Download Differences in development of coronary arteries and veins

Cardiovascular Research 36 Ž1997. 101–110
Differences in development of coronary arteries and veins
Mark-Paul F.M. Vrancken Peeters a , Adriana C. Gittenberger-de Groot
Monica M.T. Mentink a , Jill E. Hungerford b,1, Charles D. Little b,
Robert E. Poelmann a
a,)
,
a
b
Department of Anatomy and Embryology, Leiden UniÕersity Medical Center, P.O. Box 9602, 2300 RC Leiden, The Netherlands
Department of Cell Biology and the CardioÕascular DeÕelopmental Biology Center, Medical UniÕersity of South Carolina, Charleston, SC 29403, USA
Received 29 January 1997; accepted 12 May 1997
Abstract
Objective: The differentiation of the coronary vasculature was studied to establish in particular the formation of the coronary venous
system. Methods: Antibody markers were used to demonstrate endothelial, smooth muscle, and fibroblastic cells in serial sections of
embryonic quail hearts. The anti-b myosin heavy chain and the neuronal marker HNK-1 were added to our incubation protocol. Results:
In HH32, the coronary vascular network has developed into a circulatory system with connections to the sinus venosus, the aorta and the
right atrium. The connections between the aorta and the right atrium allow for direct arteriovenous shunting. Subsequently, differentiation
into coronary arteries and veins occurs with an interposed capillary network. The smooth muscle cells of the coronary arterial media
derive from the subepicardial layer, whereas the subepicardially located cardiac veins recrute atrial myocardium, as these cells express the
b-myosin heavy chain antigen. Ganglia are located in the subepicardium close to the vessels, while nerve fibres tend to colocalize with
the formed vessel channels. Conclusions: A new finding is presented in which the subepicardial coronary veins have a media that
consists of myocardial cells. The close positional relationship of neural tissue and coronary vessels that penetrate the heart wall is
explained as inductive for vessel wall differentiation, but not for invasion into the heart. q 1997 Elsevier Science B.V.
Keywords: Heart development; Coronary vasculature; HNK-1; b-Myosin heavy chain; Quail; Cardiac autonomic nervous system
1. Introduction
The development of the coronary vascular system and
its differentiation into the main coronary arteries has been
documented for the human w1–3x, as well as for the avian
embryo w4,5x. However, knowledge concerning the differentiation of coronary vessels and in particular the coronary
venous system is still inconclusive.
The initial development of the endothelium-lined coronary plexus has been reported by our group w6,7x. Also
results on the formation of a media around the main stems
of the coronary arteries using the anti-actin antibody,
HHF35 w5x, and the 1E12 antibody w7x have been reported.
In addition, an adventitial layer appears around the coro-
nary arteries, as shown with the anti-procollagen-I antibody, M38 w7x.
The subject of our present study is to single out the
formation of coronary veins from the development of the
complete coronary vasculature in the embryonic quail heart.
We have investigated the development of the coronary
venous system from HH30 onward, using the QH1 antibody w8x.
In contrast, the wall of the coronary veins did not stain
with the smooth muscle cell marker, 1E12. Therefore, we
incubated consecutive sections of quail hearts with an
anti-myosin heavy chain ŽMHC. antibody against chicken
atrial cells w9x, to explore the contribution of atrial cells to
the media of the coronary veins.
The HNK-1 antibody has been used to examine the
formation of the cardiac neural plexus, as this antibody is a
)
Corresponding author. Tel. Žq31-71. 5276691; fax Žq31-71.
5276680.
1
Current address: Department of Cell Biology, University of Virginia,
HSC Box 439, Jordan Hall 3-20, Charlottesville, VA 22908, USA.
0008-6363r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved.
PII S 0 0 0 8 - 6 3 6 3 Ž 9 7 . 0 0 1 4 6 - 6
Time for primary review 22 days.
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M.-P.F.M. Vrancken Peeters et al.r CardioÕascular Research 36 (1997) 101–110
marker for migrating neural crest cells and also for the
developing peripheral nervous system in the avian embryo
w10x. HNK-1 can be used as a marker for neural crest cells
in the avian embryo only to some extent, because non-neural crest derived structures such as the wall of the sinus
venosus and the ventricular myocardium may also be
labeled w11x. A relationship between the appearance of
cardiac ganglia within the subepicardial layer of the heart
and the presence of coronary arteries within their vicinity
has been documented w12,13x. We have used the HNK-1
monoclonal antibody to examine a possible correlation
between the presence of cardiac neural tissue and the
formation of the coronary venous system in the quail heart.
This study investigates the development of the coronary
vessel plexus, as it differentiates into the main stems of the
coronary arteries and veins. We also attempt to define the
origin of the cells of the medial layer around the endothelium of the coronary vessels. In addition, we studied the
correlation between the presence of the neural crest derived neuronal cells and the site of penetration of coronary
vessels into the aorta w4,6x and right atrium w7x.
2. Methods
2.1. Preparation of the embryos
Japanese quail embryos Ž Coturnix coturnix japonica.
were staged according to Hamilton and Hamburger for
chicken embryos w14x. Embryos ranging from HH30 to
HH44 Ž6.5 to 15 days. were used. After incubation at
37.58C Ž80% humidity., the quail embryos were carefully
removed from the yolk and transferred to a small dish with
Locke’s salt solution Ž0.94% NaCl, 0.045% KCl and
0.004% CaCl 2 .. The thorax segment ŽHH30 to HH38. or
only the heart-lung-specimen Žfrom HH38 onward. were
fixed in 2% acetic acid in 100% ethanol at 48C, for at least
48 h. After embedding in paraffin, the tissue was serially
sectioned transversally or sagitally at 5 mm and the sections were transferred to albuminrglycerin coated objective slides.
diluted in 1% ovalbumin in PBS-Tween-20. After rinsing,
the second incubation was performed for at least 1 h with
the Ž1:200. rabbit anti-mouse peroxidase conjugated antibody ŽDakopatts P260, Glostrup, Denmark.. The 1E12,
b-MHC and M38 antibody-incubated sections were rinsed
again and incubated for at least 1 h with a third antibody,
the Ž1:50. goat-anti-rabbit immunoglobulin ŽNordic,
Tilburg, The Netherlands., rinsed and incubated for one
hour with a fourth antibody, the Ž1:500. rabbit peroxidaseanti-peroxidase antibody ŽNordic.. Subsequently, all sections were rinsed twice in PBS, 10 min, and once in 0.05
M TRIS-maleic acid ŽTRIS-mal., pH 7.6, 10 min. The
peroxidase staining reaction was performed by exposing
the slides 8 min to diaminobenzidin ŽSigma. diluted in
TRIS-mal, to which was added 0.006% H 2 O 2 , followed by
washing in buffer. The sections were briefly counterstained
with Mayer’s hematoxylin, dehydrated in ethanol, covered
with Entellan ŽMerck, Darmstadt, Germany., coverslipped
and investigated by light microscopy.
2.3. Specificity of the primary antibodies
The HHF35 antibody recognizes skeletal, cardiac, and
smooth muscle alpha actin and smooth muscle gamma
actin w15x. The 1E12 antibody labels embryonic and adult
smooth muscle cells of the chick, but not the developing
cardiac muscle. Characterization of the 1E12 antigen points
towards a-actinin w17x. The quail specific QH1 antibody
reacts with endothelial cells lining a vessel lumen, and
cells that belong to the hemopoietic cell lineage. These
cells can be distinguished from one another on the basis of
time of appearance and location within the embryo w8x.
Although the QH1 epitope is unknown, it is commonly
used as a marker for endothelium. In the present study we
used a monoclonal antibody directed against the beta
isoform of the MHC molecule, recognizing both the nonspecialized atrial myocytes and the Purkinje cells of the
conduction system of the heart w9x. The HNK-1 antibody
reacts with glycoproteins and glycolipids of e.g. the central
and peripheral nervous tissue w16x.
2.2. Immunohistochemistry on serial sections
3. Results
The deparaffinized and rehydrated sections were immersed in phosphate-buffered saline ŽPBS., pH 7.4, to
which was added 0.3% H 2 O 2 to inhibit endogenous peroxidase activity. After rinsing twice in PBS for 10 min and
once in PBS with 0.05% Tween-20 ŽPBS-Tween, Sigma.
for 10 min, overnight incubation Žat room temperature.
took place with the first antibody. Consecutive sections
were incubated with either the 1:500 diluted QH1 antibody
w8x, the Ž1:1000. HHF35 antibody w15x, the Ž1:10. HNK-1
antibody w16x, the Žsupernatant, 1:10. 1E12 antibody w17x,
the Ž1:3. M38 antibody w18x, or with the Ž1:15. anti-b
MHC antibody Žcode 169-ID5. w9x. The antibodies were
3.1. The formation of the coronary Õascular system
In HH30, all the coronary vessels are endothelial-lined
tubes. The vascular network is connected to the sinus
venosus on the dorsal side of the heart ŽFig. 1A. from
which it receives its blood. These connections will develop
into the future coronary veins, connected to the coronary
sinus. At the ventriculo-arterial transition, a peritruncal
ring of vessels is present around the great arteries. In
HH32, two lumenized connections appear between the
peritruncal ring and the two semilunar sinuses of the aorta
facing the pulmonary artery. These two connecting vessels
M.-P.F.M. Vrancken Peeters et al.r CardioÕascular Research 36 (1997) 101–110
are the first Anlage of the proximal stems of the coronary
arteries ŽFig. 1B.. From this stage onward the network will
be supplied through the aorta. Other lumenized connec-
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tions appear between the peritruncal ring and the ventral
aspect of the right atrium. These vessels are the first
Anlage of the proximal stems of those coronary veins that
Fig. 1. Serial sagittal sections of embryonic quail hearts of consecutive stages, namely HH31 ŽA. and HH32 ŽB,C., stained with the QH1 antibody. ŽA. The
endothelium-lined coronary vessels Ž C . in the subepicardium Ž E . of the dorsal interventricular groove and in the ventricular myocardium Ž M . are in open
connection with the sinus venosus Ž SV .. ŽB,C. Show the arteriovenous shunt via the peritruncal ring. The two micrographs are of the same embryo in
which ŽC. is located 70 mm more to the right lateral side of the heart than ŽB.. ŽB. A proximal coronary artery Ž CA. connects the coronary vessels Ž C . of
the peritruncal ring with the aorta Ž AO .. ŽC. A proximal coronary vein Ž CV . connects the coronary vessels Ž C . of the peritruncal ring with the right atrium
Ž RA.. LV left ventricle; RV right ventricle.
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Fig. 2. Sagittal sections of consecutive stages of quail embryos, namely HH32 ŽA., HH36 ŽB., HH38 ŽC., and HH40 ŽD. in which the differentiation of
coronary vessels into arteries is illustrated. ŽA. A consecutive section of Fig. 1B is stained with 1E12. An area corresponding to the boxed area of Fig. 1B
is enlarged in panel A. The first smooth muscle cells are visible Ž arrowheads., lying close to the coronary artery endothelium and the aortic media Ž ME ..
ŽB. A media is present around a coronary artery Ž CA. in the interventricular septum Ž IS .. ŽC. A small area of the aortic sinus Ž S . where the coronary artery
invades this structure Ž arrowheads. remains 1E12-negative at all time during coronary artery differentiation. ŽD. Pro-collagen type I-positive dots
Ž arrowheads. are lying within the adventitia around the coronary arteries Ž CA. and coronary veins Ž CV .. The inset shows the same section in which the
background is mitigated by a blue filter for a better distinction of the positive dots. Note the adjoining localization of these two coronary vessels in a
combined vessel channel. Only within the subepicardium do the coronary veins attain a three-layered structure at this developmental stage. Formation of
venous media or adventitia is never observed extending into the ventricular myocardium. AO aorta; E subepicardium; M myocardium; RV right ventricle.
M.-P.F.M. Vrancken Peeters et al.r CardioÕascular Research 36 (1997) 101–110
have a separate connection to the right atrium ŽFig. 1C..
Thus, in HH32, the vascular network is supplied by the
aorta and is drained via the sinus venosus and right atrium,
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allowing for direct arteriovenous shunting via the peritruncal ring. Lumenized connections between the vascular
plexus and the left atrium have not been encountered.
Fig. 3. Sections of different stages of quail embryos, namely HH43 ŽA., HH38 ŽB., HH42 ŽC., and HH40 ŽD. in which the differentiation of coronary
vessels into veins is illustrated. ŽA. The micrograph of an anti-actin stained transversal section through the arterial orifice level shows the existence of a
coronary artery Ž CA. and a coronary vein Ž CV . in a joined vessel channel. ŽB,C. Sagittal sections through the proximal coronary veins Ž CV . connected
with the sinus venosus Ž SV . ŽB. and with the right atrium Ž RA. ŽC. reveal that the media is comprised of b-MHC-positive cells Ž arrowheads.. ŽD. A
sagittal section through a more distally located coronary vein Ž CV . reveals that the media is comprised of 1E12-positive-cells Ž arrowheads.. AO aorta; E
subepicardium; M myocardium; PA pulmonary artery.
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M.-P.F.M. Vrancken Peeters et al.r CardioÕascular Research 36 (1997) 101–110
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Fig. 5. Sagittal sections of quail embryos stage HH38 ŽA. and HH42 ŽB. stained with an anti-b-MHC antibody. Positively stained cells Ž arrowheads. are
visible in the medial layer surrounding the pulmonary vein Ž PV . endothelium ŽA. and caval vein Ž CAV . endothelium ŽB.. L liver; LA left atrium; PA
pulmonary artery; RA right atrium.
3.2. Immunohistochemistry of the media and adÕentitia
After the vascular network has contacted the aortic
lumen at HH32, the first 1E12-positive mesenchymal cells
can be noted close to the endothelium of the most proximal coronary artery stems adjacent to the aortic media
ŽFig. 2A.. These cells are the first components of the
artery medial wall. The media increases both in thickness
and in length along the arteries, located in the myocardium
of the interventricular septum and in the subepicardial
layer of the atrio-ventricular groove ŽFig. 2B.. A small part
of the wall of the aortic sinus, just surrounding the coronary orifice remains 1E12-negative ŽFig. 2C.. From HH32
onward, pro-collagen-I producing fibroblasts, demonstrated with M38, appear within the adventitia of the
proximal artery stems. The extension of adventitia formation ŽFig. 2D. occurs in concert with the arterial media
formation.
A common feature observed during vascular maturation, is the existence of an artery and a vein in a double
vessel channel ŽFig. 3A.. These channels have invaded
deep into the myocardium and nearly encircle the atrioventricular groove. At HH32, the veins still lack a media
and adventitia. In HH39, actin-expression becomes apparent in cells adjacent to the endothelium of the most
proximal part of the veins located within the subepicardial
layer of the sinus venosus and the right atrium. These
actin-positive cells also express the b-MHC antigen, as
found in the atrial cardiomyocytes ŽFig. 3B,C.. The extension of this atrial sleeve proceeds from both the right
Fig. 4. Sections of consecutive stages of quail embryos, namely HH30 ŽA., HH31 ŽB., HH36 ŽC., HH36 ŽD., and HH43 ŽE., in which the localization of
neural tissue in relation to coronary vessel formation is investigated with the HNK-1 antibody. ŽA. In this sagittal section, neuronal ganglia Ž G . are
identified within the subepicardial layer Ž E . adjacent to the site where coronary vessels Ž C . are connected to the sinus venosus Ž SV .. Neuronal fibres
Ž arrowheads. colocalize with the expanding vascular plexus. ŽB. A sagittal section through the outflow tract of the heart shows that ganglia Ž G . have
invaded the level of the peritruncal ring. At orifice level they are predominantly situated in the subepicardium adjacent to the aorta Ž AO .. ŽC. In a
transversal plane through the orifices of the great arteries of the outflow tract, the sites are indicated where ganglia and coronary vessels Ž CA, CV . are
situated. The presence of neural tissue colocalizes with the sites where the coronary arteries have invaded the aorta Ž AO . and the coronary veins Ž CV .
have invaded the right atrium Ž RA.. ŽD. Compare this photograph with Fig. 2B. In these two consecutive sections of a 1E12 ŽFig. 2B. and an HNK-1
staining ŽD., it is visible that the neuronal fibres Ž arrowheads. have invaded the subepicardium Ž E . and the ventricular myocardium Ž M . along the
developing vessel channel in close relation to the arterial media. ŽE. The neuronal fibres Žblack in this micrograph. are not limited to the vessel channel,
but are widely scattered throughout the myocardium. AS atrial septum; IS interventricular septum; L liver; LA left atrium; LV left ventricle; PA
pulmonary artery; RV right ventricle.
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atrium and sinus venosus along the ventral and dorsal
coronary veins towards the ventricular myocardial boundary, where the veins become embedded in the ventricular
mass. At HH43, we have noted that more peripherally
located cells within the venous media also stain positively
for the 1E12-antibody ŽFig. 3D.. The extension of a venous adventitia develops in concert with the venous media.
While smooth muscle cells and fibroblasts adjoin the
arteries deep into the interventricular septum, the veins that
colocalize within the myocardial vessel channel never
develop a three-layered structure. Their endothelium still
adjoins the ventricular myocardial cells until late stages of
coronary vascular development.
3.3. The location of HNK-1 positiÕe cells in relation to the
formation of coronary arteries and coronary Õeins
In HH30, HNK-1 positive cells have invaded the dorsal
mesocardium to reach the sinus venosus. These cells form
conglomerates of ganglia located in the subepicardium
adjacent to the vascular plexus connected to the venous
side of the heart. HNK-1 positive neuronal fibres run from
these ganglia over the dorsal side of the heart in close
relation to the expanding vessels ŽFig. 4A..
At HH31, HNK-1 positive cells around the outflow
tract of the embryonic heart have invaded the level of the
peritruncal ring. These ganglia are mainly situated around
the aorta, in the septum between the aorta and the pulmonary trunk and between the aorta and the right atrium
ŽFig. 4B.. They colocalize with the sites where the coronary arteries will invade the aorta and the coronary veins
the right atrium ŽFig. 4C..
From HH32 onward, nerve fibres migrate from the
outflow tract ganglia to invade the myocardium of the
interventricular septum and the subepicardium of the
atrio-ventricular groove. Migration of nerve fibres occurs
in the developing vessel channels in the direction of the
apex of the heart. A close relation between the migrating
nerve fibres and the formation of a media around the
coronary arteries of the vessel channel can be detected
ŽFig. 4D.. At HH42, nerve fibres are not only limited to
the developing main vessel channels, but they become
widely scattered throughout the myocardial and subepicardial layer ŽFig. 4D..
3.4. The extension of atrial myocardium in the media of
the Õenous inflow of the heart
The proximal part of the media of the great veins in the
body wall is also comprised of atrial myocardium, comparable to coronary venous media formation. From HH38
onward, the myocardial cells, detected with the b-MHC
antibody, extend from the left atrium around the pulmonary venous endothelium ŽFig. 5A., and from the right
atrium around the caval venous endothelium ŽFig. 5B..
Fig. 6. Schematic line drawings of the coronary vascular system of an
HH32 ŽA. and an HH42 ŽB,C. quail heart. ŽA. In this right lateral view,
the coronary vessels lying in the atrio-ventricular and those of the
peritruncal ring are shown. Connections between this vessel plexus and
the aorta Ž arrows . and the right atrium Ž arrowheads. have been made.
The coronary vessels are dorsally also in contact with the sinus venosus,
which is not depicted in this picture. ŽB. In this frontal view the fully
developed coronary arterial system is depicted. The right and left main
coronary arterial stems are each divided into an atrio-ventricular, conal
and septal branch. ŽC. In this frontal view the fully developed coronary
venous system is depicted. Several, mainly anteriorly located coronary
venous stems are in contact with the right atrial lumen Ž arrowheads.,
while one dorsally located coronary venous stem is connected to the sinus
venosus Ž arrow .. AO aorta; CA coronary artery; LA left atrium; LV left
ventricle; PA pulmonary artery; RA right atrium; RV right ventricle.
The coronary vessel development of HH32 and 42 quail
hearts is summarized in Fig. 6A–C.
4. Discussion
Extensive research has been performed on the growth
and differentiation of the coronary vascular system in
avian embryos using histological w19,20x, immunohistochemical w4,5,21x and experimental techniques to elucidate
the origin and development of the coronary arteries
w5,6,12,22–24x. However, the formation of the coronary
venous system and the origin of its components is still
unrefined.
M.-P.F.M. Vrancken Peeters et al.r CardioÕascular Research 36 (1997) 101–110
An important source of cells, the proepicardial organ, is
located between the sinus venosus and the primordial liver
and its protruding villi reach towards the dorsal surface of
the embryonic heart and contact the myocardial surface at
about HH16 w25x. The epicardial sheath derives from the
proepicardium w26–28x. Moreover, it encompasses the entire endothelial lining of both the coronary arterial and
venous system w6x, whereas the progenitor cells of the
smooth muscle cells in the media and the fibroblasts in the
adventitia of the arteries are of proepicardial origin w23x.
Although, the number of layers of which both arteries
and veins are comprised is the same, differences can be
noted in the timing of vessel wall differentiation, the origin
of media cells, and the extent of the media in the myocardium.
The arteries have started to differentiate at HH32,
whereas the veins will not attain a media or an adventitia
before HH39, which is well after the time that the vessels
of the peritruncal ring have contacted the right atrium.
The media of the veins contains other cell types than
the arteries. It stains with anti-actin, whereas the smooth
muscle cell marker for the arteries, 1E12, did not provide a
staining of the proximal part of the venous media. Here,
we showed expression of the b isoform of the atrial
specific myosin heavy chain. The media of the proximal
part of the veins derives from the myocytes of the atrial
wall. More distally the media became mixed and some
cells that were negative for the myocardial marker were
positive for 1E12. Although speculative, this variation in
phenotype could reflect the various different vasomotor
responses that these cells show to transmitters w29x. The
extension of cardiac musculature as a tunica media sleeve
around the pulmonary and caval veins has been noted
before in mammals w30–33x. However, this phenomenon
has not been described for coronary veins. We conclude
that the atrial musculature surrounds the complete venous
pole of the heart. We expect that the coronary venous
myocardial cuff will play a role in preventing backflow of
blood from the right atrium during atrial contraction.
Another important difference between arterial and venous wall development, is the extent of the venous media
and adventitia limited to the subepicardium, never entering
the myocardial wall. The endothelium of the veins within
the interventricular septum closely adjoins the ventricular
myocytes without having a differentiated vessel wall. This
is observed up to hatching in the quail. Probably, the
intramyocardial veins lack vasotonus activity, while the
venous blood is probably squeezed into the proximal veins
due to ventricular contraction.
At about HH15-HH19, neuronal parasympathetic ganglia, most likely originating from the cardiac neural crest,
invade the sinus venosus region by way of the dorsal
mesocardium w11,13x. This is the period in which the
Anlage of the coronary vasculature becomes apparent w6,7x.
The nerve fibres, that partly derive from the sympathetic
crest in later stages, colocalize with the undifferentiated
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vessels in the dorsal subepicardium. Spence and Poole w34x
stated that the formation of blood vessels preceded the
migration of neural crest cells that use the developing
blood vessels as a substratum.
Large ganglia are found in the subepicardium of the
outflow tract at HH30, when the vessels of the peritruncal
ring are not yet connected to aorta or right atrium. As in
the next stage of development, ganglia are found in areas
of connection with the aorta and right atrium, as well as
near the aortic non-facing sinus, pulmonary trunk and left
atrium, lacking connections with the peritruncal ring, we
conclude that nerve fibres apparently do not induce endothelial penetration.
The presence of ganglia in accordance with media
formation of the arteries has been reported earlier w5x and
is supposed to be essential to the survival of the definitive
coronary arteries w6,12,13x. Although the association of
ganglia with persisting coronary arteries may suggest that
chemotactic substances released by neural tissue is in some
way essential to the development of the walls of the
arteries, it can not be the only determining factor. In a
combined vessel channel nerve fibres are as closely related
to the veins as to the arteries. The veins also develop a
medial layer, however, in a different time schedule, which
seems to rule out neuronal influence. Experimental ablation of neural crest does not prohibit vascular smooth
muscle cell deployment, as coronary arteries still contain a
medial layer w5x. The presence of neuronal ganglia not
related to proximal coronary arteries in an ablation model
of persistent truncus arteriosus shows the complexity of
this matter w13x. Substances released by endothelial cells
can facilitate vascular smooth muscle recruitment w35,36x.
As increased shear stress can induce this release w37x, we
suppose that the increase in pressure and alteration in
blood flow after connecting to the aorta, is a potential
factor involved in smooth muscle differentiation around
the arterial endothelium. This phenomenon may explain
the relative late differentiation of the venous wall in the
low pressure environment of the coronary veins.
Several questions are left to be elucidated. We are
inquisitive about the origin of the vascular smooth muscle
cells around the veins that do not express the b myosin
heavy chain but stained positive for the anti-actin antibody,
HHF35, and the smooth muscle marker, 1E12. We speculate that these cells are also derived from the same extracardiac source that gives rise to the media of the arteries
w23x. We are presently transferring liver-proepicardium
pieces of the quail to the chick pericardial cavity to answer
this problem.
Acknowledgements
This work was supported in part by a Dr. E. Dekker
study grant ŽNo. D96.017. of the Netherlands Heart Foundation to M.-P.F.M. Vrancken Peeters.
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