RESEARCH ARTICLE 1475
Development 140, 1475-1485 (2013) doi:10.1242/dev.087601
© 2013. Published by The Company of Biologists Ltd
Coronary veins determine the pattern of sympathetic
innervation in the developing heart
Cardiac tissues are highly vascularized and extensively innervated
by autonomic nerves. Abnormal patterning and distribution of the
coronary vasculature is often associated with congenital heart
disease (Kayalar et al., 2009), while impairment of autonomic
functions can lead to lethal arrhythmia (Hildreth et al., 2009).
Previous studies have shown neurovascular interactions in other
tissues to be crucial in the development of both nerves and
vasculature (reviewed by Carmeliet and Tessier-Lavigne, 2005;
Larrivée et al., 2009). The importance of these structures in cardiac
development and homeostasis led us to examine the possibility of
coordinated development in the murine heart.
Coronary vasculature develops from an existing primary capillary
plexus via a remodeling process known as angiogenesis (reviewed
by Lavine and Ornitz, 2009). During angiogenic remodeling,
endothelial cells reorganize to form a hierarchical branching
network, and larger vessels recruit vascular smooth muscle cells
(VSMCs). Recent studies in mice have revealed that large diameter
coronary veins develop close to the epicardial surface layer (the
subepicardium), whereas coronary arteries arise separately in the
deeper myocardial layer (Lavine et al., 2008; Red-Horse et al.,
2010). Early studies in avian and murine models demonstrated that
the epicardium, which is derived from the proepicardium, an
extracardiac rudimentary organ, gives rise to coronary VSMCs and
provides pro-angiogenic factors such as fibroblast growth factors
(FGFs) and vascular endothelial growth factors (VEGFs) (reviewed
by Lavine and Ornitz, 2009).
Sympathetic innervation of the heart originates in the stellate
ganglia. Previous studies have shown that arterial VSMCs mediate
proximal sympathetic axon extension by secretion of artemin
(Enomoto et al., 2001; Honma et al., 2002), neurotrophin 3 (Francis
et al., 1999; Kuruvilla et al., 2004) and endothelins (Makita et al.,
2008). Although proximal extension out of the ganglia is well
characterized, the mechanisms responsible for distal extension to
reach target cells remain elusive. In distal axon extension, nerves
adopt a stereotypical pattern in target tissues prior to innervating
final target cells. Nerve growth factor (NGF) is required for terminal
sympathetic innervation of target tissues (Glebova and Ginty, 2004).
Mutants lacking Ngf and Bcl2-associated X protein (Bax) have
normal sympathetic axon extension along the extracardiac
vasculature but sympathetic innervation is drastically decreased in
the heart. This concomitant knockout of the pro-apoptotic factor
Baxallows neurons to survive in the absence of NGF, demonstrating
that NGF plays a role in distal cardiac sympathetic axon growth that
is distinct from its role in survival (Glebova and Ginty, 2004).
However, the precise origin and function of NGF during cardiac
innervation remain to be examined.
We found anatomical congruence between nerves and coronary
vessels in the developing heart. Beginning at embryonic day (E)
13.5, sympathetic axons extend along developing large diameter
coronary veins in the dorsal subepicardium of the ventricles. By
E15.5, these axons extend across the entire dorsal surface and
subsequently penetrate the dorsal myocardium while others begin to
reach the ventral subepicardium. Mutant analyses indicate that this
neurovascular association is important for proper cardiac
innervation but not for coronary vascular patterning. In vitro and in
1Laboratory of Stem Cell and Neuro-Vascular Biology, Genetics and Developmental
Biology Center, National Heart, Lung, and Blood Institute, National Institutes of
Health, Building 10/6C103, 10 Center Drive, Bethesda, MD 20892, USA.
2Development and Aging Program, Sanford-Burnham Medical Research Institute,
10901 North Torrey Pines Road, La Jolla, CA 92037, USA. 3Department of
Neuroscience, The Johns Hopkins University School of Medicine, 725 North Wolfe
Street, Baltimore, MD 21205, USA. 4Pediatric Cardiology, Stanford University School
of Medicine, 300 Pasteur Drive, Palo Alto, CA 94305, USA.
*These authors contributed equally to this work
‡Author for correspondence (email@example.com)
Accepted 25 January 2013
Anatomical congruence of peripheral nerves and blood vessels is well recognized in a variety of tissues. Their physical proximity and
similar branching patterns suggest that the development of these networks might be a coordinated process. Here we show that
large diameter coronary veins serve as an intermediate template for distal sympathetic axon extension in the subepicardial layer of
the dorsal ventricular wall of the developing mouse heart. Vascular smooth muscle cells (VSMCs) associate with large diameter veins
during angiogenesis. In vivo and in vitro experiments demonstrate that these cells mediate extension of sympathetic axons via nerve
growth factor (NGF). This association enables topological targeting of axons to final targets such as large diameter coronary arteries
in the deeper myocardial layer. As axons extend along veins, arterial VSMCs begin to secrete NGF, which allows axons to reach target
cells. We propose a sequential mechanism in which initial axon extension in the subepicardium is governed by transient NGF expression
by VSMCs as they are recruited to coronary veins; subsequently, VSMCs in the myocardium begin to express NGF as they are recruited
by remodeling arteries, attracting axons toward their final targets. The proposed mechanism underlies a distinct, stereotypical pattern
of autonomic innervation that is adapted to the complex tissue structure and physiology of the heart.
KEY WORDS: NGF, Cardiac innervation, Coronary development, Sympathetic axons, Vascular smooth muscle
Joseph Nam1,*, Izumi Onitsuka1,*, John Hatch1,*, Yutaka Uchida1, Saugata Ray2, Siyi Huang3, Wenling Li1,
Heesuk Zang1, Pilar Ruiz-Lozano2,4and Yoh-suke Mukouyama1,‡
vivo experiments further demonstrate that epicardium-derived
venous VSMCs direct the extension of sympathetic axons via
secretion of NGF during their recruitment to coronary veins. As
venous remodeling completes, subepicardial VSMCs downregulate
NGF expression. Subsequently, myocardial VSMCs begin to
express NGF during arterial remodeling, stimulating axon extension
towards final target cells in that layer. Our data suggest a model in
which large diameter coronary veins serve as an intermediate
template for sympathetic axon outgrowth. This template appears to
ensure a proper distribution of sympathetic axons for eventual
innervation of target cells in the myocardium. At the molecular
level, sequential expression of NGF in subepicardial venous
VSMCs and myocardial arterial VSMCs is responsible for a two-
step process of distal axon extension and subsequent innervation of
MATERIALS AND METHODS
Characterization of ephrinB2taulacZ/+(Wang et al., 1998), EphB4taulacZ/+
(Gerety et al., 1999), ChATBAC-eGFP (Tallini et al., 2006), SM22αlacZ/+
(Zhang et al., 2001; Walker et al., 2005), Phox2b−/−(Pattyn et al., 1999)
and Gata5-Cre; β-cateninflox/flox(Zamora et al., 2007) mice has been
reported elsewhere. NGFlacZ/+knock-in mice were generated in David
Ginty’s laboratory at Johns Hopkins University by homologous
recombination in ES cells according to standard procedures. All
experiments were carried out according to the guidelines approved by the
Animal Care and Use Committee at NHLBI.
Whole-mount immunohistochemistry of embryonic hearts
Whole-mount immunohistochemical staining of embryonic hearts was
performed essentially as described previously (Mukouyama et al., 2002).
Embryonic hearts were dissected and fixed in 4% paraformaldehyde/PBS
overnight at 4°C. The antibodies used were: anti-PECAM1 (clone
MEC13.3, BD Pharmingen, 1:300) to detect endothelial cells; anti-SM22α
(Abcam, 1:250) and Cy3-conjugated anti-αSMA (clone 1A4, Sigma, 1:500)
to detect VSMCs; anti-β-tubulin (βIII) (clone TuJ1, Covance, 1:500) to
detect nerve fibers; anti-CGRP (Millipore, 1:500~1000) to detect sensory
neurons; anti-TH (Novus Biologicals, 1:500~1000) to detect sympathetic
neurons; and anti-β-galactosidase (MP Biomedicals, 1:5000) to detect lacZ
expression. Different combinations of Alexa 488-, Alexa 568-, Cy3- or
Dylight 649-conjugated secondary antibodies (Invitrogen or Jackson, 1:250)
were used for staining. Confocal microscopy analysis was carried out on a
Leica TCS SP5 confocal microscope.
Fresh embryos were embedded in OCT compound (Sakura), followed by
cryosectioning into 6-8 µm sections and collected on precleaned slides
(Matsunami, Japan). Staining was performed using the following antibodies:
anti-NGF (Millipore, 1:200); anti-ARTN (R&D, 1:250); anti-β-gal (1:5000);
anti-EDN1 (Abbiotec, 1:250); anti-GMFβ(ProteinTech, 1:100); anti-GMFγ
(ProteinTech, 1:250); anti-NRG1 (R&D, 1:250); anti-PECAM1 (1:300);
anti-SM22α (1:250) and Cy3-conjugated anti-αSMΑ (1:500). For
immunofluorescence detection, Alexa 488-, Alexa 568-, Cy3- or Dylight
649-conjugated secondary antibodies (Invitrogen or Jackson, 1:250) were
RNA in situ hybridization analysis
In situhybridization analysis was performed as described previously (Wang
et al., 1998). The probes were amplified using the primers listed in
supplementary material Table S1. The hybridization signal was detected
using an alkaline phosphatase-conjugated anti-digoxigenin antibody and
Total RNA was purified from tissues and cultured cells using Trizol Reagent
(Invitrogen) followed by reverse transcription into first-strand cDNA using
the SuperScript III First-Strand Synthesis Supermix kit (Invitrogen)
according to the manufacturer’s instruction. Quantitative mRNA expression
analysis of chemokines and their receptors in E13.5 dorsal root ganglia and
forelimbs was performed with the Mouse Chemokine and Receptor RT2
Profiler PCR Array (Qiagen) on a 7500 real-time PCR system (Applied
Biosystems) using RT2SYBR Green qPCR Master Mix (Qiagen). The
results of mRNA expression of Ngf,Artn,Edn1,Gmfb,Gmfg andNrg1were
confirmed by semi-quantitative RT-PCR (supplementary material Table S1).
Preparation of coronary VSMCs from primary fetal epicardial
Primary fetal epicardial cells were obtained as a source of coronary VSMCs
(Rhee et al., 2009). Heart ventricles were dissected from E12.5 or E13.5
embryos and cultured in 1% collagen type I gel (rat tail collagen, BD)
containing αMEM (Invitrogen), 10% FBS (Hyclone), 10 ng/ml bFGF
(FGF2; NCI BRB Preclinical Repository) and 10 ng/ml EGF (PeproTech).
The ventricles were removed from the gel, and migrated epicardial cells
were harvested with a 0.1% collagenase treatment. Isolated epicardial cells
were further cultured on a type IV collagen-coated dish (BD) with 10% FBS
in DMEM (Invitrogen) containing 10 ng/ml bFGF and 10 ng/ml EGF.
Epicardial cells differentiated to VSMCs in response to serum and/or 50
ng/ml TGFβ1 (PeproTech). Some cultures were infected with Ngfor control
shRNA lentiviruses (3×105transducing units shRNA lentivirus particles for
1×105VSMCs; Sigma). The effect of shRNA knockdown was confirmed by
RT-PCR analysis. To generate a VSMC aggregation for co-culture with
sympathetic ganglia explants, a hanging drop culture method was used.
Primary sympathetic ganglia explant co-culture and staining
Sympathetic ganglia (SG) were dissected from E13.5 embryos and cultured
on 1% collagen type I gel (Makita et al., 2008). SG were then cultured on 1%
collagen type I gel containing αMEM, 10% FBS and 0.3% sodium
bicarbonate (Invitrogen). Some SG explant cultures were supplemented with
20 ng/ml ARTN (R&D), 100 ng/ml EDN1 (R&D), 20-100 ng/ml GMFβ
(PeproTech), 20-100 ng/ml GMFγ (PeproTech), 25 ng/ml NGF (Upstate) and
100 ng/ml NRG1 (R&D). NGF- (2.5 µg/ml in PBS) or PBS-soaked heparin-
agarose beads (Bio-Rad) were placed next to SG explants. Cultures were
incubated in the CO2incubator for 2 days. For co-culture with VSMCs, SG
explants were placed next to VSMC aggregations on 1% collagen type I gel.
Some co-cultures were treated with the neutralizing antibodies 10 µg/ml anti-
ARTN (R&D), 200 ng/ml anti-NGF (R&D) or 20 µg/ml anti-NRG1 (R&D)
and EDN1 receptor-selective antagonists (2 µM BQ123 and 200 nM BQ788,
Sigma). We established that each inhibitor successfully blocks the effect of its
factor on SG explants. Fetal epicardial and myocardial tissues were dissected
from the cardiac ventricles of E13.5 or E16.5 embryos. Both tissues were
independently cultured with SG explants on 1% collagen type I gel. Staining
was performed using anti-SM22α antibody, αSMA antibody, or anti-TUJ1
antibody in combination with the pan-nuclear marker To-Pro-3 (Invitrogen).
Volocity software (PerkinElmer) was used to quantify axon outgrowth from
confocal images of TUJ1+SG axons in the co-culture experiments. SG
explants were divided into four quadrants (see Fig. 6F), intersecting at the
center of the explant. Axon outgrowth was quantified by comparing total axon
length and number of projections between the quadrants facing and opposite
the VSMC aggregates or myocardium. Statistical significance was assessed
using Student’s t-test.
In ovo implantation
Heparin-agarose beads (Bio-Rad) were soaked for 3 hours in 10 µg/ml isotype
IgG or NGF-neutralizing antibody (R&D) or in 2.5 µg/ml NGF or BSA. The
beads were implanted on the dorsal surface of E6 chick hearts. Chick embryos
were harvested at E10 and the hearts were immunostained with anti-αSMA
antibody and anti-TUJ1 antibody in whole-mount.
Cardiac sympathetic axons associate with large
diameter coronary veins within the
In order to examine the anatomical architecture of cardiac nerves
and coronary vasculature, we developed a whole-mount
immunohistochemistry method for the mouse embryonic heart.
Development 140 (7)
Double staining with antibodies specific for PECAM1, a pan-
endothelial cell marker, and the neuronal marker class III β-tubulin
(TUJ1) revealed the structure of coronary vasculature and the extent
of cardiac innervation at E15.5 (Fig. 1A). Three large diameter
vascular branches (25-100 µm) were apparent in the subepicardial
layer of the dorsal wall of the ventricles: the right cardiac vein
(RCV), the medial branch of the left cardiac vein (mLCV), and the
lateral branch of the left cardiac vein (lLCV) [Fig. 1A?; as referred
to by Ciszek et al. (Ciszek et al., 2007)]. All three vessels associated
with TUJ1+axons (Fig. 1A,A?). Notably, magnified images showed
large diameter vessels and axons in close proximity (0-200 µm) with
strikingly similar branching patterns (Fig. 1B-D?). We quantified
the extent of nerve-vessel association: the dorsal surface of the
ventricles was divided into 17 radial sections (termed A-Q). In each
section, the relative axon density was calculated (number of axon
end points/surface area; Fig. 1E). The quantification revealed that
axons are significantly more likely to project to regions near large
diameter vessels (Fig. 1F).
We further characterized the structures found in our initial
staining. EphB4taulacZ/+embryos allowed us to visualize the venous
marker EPHB4, while ephrinB2taulacZ/+embryos were used to image
the arterial marker ephrin B2 (Wang et al., 1998; Gerety et al., 1999;
Mukouyama et al., 2002). At E15.5, EPHB4+coronary veins were
present in the subepicardial layer of the dorsal ventricular wall
(Fig. 2A-D), whereas ephrin B2+coronary arteries were found in
the myocardial layer (Fig. 2E-H). This distribution is consistent with
that reported in previous studies (Lavine et al., 2008; Red-Horse et
al., 2010). Cardiac axons were detected in close proximity to large
diameter vessels in the subepicardium but not in the myocardium,
indicating that cardiac axons associate only with EPHB4+large
diameter coronary veins at this stage (Fig. 2A,C,I).
In order to characterize the neuronal subtypes present at E15.5, we
used calcitonin gene related peptide (CGRP; CALCA – Mouse
Genome Informatics) as a marker for sensory neurons, tyrosine
hydroxylase (TH) as a marker for sympathetic neurons, and choline
acetyltransferase (ChAT) as a marker for parasympathetic neurons.
Immunohistochemical staining for these markers revealed that the
majority of axons in the dorsal ventricular subepicardium are TH+
(Fig. 2J-M). A smaller number of ventricular axons were ChAT+
(supplementary material Fig. S1), and no CGRP+axons were
detected. Consistent with these results, other studies have found that
CGRP+sensory innervation is barely detectable at E15.5 but appears
by E18.5 (Ieda et al., 2006). These data indicate that autonomic
innervation precedes sensory innervation in the developing heart, and
that the initial neurovascular interactions during cardiac development
are restricted to coronary veins and sympathetic nerves.
Patterning of cardiac nerves
Fig. 1. Cardiac nerves align with large diameter
coronary vessels in the dorsal ventricular wall of
the developing heart. (A-D? ?) The dorsal face of the
cardiac ventricles of an E15.5 mouse embryo.
Whole-mount double immunofluorescence
confocal microscopy using antibodies to the pan-
endothelial marker PECAM1 (A-D, red; A?-D?, white)
and the neuronal marker class III β-tubulin (TUJ1,
green) reveals that TUJ1+cardiac axons (A,A?) follow
three remodeled large diameter coronary vessels:
the right cardiac vein (RCV), the medial branch of
left cardiac vein (mLCV), and the lateral branch of
left cardiac vein (lLCV) (A, open arrowheads; A?,
pseudocolored in red). Magnified images (B-D?) of
the boxed regions in A clearly demonstrate the
physical proximity of TUJ1+cardiac axons (B-D?,
arrows) and large diameter vessels (B-D, open
arrowheads; B?-D?, pseudocolored in red).
(E,F) Quantification of nerve-vessel association. In 17
radial sections of the dorsal ventricular wall (termed
A-Q, see A?), axons most commonly project to
regions containing RCV, mLCV or lLCV. *P<0.01
(Student’s t-test); n=7; error bars indicate s.e.m. RV,
right ventricle; LV, left ventricle. Scale bars: 100 μm.
Coronary remodeling is required for normal
To investigate the mechanism of the interaction between large
diameter coronary veins and sympathetic nerves, we examined the
temporal pattern of development for both networks. At E13.5, a
primary capillary plexus covered the entire dorsal surface of the
heart. Remodeled vessels and sympathetic axons were present only
in a small area adjacent to the sinus venosus on the dorsal
ventricular surface (supplementary material Fig. S2A). By contrast,
the ventral surface exhibited an expanding vascular plexus and no
detectable innervation (supplementary material Fig. S2D). By
E14.5, vascular remodeling progressed dramatically (supplementary
material Fig. S2B); all three large diameter branches were
discernable and extended across the full subepicardial layer of the
dorsal ventricular wall. Sympathetic axon extension, however,
continued along large diameter vessels through E14.5 and did not
reach the distal regions of the dorsal surface until E15.5
(supplementary material Fig. S2B,C). By this stage, the ventral
surface began to display some remodeled vessels but sympathetic
axons were still mostly absent (supplementary material Fig. S1E,F).
After E15.5, sympathetic axons continued to associate with large
diameter vessels in the dorsal surface (supplementary material Fig.
S2G,H), and the ventral surface exhibited a similar pattern of
vascular remodeling followed by innervation (supplementary
material Fig. S2I,J). Importantly, vascular remodeling preceded
sympathetic innervation throughout the subepicardium.
By postnatal day (P) 5, sympathetic axons extended into the
myocardial layer of the dorsal ventricular wall. These axons
innervated large diameter coronary arteries (supplementary material
Fig. S2L; Fig. 2I). Notably, in the subepicardial layer, no obvious
sympathetic innervation of veins was detectable despite congruent
branching of sympathetic nerves and large coronary veins
(supplementary material Fig. S2K). These findings suggest that
sympathetic axons extend within the subepicardium using large
diameter coronary veins only as an intermediate template en route
to their final targets in the myocardium, such as coronary arteries.
Because vascular remodeling precedes axon extension, we
examined whether the pattern of vascular remodeling affects that of
innervation. The stereotypical pattern of the coronary vasculature is
disrupted in conditional β-catenin (Ctnnb1) mutant mice that lack β-
catenin expression in the epicardium (Zamora et al., 2007). Systemic
ablation of the β-catenin transcription factor causes lethality early in
embryonic development, but conditional deletion using Gata5-Cre
allows survival until at least E18.5 (Zamora et al., 2007). In Gata5-
Development 140 (7)
Fig. 2. Cardiac sympathetic axons associate with large
diameter coronary veins within the subepicardial
layer of the dorsal ventricular wall.
(A-H) The dorsal ventricular walls of E15.5 EphB4taulacZ/+
(A,C,E,G; the venous marker EPHB4) or ephrinB2taulacZ/+
(B,D,F,H; the arterial marker ephrin B2) hearts are shown.
Whole-mount triple immunofluorescence confocal
microscopy was performed with antibodies to PECAM1
(A,B,E,F, blue), TUJ1 (A-H, green) and β-galactosidase (A-H,
red). Boxed regions in A-F,H are magnified in insets. (A-D)
The subepicardium. Coronary veins expressing
EphB4taulacZare clearly visible in EphB4taulacZ/+embryos
(A,C). However, arteries expressing ephrinB2taulacZare
barely detectable in ephrinB2taulacZ/+embryos (B,D). TUJ1+
cardiac axons (A,C, arrows) associate with EPHB4+large
diameter veins (A,C, open arrowheads). (E-H) The
myocardium. Coronary arteries expressing ephrinB2taulacZ
cover the deeper layer in ephrinB2taulacZ/+embryos (F,H).
One large diameter artery runs from the base towards the
apex of the ventricle (E,F,H, insets, arrowheads).
EphB4taulacZ-expressing veins are barely detectable in
EphB4taulacZ/+embryos (E,G). TUJ1+cardiac axons are also
not detected in the myocardial layer (E-H). (I) Schematic
illustrating sympathetic innervation of the developing
heart. By E15.5, coronary veins develop to form large
diameter branches within the subepicardial layer (Subepi),
where cardiac axons initiate distal axon extension.
Coronary arteries develop separately, in the myocardial
layer (Myo). By P5, cardiac axons extend into the
myocardial layer (see supplementary material Fig. S2).
These axons innervate large diameter coronary arteries as
final targets. Epi, epicardial layer; V, vein; A, artery.
(J-M) Neuronal subtype characterization. E15.5 hearts
were labeled with antibodies to the sympathetic neuron
marker tyrosine hydroxylase (TH; J,K, green) or the sensory
neuron marker calcitonin gene related peptide (CGRP;
L,M, green) in addition to PECAM1 (J,L, blue) and TUJ1 (J,L,
red). TUJ1+nerves are mostly TH+, indicating that these
axons in the subepicardium are mostly sympathetic
nerves (J,K, arrows). CGRP+sensory innervation is not
detectable at E15.5 (L,M, arrows). Scale bars: 100 μm.
Cre; β-cateninflox/floxmice, the coronary vasculature is disorganized
relative to the stereotypical pattern found in control littermates
(Fig. 3A-D). In addition, SM22α+VSMCs associated less strongly
with large diameter veins in these mutants and were distributed more
uniformly throughout the subepicardium (Fig. 3E,F). The unusual
pattern of VSMCs indicates that critical steps of angiogenic
remodeling have been disrupted. Notably, these coronary defects were
accompanied by abnormal sympathetic innervation (Fig. 3G). In the
mutants, sympathetic axons failed to fully innervate the subepicardial
layer of the dorsal ventricular wall (Fig. 3A,B). Some sympathetic
axons still appeared to associate with abnormally branched large
diameter coronary veins, albeit to a lesser extent than in the control
(Fig. 3B). By contrast, examination of Phox2b−/−mutants with
defective cardiac innervation showed no vascular abnormalities
(supplementary material Fig. S3). These results suggest that proper
coronary remodeling is required for the observed neurovascular
association, whereas cardiac axon growth does not affect the pattern
of vascular remodeling.
VSMC distribution closely mirrors sympathetic
The recruitment of VSMCs to large diameter vessels is a significant
step during angiogenic remodeling. In addition, previous studies
have demonstrated that VSMCs are a likely source of growth factors
that can act on sympathetic axons (reviewed by Glebova and Ginty,
2005). Indeed, SM22α+VSMCs were found predominantly in
nerve-associated large diameter veins at E15.5 (Fig. 4A-C?). This
distribution was confirmed with immunohistochemical staining of
SM22α-lacZ embryonic hearts, which have a lacZ reporter to detect
expression of SM22α (Tagln – Mouse Genome Informatics) (data
not shown). It is also important to note that the distribution of
SM22α+VSMCs is strikingly similar to that of axons. As with
axons, the majority of VSMCs are found around remodeled vessels,
but a small number can be found in regions between these branches
(Fig. 4D,D?). These data suggest that subepicardial SM22α+
VSMCs might be involved in signaling between veins and
Coronary VSMCs secrete a diffusible signal that
influences the pattern of sympathetic axon
growth in vitro
Impairment of sympathetic innervation in Gata5-Cre; β-
cateninflox/floxmutants provides strong evidence that vascular
remodeling influences axon extension. However, abnormal signals
from VSMCs in disrupted coronary veins might not be solely
responsible for defects in innervation in these mutants. Atypical
Patterning of cardiac nerves
Fig. 3. Defective coronary development leads to
abnormal sympathetic innervation. (A-D) Dorsal
ventricular walls of Gata5-Cre; β-cateninflox/floxmutants (B,D)
and control littermates (A,C) at E15.5. Double
immunofluorescence confocal microscopy was performed
with antibodies to PECAM1 (A,B, red; C,D, white) and TUJ1
(A-D, green). In Gata5-Cre; β-cateninflox/floxmice the pattern
of coronary remodeling appears disorganized compared
with control littermates (A versus B, open arrowheads; C
versus D, pseudocolored in red). The mutants also exhibit
abnormal sympathetic innervation (A versus B, arrows).
Both large diameter veins and sympathetic axons fail to
fully develop in the subepicardium. (E,F) Vascular smooth
muscle cell (VSMC) recruitment. Triple staining with
antibodies to the VSMC marker SM22α (E,F, red) in addition
to PECAM1 (E,F, blue) and TUJ1 (E,F, green) revealed that
SM22α+VSMCs associate less strongly with large diameter
veins in these mutants; SM22α+VSMCs are distributed
more uniformly throughout the subepicardium, indicating
defects in angiogenic remodeling (E versus F, open
arrowheads, G). (G) Quantification of nerve-vessel
association. The length of large diameter vessels and of
sympathetic axons is significantly affected in Gata5-Cre; β-
cateninflox/floxmutants. Length is measured as a percentage
of distance from base to apex. Control littermates, n=3;
Gata5-Cre; β-cateninflox/floxmutants, n=3; error bars indicate
s.e.m. Scale bars: 100 μm.
sympathetic innervation might instead be secondary to signal
defects caused by leaky activity of Gata5-Cre expression in the
myocardium. Therefore, we turned to in vitro culture experiments
to directly examine whether coronary VSMCs guide sympathetic
We initially isolated coronary VSMCs and sympathetic ganglia
(SG) from embryos at E13.5, the stage at which sympathetic axons
begin to innervate the subepicardial layer (supplementary material
Fig. S2A). Since coronary VSMCs originate from epicardial cells
(Mikawa and Fischman, 1992; Cai et al., 2008; Zhou et al., 2008),
we dissected cardiac ventricles from E12.5 or E13.5 embryos and
cultured them to isolate migrating epicardial cells on a collagen gel
(Fig. 5A). VSMCs were obtained from the differentiation of isolated
epicardial cells and VSMC aggregates were generated in hanging
drop culture (Fig. 5A). We confirmed the identity of the cultured cells
by immunostaining for VSMC markers such as SM22α, αSMA and
SM-MHC (Fig. 5B,C). SG explants cultured alone demonstrated
minimal axon outgrowth (supplementary material Fig. S4A-D). By
contrast, SG explants cultured with VSMC aggregates showed robust
axon outgrowth (Fig. 5D). Axons projected extensively and
preferentially towards VSMC aggregates (Fig. 5D-H, compare 5E
with 5F). These results demonstrate that coronary VSMCs secrete a
diffusible factor that promotes sympathetic axon outgrowth.
Additionally, we examined whether myocardial tissue was also
able to induce directional sympathetic axon outgrowth. Explants of
E13.5 myocardial tissue failed to stimulate axon outgrowth from
both E13.5 and E16.5 SG explants (Fig. 5I,J,M,N). By contrast,
E16.5 myocardial tissue explants successfully induced directional
axon outgrowth from both E13.5 and E16.5 SG explants (Fig. 5K-
N). These results are consistent with our timecourse analysis, which
shows that sympathetic innervation of the myocardium begins after
E15.5 (supplementary material Fig. S2A). Furthermore, primary
epicardial tissue explants did not promote directional axon extension
from SG explants (supplementary material Fig. S4E). These
experiments demonstrate that coronary VSMCs, but not early
myocardial or epicardial tissues, secrete a neurotrophic signal that
mediates sympathetic axon extension in the subepicardium.
Subsequently, cells in myocardial tissue provide a signal that directs
sympathetic axons into the deeper myocardial layer.
Coronary venous VSMC-derived NGF mediates
sympathetic axon extension
We next used our SG and VSMC co-culture system to identify a
coronary VSMC-derived signal that is responsible for sympathetic
axon growth. First, we surveyed differential expression of candidate
neurotrophic factors and receptors (84 genes in total) from coronary
VSMCs relative to myocardial tissues from E13.5 hearts using an
RT-PCR array method. We found that artemin (Artn), endothelin 1
(Edn1), glia maturation factors β and γ (Gmfb and Gmfg),
neuregulin 1 (Nrg1) and nerve growth factor (Ngf) were more highly
expressed in VSMCs than in myocardial tissues (data not shown).
Further, a semi-quantitative RT-PCR analysis confirmed the
differential expression of Artn, Gmfb, Gmfg, Nrg1 and Ngf between
VSMCs and myocardial tissues (Fig. 6A). Of these six candidate
factors, NGF expression was clearly detected in coronary VSMCs
in the subepicardium by immunohistochemical staining
(Fig. 6B,D,E) and in situ hybridization (Fig. 6C). These
observations were supported by the analysis of an NGFlacZ/+
reporter strain to identify NGF-expressing cells (Fig. 6F).
Expression of ARTN and NRG1 was also detected in coronary
VSMCs (supplementary material Fig. S5A-D,Q-T; data not shown).
The remaining candidates
immunohistochemical staining or in situ hybridization analysis
(supplementary material Fig. S5E-P; data not shown).
We next examined which factors can promote axon outgrowth
from SG explants in vitro. E13.5 SG explants were responsive to
ARTN, EDN1, NRG1 and NGF, but displayed little or no axon
outgrowth when exposed to GMFβ or GMFγ (supplementary
material Table S2). To inhibit the action of these four candidates on
SG explants, we employed EDN1 receptor-selective antagonists
(BQ123 for endothelin receptor type A; BQ788 for endothelin
receptor type B) and neutralizing antibodies against ARTN, NRG1
or NGF. Each inhibitor successfully blocked the effect of its factor
on SG explants (supplementary material Fig. S6).
We next tested whether these inhibitors could block VSMC-
mediated axon outgrowth from SG explants in vitro. Among them,
only the NGF-neutralizing antibody (anti-NGF NZAb) could
selectively inhibit directional axon outgrowth as compared with a
control isotype IgG (Fig. 6G-I). When the co-culture system was
were not detected by
Development 140 (7)
Fig. 4. VSMCs cover nerve-associated large diameter coronary veins.
(A-D? ?) Visualization of coronary VSMCs in the subepicardium. Whole-
mount triple immunofluorescence confocal microscopy was performed
with antibodies to the VSMC marker SM22α (B-D?, red) in addition to
PECAM1 (A, red; A?, white; B-D, blue) and TUJ1 (green). Magnified images
(C-D?) show the boxed regions in B,B?. At E15.5, SM22α+VSMCs are found
predominantly around nerve-associated large diameter coronary veins
(A-C?, open arrowheads), and a smaller number of SM22α+VSMCs are
located between remodeled veins (D,D?, arrowheads). Scale bars: 100 μm.
treated with this antibody, we observed random and non-directional
axon outgrowth, and axons appeared more fasciculated (Fig. 6H).
Further, the level of inhibition varied with the concentration of the
antibody (Fig. 6I). The other inhibitors, alone or in combination,
showed no effect at any tested concentrations (supplementary
material Fig. S6D-Z).
To further confirm that directional axon outgrowth depended on
VSMC-derived NGF, we employed a knockdown of Ngfin VSMCs
using a lentiviral vector carrying NgfshRNA during primary culture.
Compared with control shRNA-infected VSMCs, the Ngf
knockdown reduced NGF expression by more than 50% (Fig. 6J).
These Ngf-deficient VSMCs failed to induce preferential directional
axon outgrowth from the SG explants (Fig. 6K-M). These data
suggest that VSMCs secrete NGF to promote directional axon
outgrowth from sympathetic nerves. Indeed, NGF-soaked beads
successfully promoted directional axon outgrowth from SG explants
(supplementary material Fig. S7A-B?), demonstrating that NGF is
both necessary and sufficient for VSMC-mediated guidance in vitro.
NGF is required for cardiac sympathetic
innervation in vivo
An in vivo demonstration in support of these in vitro results would
require a coronary VSMC-specific knockout of Ngf, but the floxed
Ngf allele is not currently available. A definitive test of whether
NGF serves as a chemotactic factor for directional axon growth in
vivo was accomplished by implantation of beads coated with anti-
NGF NZAb on the dorsal surface of chick heart. In E10 hearts
implanted with control isotype IgG beads, TUJ1+axons fully
extended along with αSMA+VSMC-covered large diameter vessels
(Fig. 6N). In hearts with anti-NGF NZAb beads, the axons failed to
extend into the regions where the beads were placed (Fig. 6O),
despite the normal formation of large vessels (Fig. 6N).
Quantification indicated an ~50% reduction in axon extension along
the large vessels in the hearts implanted with neutralizing antibody
We next examined whether the ectopic presence of NGF causes
precocious innervation on the dorsal surface of chick heart. At
E10, the distal portion of αSMA+VSMC-covered large diameter
vessels did not accompany TUJ1+axons in the control (data not
shown) or in the presence of BSA-soaked beads (supplementary
material Fig. S7C). By contrast, NGF-soaked beads successfully
recruited TUJ1+axons (supplementary material Fig. S7D).
Cumulatively, these results suggest that local NGF secretion from
coronary VSMCs in the subepicardium is required for sympathetic
axon growth along large diameter coronary vessels in the
Patterning of cardiac nerves
Fig. 5. Fetal epicardium-derived VSMCs
promote axon outgrowth from fetal
sympathetic ganglia in vitro. (A) Schematic
illustrating the preparation of coronary VSMCs.
E12.5 or E13.5 cardiac ventricles were cultured in
collagen gel for 2 days. The ventricles were
removed from the gel, and migrated epicardial
cells were harvested and further expanded on a
type IV collagen-coated dish. VSMC aggregations
were obtained using a hanging drop culture
method. (B,C) Primary culture of epicardial-derived
VSMCs stained with antibodies to VSMC markers
SM22α (B,C, green) and αSMA (B, red), and SM-MHC
(C, green), together with the nuclear dye To-pro-3
(blue). (D-H) Co-culture of coronary VSMCs and
sympathetic ganglia (SG). E13.5 fetal SG were
cultured with VSMC aggregations obtained as in A
and stained with anti-TUJ1 antibody (green) and
To-pro-3 (blue). Magnified images (E,F) show the
boxed regions in D. Note that directional axon
outgrowth towards VSMCs was observed (E). The
total lengths of axons in the forward and reverse
quadrants (G) were calculated using Volocity (H).
n=19. (I-N) Co-culture of myocardium explants and
SG. SG were cultured with fetal myocardial tissue
explants and labeled with anti-TUJ1 antibody
(green) and To-pro-3 (blue). E13.5 myocardial tissue
explants failed to stimulate axon outgrowth from
E13.5 and E16.5 SG explants (I,K), whereas E16.5
myocardial tissue explants successfully induced
directional axon outgrowth from both E13.5 and
E16.5 SG explants (J,L). The total number of axons
in the forward region (M) for each sample was
quantified using Volocity (N). E13.5 SG with E13.5
myo, n=7; E13.5 SG with E16.5 myo, n=8; E16.5 SG
with E13.5 myo, n=5, E16.5 SG with E16.5 myo, n=6.
*P<0.01 (Student’s t-test); error bars indicate s.e.m.
1482 RESEARCH ARTICLE
Development 140 (7)
Fig. 6. Coronary VSMCs stimulate directional axon growth by NGF in vitro. (A) Semi-quantitative RT-PCR analysis showing differential expression of
neurotrophic factors as indicated between VSMCs and E13.5 myocardial tissues. A ratio exceeding 1.0 indicates that the factor is more highly expressed
in VSMCs than in myocardial tissues. (B-F) NGF expression in coronary VSMCs in the subepicardium. Triple immunofluorescence confocal microscopy of
E15.5 heart sections was performed using antibodies to SM22α (B,D, green) and NGF (B,E, red) as well as PECAM1 (B, blue). Magnified images (D,E) show
the boxed region in B. NGF expression was detected in venous VSMCs in the subepicardium (B,D,E, arrows). In situ hybridization with Ngf RNA probes on
E15.5 heart section shows that Ngf mRNA is expressed in coronary veins in the subepicardium (C, arrows). The NGF-expressing cells were also detected
in coronary veins by triple staining of E15.5 NGFlacZ/+reporter heart sections with antibodies for β-gal (green), PECAM1 (red) and the myocardial cell
marker α-actinin (blue) (F, arrows). CV, coronary vein; Se, subepicardium; Myo, myocardium. (G-I) VSMC-mediated directional axon outgrowth is
attenuated by anti-NGF neutralizing antibody (NZAb). E13.5 SG were cultured with VSMC aggregations in the presence of control isotype IgG (G) or 200
ng/ml anti-NGF NZAb (H), and were labeled with anti-TUJ1 antibody (green) and To-pro-3 (blue). Anti-NGF NZAb selectively inhibited directional axon
outgrowth as compared with control isotype IgG. The level of inhibition varies with the concentration of anti-NGF NZAb (I). The total lengths of axons in
the forward and reverse quadrants (see Fig. 5M) were calculated using Volocity. The ratios of the total lengths of axons in the forward versus reverse
quadrants are shown (I). A ratio above 1.0 indicates directional axon outgrowth towards VSMC aggregates. *P<0.05 (Student’s t-test); isotype IgG, n=8;
100 ng/ml anti-NGF NZAb, n=5; 200 ng/ml anti-NGF NZAb, n=6. (J-M) Ngf-deficient VSMCs fail to induce directional axon outgrowth. VSMCs were
infected with a control or Ngf shRNA lentivirus during primary epicardial culture. The expression levels of Ngf were assessed by RT-PCR analysis (J). E13.5
SG were cultured with control VSMCs (K) or Ngf-deficient VSMCs (L) and labeled with anti-TUJ1 antibody (green) and To-pro-3 (blue). Ngf-deficient
VSMCs failed to induce preferential directional axon outgrowth. Directional outgrowth was quantified as in Fig. 5G (M). *P<0.05 (Student’s t-test); control
shRNA-infected VSMCs, n=3; Ngf shRNA-infected VSMCs, n=5. (N-P) Effect of anti-NGF NZAb on coronary VSMC-mediated sympathetic axon growth in
chick embryonic hearts. Control isotype IgG-soaked beads or anti-NGF NZAb-soaked beads were implanted on the dorsal surface of E6 chick hearts.
After 4 days of incubation, the hearts were dissected for whole-mount double immunofluorescence confocal microscopy using antibodies to TUJ1 (N,O,
green) and αSMA (N,O, red). Control beads have no effect (N, white dashed circle) and anti-NGF NZAb beads show no distal extension of sympathetic
axons (O, white dashed circle). Distal axon extension was quantified (P). *P<0.01 (Student’s t-test); isotype IgG, n=5; anti-NGF NZAb, n=5. Note that anti-
NGF NZAb beads do not inhibit the formation of large diameter coronary vessels (O, arrow). Error bars indicate s.e.m.
Arterial VSMC-derived NGF is responsible for
subsequent axon penetration into the
myocardium at a late stage of cardiac
We next sought to determine what controls axon penetration into
the myocardium after distal extension in the subepicardium. Unlike
at E13.5, E16.5 myocardial explants secrete chemoattractants to
stimulate sympathetic axon growth. Indeed, average NGF
expression is highly enhanced in E16.5 myocardial tissue compared
with E13.5 myocardium (Fig. 7A). To more precisely localize NGF-
expressing cells in the myocardium, we utilized an NGFlacZ/+
reporter strain that allows us to detect NGF expression with greater
spatial resolution than previously possible. Consistent with the
analysis by whole-mount staining (Fig. 2), section staining of
EphB4taulacZ/+and ephrinB2taulacZ/+cardiac ventricles revealed that
EPHB4+coronary veins and ephrin B2+arteries were present in the
subepicardium and myocardium, respectively (Fig. 7B-D).
Surprisingly, NGF expression appeared to be restricted to VSMCs
throughout the heart (Fig. 7E-L). Based on the distinct localization
of cardiac arteries and veins, myocardial NGF expression is
therefore limited to arterial VSMCs. At E15.5, NGF expression in
venous VSMCs proceeds distally in parallel with angiogenic
remodeling and immediately preceding axon extension within the
subepicardium (Fig. 7E,G,H). NGF expression was downregulated
in venous VSMCs but not in arterial VSMCs near the sinus venosus,
where axons had already completed their extension along the veins
(Fig. 7E,F,H). By E17.5, no venous VSMCs expressed NGF at
detectable levels, but arterial VSMCs in the myocardium
demonstrated persistent NGF expression as axons extended into that
layer (Fig. 7I-L). This dynamic pattern of NGF expression in
cardiac VSMCs suggests that NGF might have a functional role as
We next examined whether VSMC-derived NGF mediates the
chemotactic effect of the E16.5 myocardium explants on
sympathetic axons. Anti-NGF NZAb clearly blocked E16.5
myocardium-mediated directional axon outgrowth in vitro(Fig. 7M-
O). These data suggest that arterial VSMCs secrete NGF to
stimulate sympathetic axon growth into the myocardial layer.
Furthermore, this sequential expression pattern of NGF in venous
VSMCs in the subepicardium and arterial VSMCs in the
myocardium is responsible for a coordinated process of distal axon
extension in the developing heart (Fig. 8).
In this study, we show that cardiac sympathetic axons are
preferentially aligned with large diameter coronary veins in the
Patterning of cardiac nerves
Fig. 7. Dynamic expression of NGF in VSMCs from
the subepicardium to the myocardium is
responsible for myocardial innervation. (A) RT-PCR
analysis of E13.5 and E16.5 myocardium explants.
(B-D) Location of EPHB4+coronary veins (B) and ephrin
B2+arteries (C) in the cardiac ventricles. Sections of
EphB4lacZ/+(B) and ephrinB2lacZ/+(C) hearts were stained
with antibodies for β-gal (green) and PECAM1 (red). The
relative location of coronary veins (CV) and arteries (CA)
in heart ventricle is shown in D. (E-L) NGF expression
analysis on NGFlacZ/+reporter heart sections. E15.5 (E-G)
or E17.5 (I-K) heart sections of NGFlacZ/+reporter
embryos were stained with antibodies for β-gal (green),
PECAM1 (red) and α-actinin (blue). Magnified images
(F,G,J,K) show the boxed regions in E,I. Schematic
models illustrating dynamic expression of NGF in
VSMCs in parallel with sympathetic axon extension at
E15.5 (H) and E17.5 (L). (M-O) SG and myocardium co-
culture with anti-NGF NZAb. E16.5 SG were cultured
with E16.5 myocardial explants in the presence of 200
ng/ml isotype IgG (M) or 200 ng/ml anti-NGF NZAb (N).
The culture was stained with anti-TUJ1 antibody (green)
and To-pro-3 (blue). (O) Directional axon growth was
quantified as in Fig. 5G. *P<0.02 (Student’s t-test);
isotype IgG, n=5; 200 ng/ml anti-NGF NZAb, n=5; error
bars indicate s.e.m. CV, coronary vein; CA, coronary
artery; Myo, myocardium; Se, subepicardium.
subepicardial layer of the dorsal ventricular wall in the developing
heart. Our results suggest that sympathetic innervation of myocardial
arteries proceeds by a two-step process (Fig. 8). First, coronary
venous VSMC-derived NGF mediates sympathetic axon extension
along large diameter coronary veins within the subepicardium. After
axons complete their extension within that layer, they penetrate into
the myocardial layer guided by arterial VSMC-derived NGF.
Cardiac sympathetic axons associate with large
diameter coronary veins
Our whole-mount immunohistochemical analysis of the developing
heart has revealed a novel neurovascular association. In the
subepicardium, sympathetic axons branch alongside large diameter
veins. By contrast, earlier studies have demonstrated that arteries
align with sensory nerves in the developing limb skin (Mukouyama
et al., 2002) and that sympathetic axons extend from SG along
arteries (Luff, 1996; Glebova and Ginty, 2005). To our knowledge,
no studies have yet reported an interaction between sympathetic
nerves and veins. Our observations reflect the characteristic
distribution of coronary veins and arteries in the ventricular wall of
the developing heart.
Large diameter coronary veins serve as
intermediate conduits for distal sympathetic axon
extension via VSMC-derived NGF
Our findings suggest that coronary VSMCs induce sympathetic
axon extension via local secretion of NGF. This is surprising
because NGF has primarily been implicated in the innervation of
final target cells, rather than distal axon extension (Levi-Montalcini,
1987). The dynamic pattern of NGF expression in coronary VSMCs
provides strong evidence that NGF directs sympathetic axon
growth, as previous work has demonstrated similar patterns in other
chemoattractants. For example, ARTN expression in VSMCs shifts
from central to peripheral blood vessels in parallel with sympathetic
axon extension along these vessels (Honma et al., 2002). In the
developing heart, NGF is important for sympathetic innervation of
the myocardium (Hassankhani et al., 1995; Ieda et al., 2004). In
Ngf−/−; Bax−/−embryos, there appears to be normal sympathetic
axon extension along the extracardiac vasculature but a drastic
decrease in sympathetic innervation of the heart (Glebova and
Ginty, 2004). These studies suggest that NGF is required for the
sympathetic innervation of target organs but not for proximal axon
extension along blood vessels. However, these studies did not
address the role that NGF plays in determining the pattern of
sympathetic innervation within the heart. We find that NGF is
transiently expressed by VSMCs in coronary veins at the stage when
sympathetic axons extend throughout the subepicardium
(supplementary material Fig. S2I versus S2L). This brief period of
expression might explain why axons follow veins during
remodeling but fail to innervate them as final targets. Together, these
results indicate that local secretion of NGF by VSMCs in coronary
veins provides a template for sympathetic axon extension in the
Sympathetic innervation of the heart is a two-
Beginning at E13.5, angiogenic remodeling moves outward from the
sinus venosus in the subepicardial layer. As VSMCs are recruited to
newly formed veins, they transiently express NGF, stimulating axon
extension along the vessels as they form. While subepicardial VSMCs
downregulate NGF expression after venous remodeling, myocardial
VSMCs begin to secrete NGF during arterial remodeling. These
observations suggest that a two-step process is responsible for
sympathetic innervation of the developing heart (Fig. 8). The
mechanisms that control spatiotemporal changes in NGF expression
by VSMCs merit further study, as precisely localized expression is
crucial to cardiac innervation. Transient expression of NGF by venous
VSMCs enables veins to function as a template but avoid innervation.
Later, persistent NGF expression by arterial VSMCs allows for final
target innervation in the myocardium.
The involvement of other signals in final target innervation of the
myocardium remains under investigation. Previous work has
provided a supportive mechanism in which SEMA3A, a myocardial
cell-derived chemorepellent, may establish a restrictive environment
that prevents sympathetic axon growth in the myocardium during
subepicardial extension (Ieda et al., 2007). Whether cells other than
VSMCs in the myocardium provide distinct innervation cues in a
time-specific fashion remains to be addressed. Our results suggest
that target organs of SG might possess stereotypical templates for
distal extension and innervation that are adapted to their complex
tissue structure and physiology.
We thank C. Lo and D. Alpert for breeding SM22αlacZ/+mice; J. F. Brunet and
M. German for Phox2b mutant mice; M. Kotlikoff for ChATBAC-eGFP mice; D.
Ginty for NGFlacZ/+mice; J. Hawkins and the staff of the NIH Bldg50 animal
facility for assistance with mouse breeding and care; C. Combs and D. Malide
for digital video editing; K. Gill for laboratory management and technical
support; Y. Carter and R. Reed for administrative assistance; A. M. Michelson,
Development 140 (7)
Fig. 8. A two-step process is responsible for sympathetic innervation of the developing heart. Model for sympathetic axon innervation of the
developing mouse heart. At E13.5, coronary veins undergo angiogenic remodeling in the subepicardium. Venous VSMCs transiently secrete NGF as they
associate with remodeled veins. Sympathetic axons start to extend into the subepicardium. By E15.5, NGF expression in venous VSMCs proceeds distally
in parallel with vascular remodeling, and axons extend along newly formed veins. Some arterial VSMCs begin to express NGF in the myocardium. By
E17.5, which is the stage when sympathetic innervation penetrates the myocardium, NGF expression is detected only in arterial VSMCs.
R. S. Balaban, R. S. Adelstein and X. Ma for invaluable help and discussion; M.
Conti for editorial advice on the manuscript; and other members of the
Laboratory of Stem Cell and Neuro-Vascular Biology for technical help and
This work was supported by the Intramural Research Program of the National
Heart, Lung and Blood Institute, National Institutes of Health (NIH) [HL006116
to Y.-s.M.] and was also supported by NIH grants [HL065484 and HL086879 to
P.R.-L.]. Deposited in PMC for release after 12 months.
Competing interests statement
The authors declare no competing financial interests.
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