Cardiovascular Research (2011) 91, 260268 SPOTLIGHT REVIEW

doi:10.1093/cvr/cvr035

Vascularizing the heart

Paul R. Riley * and Nicola Smart
Molecular Medicine Unit, UCL-Institute of Child Health, London WC1N 1EH, UK

Received 30 November 2010; revised 26 January 2011; accepted 27 January 2011; online publish-ahead-of-print 31 January 2011

Downloaded from http://cardiovascres.oxfordjournals.org/ at rouen university hospital on February 9, 2013

Abstract As the developing heart grows and the chamber walls thicken, passive diffusion of oxygen and nutrients is replaced by
a vascular plexus which remodels and expands to form a mature coronary vascular system. The coronary arteries and
veins ensure the continued development of the heart and facilitate cardiac output with progression towards birth.
Many aspects of coronary vessel development are surprisingly not well understood and recently there has been
much debate surrounding both the developmental origin and tissue contribution of cardiovascular cells alongside
the specific signals that determine their fate and function. What is clear is that an understanding of the cellular
and molecular cues to vascularize the heart of the embryo has significant implications for adult heart disease and
regeneration, as we move towards targeted cell-based therapies for neovascularization and coronary bypass engraft-
ment. This review will focus on the proposed cellular origins for the coronary endothelium with due consideration to
the pro-epicardial organ/epicardium, sinus venosus and endocardium as potential sources, and we will explore the
outstanding questions and technical limitations with respect to accurate labelling and lineage tracing of the developing
coronaries. We will briefly document canonical vascular signalling that induces vessels in the heart alongside a focus
on the potential for developmental reprogramming and putative mechanisms underpinning venous vs. arterial cell fate.
Finally, we will extrapolate directly from development to address adult maintenance of the coronaries, vascular homeo-
stasis and remodelling in response to pathology, aligned with the potential for revascularizing the injured adult heart.
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Keywords Coronary vasculogenesis Endothelial cells Proepicardial organ Sinus venosus Endocardium
Neovascularization
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This article is part of the Spotlight Issue on: Cardiac Development

there is the distinct possibility of developmental studies uncovering

1. Introduction
The development of the coronary vessels, and subsequent attachment
of the main coronaries to the closed vascular system of the embryo, is
fundamental for continued foetal growth and survival. Traditionally,
coronary vessels were thought to have a unique derivation from
mesothelial cells arising from the proepicardium or proepicardial
organ (PEO) and yet retain a high level of functional and molecular
similarity to the systemic vasculature. Proepicardial cells are trans-
ferred to the heart by complex signalling and epithelial mesenchymal
transition (EMT) to form the epicardium and give rise to endothelial
cells (ECs), smooth muscle cells, and cardiac fibroblasts (reviewed in
Smart et al.1). Endothelial cells derived from the epicardium form a
vascular plexus by combined vasculogenesis and angiogenesis to
remodel and expand the vascular network, which subsequently
attaches to the aorta and recruits epicardially derived mesenchyme
to become vascular smooth muscle cells (VSMCs) through arterio-
genesis. Interest in coronary vessel formation stems not only from
the fact that it is essential for embryonic survival, but also because
cellular and molecular cues towards neovascular repair of the dis-
eased adult heart.
2. Cellular origins of the coronary
vasculature
Despite the fact that the development of the vessels of the heart has
been a subject of relatively intense study for more than a century,
there is still ongoing debate regarding the cellular origin and tissue
sources for the coronary vasculature.
2.1 The PEO/epicardium: a historical
perspective
Early anatomical observations documented that the coronary vessels
simply bud off from the aorta but these were challenged by studies in
which coronary arteries were observed to grow into the aorta.2 More

* Corresponding author. Tel: +44 20 7905 2345, fax: +44 20 7404 6191, Email: p.riley@ich.ucl.ac.uk
Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2011. For permissions please email: journals.permissions@oup.com.
Vascularizing the heart
recent retroviral tagging, adenoviral cell lineage tracing, and quail
chick chimera studies have proposed the PEO as a source of the cor-
onary vasculature.3 5 The PEO is a transient extracardiac mesothelial
cell population situated between the sinus venosus and the liver in
chick and on the surface of the septum transversum in mouse.6
PEO cells migrate to the developing heart and envelope the surface
to give rise to the epicardium. A subpopulation of epicardium-derived
progenitor cells delaminate from this primitive epithelium and
undergo EMT to generate a population of mesenchymal cells which
migrate into the underlying myocardium and give rise to VSMCs, peri-
cytes, fibroblasts, and cardiomyocytes.3,7 9 That the coronary ECs are
also EPDC-derived is controversial. It is widely accepted that not all
vascular ECs are contributed by the epicardium,10 and an increasing
number of studies now argue that very few, if any, coronary ECs
are PEO epicardium-derived. In chick quail chimera studies with
anti-endothelial immunohistochemistry, prospective coronary ECs
were mapped back to the liver sinusoids extending into the sinus
venosus lumen via the PEO11 with the liver subsequently proposed
to be a primary source of endothelial progenitor cells (EPCs).12 In
mouse, lineage tracing and fate mapping of epicardium derived cells
(EPDCs) during development have either failed to identify
epicardium-derived ECs8 or observed that the epicardium gives rise
to only a few isolated ECs.9
2.2 A sinus venosus and endocardium
contribution?
More recently, in mouse, coronary vessels have been shown to arise
from angiogenic sprouts of the sinus venosus, the endothelial-lined
cavity at the inflow region of the embryonic heart.13 The sinus
venosus was identified as a site for the earliest appearance of
vessels in the heart in classical embryological studies in dogfish14
and, in mouse, a connection of the undifferentiated coronary vascular
network to the sinus venosus was also previously demonstrated.15
However, there was no historical insight into a sinus venosus origin
for coronary artery ECs until the recent study by Red-Horse
et al.13 Here, the authors fate mapped lacZ+ ECs via an
ephrinB2-LacZ reporter to determine that coronary sprouts were
continuous with the sinus venosus and did not arise from the PEO
or other vasculogenic sources. This was complimented by organ
culture and tissue recombination to demonstrate EC outgrowth
restricted to sinus venosus and atrium, which in turn was dependent
upon unknown signals from the ventricle and epicardium, and clonal
analysis with an inducible VE-cadherin-CreER transgene to map the
origin and fate of coronary endothelial progenitors in vivo. The
clonal analysis, via an early (E7.5) induction of VE-cadherin-CreER,
suggested that coronary artery progenitors arise from differentiated
veins and this was confirmed by documenting a switch in molecular
signature from a venous (EphB4+) to arterial (ephrinB2+) identity
within the coronary sprouts as they emerged from the sinus venosus.
What the clonal analysis also revealed13 was a secondary source of
coronary ECs from the endocardium. In the mouse, endocardial cells
are among the earliest ECs that differentiate from Vegfr-2-positive
(Vegfr-2+) multipotent progenitors within the heart field.16 They
form an endocardial tube by de novo growth or vasculogenesis and
later become the inner epithelial lining or endocardium of the
heart,17 subsequently contributing to the formation of the cardiac
valves and membranous septa by endocardial-to-mesenchymal trans-
formation.18 Relative to the endocardial ECs, coronary ECs arise later
261
in the developing heart which establishes a potential for contribution
of the former to the latter population, although, to date, this has again
proven somewhat controversial. That the primitive vascular bed might
arise as a continuation of the endocardium was first described in the
developing rat heart19 and subsequently in mouse, where the first
vessels of the heart were described as appearing by the invagination
of the endocardium.6 In contrast, retroviral tracing in chick revealed
ECs of the coronary vessels to have a different clonal origin than
ECs of the endocardium.7 Red-Horse et al. 13 were the first to geneti-
cally trace and clonally map an endocardial origin for coronary ECs,
albeit a relatively minor contribution with the tools they employed
and described a process of endocardial budding to form endothelial
blood islands which they hypothesized might join the sinus venosus-
derived plexus. A more autonomous and potentially more extensive
contribution of endocardial ECs to the coronary vasculature is yet
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to be determined, but since endocardial cells closely resemble endo-
thelial precursor cells in terms of their molecular profile, unequivocal
lineage tracing and chimera analyses are somewhat confounded.12
2.3 Reconciling a common source
for coronary ECs
So what is the current state of play and what if any is the consensus on
the origins of the coronary vasculature? In attempting to reconcile all
of the above, it is important to take account of species differences,
with a focus on avian vs. rodent as the most studied vertebrate
models.
It would appear that there are two schools of thought derived from
avian studies. The first advocates that the PEO/epicardium contributes
cells to the developing coronary vessels,5 although the PEO is not the
sole source20 and indeed it is apparent that other cell types may par-
ticipate in the building of the coronary tree. The second focuses on
the other cell types and in particular proposes a non-PEO source
favouring the liver sinusoids and sinus venosus.11 In this respect, the
quail-to-chick and/or retroviral techniques used to tag the develop-
mental fate of avian PEO or sinus venosus cells cannot guarantee
that all the coronary progenitors are properly labelled and, therefore,
not only is it difficult to encompass different contributions from differ-
ent sources in a single experiment, but it is equally difficult to make an
accurate quantitative assessment between experiments for cellular
contribution in coronary morphogenesis.
In rodents, and in particular mice, where genetic lineage tracing and
fate mapping tools are available, there has been a recent movement
away from a single PEO/epicardium source for coronary ECs. New
insight has informed on contributions from dedifferentiation of the
sinus venosus endothelium at least giving rise to the coronary arteries.
This of course begs the question as to the origin of the ECs that com-
prise the early coronary veins and in particular the endothelial lining
of the sinus venosus? Moreover, the endocardium has been implicated
as a source of ECs that may further contribute to the developing cor-
onary plexus, and indeed, the precise extent of endocardial contri-
bution remains unknown. It would appear that regardless of source,
the coronary ECs in mammals represent a specified lineage within
the vasculature arising in part from a subpopulation of venous ECs
at defined stages in development. Furthermore, as far as the PEO
and sinus venosus are concerned, their adjacent location at the
inflow region of the developing mouse heart and proximal to the
liver primordium (Figure 1A) means that it is extremely difficult with
the current tools available (specific Cre-expressing lineage mouse
262 P.R. Riley and N. Smart

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Figure 1 A schematized heart during the initial stages of coronary vasculogenesis. The relative positions of the putative tissue origins of coronary
ECs are shown in (A), with the liver sinuses (ls; blue), pro-epicardial organ/epicardium (peo; green), and sinus venosus (sv; blue) highlighted; ra, right
atrium; rv, right ventricle. Potential cellular contributions between the sources and ultimately to the developing venous and arterial sprouts are high-
lighted in (B), arising from the ls (blue), peo/epicardium (green), sv (blue), and endocardium (red); arrowheads indicate fate (blue, sv or sv venous
sprout; green, peo/epicardium; and red, arterial sprout).

lines) to unequivocally rule out common progenitor pools. Equally,
we are unable to discern early contribution of prospective liver
sinusoid progenitors directly to the sinus venosus, or via the PEO
which in turn contribute to the sinus venosus and ultimately the
coronary vasculature (Figure 1B). In the principal cell lineage tracing
adopted by Red-Horse et al.,13 namely the crossing of
VE-Cadherin-CreER;ROSA26 animals, while they could rule in the
sinus venosus source for coronary ECs, the selective expression of
Cre under the control of VE cadherin and the relative timing of induc-
tion meant that they were unable to rule out the possibility that ECs,
either from a distinct source (PEO, endocardium) or arising as earlier
progenitors elsewhere and migrating to the sinus venosus, may con-
tribute to the building of the coronary tree.
2.4 Confounding avian rodent
species differences
Differences between the chick quail and mouserat studies may
reflect not only the outcome of different technologies applied, retro-
viral tracing in chick vs. genetic fate mapping in mouse, but also may be
an indictment on differences in the formation and patterning of the
vascular beds in avian compared with rodent hearts. A potentially uni-
fying theme, however, is that most, if not all, of the reports found in
the literature support the concept that the avian and mammalian PEO
contains some sort of vascular progenitor cell type or (haem-) angio-
blast. The specific embryological origin of this angioblast population
and whether it contributes as a whole or in part to the sinus
venosus ECs, or the endocardium, as an intermediate stage prior to
coronary vascularization (Figure 1B), remain to be determined and
are still open to speculation.
2.5 Cre-lox technology to dissect out
coronary origin
When addressing the question of origin, it is imperative to consider
the possible vagaries of fate mapping alongside the pitfalls of using
the favoured Cre-lox system in mouse, as weighted against species-
specific differences and possible heterogeneity in the origins of the
vascular lineages. In addition, there is a requirement to establish cri-
teria for optimal assessment of the progressive contribution of coron-
ary vascular cells from the earliest angioblast cell (if such a cell exists?)
to the differentiated arterial EC. From the outset, we require a wider
range of markers of the prospective tissue sources for coronary cells,
both to distinguish between alternate origins (Figure 1) and to facilitate
fate mapping at the earliest time points, thus addressing the question
of a common progenitor pool. In this regard, the so-called natural
history of genes we might adopt as tools for fate mapping becomes
important. Within a specific source, we require finer mapping to
Vascularizing the heart
identify progenitors which may have overlapping and/or distinct
expression profiles for specific markers and to identify a more com-
prehensive gene-set with which to speak to the entire heterogeneity
of cells within a source and to resolve the complete fate map.
In the case of the PEO, genetic markers which are expressed at an
early stage in PEO development and in cells entirely or partially dis-
tinct from previously described Tbx18-positive and Wt1-positive
populations are required to trace distinct subcompartments. One
approach towards identifying novel factors around the model of a
venous origin might be to build on the transcriptional code for sys-
temic vascular ECs defined by a composite cis-acting element, the
FOX:ETS motif.21 Thus, a suitable screen for coronary EC-specific
enhancers, which might act in concert with the FOX:ETS/ets
cluster, could be utilized to establish reporter lines and to identify
trans-acting factor(s) for use in Cre-lox lineage tracing. Subsequently,
selection of a specific gene locus and/or enhancer region to drive Cre
should be set against high-resolution in situ expression for the gene of
interest to ensure that there is no possibility of Cre being expressed
within the descendant ECs or in more than one putative source for
ECs to cloud interpretation on origin. Ideally, in any single study, mul-
tiple Cre lines should be employed to identify potentially distinct
sources for ECs and one must consider the efficacy of the Cre.
With variable efficacy comes differential sensitivity of individual
alleles to Cre-mediated recombination which limits the utility of
reporter alleles.22 This has important implications for interpreting
Cre fate mapping experiments with the potential for alternate repor-
ters defining narrower or broader daughter cell domains. Recently,
the widely used R26R reporter, based on the Rosa-26 locus, was
shown to be less susceptible to Cre in studies tracking Isl1- and
Nkx2.5-expressing progenitors in cardiogenesis than an alternative
Gata4-flap reporter, providing unique insight into the contribution
of Isl1 descendants to both primary and secondary heart fields and
Nkx2.5 descendants to the coronary endothelium and smooth
muscle.23 A further generic challenge with Cre-lox technology is
the issue of potential toxicology of Cre recombinase arising from
recombination at cryptic or pseudo-loxP sites within the genome hin-
dering the cellular DNA repair machinery and ultimately resulting in
apoptosis.22
As a minimum requirement, the Cre drivers should be inducible to
avoid issues with (i) spurious activation of Cre due to prior expression
in founder populations, (ii) detecting descendants of rare cells that
have activated Cre at an earlier time point, and (iii) ectopic expression
of Cre in ECs that does not reflect lineage contribution. Finally, the
timing of induction needs to be early enough so as to trace progeni-
tors which may contribute transiently to intermediate sources (e.g. a
potential PEO/epicardium contribution to the endocardium and sinus
venosus) en route to the coronaries (Figure 1).
2.6 Classical embryology, clonal
and transplant analyses
Even if safeguards are imposed against a misinterpretation of the
Cre-lox labelling, evidence that the coronary cells are indeed the
linear descendents of cells that were expressing the gene (employed
to drive Cre) in the specific source at earlier stages must be provided
to claim lineage tracing and fate mapping. Here, Cre-lox can be com-
plimented by other analyses. Returning to more classical embryology,
in vivo vital dye (DiI) injection and physical labelling of progenitors in
conjunction with whole-embryo culture remains a valid approach
263
and has been used successfully elsewhere in both chick and
mouse.24 Organ culture and tissue recombination, as previously
described,13 is potentially informative regarding a single origin but is
restricted against the breadth of potential sources in vivo as it is not
possible to ascertain whether all coronary ECs might arise from the
specific cultured tissues. Additional insight can arise from the
culture of neighbouring tissues where there may be a requirement
for secreted signals from one tissue to induce coronary EC outgrowth
and remodelling in another and, in such cases, co-culture may facilitate
the identification of signals by mass spectrometry or via a candidate
approach.25
Clonal analysis is a potentially powerful tool to map origin and
determine proliferation, outgrowth and fate of the coronary progeni-
tors in vivo. This analysis is, however, open to over-interpretation and
dependent upon inducible genetic labelling which can be biased from
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the outset, so as not to be inclusive of all possible sources.13 Here, the
solution might be to perform a time course of inductions to poten-
tially encompass all clones of vascular progenitors. A further issue
with clonal analysis is the documenting of a rare genetic recombina-
tion event and the tracking of a discrete number of clones on the
assumption that this stochastically reflects the entire lineage. In the
case of venous-to-arterial reprogramming, the frequency of arterial
EC clones arising from venous ECs was in fact quite low (around
13%)13 which could be interpreted to the effect that venous ECs
are not the primary source of coronary arteries, leaving open the
identity of the arterial antecedents.
A further complementary approach is engraftment, such that
labelled endothelial progenitors are transplanted into the embryo
to determine lineage potential and an EC fate within the developing
heart. This is, however, compounded by technical difficulties regarding
the isolation and transplantation procedure, propensity for phenoty-
pic drift during culture, and mode of delivery. Further caveats
include subsequent graft survival alongside a reduced exposure to
the appropriate in vivo signals which may be critical to determine
EC fate.
2.7 Defined sources of coronary vascular
smooth muscle
In stark contrast to the coronary endothelium, the VSMCs have fairly
well-defined origins, dependent on location within the developing
heart and function (reviewed in Luttun and Carmeliet26). Simply
put, VSMCs of the proximal and major anterior coronary arteries
arise from the neural crest and the rest derive from the PEO and
epicardium.27 In contrast, coronary vein VSMCs arise from an atrial
cardiomyocyte origin.28
3. Developmental programming
of vessels in the heart
In keeping with the prevailing dogma of a PEO/epicardium origin for
the coronary vasculature, there has been considerable focus on the
communication of signals between the PEO/epicardium, subepicardial
mesenchyme, and myocardium. These signals regulate coronary
development and myocardial growth and are mediated in part by
secreted growth factors which originate from the epicardium and
EPDCs or from cardiomyocytes and which sit at the top of hierarch-
ical signalling pathways to act in an autocrine or paracrine fashion.
Although some growth factors have been implicated in the formation
264
of vascular beds elsewhere during development, the pathways they
mediate within the heart are largely distinct from those that govern
vasculogenesis in the rest of the embryo (reviewed in Olivey and
Svensson29). What is evident is that reciprocal signalling between
lineages such as the PEO/epicardium, endocardium, and myocardium
ensures developmental coupling of coronary vessels with that of myo-
cardial growth (reviewed in Bhattacharya et al.30). Suffice to say that
TGF, FGF, VEGF, PDGF, BMP, Wnt/b-catenin and retinoic acid path-
ways have all been implicated in epicardialmyocardial or myocar-
dialepicardial signalling principally in support of PEO/epicardial EMT,
outgrowth, protrusion and adhesion of EPDCs plus the induction and
remodelling of the coronary plexus. For detailed accounts of growth
factor function and downstream signalling in coronary development,
refer to reviews elsewhere1,26,29 31 and references therein.
3.1 Signalling to specify arteriovenous
identity?
When considering the molecular control of vessel formation in the
developing heart, a key question is what are the signals which
specify venous vs. arterial identity? Physical forces such as blood
flow have been implicated in the regulation of EC fate. Coronary
veins develop before arteries and the latter have been considered
simplistically as a by-product of the recruitment of a medial layer of
VSMCs in response to pressure increase and blood flow alteration
after connection to the aorta.26 Hence, direct stretch and sheer
forces acting on the endothelium or VSMC differentiation and sub-
sequent smooth muscle paracrine signalling may instruct coronary
ECs to adopt an arterial fate, or at later stages ensure appropriate
arterial EC differentiation and functional maturation. A robust argu-
ment against altered haemodynamics as determinants of arterial EC
fate arises in the study proclaiming coronary artery formation by
developmental reprogramming of venous cells.13 In the model pro-
posed, sprouting sinus venosus ECs are initially differentiated with a
venous identity characterized by Ephb4 expression and other
venous markers such as Vegfr3, Np2, Aplnr and COUP-TFII but, as
angiogenesis extends beyond the sinus venosus, those vessels which
invade the myocardium begin to express ephrinB2 and other arterial
markers such as Dll4, Hey1, Notch4 and Depp. The timing of the
initial down-regulation of Ephb4 as the first stage in venous repro-
gramming towards arterial fate is E11.5 in the mouse which signifi-
cantly precedes arteriogenesis and the connection of the vascular
network to the aorta. This suggests that arterial specification is a pre-
programmed spatial event occurring in response to either positive
induction via signals from the myocardium/endocardium or reduced
exposure to inhibitory signals from the PEO/epicardium and sinus
venosus. However, this does not rule out a role for haemodynamics
in ensuring subsequent assembly into mature coronary arteries. The
identity of flow/pressure-dependent signals is currently unknown
but, outside of the coronaries, molecular readouts have been ele-
gantly defined in response to haemodynamic changes in the develop-
ing branchial arch arteries (BAAs). Blood flow in the left sixth BAA is
sufficient to stimulate PDGFR and VEGFR2 signalling to maintain
arterial structure whereas, with decreased blood flow in the right
sixth BAA, the consequence is arterial regression.32
3.2 Coronary artery induction by Notch
The molecular profile of coronary arterial ECs, which includes Dll4,
Hey1 and Notch4, implicates Notch signalling as a potential inducer
P.R. Riley and N. Smart
of arterial cell fate. Delta-Notch plays a central role in vertebrate
organogenesis per se and regulates multiple processes during cardiac
development, where it functions both cell autonomously and
non-cell autonomously pointing to a role as a signal coordinator
during cardiogenesis (reviewed in MacGrogan et al.33). Notch signal-
ling within the myocardium is known to be important for cardiomyo-
cyte differentiation, ventricular trabeculation and outflow tract
development, and there is also a critical role for Notch in the endo-
cardium, such that Notch1, via interplay with myocardial Bmp2,
restricts endocardial EMT to a valve-forming field.34 In avian coronary
vasculogenesis, active Notch1 (the Notch1 intracellular domain;
N1ICD) is present within the mesothelial cells of the PEO, the
mesenchymal cells of the epicardium and in the ECs within the
nascent vessel plexus at the atrioventricular junction, suggesting that
Notch1 is important in the chick for epicardial EMT and coronary
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progenitor cell differentiation.35 In mice, which lack Notch1 or have
a combined loss of the Notch targets, Hey1 and Hey2, the mutant
phenotype is characterized by cardiac defects and coronaries which
fail to express arterial markers, including ephrinB2. This suggests
that Hey1 and Hey2 are essential transducers of Notch signals and
may mediate arterial fate decision.36 Notch also functions at later
stages in coronary maturation and is up-regulated in response to
VEGF signalling to establish a Notch/ephrinB2 cascade and ensure
coronary arteriogenesis.15
3.3 Determinants of venous identity
and reprogramming?
Although Notch signalling may define coronary arterial ECs, we have
no current insight into the determinants of coronary venous fate or
the signals which instigate venous reprogramming in the process of
coronary artery formation. During remodelling of the systemic
primary capillary plexus in the early embryo, arteriovenous differen-
tiation involves suppression of Notch signalling in venous ECs by
the nuclear orphan receptor COUP-TFII which permits expression
of the venous marker EphB4.37 This seems to indicate a default arter-
ial EC fate in systemic vasculogenesis which requires active suppres-
sion. It is not known whether this is the case in early coronary
vessel development but is somewhat unlikely, given the recent pre-
cedent for venous EC differentiation as the earlier event within the
sinus venosus, followed by subsequent reprogramming towards an
arterial fate.13
In terms of venous reprogramming, although again untested in
terms of the coronary vasculature, during systemic vasculogenesis,
it is clear that the EphB4 receptor tyrosine kinase and its plasma
membrane-spanning ligand, ephrinB2, demarcate venous and arter-
ial domains, respectively.38 Moreover, a number of studies of
EphB4- and ephrinB2-null mice have revealed roles for forward
and reverse EphB4 ephrinB2 signalling in vascular morphogen-
esis.38 40 This establishes a molecular basis for bidirectional pro-
gramming of venous arterial EC fate; however, the exact
function and mode of action of bidirectional EphB4 ephrinB2 sig-
nalling remains unknown (reviewed in Adams and Alitalo41). An
understanding of the molecular signals which instruct dedifferentia-
tion and reprogramming of coronary vessels is an obvious priority
in terms of not only identifying pathways underlying coronary
development but towards the induction of new vessels in adult
cardiac repair.
Vascularizing the heart
4. Adult maintenance and
pathological remodelling
4.1 Maturation of the post-natal coronaries
The coronary vasculature continues to mature post-natally, growing
significantly in the first 2 weeks after birth to keep pace with 80%
increase in cardiac mass.42 Indeed, a 2.8-fold increase in capillary
growth occurs during the first 5 days of post-natal life, predominantly
by intussusceptive growth, i.e. splitting of pre-existing capillaries.43
FGFs play a key role in perinatal arterial development, such that pre-
cocious expression of FGF-2 following retroviral epicardial injection in
chicks causes abnormal branching of coronary vessels,44 whereas
anti-FGF-2-neutralizing antibodies, administered between 5 and 12
days after birth, inhibited arteriolar growth.42 Coronary artery remo-
delling is necessary to establish an effective hierarchy within the arter-
ial tree. Initiation of blood flow through the coronary system provides
the stimulus for remodelling the main arteries, inducing EC prolifer-
ation and up-regulating VEGF, integrin aVb3, PECAM-1 and
VE-cadherin45 to increase vessel diameter. Anti-VEGF neutralizing
antibody disturbed the normal arteriolar hierarchy, implying that
although FGF-2 appears to direct arteriolar growth, its interaction
with VEGF serves to establish a normal hierarchy of the coronary
tree, limiting growth of terminal arterioles and facilitating growth
and remodelling of upstream arterioles.42
4.2 Homeostatic maintenance and
turnover of adult endothelium
Once the mature coronary architecture is established, appropriate
angiogenic signalling is required thereafter for its maintenance.
Renewal of vascular cells, as and when required, is facilitated by pro-
genitor cell populations, derived either from bone marrow or resident
within the vessels or myocardium46 (further reviewed in Hibbert
et al.47). Even the healthy endothelium is turned over approximately
every 3 years48 and the cellular source for its renewal may not necess-
arily derive from the same embryonic lineage as the original. The
notion that vessels in the adult are derived only by angiogenesis,
and not by vasculogenesis as in the embryo, has now been superseded
following the acceptance that circulating EPCs, derived from bone
marrow, represent at least one source of vascular progenitors that
actively contribute to new vessel growth in the adult. Analogous in
many respects to the embryonic angioblast, EPCs are mobilized,
when required, from the bone marrow and incorporate into new
or injured vessels where they develop into mature ECs during the
processes of re-endothelialization and neovascularization (reviewed
in Smart and Riley49). The precise molecular definition of EPCs
across animal models, and including humans, is somewhat controver-
sial, although there is a consensus that their beneficial effects may also
be mediated, in part, through paracrine secretion of angiogenic factors
(Figure 2).
ECs ordinarily reside in a non-proliferative state throughout their
average lifespan of between 1 and 3 years.50 Despite their quiescent
appearance, ECs are highly metabolically active, playing key roles in
controlling vasomotor tone, blood cell trafficking, haemostatic
balance, permeability and immunity. The endothelium exerts effects
on both the surrounding VSMCs and blood cells to coordinately regu-
late tissue blood supply balancing vasodilation with vasoconstriction,
VSMC differentiation, inflammatory responses, maintenance of
blood fluidity and prevention of bleeding.51 A critical balance
265
between endothelium-derived paracrine factors appears to regulate
vascular homeostasis, and similarly, VSMCs rarely proliferate under
normal physiological conditions, rather they remain in a differentiated
contractile state.
4.3 Consequences of endothelial
dysfunction in vascular disease
In disease states leading to deterioration in endothelial function, a pro-
portion of VSMCs adopt a dedifferentiated, proliferative state leading
to pathological changes in the vascular walls, including a loss of cyto-
skeletal markers and their ability to respond to contractile stimuli. If
the processes of endothelial homeostasis and repair both fail, endo-
thelial damage ultimately leads to arterial stiffness (arteriosclerosis)
and lipid accumulation (atherosclerosis). Atherosclerosis is not
Downloaded from http://cardiovascres.oxfordjournals.org/ at rouen university hospital on February 9, 2013
simply an inevitable degenerative consequence of ageing, rather a
form of chronic inflammation.52 The inflammatory cytokines
interleukin-1, tumour necrosis factor-a and g-interferon transform
ECs to an active state. Activated ECs express leukocyte adhesion mol-
ecules and produce tissue factor to create a procoagulant environ-
ment which is augmented by accompanying vasoconstriction
through an up-regulation of endothelin-1 in a setting of diminished
nitric oxide (NO) production. This in turn is permissive for neointimal
proliferation, due to loss of the usual VSMC growth inhibitory factors,
and vascular occlusion.
Coronary artery occlusion may not always lead to infarction since
in two-thirds of patients with CAD, collateral arteries develop from
pre-existent interconnecting arterioles, by proliferation of VSMCs
and ECs, to bypass the stenosis.53 Fluid shear stress (FSS) is the
primary trigger for collateral growth and increased pulsatile FSS
leads to deformation of the endothelium and activation of all NO
synthase isoforms. NO production, followed by VEGF secretion,
induces monocyte chemoattractant protein-1 synthesis that attracts
monocytes and T cells to attach to the endothelium; these produce
proteases to digest the internal elastic lamina and extracellular
matrix and simultaneously induce a phenotypic change of ECs and
VSMCs into the synthetic and proliferative types. Following prolifer-
ation, vessels mature via orderly arrangement of the VSMCs in circular
layers, establishment of cell-to-cell contacts and synthesis of elastin
and collagen to provide scaffold for the larger vessel.
5. Neovascularization and bypass
engraftment
Although the phenomenon of collateral growth has long been recog-
nized, attempts to therapeutically stimulate these processes have
lagged behind. Capillaries that form by vasculogenesis, without
smooth muscle support, are unstable and regress over time.
Compared with vasculogenesis, arteriogenesis is more complex and
has proven more recalcitrant to therapy. Arteriogenesis is not
stimulated by the same signals, such as hypoxia, but rather by
haemodynamic forces, principally shear stress54; therefore, although
single genes or proteins successfully induce coronary angiogenesis,
the simultaneous activation of multiple growth factors is seemingly
required to stimulate arteriogenesis.55 Therapeutic methodologies
have, therefore, evolved to concomitantly activate several growth
factors, for example, by providing a metabolic stimulus, such as the
thyroxine analogue 3,5-diiodothyropropionic acid (DITPA).56 DITPA
administration to rats post-MI enhanced levels of VEGF, FGF-2,
266 P.R. Riley and N. Smart

Downloaded from http://cardiovascres.oxfordjournals.org/ at rouen university hospital on February 9, 2013

Figure 2 Cellular maintenance and repair of the post-natal coronary vasculature. Under conditions of vascular homeostasis (maintenance) in the
adult heart, circulating EPCs respond to a plethora of growth factor and chemokine signalling to home to the coronaries and replenish ECs. Following
arterial occlusion leading to vessel regression and MI, a subepicardial graft of c-kit+ cardiac progenitor cells (CPCs) is able to migrate to the site of
vascular injury and differentiate into de novo ECs and VSMCs to establish a bypass of the occlusion and reperfuse the injured myocardium.67 lad, left
anterior descending artery; mi, myocardial infarction; ANG1, angiopoietin 1; EPO, erythropoietin; FGF, fibroblast growth factor; HGF, hepatic growth
factor; HIF-1a, hypoxia factor-1a; SDF-1, stromal cell-derived factor-1; VEGF, vascular endothelial growth factor.

Ang-1, and Tie-2, leading to increased arteriolar density and improved
left ventricular function.
A natural consequence of arterial occlusion and ischaemia is the
establishment of a hypoxic environment which has significant effects
on the neovascular response. The regulation of angiogenesis by
hypoxia depends on a precise balance between positive and negative
angiogenic regulatory molecules. Increasing evidence points to the
existence of complex feedback networks such that factors generally
considered pro-angiogenic, such as HIF-1, additionally activate angio-
genic inhibitors including Regulator of G protein signalling 5 (RGS5)57
and Delta-like ligand 4 (Dll4)58 downstream of VEGF. The negative
feedback regulation of angiogenesis during hypoxia may explain not
only the inadequate natural collateral circulation in ischaemic heart
disease but also the disappointing and inconsistent results of clinical
trials.59 Consequently, identifying key feedback inhibitors may reveal
new targets for angiogenic therapy.
embryonic programme that created the tissue in the first instance.
As such, there is much to gain from understanding the embryonic
mechanisms of vasculogenesis (recently reviewed).1,49 The possibility
of a post-natal contribution from cardiac populations that play a
role during embryonic development is provided from studies in zeb-
rafish which suggest that EPDCs are incorporated into the heart not
only during regeneration, but also during continuous growth of the
adult heart.60 It remains to be determined whether the mammalian
epicardium contributes to coronary homeostasis but, if so, the con-
tribution is likely to be much reduced compared with the fish for at
least two reasons. The epicardial contribution in zebrafish may be
peculiar to the continued organ growth and hyperplasia which is
an adaptive precedent in this species and, moreover, in mammals
even in the injury setting, an exogenous stimulus, such as Thymosin
b4, is required to fully reinstate embryonic potential to adult
EPDCs61 and enable neovascularization of the ischaemic adult
heart.62

5.1 Developmental programming to 5.2 Adult progenitor cell vascular repair

facilitate cell-based neovascularization Recent studies have recognized the ability of a multitude of cardiac-
An evolving paradigm in regenerative medicine is that tissue repair and vessel-resident progenitor cell populations to engraft into sites
in the adult is frequently underpinned by a re-activation of the of vascular injury or homeostatic replenishment (reviewed in
Vascularizing the heart
Hibbert et al.47). Along similar lines, the discovery that circulating
EPCs participate in homeostatic neovascular repair has led to many
animal studies (reviewed in Miller-Kasprzak and Jagodzinski63) and ulti-
mately clinical trials to enhance EPC mobilization from bone marrow
to effect neovascularization, with promising preliminary results.64,65
With regard to arteriogenic therapy, EPCs would appear inferior to
those progenitor cells that possess a broader differentiation potential
that also includes pericytes or smooth muscle cells, given the need for
mural cell stabilization of vessels. Smooth muscle progenitor cells
were more recently identified in bone marrow, circulating blood
and vascular adventitia66 and may prove more successful for coronary
neovascularization. More recently, c-kit+ cardiac progenitor cells
were shown to differentiate into ECs and SMCs to form conductive
(up to 250 mm) and intermediate-sized coronary arteries together
with resistance arterioles and capillaries. These new vessels con-
nected with the primary circulation to more than double myocardial
flow following MI and thereby constitutes an example of a biological
coronary bypass67 (Figure 2).
5.3 Tissue engineering of vascular grafts
A natural progression from cell-based therapy has been the develop-
ment of tissue engineering techniques. New vessel formation and
improvements in neovascularization have been reported in hindlimb
ischaemia using human smooth muscle cell sheets68 or Matrigel
plugs seeded with a combination of human endothelial and cord
blood-derived mesenchymal progenitor cells.69 For coronary angio-
genesis, in vitro engineered three-dimensional neonatal cardiomyocyte
sheets containing preformed EC networks were transplanted onto
infarcted rat hearts, resulting in a significantly elevated capillary
density with blood vessels originating from the cardiac patch connect-
ing with host capillaries.70
6. Future perspectives
Our understanding of embryonic and post-natal coronary vasculature
development has brought us some way towards developing effective
strategies to promote neovascularization in cardiovascular repair.
Numerous outstanding questions remain regarding the lineages that
contribute the cells of the coronary vasculature, the precise mechan-
isms that govern vascular cell fate, and the regulation of arteriovenous
identity, vessel growth, and maintenance. However, further insight
into vascularizing the heart during development should pave the
way for significant advances in neovascular therapy.
Conflict of interest: none declared.
Funding
P.R.R. is supported by grants from the British Heart Foundation and
Medical Research Council, and N.S. is a recipient of a British Heart Foun-
dation Intermediate Basic Science Research Fellowship.
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