promote vessel maturation
through the secretion of different paracrine factors, notably
Ang1, TGFβand PBGF-BB [33]. Therefore the authors
proposed a model in which mature vessel formation relies on
the occurrence of two events: the activation of endothelial
cells, through the canonical VEGF pathway, and the
recruitment of bone-marrow-derived cells through the NRP1
receptor. These cells in turn facilitate pericyte or SMC
(smooth muscle cell) recruitment to the new vessels by
PDGF-BB production [33].
Vascular maturation as a therapeutic
target
Targeting vascular maturation is an attractive strategy to
overcome VEGF limitations that became evident in the
first generation of clinical trials. The complexity and
heterogeneity of factors involved in vascular maturation,
described above, suggest different strategies to target this
process. To date, investigations have addressed approaches
acting either directly on endothelial cell receptors, for
example through the co-delivery of Ang1, or indirectly
through the recruitment of pericytes by PDGF-BB co-
delivery.
Systemic administration of Ang1 through intravenous
adenoviral delivery has been shown to protect adult
vasculature from the lethal vascular leakage induced by
systemic VEGF expression [34]. Local co-expression of
VEGF and Ang1 has been described to reduce VEGF-
induced leakage also in rat hindlimb ischaemia [35] and
in normal rat muscle 3 months after AAV injection [36].
Moreover, adenoviral delivery of VEGF and Ang1 induced
vessels that were more mature and persisted after 4 weeks,
whereas the effect induced by VEGF alone disappeared at
the same time point [37]. Although these results clearly
demonstrate the role of Ang1 in preventing VEGF-associated
oedema, whether Ang1 co-expression might correct the
development of aberrant angioma-like structures caused by
uncontrolled VEGF levels has not been investigated.
The approach of co-delivering PDGF-BB is based on the
rationale that increasing the recruitment of pericytes could
provide a comprehensive stimulation of several pathways
in a co-ordinated fashion, thereby better reflecting the
physiological process. Delivery of recombinant VEGF and
PDGF-BB proteins from a polymeric biomaterial has been
shown to induce larger and more mature vessels compared
with VEGF alone subcutaneously and in ischaemic skeletal
muscle [38]. The combination of recombinant PDGF-BB
with FGF-2 (fibroblast growth factor-2) has been reported
to increase vascular stabilization in the mouse cornea and
in rat and rabbit hindlimb ischaemia [39]. However, the
combination of PDGF-BB was found not to correct VEGF-
induced abnormalities in the cornea and was therefore not
pursued further in the more clinically relevant muscle tissue.
Co-delivery of the VEGF and PDGF-BB genes to
ischaemic muscle and myocardium by venous retroinfusion
of AAV vectors has recently been found to significantly
improve the effects of a low VEGF vector dose, in
terms of both perfusion and collateral arteriogenesis [40].
Korpisalo et al. [41] studied the effects of VEGF and
PDGF-BB co-expression in skeletal muscle by co-delivering
two separate adenoviral vectors. They found that the
combination of VEGF and PDGF-BB did not significantly
increase angiogenesis and failed to reduce VEGF-induced
acute oedema 6 days after delivery. In addition, vascular
structures induced by the combination of the two viruses
showed an impaired mural cell coverage, since pericytes
were recruited away from the vascular structures, probably
due to the generation of discordant gradients from the
two separate vectors. Nevertheless, vessels induced by co-
expression persisted longer than those induced by VEGF
alone, through pericyte-independent paracrine signals from
recruited myeloid cells [41]. These results underline the
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Advances in the Cellular and Molecular Biology of Angiogenesis 1657
importance of a proper co-localization of VEGF and
PDGF-BB gradients in target tissue in order to stimulate
physiological angiogenesis. In fact, using myoblast-mediated
gene transfer, we recently found that only VEGF and
PDGF-BB co-expression from a single retroviral bicistronic
vector, which ensures the generation of coincidential factor
gradients around each producing cell in vivo, completely
prevented aberrant angiogenesis induced by uncontrolled
VEGF levels and induced homogeneous mature capillary
networks instead (A. Banfi, G. von Degenfeld, R. Gianni-
Barrera, M.J. Merchant, D.M. McDonald and H.M. Blau,
unpublished work). Furthermore, in the same system, we
also recently found that PDGF-BB co-expression accelerated
stabilization of vessels induced by heterogeneous VEGF
levels, so that 50% of new vessels were already VEGF-
independent after 2 weeks, whereas none was stable when
VEGF was expressed alone (S. Reginato, E. Groppa,
R. Gianni-Barrera, M. Herberer and A. Banfi, unpublished
work).
Conclusions
The increasing understanding of the biological mechanisms
that regulate blood vessels growth and maturation highlights
the complexity of molecular and cellular interactions taking
place within the angiogenic microenvironment. Therefore
targeting both vascular induction and maturation could
provide a promising strategy to overcome some limitations
related to the use of VEGF as a single factor in gene-delivery
approaches for therapeutic angiogenesis.
However, the heterogeneity of delivery systems, of
functional readouts and of target tissues considered so far in
promoting vascular maturation, generated sometimes unclear
or contradictory results. A systematic determination of both
efficacy and safety of specific combinations of factors in
clinically relevant target tissues, such as skeletal muscle for
the treatment of peripheral artery diseases, would greatly
benefit the rational design of novel therapeutic angiogenesis
strategies.
Funding
This work was supported by the Swiss National Science Foundation
[grant number 310030_127426 (to A.B.)].
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Received 14 July 2011
doi:10.1042/BST20110652
C
The Authors Journal compilation C
2011 Biochemical Society