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Cardiovascular Research 41 (1999) 663–671
Rate of coronary vascularization during embryonic chicken development is
influenced by the rate of myocardial growth
a,
1 ,b
a
2 ,b
Robert J. Tomanek *, Norman Hu , Bick Phan , Edward B. Clark
a
Department of Anatomy and Cell Biology, and The Cardiovascular Center, Bowen Science Bldg, University of Iowa, Iowa City IA 52242, USA
b
Strong Children’ s Research Center, Department of Pediatrics, University of Rochester, Rochester NY 14642, USA
Received 19 January 1998; accepted 11 August 1998
Abstract
Objective: We tested the hypothesis that the degree of coronary microvessel formation in the embryonic heart is regulated by the
magnitude of myocardial growth. Methods: The outflow tract of Hamburger–Hamilton stage 21 chicken hearts (prior to the onset of
coronary vasculogenesis) was constricted in ovo with a loop of 10-0-nylon suture, and the hearts were studied at stages 29 and 36.
Results: At stage 29 ventricular mass was 64% greater in the pressure-overloaded than in the hearts of sham-operated controls, but
vascular volume density and numerical density, determined by electron microscopic morphometry, were identical. As demonstrated by
histological morphometric evaluation, the compact region of the left ventricle at stage 29 was 43% thicker than the shams. However, by
stage 36 heart mass, thickness of the compact region, and overall wall thickness (demonstrated by scanning electron microscopy) were
significantly less than in the sham group of this stage, but vascular volume density was virtually identical in the two groups. Formation of
the two main coronary arteries was clearly impeded in the banded hearts, i.e., the coronaries were stunted in their development or failed to
completely form coronary ostia. Conclusions: Vascular growth is proportional to myocardial growth in the embryonic, overloaded heart,
but the persistence of the pressure overload results in a failure of or severe limitations in coronary artery development. These data support
the hypothesis that vascular growth during this period of development is regulated, at least in part, by the rate and magnitude of
myocardial growth.  1999 Elsevier Science B.V. All rights reserved.
Keywords: Pressure overload; Chicken; Electron microscopy; Angiogenesis; Vasculogenesis
1. Introduction
The vascular response to cardiac enlargement in the
adult is variable and dependent upon several factors
including the stimulus evoking the increase in ventricular
mass, age, and the duration of hypertrophy [1,2]. Coronary
angiogenesis (sprouting of vessels from pre-existing vessels) may be stimulated by mechanical factors such as
enhanced flow and shear stress, or stretch resulting from
increased ventricular filling [3,4]. During early normal
postnatal growth a marked capillary proliferation occurs,
*Corresponding author. Tel.: 11-319-335-7740; fax 11-319-3357198.
1
Current address: Department of Pediatrics, Primary Children’s Medical
Center, 100 N. Medical Drive, Salt Lake City UT 84113, USA.
2
Current address: Department of Pediatrics, Primary Children’s Medical
Center, 100 N. Medical Drive, Salt Lake City UT 84113, USA.
and then is attenuated as capillary growth does not keep
pace with myocyte growth, and capillary density becomes
stabilized to adult values [5]. Since vasculogenesis (vessel
formation by endothelial cell precursors) constitutes the
initial phase of neovascularization in the embryo, comparisons to postnatal growth are not entirely appropriate.
Moreover, little is known about the role of ventricular
mass in determining vascular parameters in the embryo
because coronary vascular growth during the prenatal
period has not been as extensively studied.
We previously demonstrated that when ventricular pressure is increased in the chicken embryo the consequential
increase in cardiac mass is due to hyperplasia rather than
hypertrophy of myocytes [6]. In our model, the conotruncus is constricted with a loop of 10-0 nylon suture at
Hamburger–Hamilton (HH) stage 21, which results in a
Time for primary review 25 days.
0008-6363 / 99 / $ – see front matter  1999 Elsevier Science B.V. All rights reserved.
PII: S0008-6363( 98 )00330-7
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R. J. Tomanek et al. / Cardiovascular Research 41 (1999) 663 – 671
substantial increase in the ventricular dry weights of the
constricted hearts compared to sham controls at stage 29.
Thus, the heart is able to adjust to loading conditions by
increasing its mass even during the embryonic period.
Since this period of development is characterized by the
onset of vasculogenesis (at approximately stage 22–23) the
increase in cardiac mass occurs during myocardial vascularization. In humans, congenital cardiac enlargement due
to pressure overload is characterized by normal vascular
densities, while cardiac enlargement acquired in adults is
not [5]. However, the early onset of accelerated growth
described in the chick model has not been replicated or
studied in other species. Accordingly, the response of the
vasculature at this early age is not predictable. To our
knowledge the only study to explore loading conditions in
the prenatal heart is our work on embryonic day 12 rat
hearts which were grafted in oculo in adult rat hosts [7].
Data from that study showed that 3 weeks after grafting
this unloaded, beating heart had vascular densities which
were similar to a full term (21 days) heart developing in
utero. Thus, the slow myocyte growth in this model was
accompanied by a slow but proportional growth of the
vasculature. However, this approach is limited, since the
ventricles are not operating in a normal hemodynamic
state.
The central question addressed in this study was: does
an accelerated growth and enhanced ventricular mass due
to an increased afterload in the embryo induce a comparable accelerated coronary vascular response? The second
question concerned the morphogenic changes in the ventricles. The ventricular walls are initially heavily trabeculated
with the intervening spaces constituting a sinusoidal
system which provides oxygen and nutrients to the
myocytes prior to the establishment of coronary arteries
[7–10]. Thus, we questioned whether pressure overload
and the consequential accelerated growth influences the
modeling of the trabecular and compact regions of the
ventricles.
2. Methods
2.1. Experimental protocol
All protocols used in this study conform with the Guide
for the Care and Use of Laboratory Animals published by
the US National Institutes of Health (NIH Publication No.
85-23, revised 1996). The model of pressure overload used
in this study has been previously described [6,10] and is
summarized as follows. Fertilized White Leghorn chicken
eggs were incubated, blunt end up, to HH stage 21 (3.5
days) of a 21-day incubation period. We reached the
embryo by making a hole in the shell, and incising the
inner shell membrane. The outflow tract of the heart was
constricted by tying a loop of 10-0 nylon suture at the
midpoint of the conotruncus. At stage 21 the outflow tract
and vessels are semitranslucent and thus we were able to
distinguish the conotruncal valves, which mark the root of
the conotruncus. The outflow tract ends at the base of the
aortic arches, which emerge at the neck region. The
procedure was performed under a photomicroscope attached to a videocassette recorder. Sequential video fields
of the loop diameter and the diameter of the conotruncus at
the suture region were planimetered using a video frame
grabbing board and video software. Accordingly, the loop
diameter was adjusted to the diameter of the outflow tract.
In sham-operated embryos the suture was placed in
position but not tied. Normal embryos were unoperated.
The window in the shell was sealed with paraffin film, and
the egg was returned to the incubator. Hearts from
embryos of various incubation periods were perfuse-fixed
in diastole, via a microcannula placed into the ventricle.
The diastolic state was maintained by perfusing 0.025 mg
verapamil in a 2.5% glutaraldehyde solution buffered with
0.1 M sodium cacodylate. The heart was then excised, and
the ventricles were weighed, postfixed in 1% osmium
tetroxide and embedded in Spurr’s plastic.
2.2. Light microscopy and morphometric analysis
Sections, 1 mm in thickness, were cut on a ultramicrotome and stained with Richardson’s solution. Sections from several levels of the ventricles were used for a
subjective analysis of morphological characteristics, while
sections from the midpoint between the base and apex
were used to assess the thickness of the compact layer of
the myocardium. This was accomplished by projecting
images of the left ventricular free wall onto drawing paper
with a microprojector and tracing the outlines of the
ventricular wall. Subsequently, equidistant points were
placed along the epicardial–myocardial border and distances from these points and the nearest points of the
endocardial endothelium were measured. The mean distance for each specimen was based on an average of 33
measurements representing over 1 mm of the epicardial–
myocardial border.
Serial sections of normal hearts were prepared from
JB-4 blocks on a Reichert microtome and stained with
eosin and hematoxylin and representative sections were
photographed. Particular attention was paid to the basal
half of the heart and the formation of the coronary arteries
and the time of coalescence with the aorta. Coronary vessel
diameters in the stage 36 hearts were obtained from
tracings (3552) of microprojected images.
2.3. Electron microscopy and morphometric analysis
Ultrathin sections were prepared with a diamond knife
on a Reichert Ultratome, placed on either single oval slot
or 100 mesh cooper grids coated with a formvar film. The
sections were stained with uranyl acetate and lead citrate
and examined with a Hitachi H-7000 electron microscope.
R. J. Tomanek et al. / Cardiovascular Research 41 (1999) 663 – 671
For quantification of vascular density, specimens were
photographed at 31000 and micrographs prepared at a
total magnification of 32500. At least eight fields (of the
compact region), representing over 10 000 mm 2 of tissue,
were photographed from two or more blocks. Volume
density of vessels ,12 mm was determined, as previously
described [4], by point counting of vessels using an
overlay grid with intercepts every 6 mm (1008 points /
micrograph). Nonvascular cells were point-counted using
an overlay grid with intercepts every 12 mm (252 / micrograph) and the counts multiplied by 4 in order to
express vascularity as percent of tissue (excluding extracellular space). Vascular volume density (Vvas /Vt ) is derived
from the relationship: Vvas /Vt 5Pvas /Pt , where Pvas is the
number of points falling on vessels and Pt is the total
number of points falling on various cells plus vessels. The
basal half of one group of overloaded and sham HH 36
hearts was dehydrated, sputter-coated with gold and
studied with a scanning electron microscope (Hitachi S4000). The hearts were mounted so that they could be
viewed from their cut surface. Micrographs from these
hearts were used to assess intergroup differences in the
spongy and compact regions of the ventricles and to
measure the thickness of the compact region.
2.4. Statistical analysis
The data were analyzed by analysis of variance and a
Bonferroni adjustment for multiple comparisons. A p-value
,0.05 was selected to test for significant differences
between groups.
3. Results
3.1. General effects of conotruncal banding
Data in this study are based on 42 embryos. The survival
rate of the chicks banded at stage 21 was 60% at stage 29
and 35% at stage 36. As in previous studies [6,10], we did
not note any general abnormalities in the embryos which
were banded. We verified the position of the ligature in
each embryo studied. All of the loops were positioned well
above the conotruncal valves.
3.2. Ventricular morphogenesis
As previously shown by scanning electron microscopy,
the smooth walled chick ventricle at stage 16 develops
myocardial trabeculae by stage 18 which grow to form a
mesh by stage 21 [11]. Thus, the ventricular wall at this
point consists of an outer compact layer and an inner
spongy layer. The spaces between the trabeculae are
termed ‘‘sinusoids’’ and serve to minimize diffusion
distances between blood in the ventricle and myocardial
cells. The formation of the coronary vasculature is initiated
665
when angioblasts form tubes by joining together in either
(1) blood islands, or outside blood islands, or (2) by
forming large vacuoles and thereby ‘‘hollowing out’’ to
form a channel. These microvascular channels coalesce
and branch throughout the compact region. Attachment to
the aorta occurs via ingrowth of coalescing capillary
plexus at about stage 32 or 33, as indicated by analysis of
our serial sections and as previously demonstrated by
others [12]. Formation of a media in the arterial system
begins at this time, and follows a proximal to distal
sequence from the root of the aorta. This pattern was
confirmed in this study and is similar to our previous
observations in rats [8]. At stage 36 the arterial tree is
limited to the basal ]13 of the heart. Such channels were not
encountered in sections used for quantitative analysis. At
the time of banding (stage 21) the avascular left ventricular
compact myocardium is about two cell layers thick in the
compact zone, has numerous long trabeculae, and the
epicardium is thinner than that observed at successive
stages (Fig. 1A). By stage 24 the compact myocardium is
about 3–4 cells in thickness and the trabecular (spongy)
layer has also expanded. Large vascular plexuses overlay
the AV groove. By stage 29 the myocardium is about 5–7
layers thick, the trabeculae are relatively shorter and the
epicardium is considerably thicker (Fig. 1B). The myocardium grows rapidly between stages 29 and 36 as the
compact myocardium increases to 12–20 cells in thickness
and the trabeculae thicken and shorten (Fig. 2A). By stage
36 the spongy region of the ventricle is markedly reduced.
Capillaries are seen only within the compact region and the
thicker trabeculae. The coronary microvasculature is connected to the aorta by stage 33, as previously noted [12],
and as confirmed by us in this study by analysis of serial
sections.
3.3. Ventricular mass and morphology in overloaded
heart
In the banded hearts, at stage 29, the increase in
thickness of the compact region (Fig. 1C) was due to an
increase in the number of cell layers in the compact region,
i.e., about 7–8 cells were not uncommon. Thus, at least
part of the increase in cell number, previously documented
in this model [6], occurred transmurally in the left ventricle
and contributed to the substantial (64%) increase in
ventricular mass (Table 1).
In contrast, the pressure-overloaded hearts harvested at
stage 36 had a smaller mass than the shams (Table 1). The
compact region of the heart did not develop further, thus a
major portion of the left ventricular wall remained as a
spongy layer, as seen in both histological sections (Fig.
2B) and in scanning electron micrographs (Fig. 3). The
thickness of the compact region was quantified by measuring the shortest distance from the epicardium (the border
of the epicardium and myocardium) to the endocardium
(Fig. 4A). At stage 29 the mean thickness of the compact
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R. J. Tomanek et al. / Cardiovascular Research 41 (1999) 663 – 671
percentage of the myocardium remaining as the spongy
(trabecular layer). In sham hearts at this stage only 24% of
the left ventricular wall consists of spongy myocardium,
while in overloaded hearts 33% remains spongy ( p5
0.002). Moreover, the ventricles in the stage 36 overloaded
hearts were more ellipsoid that the shams, and mean
overall ventricular thickness, measured in scanning electron microscopic micrographs, was 24% less in the overloaded group at HH stage 36 (mean6SEM: 116610 vs.
15368 mm). Accordingly, the persistence of the pressure
overload was associated with reduction of ventricular
growth between stages 29 and 36.
3.4. Vascular growth
Fig. 1. Light micrographs of the left ventricular wall of hearts from
normal HH stage 21 (A), sham HH 29 (B), and overloaded HH 29 (C)
chick hearts. In normal (nonoverloaded) hearts the thickness of the
compact region (between arrowheads) increases approximately threefold
between stages 21 and 29. In the overloaded hearts this increase is
significantly greater (see Fig. 4) as a consequence of more cells. Bar5
100 mm.
region is 27% greater in the overloaded than in the sham
hearts. In contrast, by stage 36 the compact region mean
thickness is 31% lower in the overloaded ventricles
compared to the shams. Thus, the compact region of left
ventricle expanded to a much greater extent in the sham
than in the overloaded group. As illustrated in Fig. 4B, the
lower growth rate of the compact region between stages
29–36 in the overloaded hearts resulted in a higher
Fig. 5 illustrates a capillary as seen with the electron
microscope, and represents a portion of a field used to
quantify vascularity. We selected fields from the compact
region which bordered the epicardium. In order to minimize the inclusion of sinusoids in our measurements we
excluded any region with an endothelial-lined channel with
a diameter above 12 mm. This criterion was based on
preliminary experiments in which we traced structures
which appeared to be vascular channels of this size or
larger in serial sections and determined that they were
continuous with the ventricular lumen. Our data on vessel
volume percent (Fig. 6) and vessel numerical density (Fig.
7) indicate that vascularity was proportional to ventricular
mass, i.e., vascular growth in the overloaded hearts was
accelerated between stages 21 and 29 in order to compensate for the greater increase in ventricular mass in this
group. Thus, vascular growth necessarily increased proportionally to the accelerated growth of the myocardium.
The vasculature was qualitatively similar in overloaded
and sham hearts, and evidence of cell damage or degeneration was lacking. Vascular volume percent in the overloaded and sham hearts in the stage 36 chicks was also
similar (Fig. 6), but numerical density was higher in the
overloaded compared to the sham hearts (Fig. 7). This
finding indicates that the vessels in the overloaded hearts
were smaller than in shams.
In order to assess the formation of the coronary arteries,
we examined serial sections through the basal ]13 of the
heart. The coronary arteries in sham hearts are continuous
with the aorta at stage 32 or 33. In stage 36 sham hearts
the coronary arteries extend down about ]15 of the distance
between base and apex (Fig. 8A). They appear to branch
only once or twice. The three banded hearts which were
serially sectioned at stage 36 revealed defects in coronary
artery development. In one heart the coronary ostia did not
form. A second heart revealed an incomplete channel
through the aortic wall. In a third heart both coronary
arteries were formed. However, arteries found in any of the
three hearts were very small compared to the shams. In the
latter (n54) coronary lumen diameters were 69–81 mm,
while in the three overload HH 36 hearts they ranged from
R. J. Tomanek et al. / Cardiovascular Research 41 (1999) 663 – 671
667
Fig. 2. Light micrographs of the left ventricular wall of hearts from HH stage 36 sham (A) and overloaded (B) chick hearts. The compact region of the
shams is much thicker than that of the overloaded hearts due to a lack or slowing of growth in the latter. Note the sinusoids of the spongy layer (arrows)
generally extend deeper into the ventricle compared to those of the shams. Bar5100 mm.
15–34 mm. The smallest diameters were in the heart in
which coronary ostia were totally absent.
Finally, we have shown that development of the coronary
arteries is stunted in hearts which were banded.
4.1. Ventricular adaptations to embryonic pressure
overload
4. Discussion
This study provides three major findings. First our data
document that myocardial vascularization is accelerated
and fully compensates for the marked increase in ventricular mass resulting from embryonic pressure overload.
Although many studies have addressed angiogenesis, or its
lack, in postnatal hearts, this is the first study to consider
the vascular consequences of the influences of increased
load and ventricular mass in embryonic hearts. A second
major finding is that subsequent to the substantial adaptive
myocardial growth which occurs with pressure overload
between HH stages 21 and 29, myocardial growth in the
pressure overloaded hearts is severely compromised between HH stages 29 and 36, and as a consequence is much
less than the growth occurring in sham chick hearts.
Vascular growth during this period is proportionately
decelerated, thus vascular volume is similar in shams and
overloaded hearts. Left ventricular mass increased more
than sixfold in the sham group between stages 29 and 36,
but increased only 2.5-fold in the overloaded group. These
data support the hypothesis that vascular growth is influenced by the magnitude and rate of myocardial growth.
Table 1
Ventricular weights for overloaded and sham hearts
HH stage 29
Weight (mg)
% Difference
n
p-Value
HH stage 36
Sham
Overloaded
Sham
Overloaded
2.6160.28
4.2860.43
164%
9
,0.005
16.1760.80
10.5760.35
235%
7
,0.001
9
Values are means6SEM.
7
Previous work by our group showed that between stages
21–29 ventricular pressure increased more than twofold,
cardiac work increased about sevenfold, and left ventricular mass increased ninefold [13,14]. Subsequently, we
found that when the outflow tract was banded at stage 21
ventricular dry weight increased about 40% above the
sham-banded controls, and that this compensatory growth
was due to myocyte hyperplasia since myocyte size and
DNA:protein ratio were similar to the controls [6]. Moreover, overload did not affect embryo dry:wet weights,
heart rate, cardiac output, or overall ventricular growth.
Thus, accelerated ventricular growth during this period of
time did not result in abnormalities in these structural and
functional parameters.
In the current study we found that pressure overload
during stages 21–29 increases the thickness of the compact
region of the left ventricle as evidenced by an increase in
epicardial to endocardial distance. In sharp contrast, persistence of the pressure overload to stage 36 alters left
ventricular growth and morphology, i.e., the compact
region grows more slowly and usually develops a more
ellipsoidal shape than that of hearts with a normal afterload. This reduction in growth in the compact region
resulted in a smaller area of compact myocardium and a
greater area of spongy myocardium compared to the
shams. Growth between stages 29 and 36 in the banded
hearts was so markedly depressed that their ventricles were
smaller than the shams. Thus, our experimental design has
included two stages of pressure overload: (1) a compensated stage with acceleration of ventricular growth, and (2)
a decompensated stage in which ventricular growth is
decelerated compared to the nonoverloaded sham hearts.
The reason for the decelerated ventricular growth in
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R. J. Tomanek et al. / Cardiovascular Research 41 (1999) 663 – 671
Fig. 3. Scanning electron micrographs of hearts from HH stage 36 hearts. Note that the compact region between arrowheads of the sham heart (A) is thicker
than than that of the pressure-overloaded heart (B). Sinusoids are indicated by arrows. The left ventricles of overloaded hearts are usually more ellipsoid
than those of shams, as seen in these micrographs. At a higher power view of sham (C) and overloaded (D) hearts, the presence of sinusoids in the
ventricular wall can be more clearly visualized (arrows). Note that sinusoids are closer to the epicardium in the stage 36 overloaded hearts (compared to
shams), i.e., the compact layer is less developed while the spongy layer persists to a greater extent. In A, Bar5 1 mm; in B, Bar5100 mm.
banded hearts between stages 29 and 36 is not evident.
One possible reason could be the poor development of the
coronary vessels and as a consequence limited O 2 availability to the compact region. Thus, the compact myocardium failed to expand because coronary perfusion was
inadequate or failed to develop. Another possible reason
for the decelerated growth is that the pressure overload
increased as the band became more restrictive as the
outflow tract increased in diameter, thereby exhausting the
adaptability of the myocytes. This explanation, however, is
less likely since myocytes maintained normal ultrastructural features.
Fig. 4. (A) The mean thickness (6SEM) of the compact region of the left
ventricular wall is illustrated for each of the four groups. Mean thickness
was based on measurements of the shortest distance from the epicardial–
myocardial border to the endocardium as derived from projected images
of tissue sections. (B) Percent of the left ventricle composed of spongy
myocardium. These values are based on image analysis of slides of
cross-sectioned left ventricle. Five to seven hearts were included in each
group.
4.2. Accelerated vasculogenesis /angiogenesis with
accelerated ventricular growth
The accelerated ventricular growth between stages 21
and 29 was substantial (64% increase in wet weight) and
occurred in a relatively short time span (2.5 days). The
finding that coronary vascular growth matched the increase
in mass indicates that the latter, by some yet undefined
R. J. Tomanek et al. / Cardiovascular Research 41 (1999) 663 – 671
669
Fig. 5. Electron micrograph of capillaries (between arrows). This micrograph represents a portion of one field used for analysis of vascular volume density
and numerical density.
mechanism, dictates the degree of vessel formation. In
postnatal hearts the vascular response to ventricular hypertrophy is usually limited and / or requires a long period
of time, and is dependent upon the stimulus evoking the
ventricular enlargement (reviewed in Refs. [1,2]). The
current study is the first to address adaptive vascular
growth in embryonic hearts.
Vasculogenesis occurs as the compact region of the
ventricle thickens, and is not seen in trabeculae until much
later and only when they reach a certain critical thickness.
Thus, vascularization occurs as myocardial cells become
remote from the endocardium and their initial source of
oxygen. Accordingly, hypoxia is one possible trigger of
vascularization in the embryonic heart. Consistent with this
hypothesis is the work of Hoper and Jahn [15] indicating
that oxygen levels influence the vascular density of the
Fig. 6. Vascular volume percent. Means and SEM are given. Number of
hearts is indicated in parentheses.
Fig. 7. Numerical vascular density (means6SEM). Number of hearts is
indicated in parentheses.
670
R. J. Tomanek et al. / Cardiovascular Research 41 (1999) 663 – 671
Fig. 8. Light micrographs of coronary arteries (*) from sham (A) and overloaded (B and C) chick hearts at stage 36, from sections at the base of the aorta
(V5valve). The coronary arteries from the overloaded hearts are considerably smaller than those of the sham hearts. The arteries in B were not completely
confluent with the aorta (the aortic wall was partially penetrated). All micrographs are at the same magnification. Bar525 mm.
yolk sac of chick embryos, i.e., hypoxia increases vascular
density, while hyperoxia decreases vascular density. Vascular endothelial cell growth factor (VEGF) mRNA is
upregulated by hypoxia [16,17] and accordingly could
serve as a primary inducer of coronary vasculogenesis.
This growth factor and its receptors are expressed in early
embryos [18]. Flk-1 (a VEGF receptor) mRNA expression
has been found in all endothelial cells at all stages of
development [19]. Thus, one possible explanation of the
accelerated coronary growth in the hearts of pressureoverloaded embryos is that a relative hypoxia enhances
VEGF expression and accelerates coronary growth in the
compact region which initially expands more rapidly then
that of the shams. Indeed, unpublished data on embryonic
rat hearts from our laboratory show that the highest
expression of VEGF mRNA is initially near the epicardium and corresponds to the first sites of vascular tube
formation. While VEGF is a key vasculogenic / angiogenic
factor, other growth factors may also play a regulatory role
in accelerated myocardial vascularization. For instance,
basic fibroblast growth factor is upregulated in the early
stage of embryonic myocardial vascularization [8]. We
have shown that both of these growth factors play a role in
coronary vessel formation in vitro in the rat [20].
An unexpected finding was the decreased myocardial
and vascular growth in the overloaded hearts between
stages 29 and 36, i.e. heart mass increased only about
2.5-times compared to 6.2-times in the sham. Thus,
vascular volume growth paralleled the increase in ventricular mass. This is evidenced by the fact that vessel volume
density of the overloaded hearts was virtually identical to
the shams at both stages of development. Consistent with
this finding are data on capillary growth in normally
loaded prenatal sheep hearts [21], which indicate that
during the last 4 weeks of prenatal development a con-
sistent intercapillary distance is maintained as capillary
proliferation compensates for the myocardial cell hyperplasia and hypertrophy. Thus, cardiac mass and myocardial
vascularity are closely linked during the embryonic period.
This relationship is also evident in the distribution of the
two main coronary arteries in humans [22]. Regardless of
left:right ventricular mass ratio, the right coronary artery
perfuses approximately the same fraction of the ventricular
mass. Thus, the perfusion territory of a given vessel adjusts
to the cardiac mass.
Although vessel volume percent was similar for overloaded and sham chicks at each stage studied, vessel
numerical density was higher in the overloaded group at
stage 36 compared to the shams. Normal growth between
stages 29 and 36 had no effect on numerical density, and
overload between stages 21 and 29 also failed to influence
this parameter. We interpret the increased vessel numerical
density in the overloaded hearts at stage 36 as evidence
that vascular proliferation was not blunted to the same
degree as myocardial growth between stages 29 and 36,
while vessel size was sufficiently blunted to maintain a
normal volume percent. In contrast, the coronary arteries,
which form at stages 32 and 33, were compromised in the
banded hearts. Their failure to become confluent with the
aorta, or their failure to develop may be the consequence
of (1) the enhanced pressure within the aorta and ventricle
or (2) interference with migrating cells along the outflow
tract or signals originating above the site of ingrowth of
the vascular plexuses which merge to form the coronary
arteries. While neural crest cell ablation leads to variance
in their sites of origin, they still form at the appropriate
time [23]. Therefore, defects in neural crest migration do
not appear to be the cause of coronary artery developmental failure or inadequacy. The tunica media of coronary
arteries is not derived from the cardiac neural crest,
R. J. Tomanek et al. / Cardiovascular Research 41 (1999) 663 – 671
although neural crest cells appear around the origin of the
coronary arteries, but not until embryonic day 14 or about
stage 40 [24].
In conclusion, our data define an interrelationship between myocardial mass and vascular growth. Vascular
volume growth (1) accelerates when myocardial growth is
increased and (2) decelerates when the rate of myocardial
growth decreases. These growth modifications seen in this
model of prenatal pressure overload affect primarily the
compact region of the myocardium where most of the
vascularization occurs. If similar mechanisms operate in
human development, changes in embryonic cardiovascular
function may have long term effects on myocardial formation and vascularization. Our second major finding indicates that outflow tract banding compromises the development of the coronary arteries.
[9]
[10]
[11]
[12]
[13]
[14]
[15]
Acknowledgements
[16]
This work was supported by NIH grant HL 48961. Bick
Phan was a Fellow in the University of Iowa Summer
Research Opportunity Program.
[17]
[18]
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