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Vascular patterning of the quail coronary system during development.

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THE ANATOMICAL RECORD PART A 288A:989–999 (2006)
Vascular Patterning of the Quail
Coronary System During Development
ROBERT J. TOMANEK,* HEIDI K. HANSEN, AND EDUARD I. DEDKOV
Department of Anatomy and Cell Biology and Cardiovascular Center,
University of Iowa Carver College of Medicine, Iowa City, Iowa
ABSTRACT
Recent studies have provided insights into specific events that contribute to vasculogenesis and angiogenesis in the developing coronary
vasculature. This study focused on the developmental progression of coronary vascularization beginning with tube formation and ending with the
establishment of a coronary arterial tree. We used electron microscopy,
histology of serial sections, and immunohistochemistry in order to provide
a comprehensive view of coronary vessel formation during the embryonic
and fetal periods of the quail heart, a species that has been used in a
number of studies addressing myocardial vascularization. Our data reveal
features of progenitor cells and blood islands, tubular formation, and the
anatomical relationship of a transformed periarterial tubular network
and sympathetic ganglia to the emergence and branching of the right and
left coronary arteries. We have traced the pattern of coronary artery
branching and documented its innervation. Finally, our data include the
relationship of fibronectin, laminin, and apoptosis to coronary artery
growth. Our findings bring together morphological events that occur over
the embryonic and fetal periods and provide a baseline for studies into the
mechanisms that regulate the various events that occur during these time
periods. Anat Rec Part A, 288A:989–999, 2006. Ó 2006 Wiley-Liss, Inc.
Key words: vasculogenesis; angiogenesis; arteriogenesis; epicardium
The coronary vasculature develops via closely regulated events that involve vasculogenesis, angiogenesis,
and arteriogenesis. The cells that contribute to the various vessel types of the coronary system arise from the
epicardium, which in turn is derived from the proepicardium. Development of the coronary vasculature has
recently been reviewed (Majesky, 2004; Tomanek, 2005).
Numerous studies have provided insights into signaling
events and the process of epicardium development and
cell differentiation of coronary precursor cells (reviewed
in Olivey et al., 2004). The epicardial-derived cells are a
source of endothelial, smooth muscle, and pericytic cells.
Formation of a capillary network occurs as the compact
region of the left ventricle expands and subsequently
vascular tubes penetrate the aorta at the left and right
coronary cusps, thus establishing the two coronary ostia.
This pattern has been documented in mammalian and
avian species.
Quail and chicken have been used in studies that have
addressed various aspects of myocardial vascularization,
Ó 2006 WILEY-LISS, INC.
including the roles of VEGF and FGF2 (Tomanek et al.,
1998). Several key findings, published between 1989 and
1996, are based on these species. The in-growth of a capillary plexus into the aorta was first documented in 1989
(Bogers et al., 1989). Mikawa and Fischman (1992) used
retroviral tagging to establish independent precursors
for cardiac myocytes, endothelial cells, smooth muscle
cells, and perivascular fibroblasts. Poelmann et al.
Grant sponsor: The National Institutes of Health; Grant
number: R01 HL075446.
*Correspondence to: Dr. Robert J. Tomanek, Department of
Anatomy and Cell Biology, 1-402 BSB, University of Iowa, Iowa
City, IA 52242. Fax: 319-335-7198.
E-mail: robert-tomanek@uiowa.edu
Received 4 May 2006; Accepted 24 May 2006
DOI 10.1002/ar.a.20365
Published online 4 August 2006 in Wiley InterScience
(www.interscience.wiley.com).
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TOMANEK ET AL.
(1993) verified that an extracardiac source provided
these precursors and described the path of the subepicardial precursors in forming vessels at the sinus venosus and myocardium. Subsequent to the finding that the
proepicardium provides cells for the coronary vasculature
(Viragh et al., 1993), proof that the proepicardium is a
source of endothelial and vascular smooth muscle cells
was provided by Mikawa and Gourdie (1996). Recent
studies have used whole mounts to describe arterialization of the myocardium (Kattan et al., 2004) and endothelial cell penetration of the aorta to form the proximal coronary arteries (Ando et al., 2004). Most recently, we have
documented the role of key VEGF family members as regulators of coronary artery formation (Tomanek et al.,
2006). This study also found that red blood cells of cardiac blood islands are derivatives of the proepicardium.
Because of the existence of an endothelium-specific
marker for quail (QH1 antibody), this species is a good
model of coronary vessel formation, as the vasculature is
easily and reliably identifiable. We have utilized quail
heart explants in order to document the regulation of
vasculogenesis and angiogenesis by specific growth factors and their interactions (Yue and Tomanek, 1999,
2001; Tomanek et al., 2001, 2002; Tomanek and Zheng,
2002; Holifield et al., 2004). Other studies have used
chicken-quail chimeras to answer questions regarding
the progenitor cells that contribute to the coronary circulation (Vrancken Peeters et al., 1997, 1999; Bergwerff
et al., 1998; Gittenberger-de Groot et al., 1998, 2004;
Perez-Pomares et al., 1998; Manner, 1999; Eralp et al.,
2005; Van Den Akker et al., 2005) or have examined specific aspects of coronary vessel (Bogers et al., 1989; Dettman et al., 1998; Perez-Pomares et al., 2002; Ando
et al., 2004; Kattan et al., 2004; Pennisi and Mikawa,
2005). These studies, together with our in vitro studies,
have precipitated a number of questions regarding the
sequence of events encompassing vasculogenesis, angiogenesis, and arteriogenesis. Accordingly, the current
study addressed the temporal and spatial aspects of
myocardial vascularization. The data, based on serial
microscopic sections, immunohistochemistry, and electron microscopy, were obtained in order to define vascular patterning in the coronary system during development in this species.
ter paraformaldehyde fixation, then frozen in isopentane
(precooled with liquid nitrogen). Sections were then cut
with a cryostat and used for double immunostaining for
smooth muscle a-actin and either neurofilaments, fibronectin, laminin, or the TUNEL technique.
A few hearts were injected with India ink (Higgins,
Bellwood, IL) and then fixed by immersion in glutaraldehyde. These hearts were processed, embedded in Spurrs,
and ultrathin sections were prepared for electron microscopy. The sections were viewed with a Hitachi 7000
electron microscope.
Immunohistochemistry
The following antibodies were purchased from the Developmental Studies Hybridoma Bank, University of
Iowa (Iowa City, IA): fibronectin (B3/d6) and quail endothelial cell surface marker (QH1). Cy-3-conjugated
monoclonal smooth muscle a-actin (Clone 1A4) and polyclonal laminin antibodies were purchased from Sigma
(St. Louis, MO). Neurofilament polyclonal antibody
(150 KD) was from Chemicon (Temecula, CA). Antibodies
for VEGF and FGF-2 (bFGF) were purchased from
Santa Cruz Biotechnology (Santa Cruz, CA). Both paraffin-embedded and frozen hearts were used for immunohistochemical staining procedures. Nonspecific binding
was blocked with 5% normal goat serum, 1% BSA in
PBS. Incubation with primary and secondary antibodies
was in the dark at room temperature. For fibronectin
and QH1, we used Alexa Fluor 488 antimouse-conjugated secondary antibodies (Molecular Probes, Eugene,
OR). For neurofilaments and laminin, we used Alexa
Fluor 488 antirabbit secondary antibodies. Staining for
VEGF and FGF-2 utilized the alkaline phosphatase-conjugated to the secondary antibody (goat, antirabbit).
TUNEL Technology for Apoptosis
Paraformaldehyde-fixed frozen sections were used to
detect in situ apoptosis. We employed an ApopTag Fluorescein In Situ Apoptosis Detection Kit (Chemicon)
according to the manufacturer’s instructions. The
method, TUNEL, is based on the modification of genomic
DNA utilizing deoxynucleotidyl transferase for detection
of positive cells. All nuclei were revealed by nuclear
counterstain with propridium iodide (DAPI).
MATERIALS AND METHODS
Fertile quail embryos (Northwest Game Girds, Kennewick, WA) were incubated under humified conditions at
388C. We staged embryos either by days of incubation or
by the system of Hamburger and Hamilton (1951).
Tissue Preparation
Hearts from embryos incubated (in ovo) for 5–18 days
were fixed by perfusing 4% paraformaldehyde buffered
with PBS via the apex of the left ventricle. The basal
half of the heart was processed, embedded in paraffin,
and serial sections (6 mm thick) were placed on glass
slides. Slides were then deparaffinized, rehydrated, and
stained with hematoxylin and eosin, or the sections were
viewed prior to deparaffinization under dark-field microscopy and slides representing select areas of the
heart and great vessels processed for immunostaining. A
subset of hearts was cryoprotected in 5% sucrose/PBS af-
RESULTS
Coronary Cells and Precursors:
Electron Microscopy
Coronary vascular cells are derivatives of the epicardium/subepicardium. As documented by electron microscopy, delamination of epicardial cells (Fig. 1A) necessitates
the breaking of tight junctions (Fig. 1B). The subepicardial population includes a variety of cells, some of which
migrate to the myocardium and are active in protein synthesis, as indicated by a rich endoplasmic reticulum (Fig.
1B). Phagocytosis occurs in this region and less commonly
in the myocardium (Fig. 1D and E) and frequently cellular remnants are enclosed in a cytoplasmic ring. In some
instances, glycogen is present, indicating that the degenerated cells were cardiomyocytes. Some areas of the subepicardium, which vary greatly in thickness depending on
the region, also include erythrocytes (Fig. 1C). These cells
DEVELOPMENT OF CORONARY VASCULATURE
991
Fig. 1. Electron micrographs of epithelial-derived coronary vascular
cells. A: Two mesothelial cells (arrows) delaminating from the epicardium (E8 heart). B: Tight junction (arrow) between two mesothelial
cells (asterisk) in E10 heart. Note the large subepicardial cells with
rough endoplasmic reticulum indicative of protein synthesis. C: Mesothelial cells (Mes) of the epicardium with underlying (subepicardial)
cells (E7 heart). The latter often include erythrocytes (asterisk).
D: Subepicardial region with cellular remnants surrounded by a cell
ring with thin cytoplasm (between arrows) in an E7 heart. E: A similar
degenerative profile (between arrows) consisting of cell elements
including contractile filaments (E9 heart). The debris lies within a tubular structure just under the mesothelium (Mes) and is another example
of phagocytosis in this region. F: Subepicardial network of cells with
multiple thin processes. A cell remnant with glycogen is seen (arrow).
Note the capillaries (asterisk) in this E6 specimen. G and H: Blood
islands (demarcated by arrowheads) in an E10 heart. They are seen in
the subepicardium (G) and in the myocardium (E10 heart) with adjacent mature capillaries (asterisk). Scale bars ¼ 5 mm.
are present prior to establishment of the coronary circulation and are also found in blood islands before and
after coronary arteries are formed (Fig. 1G and H). Blood
islands occur most frequently in the subepicardium and at
the base of the aorta, but may also be seen within the
myocardium (Fig. 1H). The expanded subepicardium, typically seen at the aortic root and A-V groove, contains a
network of cells with long multiple cellular processes
abutting one another (Fig. 1F). The earliest signs of tubulogenesis, i.e., formation of capillaries, is observed in this
network of cells (Fig. 1F).
Vascular tubes (capillaries) vary in their morphology,
which may be in part accounted for by their stage of development. Cells may be relatively thick or very narrow
(Fig. 2). A tube profile may consist of two or more cells or
a single cell with a large vacuole (not shown). Mature
forms have a scant cytoplasm and usually an adjacent
pericyte (Fig. 2C, E, and F). The development of a myocardial tubular (capillary) network develops between E6
and E8, prior to the establishment of the coronary ostia
and a functional coronary circulation at E8–E9 (Tomanek
et al., 2006). We injected India ink via the apex of beating
E7 or E9 hearts to determine the extent of perfusion of
the compact region of the left ventricle. As seen in Figure
2, we failed to find ink carbon particles in vascular tubes
at E7, even when erythrocytes were present (Fig. 2C). In
contrast, nearly all vessels in E9 hearts contained the
tracer (Fig. 2D–F). These data support the conclusion that
the tubular network of the ventricles is not, to a significant extent, open to the sinusoidal system of the trabecular spongy portion of the developing ventricles.
Temporal Aspects of
Myocardial Vascularization
We double-immunostained tissue sections with antibodies against QH1 and smooth muscle a-actin to
reveal endothelial lined channels and arteries/arterio-
992
TOMANEK ET AL.
Fig. 2. Election micrographs of capillaries from E7 (A–C) and E9
(D–F) beating hearts perfused via the ventricular apex with India ink.
Capillaries are in various stages of development. Arrows indicate ink
carbon particles in E9 capillaries, but not in those of E7. Note that
even when erythrocytes are present in E7 capillaries, they are devoid
of carbon particles. Intraluminal erythrocytes are likely derived from
blood islands at this stage of development. Scale bar ¼ 5 mm.
les and veins (Fig. 3). These micrographs show the
early stage of tubulogenesis in the compact region of
the myocardium (E6.5) and its progression through
E18, which corresponds to the end of the fetal period.
Tubular density increases during the next few days
(E8, E9, E10). Although both coronary stems are
formed by E9, the coronary arteries develop their tunica media gradually in a base-apex direction and
accordingly smooth muscle is not seen at the base-apex
midpoint in the E9 specimen. After this time, coronary
vessels increase in diameter by remodeling and more
branches are formed, as evidenced by muscle cell
recruitment to endothelial-lined channels (E10–E18).
The growth of the vasculature parallels the growth of
the ventricular wall, as previously documented by us
in chicken embryos (Tomanek et al., 1999). In the
quail, wall thickness increases sharply from 60 mm at
E7 to over 200 mm at E10 (Fig. 3). During the next
6 days, the increase is more gradual as wall thickness
reaches about 340 mm at E16. The diameter of the left
coronary artery in general increases in proportion to
the expansion of the ventricle’s compact region.
Coronary Ostia and Coronary
Artery Growth
As previously described (Ando et al., 2004), an endothelial network develops around the aortic root at E6–
E7, as seen in Figure 4A. Penetration of the aortic wall
by a tubular network to form the two coronary ostia
occurs between E8 and E9 (Fig. 4B) as previously documented (Ando et al., 2004; Tomanek et al., 2006). At the
site of developing coronary ostia, we have consistently
noted the presence of a blood island. The formation of
the two coronary ostia is not simultaneous as indicated
by the observation that E8 hearts frequently have only
one coronary artery.
The onset of coronary artery formation in E8 embryos
(Fig. 4B) is characterized by a narrow channel through
the aortic wall. The aortic smooth muscle cells realign
themselves at this site from a circumferential alignment
to one more in an outward direction, i.e., parallel to the
coronary artery. This pattern is maintained through the
embryonic/fetal period as seen in an E16 specimen (Fig.
4C). Micrographs of coronary arteries at their site of origin during development in E14 and E16 hearts are seen
in Figure 4D–H. We consistently noted three features at
this site. First, multiple branches occur close to the root
of the coronary artery. Second, a vascular plexus consisting of capillaries and venules surrounds or is in close
proximity of the coronary artery. This appears to be an
expansion of the tubular plexus noted in the stages prior
to the development of the coronary ostia. Thus, the tubular plexus, seen prior to coronary artery formation
and contributing to the artery’s formation, does not
regress in its entirety. Rather, the portion of the plexus
at the coronary artery stems enlarges and surrounds
these vessels. We noted this as a consistent feature in
all hearts (E9–18). Third, parasympathetic ganglia are
adjacent to the root of the coronary artery and present
along its sites of branching.
The patterns of coronary artery growth and spatial
distribution were examined by following their course in
serial sections, and a summary is illustrated in Figure 4I.
The left coronary artery develops numerous branches, as
is seen at the interventricular groove. These course toward the apex and posteriorly. The right coronary artery
divides at its root into a large septal artery and a smaller
right coronary artery division. The septal courses through
the middle of the septum and provides branches anteriorly and posteriorly. Subsequently, with further development and an increase in LV wall thickness, the large
branches of the left coronary artery are positioned somewhat closer to the epicardium. Vascular remodeling of
major branches occurs simultaneously with muscularization of the endothelium-lined channels. Thus, both the
two main coronary arteries and their major branches
increase in diameter. For example, as the lumen of the
left coronary artery is formed at E8, it is only 5–15 mm in
diameter, but enlarges to about 30–40 mm at E9.
We immunostained selective serial sections for VEGF
and FGF-2 to determine preferential staining of various
cells and structures (Fig. 5). Although both angiogenic
growth factors are expressed throughout the myocardium, we noted selective staining of some subepicardial
cells. VEGF immunoreactivity was intense in the cells of
the epicardium and subepicardium, and in a select population of cells near the root of coronary arteries, and in
blood islands (Fig. 5A–C). Most were spindle-shaped;
they appear to be specialized components of the subepicardial population. At the root of the aorta, we noted
that some smooth muscle and endothelial cells of small
vessels stained distinctly for FGF-2, while larger vessels
displayed a very weak reactivity (Fig. 5D).
DEVELOPMENT OF CORONARY VASCULATURE
993
Fig. 3. Micrographs of left ventricular (LV) cross-sections immunoreacted for QH1 to label endothelium and smooth muscle a-actin to
label arterioles and arteries. The epicardium is on the left and endocardium on the right of all micrographs. Tubulogenesis progresses as
LV wall thickness increases from early onset at E6.5 through subsequent stages. Mean wall thickness 6 SE (n ¼ 4–7 per gestation day)
of the compact myocardium is shown in the graph (lower right corner).
Formation of the coronary ostia occurs at E8–E9, after which time coronary artery branches develop progressively from base to apex
(stained red in these micrographs). Most of these vessels are situated
intramurally in the ventricular wall and increase in size over time. Note
major arteries and veins at E18 (seen in the bottom of the figure).
V, vein; S, sinusoid. Scale bars ¼ 100 mm in E6.5 through E16 and
200 mm in E18.
Development of the Arterial Wall: Innervation,
Extracellular Matrix, and Apoptosis
seen in later stages (E12 and E15), the nerve fibers can
be seen in contact with the tunica media. The numbers of
nerve fibers associated with vessels increased notably
between E10 and E15. Thus, nerve fiber growth appears
to follow arteries, with innervation becoming more evident
in the later stages of development.
Our data indicate a close association between vascular
smooth muscle cells and two key extracellular components: fibronectin and laminin (Fig. 5H–M). Developing
arterioles/arteries are characterized by an intermingling
Having defined the temporal/spatial patterning of the
coronary arterial system, we focused on development of
the arterial wall. Based on immunohistochemical reactivity of neurofilaments, we show that innervation of the coronary arteries and arterioles is progressive (Fig. 5E–G).
In smaller vessels, as seen in E10, the nerve fibers tend
to lie near the vessel, while with further development,
994
TOMANEK ET AL.
Fig. 4. Micrographs of coronary artery development. A: Immunostained with QH1. B: H&E-stained. C: 1 mm section of Spur’s embedded heart stained with Richardson’s solution. D–H: Immunostained
with QH1 and smooth muscle a-actin. A: Base of aorta (ao), in an E7
specimen surrounded by capillary plexus (arrows) immunostained for
endothelium. This capillary plexus will penetrate the aorta at E8–E9 to
form the coronary artery stems. B: Early formation (E8) of a coronary
artery (arrows) whose lumen is confluent with the lumen of the aorta
(20). C: Aortic wall (E16) near coronary artery ostia (not seen in this
field). Coronary artery branch on surface of aorta (arrow). The aortic
smooth muscle cells are disorganized around the site of coronary
ostia (within broken lines) in contrast to their characteristic circumferential arrangement (asterisk). D–G: Coronary artery roots and
branches in E14 (D and E) and E16 (F and G) embryos. D: Coronary
artery (ca) at the level of the aortic valve (av) in close proximity to a
plexus of capillaries and venules. pa, pulmonary artery. E: Coronary
artery (ca) stem emerging through the wall of the aorta. Again, note
the capillary/venous plexus closely opposed to the artery (arrow
heads). Some of these vascular channels are associated with cells
positive for smooth muscle a-actin. F: A coronary artery stem at the
aorta. Note that immunostaining for smooth muscle a-actin is more
intense in the coronary artery than in the aorta. Branching of the main
coronary artery (as indicated by the arrow) occurs close to the aorta.
G: Branching of the main coronary artery at its root. Note the capillary/venous plexus (arrow heads) surrounding the coronary artery;
most of its components are in the venular diameter range (> 10 mm).
H: Major coronary artery branches (E16). Several branches of the left
coronary artery are seen in the left ventricular free wall at the lower left
coroner of this micrograft. The three arteries seen in the septum are
divisions of the large septal artery, which is the main stem of the right
coronary artery. rv, right ventricle; lv, left ventricle; sep, septum. I: Diagram illustrating the branching and distribution of the right and left
coronary arteries. Note the periaortal cell and tubular network and its
presence as a venous-capillary plexus at the coronary stems; the parasympathetic ganglia are consistently seen at the coronary artery
stems and their branches. Scale bars ¼ 25 mm.
of smooth muscle cells with a fibronectin network. The
latter is generously distributed at sites of vessel formation. As seen in Figure 5H, smooth muscle cells align
themselves with fibronectin, which appears to form a
tract for them as they are recruited to vascular tubes.
Regions containing early vessel formation are characterized by a generous fibronectin network. We previously
showed that fibronectin in the developing rat heart pre-
cedes capillary formation (Rongish et al., 1996). Thus,
the origin of the extracellular component is likely from
fibroblasts. With further development, fibronectin, as
seen in E12 and E15 hearts, forms a fine network within
the tunica adventitia and extends into the tunica media.
Laminin is somewhat similarly distributed, i.e., closely
associated with smooth muscle cells, but additionally
forms a ring somewhat removed from the media. This
DEVELOPMENT OF CORONARY VASCULATURE
995
Fig. 5. Immunostaining for VEGF (A–C), FGF-2 (D), neurofilaments
(E–G), fibronectin (H–J), laminin (K–M), and apoptosis (N–P). A: VEGF
immunoreactivity is evident throughout the myocardium, but intense in
the epicardial and subepicardial cells (E9 heart). B: VEGF expression
is seen in a select population of cells (arrows) at the root of the coronary artery (ca), as well as the myocardium (left bottom of micrograph)
in this E15 heart. C: Some cells (arrows) in a blood island stain
intensely for anti-VEGF antibody (E11 specimen). D: FGF-2 expression
is selectively intense in small vessels (arrows) at the aortic root (E14
specimen). Note that 3 embryonic days are represented in the micrographs as follows: E, H, K, N (E10), F, I, L, O (E12), and G, J, M, P
(E15). All of these specimens were immunoreacted for smooth muscle
actin (shown in red). Neurofilament reactivity (green profiles), a marker
for nerve fibers, follows arterioles and arteries (E–G). Nerve fibers are
seen at the junction with smooth muscle (arrows) in E10 specimen; as
vessels mature, nerve fibers extend into vessels’ tunica media. Arterial/arteriolar development is closely associated with fibronectin (H–J).
Smooth muscle cells (arrows) are intermingled with a relatively dense
fibronectin network during early formation of the media in arterioles
(H). In more mature vessels (I and J), fibronectin fibers surround
smooth muscle cells in the media and also continue into the adventitia
(arrows). Laminin also exists as a network within the media (K–M), but
in addition encircles the vessels (arrows). N–P: Apoptotic nuclei (green)
are found scattered throughout the myocardium. In arterioles/arteries,
they can be seen in both the adventitia and media in various sized vessels. In O, several apoptotic nuclei are seen at a branching point.
characteristic circle of laminin was noted in both developing and more mature vessels (Fig. 5K–M).
Finally, we used TUNEL to localize apoptotic cells in
the arterial wall. Apoptotic nuclei were encountered
within the myocardium, subepicardium, and in arterioles/arteries. Since remodeling of vessels is necessary for both increases in their diameters and pruning
of the tubular network, it is not surprising to find
996
TOMANEK ET AL.
apoptotic figures in the media and adventitia. They
were seen in small as well as large arterioles/arteries
(Fig. 5N–P).
tubulogenesis. The magnitude of growth of the compact
component of the left ventricle during the embryonic period, as noted by wall thickness, is very similar in quail
(the current study) and chicken (Sedmera et al., 2000).
DISCUSSION
Since formation of the coronary vasculature is strikingly similar in avian and mammalian species (Tomanek, 2005), data obtained from quail and chicken
reflect developmental patterns and mechanisms in man
and other mammals. Experiments on avian hearts provide two distinct advantages over those using mammalian species, namely, accessibility and cost. The current
study provides the first temporal-spatial documentation
of coronary vascularization throughout the entire embryonic/fetal period.
Tubulogenesis
Hirakow (1983) noted that the first definitive blood
vessels in the developing human heart resembled blood
islands, with ‘‘primitive erythroblasts.’’ Perez-Pomares
et al. (1998) found that 20% of the cells within the
expanding subepicardium in the embryonic hamster
heart were VEGFR-2-positive, suggesting that they were
endothelial or erythrocyte progenitors. Subsequently,
CD45-positive hematopoietic precursors were shown to
precede blood vessel formation in the quail heart
(Kattan et al., 2004). Most recently, using retroviral tagging, we documented that erythrocytes in blood islands
are derivatives of the proepicardium (Tomanek et al.,
2006). Thus, the proepicardium is a source of not only
all vascular components (Mikawa et al., 1992, 1996), but
also erythrocytes that associate with endothelial cells
that form vascular channels. Our current study and previous work (Tomanek et al., 2002) also document a very
strong expression of VEGF protein in epicardial and
subepicardial cells. Thus, this ligand is available in high
quantity at the site of cells that delaminate from the mesothelium of the epicardium and that express the
VEGFR-2 receptor. The earliest tube formation (capillaries) occurs in the subepicardium, where there is the
highest level of VEGF expression.
Vrancken Peeters et al. (1997) noted that the myocardial tubular network has nonluminized strands that connect to the sinusoids that are continuous with the ventricular lumen. They suggested that flow through the
capillary bed prior to connections to the aorta is possible. Subsequently, Manner (2000) grafted quail proepicardium to chick hearts at E3 and incubated the chimeras until E10. He found small patches of quail-derived
endocardium in communication with the coronary vasculature, which he described as ‘‘ventriculo-coronary communications.’’ Although the question of flow between
the ventricular lumen and the coronary system is not
entirely resolved, our electron micrographs of ink-injected
beating hearts show that capillary profiles lack carbon
particles prior to the establishment of the coronary
arteries. Even when erythrocytes were present in the vascular tubes of E7 hearts, carbon particles were lacking.
Thus, blood islands lacked connections with the ventricular lumen. As the compact region of the ventricle expands,
tubulogenesis keeps pace, a finding consistent with our
previous work on embryonic chicken hearts (Tomanek
et al., 1999). In that study, we showed that experimentally
accelerated myocardial growth is matched by enhanced
Coronary Arterial System: Arteriogenesis
Since the first evidence that the coronary arteries are
formed by ingrowth, rather than outgrowth, into the
aorta (Bogers et al., 1989), this concept has been corroborated by a variety of approaches in several species
(Waldo et al., 1990; Poelmann et al., 1993; Tomanek
et al., 1996). Ando et al.(2004) showed that endothelial
strands in the quail penetrated the aorta at E6 and E7
and fused by E8 to form the coronary stems. Our own
findings agree with this temporal sequence, but we also
provide evidence that the tubular network at the coronary ostia expands into a venous-capillary plexus, surrounds the coronary artery stems, and persists through
venous-capillary plexus, and the coronary arteries and
the parasympathetic ganglia remain in close proximity
throughout this period (Fig. 4).
It is not surprising, as documented in this study, that
parasympathetic ganglia are closely associated with the
roots of the two coronary arteries and also with their
immediate branches. The presence of these ganglia has
been documented to be necessary for normal development of coronary artery vascular smooth muscle (Hood
and Rosenquist, 1992). Their study used neural crest
ablation to demonstrate a spatial disordering of smooth
muscle, the presence of only one coronary ostium and
anomalous coronaries from the subclavian artery. Moreover, cardiac ganglion cells in the chick have been
shown to originate from the cardiac neural crest (Verberne et al., 1998). The latter are not coronary artery
smooth muscle precursors, but rather are positioned at
the base of coronary arteries (Waldo et al., 1994), a
finding that suggests their signaling may contribute to
formation of the coronary ostia. The locations of the
intracardiac ganglia in human neonate (Smith, 1971)
and adult (Pauza et al., 2000) have been described in
detail.
The formation of the coronary ostia at E8–E9 is dependent on a variety of factors (reviewed by Tomanek,
2005). Foremost is a functional epicardium, including
the timing of its outgrowth (Eralp et al., 2005). Our
recent work has shown that VEGF family members,
especially VEGF-B, play a critical role in the formation
of coronary ostia (Tomanek et al., 2006). Further growth
and remodeling of the coronary arteries have not
received much attention. That arteriolar growth is dependent on FGF-2 has been demonstrated in neonatal
rats (Tomanek et al., 2001). In that study, we showed
that arteriolar growth was attenuated in response to
anti-FGF-2 neutralizing antibodies. When both FGF-2
and VEGF are experimentally decreased, there occurs a
shift in the arteriolar hierarchy, i.e., length density of
the smallest arterioles is decreased and increased in the
largest arterioles. Moreover, precocious expression of
FGF-2 induces abnormal coronary artery branching
(Mikawa, 1995). Current studies in our laboratory on
embryonic quail indicate that PDGF-B, which is expressed in coronary endothelial cells (Van Den Akker
et al., 2005), in combination with FGF-2 are critical for
coronary artery formation (data not shown).
DEVELOPMENT OF CORONARY VASCULATURE
As noted in the current study, coronary artery growth
is rapid. Ratajska et al. (2000) noted that coronary artery diameter in the rat increases fourfold in a 3-day period prior to birth. Our findings in the quail show a progressive increase in coronary artery diameter that follows the increase in ventricular thickness. By analysis
of serial sections, we have also noted a consistent pattern of branching and distribution of the coronary arterial tree, including a dominant septal artery. The substantial increases in artery diameter must involve a
rapid remodeling process, which occurs with increased
flow, as shown in adult models of remodeling (Gibbons
and Dzau, 1994; Sho et al., 2003). It has been reported
that coronary artery formation predates the periarterial
Purkinje fibers (Harris et al., 2002). The periarterial
Purkinje fibers thus follow the course of coronary
arteries. Interestingly, the large main septal artery lies
between the two main bundle branches of the conduction system. A recent review of apoptosis in the chick
heart notes that the greatest numbers of apoptotic figures occur in the outflow tract in HH 19–26 hearts,
with a concentration noted just below the aorta at HH
31 (Martinsen, 2005). This concentration, also noted by
Schaefer et al. (2004), at this developmental stage corresponds to the site of coronary ostia formation. Velkey
and Bernanke (2001) documented apoptosis within the
aorta at the site of penetration by the peritruncal capillary plexus, as well as pruning of the capillary plexus
around the aorta, a finding that is consistent with
observations on quail (Ando et al., 2004). Although it is
well known that apoptosis functions in vascular pruning and reduction in vessel diameter (reviewed by
Fisher et al., 2000), less attention has been paid to its
role in angiogenesis and arteriogenesis. In this regard,
it has been shown that inhibition of apoptosis attenuates capillary formation in vivo (Segura et al., 2002). It
has been noted that apoptosis occurs mainly in the
‘‘nonmyocardial compartment’’ (Poelmann et al., 2000),
with a high rate in neural crest cells (Poelmann et al.,
2005).
Apoptotic figures were noted in coronary arteries of
HH 40 (E14) chick hearts (Cheng et al., 2002). Our
study documents apoptosis in various-sized arterial vessels at E10 and later in both the medial and adventitial
tunics. This suggests a role for apoptosis in coronary
vessel remodeling. It is important to understand apoptosis in the developing heart because it plays a role in cardiac malformations (Fisher et al., 2000).
The importance of the extracellular matrix in endothelial morphogenesis and stabilization has been reviewed
(Davis and Senger, 2005). Evidence is provided to support the idea that fibronectin and collagen matrices
stimulate tubulogenesis, whereas laminin-rich matrices
affect stabilization. This supports our previous finding
on rat embryonic development of the coronary vasculature (Rongish et al., 1996). In that study, we documented
a role for fibronectin as scaffolding for vascular cells and
suggested that laminin deposition coincides with tube
formation. The current study, which focused on the arterial system, also indicates the importance of fibronectin
for cell migration as evidenced by smooth muscle cell
contact with fibronectin strands. We show that in addition to the presence of fibronectin and laminin in the
artery’s media, the latter forms a distinct ring outside of
the tunica adventitia. Both of these noncollagenous gly-
997
coproteins are cell adhesion molecules that are highly
expressed in the embryonic human coronary arteries
(Kim et al., 1999). However, our findings should not be
interpreted to mean that fibronectin and/or laminin are
the only extracellular matrix components regulating vessel development. For example, fibulin-1 and fibulin-2 are
expressed in tunica adventitia and basement membrane
of the endothelium in human embryonic blood vessels
(Miosge et al., 1996).
Perspective
Myocardial vascularization includes many phenomena
that are spatially and temporally regulated. The current
study illustrates the progression of myocardial vascularization in the quail during the embryonic and fetal periods, i.e., E6–E18. Most of our data concern the development of the arterial hierarchy, i.e., arteriogenesis, a component of vascular development that has received less
attention than the earlier stages of vascularization, i.e.,
vasculogenesis and angiogenesis.
Our findings, some of which confirm and complement
data from other studies, provide new insights into several developmental events. Using electron microscopy,
we show some features of coronary precursors, including
blood islands, and demonstrate that the tubular network
is almost entirely without connections to the endocardium. The hierarchy of the arterial system, based on serial sections, has been revealed and its development
noted with respect to innervation, extracellular matrix,
and apoptosis. Thus, the findings presented here provide
an overview of a relatively broad period of vascular development. Our goal in providing these data was to
chart the components of vascular development and provide a foundation for future studies on the mechanism
underlying formation of the coronary vasculature.
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