© 1997 American Heart Association, Inc.
Articles |
Unilateral Vitelline Vein Ligation Alters Intracardiac Blood Flow Patterns and Morphogenesis in the Chick Embryo
From the Department of Anatomy and Embryology, Leiden University (the Netherlands).
Correspondence to Dr R.E. Poelmann, Department of Anatomy and Embryology, Leiden University, PO Box 9602, 2300 RC Leiden, the Netherlands.
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Abstract To study the role of blood flow in normal and abnormal heart development, an embryonic chicken model was developed. The effect of altered venous inflow on normal intracardiac blood flow patterns was studied by visualization of blood flow with India ink. At stage 17, India ink was injected into a capillary or small venule within a specific yolk sac region. After determination of the normal intracardiac flow pattern, the right lateral vitelline vein was ligated, and the new intracardiac flow pattern was studied. Ligation resulted in disturbance of normal intracardiac flow patterns, which was most obvious in the conotruncus. The long-term effect of these abnormal intracardiac flow patterns on the development of the heart and pharyngeal arch arteries was investigated by permanent ligation in ovo with a microclip at stage 17 and subsequent evaluation at stages 34, 37, and 45. These experiments revealed anomalies of the vascular system in 58 of the 91 ligated embryos studied. We observed intracardiac malformations consisting of subaortic ventricular septal defects (n=52), semilunar valve anomalies (n=19), atrioventricular anomalies (n=7), and pharyngeal arch artery malformations (n=32). It is concluded that abnormal intracardiac blood flow, resulting from hampered venous inflow, may result in serious intracardiac and pharyngeal arch artery malformations comparable to defects observed in embryonic chicken models subjected to neural crest ablation, cervical flexure experiments, and excessive retinoic acid treatment.
Key Words: blood flow visualization • hemodynamic interference • heart development • cardiac malformation
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Introduction |
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The role of blood flow in heart morphogenesis has been a subject of interest for a long time. The description of two spiral blood streams in the tubular heart after observing moving blood cells led to the hypothesis that septa would arise between these two streams because of less internal pressure.1 2 3 Unfortunately, these two streams appeared to be an optical illusion caused by the nonconcentric cardiac contraction pattern.4 In addition, subsequent visualization of blood flow with dye indicators did not demonstrate the spiral blood streams described earlier, although specific intracardiac blood flow patterns during development were demonstrated.5 6 7 Because these stage-specific patterns are related to the development of the yolk sac circulation,7 blood flow might be an important physical factor in heart development during specific stages of embryonic development.
The first phase of heart looping seems to be independent of blood flow, as Manasek and Monroe8 have shown with in vitro–cultured disconnected hearts. This is currently supported by studies involving gene regulation of heart looping.9 Since absence of blood flow results in cardiac lumen obstruction by cardiac jelly proliferation,10 an additional role for blood flow is indicated in the refined modeling of the endocardial cushions and ridges.
The long-term effect of blood flow on heart development was extensively studied by mechanical obstruction of various arterial vessels and heart segments leading to a variety of malformations, like double-outlet right ventricle (DORV), ventricular septal defects (VSDs), and pharyngeal arch artery anomalies.11 12 13 14 15 16 17 18 19 20 However, in addition to altered blood flow, normal morphogenetic processes in the pharyngeal arch area could have been disturbed by mechanical interference in this area. Impaired cardiac neural crest cell migration as well as suppression of cervical flexure and failure of thoracic wall closure lead to similar cardiac malformations.21 22 23 24 25 26
To avoid the problems of intervention at the arterial side of the heart, venous blood flow has also been manipulated by transection,27 28 29 ligation,10 12 and cauterization.12 However, the survival rate of these embryos was very low, and the survival time was rather short. The resulting malformations consisted of abnormal endocardial cushions,12 29 VSD (1 of 24),12 DORV (no percentage),12 and left microphthalmia.27 The effect of decrease in oxygen tension and blood pressure as a result of hemorrhage following transection cannot be distinguished from the effect of changed venous inflow. It is also not known whether cauterization results in the release of certain factors like cytokines, as demonstrated in burning wounds.30
Although human placental anomalies are associated with intrauterine growth retardation31 and human cardiovascular malformations are related to lower birth weight, smaller body length, and head circumference,32 it is, to date, not possible to distinguish any causal relation between placental anomalies, growth retardation, and cardiovascular malformations. It is suggested that embryos with intrinsic growth disturbances may be at risk for errors resulting in cardiac malformations.33 On the other hand, abnormal circulation due to an abnormal functioning heart may result in growth retardation.32 With use of a Doppler velocity system, it was demonstrated that fetuses with intrauterine growth retardation displayed increased umbilical placental and uteroplacental resistance, decreased end-diastolic flow velocities in the descending aorta and umbilical artery, and decreased peak systolic flow velocities at the cardiac level,34 indicating a role for hemodynamic factors in growth retardation. To fully understand the effect of placental blood flow on human heart development, animal models are needed that change blood flow without changing neural crest, cervical flexure, blood volume, or oxygen content. We developed a chicken model in which intracardiac blood flow was changed as a result of a rerouting of venous inflow by ligation of a vitelline vein with a microclip. The present study was designed to investigate both immediate and long-term effects of this hemodynamic manipulation. Ligation was performed at stage 17, after completion of the early phase of looping. Normal blood flow, as described earlier,7 was visualized by India ink injections into defined yolk sac regions and subjected to short-term ligation of the right lateral vitelline vein. Ligation forced the blood to make a detour toward the heart, and the effect of ligation on intracardiac flow patterns was studied. The long-term effect of venous ligation was determined by permanent obstruction of the right lateral vitelline vein with a microclip. The embryos were evaluated both macroscopically and microscopically at stages 34, 37, and 45, with special attention to the morphology of the heart and pharyngeal arch arteries.
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Fertilized white leghorn eggs (Gallus domesticus) were incubated at 37°C and 60% to 70% relative humidity for 70 hours to yield embryos at stage 17.35
Procedure: India Ink Injections During Short-term Ligation
Because India ink injections have been described
earlier,7 only a brief description will be given. The
chick embryos were carefully removed from their yolks, leaving the
vitelline circulation intact. Each embryo was transferred to an
agar-covered plastic Petri dish and immersed in Locke's solution
(0.94% NaCl, 0.0045% KCl, and 0.004% CaCl2) at 37°C.
The yolk sac was flattened and adhered to the agar. A pair of forceps
was connected to a micromanipulator and positioned as close as possible
to the embryo around the right lateral vitelline vein (Fig 1
). The extraembryonic vasculature was injected by means
of a glass needle (tip diameter, 2 to 6 µm) connected by a
pressure-insensitive oil-filled tube to a Hamilton syringe, allowing
careful and controlled administration of minute amounts of india ink
(diluted 1:5 in Locke's solution). The embryo was turned to the right
to reveal a ventral view of the heart. The route of india ink through
the heart was recorded with a video camera (model DXC-151P, Sony)
and a U-matic videocassette recorder (model VO-5630, Sony) and
traced through the following segments of the heart: sinus venosus,
atria, atrioventricular canal, ventricle, and
conotruncus. For each segment of the heart, it was determined whether
the inner, central, or outer curvature was used. Moreover, for the
conotruncus, it was determined whether the current was localized
ventrally, centrally, or dorsally. After registration of the normal
intracardiac route, the forceps were closed, and the altered
intracardiac route was recorded. Each embryo was injected only
once. The injection volume is estimated at 0.002 µL, which is less
than the volume measured to be effective in raising the stroke volume
index in stage-18 embryos.36 Only experiments with a
complete ligation of the right lateral vitelline vein that demonstrated
a clear intracardiac flow pattern before and after ligation were
included. A total number of 28 embryos were used (5 embryos injected in
the anterior yolk sac region, 5 embryos injected in the left lateral
yolk sac region, 8 embryos injected in the right lateral yolk sac
region, and 10 embryos injected in the posterior yolk sac region).
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Ligation In Ovo
The egg shell was cleaned with 70% ethanol and windowed. The
egg was kept warm in isolating foil. All manipulations were performed
using a dissecting microscope. Above the intended ligation site (Fig 1
), the vitelline membrane was removed, and a small incision was made
with a tungsten needle in the yolk sac membrane, adjacent to the
vitelline vein. A microclip, devised from a transmission electron
microscopy carrier grid, was clamped around the right lateral vitelline
vein (Fig 2). The cessation of blood flow proximal to
the microclip was confirmed. Eggs were resealed with Scotch tape. From
the 120 ligated embryos, 25 did not survive, leaving a survival rate of
79% (95 of 120). Only 4 of all ligated embryos that survived had an
open thorax, due to the accidental clamping of a small part of the
still open body wall together with the vitelline vein. These embryos
were excluded from further study. So a total of 91 embryos were
successfully ligated. Sham-operated embryos (n=15), in which all
procedures were similar except for the ligation with the microclip, and
normal eggs (n=10) served as controls. The eggs were reincubated until
stage 34 (embryonic day 8), stage 37 (embryonic day 11), and stage 45
(hatching). The embryo and the heart were macroscopically evaluated
before fixation in a mixture of 2% glacial acetic acid in 100%
ethanol at 4°C. After they were embedded in paraffin, the embryos
were serially sectioned. The 5-µm sections were alternately stained
with hematoxylin-eosin and a modified van Gieson stain.
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In addition, some ligated embryos were prepared for scanning electron microscopy. Half-strength Karnovsky's37 perfusion-fixed hearts were opened frontally with iridectomy scissors. They were rinsed in 0.1 mol/L sodium cacodylate buffer (pH 7.2) and postfixed for 2 hours at 4°C in 1% OsO4 in the same buffer, followed by dehydration in graded ethanol solutions. The preparations were critical point–dried over CO2 by conventional methods, sputter-coated with gold for 3 minutes (Balzers MED 010), and studied in the scanning electron microscope (Philips SEM 525M).
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Extracardiac Blood Flow
Ligation did not change the absolute circulating blood volume but altered the left/right balance of venous inflow to the heart. This was concluded from the observation that blood from the ligated right vitelline vein nearly immediately followed a detour via the caudal capillary plexus present underneath the embryonic trunk. Within minutes, this plexus was remodeled into a new vessel, connecting the right vitelline vein to the left vitelline vein via the posterior vitelline vein (arrow in Fig 1
).
Intracardiac Flow Patterns
Normal intracardiac blood flow patterns and experimentally altered
flow patterns caused by the ligation of the right lateral vitelline
vein are shown in Fig 3. Blood from the anterior yolk
sac region shifted after ligation from the inner curvature (Fig 3a)
toward the outer curvature of the conotruncus (Fig 3f). Moreover,
within the conotruncus, the course changed from dorsal (Fig 3a) to
central (Fig 3f). Blood from the right lateral yolk sac region shifted
from a central position in a nonligated situation (Fig 3b) toward a
more inner curvature (Fig 3g) after ligation. Blood from the left
lateral yolk sac region (Fig 3c) coursed slightly more outward in the
conotruncus after ligation (Fig 3h). Without manipulation, blood from
the posterior shows two possible patterns, either a single current (Fig 3d) or a double configuration (Fig 3e). After ligation, the single
current split into a double one (Fig 3i), whereas the double
configuration only changed its position within the conotruncus, ie,
from dorsal to ventral (Fig 3j).
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In summary, the distribution of the different intracardiac currents
changed after ligation of the right lateral vitelline vein. The most
prominent change occurred in the conotruncus, where basically the outer
curvature was used after clipping. The transverse sections (Fig 3f to
3j) showed that these newly acquired currents through the outer
curvature were all localized ventrally, instead of being evenly
distributed in the normal situation.
Long-term Ligation In Ovo
Permanent ligation of a vitelline vein with a microclip resulted
in anomalies of the heart and pharyngeal arch arteries in 58 cases
(Table 1). It appeared that only the
cardiovascular system was affected. The extremities,
head, and eyes developed morphologically normally. Both shams and
control embryos were macroscopically and microscopically normal except
for one sham embryo, which had a small subaortic VSD. Ligation of the
right lateral vitelline vein resulted in a spectrum of conotruncal
malformations, ranging from a mild form with a low subaortic VSD (Fig 4c) with a muscular outflow tract septum to a severe
form with a high VSD directly below the semilunar valve level (Fig 4b).
In the latter orifice, septation was only brought about by mesenchyme;
myocardium was not observed. In both the mild and severe
forms, semilunar valve abnormalities were common. These varied from an
additional leaflet and fused commissures to bicuspid aortic valve
leaflets and quadricuspid pulmonary valve leaflets. Although
the intracardiac malformations mainly consisted of conotruncal
malformations, we did observe some minor
atrioventricular anomalies (in 7 of 51 embryos), which
always occurred in combination with a VSD. Because of impaired wedging
of the arterial trunk between the
atrioventricular orifices, the tricuspid orifice was
sometimes situated dorsal in relation to the aorta instead of to the
right of the aorta. Also, relatively immature
atrioventricular valves were observed at these
developmental stages. All hearts demonstrated fused
atrioventricular cushions.
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There were also a substantial number (32 of 58 embryos) of pharyngeal
arch artery malformations (Table 1), like abnormal obliteration or
persistence of the fourth pharyngeal arch artery, eg, interruption of
the aorta (Fig 5) or an additional (atretic) left aorta,
respectively, and reduction in diameter of the right third or sixth
pharyngeal arch artery. Except for a reduction in diameter, we never
observed anomalies of the derivatives of the sixth pharyngeal arch
artery, eg, the pulmonary trunks and ducti arteriosi. We
observed within the group of 32 embryos with pharyngeal arch artery
malformations 28 embryos with additional heart malformations, leaving
only 4 embryos with pharyngeal arch artery malformation alone.
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Malformed hearts often coincided with a dextroposed aorta (Table 2). A dextroposed aorta in combination with a subaortic
VSD is defined as DORV. At stage 34, 14 of the 38 abnormal hearts were
DORV; at stage 37, 5 of the 13 abnormal hearts were DORV; and at stage
45, 3 of the 7 abnormal hearts were DORV.
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In the normal heart, the condensed mesenchyme of the
aorticopulmonary septum has a central position between the
aorta and pulmonary trunk at the semilunar valve level,
enclosed by myocardium and vessel wall (Fig 4a). This
central mass of condensed mesenchyme has a double concave shape, from
which the small concave surfaces make contact with the
myocardium and result in myocardial invagination. Directly
below the orifice level, the central mass of the aortopulmonary
septum splits into two prongs that retain their myocardial contact.
Normally, the ventral one is somewhat larger than the dorsal one, with
the latter being flatter. The severe form of subaortic VSD showed a
remarkable ventral position of the condensed mesenchyme. The two prongs
were markedly asymmetric, with the ventral one being much larger than
the dorsal one. At the ventral side, normal contact was established
with the myocardium. The small dorsal prong was connected
to the myocardium at the dorsal side. In these cases,
myocardial invaginations remained widely apart, resulting in a very
short fibrous septum between the aorta and pulmonary orifice
and a high VSD at the semilunar valve level (Fig 4b). There was no
substantial conus septum. In the milder form of subaortic VSD, there
was septation at the orifice level by the condensed mesenchyme of the
aortopulmonary septum. Below the orifice level, a more or less
extensive muscular conus septum was present, sometimes still
showing a fine central band of mesenchyme (Fig 4c), in contrast to
normal hearts, where the conus septum is completely muscular. Failure
of the muscular conus septum to contact the ventricular
septum resulted in a VSD (Fig 4c).
Fig 6 shows a scanning electron micrograph of the severe
VSD variation with DORV. Absence of proper outflow septation at the
valvular level results in continuous semilunar valve leaflets.
Both arterial orifices are seen above the right ventricle
(Fig 6b and 6d).
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During normal development, there are stage-specific intracardiac blood flow patterns, which are considered to play an important role in heart development.7 A consistent but continuously changing pattern of intracardiac blood flow during development is related to the growth of the yolk sac to meet the increasing metabolic demands of the embryo, which in turn is accompanied by increased atrial and ventricular blood pressure.38 In the described model of hemodynamic interference, by clipping the right lateral vitelline vein, we were able to disturb these normal flow patterns, as visualized by india ink. The most prominent blood flow changes were seen in the conotruncus. Ligation forced blood from each yolk sac region to follow a more ventral course along the outer curvature of the conotruncus. Permanent ligation of the right lateral vitelline vein with a microclip proved to be a good method to produce specific cardiovascular malformations, like subaortic VSD, semilunar valve anomalies, atrioventricular valve anomalies, dextroposed aorta, and pharyngeal arch artery malformations. The observed intracardiac malformations are believed to be part of a spectrum. The VSD is the common factor, and the range is determined by the localization of the VSD, varying in extent from the top of the interventricular septum to the attachment sites of the semilunar valves. A possible explanation for the range in anomalies found after equally performed ligations could be the small differences in intracardiac flow patterns after ligation, as demonstrated with India ink. It is likely that not every ligation produces a flow change of the magnitude necessary to alter morphology, which explains the 36% normal embryos. When the magnitude of flow change exceeds a certain threshold, abnormal cardiac development will occur.
Although we were not able to perform both India ink experiments and long-term ligation experiments in the same embryos, we postulate that the observed cardiovascular malformations are due to the changed intracardiac flow patterns. Both the introduction of India ink into the vascular system and the leakage of blood after withdrawal of the glass needle were thought to influence normal embryonic development. Therefore, we were forced to study direct and long-term effects separately. By determining average flow patterns after ligation of a relatively large group of embryos and comparing these results with a group of embryos that survived after clipping, instead of studying individual cases, we could deduce a causal relationship.
Compared with experimental procedures generating cardiac malformations16 22 23 24 39 that resulted in a mean survival rate of 30%, venous clipping resulted in a relatively high survival rate of 79%. In addition, 64% of the surviving embryos had cardiovascular malformations that were not lethal during embryonic life. In stage-41 chick embryos, the spontaneous frequency of VSD was 11.7%,40 which is considerably lower than the 56% observed in the present study. In addition, these spontaneous VSDs were due to delayed interventricular septal closure, whereas the VSDs in the clip model were the result of malalignment of septal components due to impaired conotruncal septation and/or disturbed looping. Also, the spontaneous frequency of pharyngeal arch artery malformations was lower than that observed after venous clipping (7.1%40 and 35%, respectively).
In contrast to Orts Llorca et al,27 who changed venous inflow by transection of vitelline veins, we did not obtain embryos with left microphthalmia, nor did we see any eye malformation. The eyes of ligated embryos in the present study were symmetrical and normal. This discrepancy is likely the result of a different method. Probably, complete section of a vein causes hemorrhage, resulting in temporary hypoxia, which could produce malformations during a critical period of eye development.
Similar to other long-term hemodynamic interference studies,11 12 15 20 we observed DORV, VSD, and pharyngeal arch artery anomalies. Therefore, it is justified to link hemodynamic alterations during development and the occurrence of cardiac abnormalities.
Surprising was the similarity in malformations obtained in nonhemodynamic models, like cardiac neural crest ablation,22 41 42 43 excessive retinoic acid treatment,44 45 and cervical flexure experiments.24 46 All had dextroposed aortas, VSDs, and pharyngeal arch artery malformations. With the venous clip model, we were not able to produce the complete spectrum of cardiac maldevelopment that is obtained after neural crest ablation. We never see the most severe case, which is persistent truncus arteriosus. Because venous clipping is performed at stage 17, in contrast to stages 8 to 10 of neural crest ablation, we are likely to interfere with later morphogenetic processes. It would be interesting to speculate about the possibility that we might even produce persistent truncus arteriosus if clipping is performed at the appropriate stage, which offers a challenge for further research. But until that time, our model can be compared with the DORV group after ablation,41 47 which is thought to be the result of altered hemodynamics caused by decreased number of neural crest cells in the pharyngeal arches.22 42
In the venous clip model, the pharyngeal arch artery anomalies and intracardiac malformations are highly correlated, but both can exist as a solitary event. It is interesting to speculate on the mechanism that leads in our model to pharyngeal arch artery anomalies, also an inherent part of the neural crest ablation. As at the onset of our hemodynamic manipulation, the neural crest cells are still positioned in the pharyngeal arch region; it cannot be ruled out that they are delayed in their arrival and misdirected, as indicated by the eccentric condensed mesenchyme of the aorticopulmonary septum.
Part of the cardiac malformations obtained in all models, including ours, can be attributed to lack of proper looping, especially in the later stages.24 41 45 47 Impaired looped hearts are characterized by an increased distance between the inflow (atrioventricular canal) and outflow (conotruncus), a dextroposed position of the arterial trunk, and disturbed atrioventricular and conotruncal cushion formation. Further development will lead to incomplete or absent fusion of the septal components.24 Although we initially expected to induce inflow malformations (eg, as described in two mouse models presenting disturbed looping, the bulboventricular iv/iv mouse48 and the trisomy-16 mouse49 50 ), such as persistent sinus venosus, common atrium, and common atrioventricular canal, we never observed these cardiac malformations. However, both the mouse models and our venous clip model share the ventricular septal defects, often in combination with DORV. The explanation suggested by Webb et al49 that impaired placental circulation in trisomy-16 mouse embryos influenced the cardiac malformations is in line with our observations in the chick. But, although malformations in these mouse and chick models are believed to arise from improper looping, it is very likely that there is a different primary event, because there is only partial overlap.
How can changes in intracardiac blood flow patterns lead to cardiac malformations? So far, a molecular biological explanation can only be given through indirect evidence derived, for example, from studies on endothelial cell signaling. As a result of flow, endothelial cells are subjected to fluid shear stress and are aligned in the direction of blood flow.51 52 53 54 Because of abnormal blood flow, shear stress patterns might change. Direct response of endothelial cells to changing shear stress has been observed in vitro. There is a calcium-dependent pathway in which phospholipase C is activated, followed by an increase in intracellular calcium.55 56 This rapid pathway requires phospholipase C and phospholipids (phosphatidylinositol diphosphate)57 58 and ion channels (K+)59 and will result in nitric oxide synthase activation, ion transport, and secretion of stored proteins like endothelin-1.60 There is also a slower calcium-independent pathway, which acts via mitogen-activated protein kinase, tyrosine kinases (such as src and focal adhesion kinase), and calcium-independent protein kinase C. This pathway requires the G protein and results in sustained nitric oxide synthase activation, cytoskeletal rearrangement,61 62 63 and gene expression.54 64 65 Recently, a shear stress–responsive element has been defined in the promoter region of many genes, including the platelet-derived growth factor-ß gene.66
Each of these factors, alone or in combination, can be involved in cardiac morphogenesis, and disturbed regulation could result in cardiac malformations. In this respect, endothelin-1 deserves special attention. In addition to vasoconstrictive effects on vascular smooth muscle cells, endothelin-1 can also stimulate proliferation of mesenchymal cells and cardiomyocytes.67 During normal embryonic development, endothelin-1 gene expression is also localized in the ectoderm of pharyngeal arches, endothelium of the pharyngeal arch arteries, conotruncus, and endocardial cushions68 and in the cardiac myocytes.69 Endothelin-1 is thought to be involved in the epithelial-mesenchymal interaction necessary to induce the neural crest cells to differentiate into ectomesenchymal cells and might thus play a role in normal neural crest deposition, which is known to be of importance for pharyngeal arch development and conotruncal septation.26 Homozygous knockout mice for the endothelin-1 locus display pharyngeal arch artery malformations and ventricular septal defects.68 Because shear stress causes downregulation of endothelin-1 mRNA in vascular endothelium in vitro,70 this might indicate a common mechanism, explaining the similarity in malformations seen in several models, such as the endothelin-1 knockout mice,68 the neural crest–ablated embryos,47 and our venous clip model.
In conclusion, the cascade of events leading to the cardiac malformations observed after ligation of the right lateral vitelline vein is still not clear. The intracardiac and pharyngeal arch malformations observed after venous clipping show high similarity with malformations seen in several other models, such as those with neural crest ablation,22 42 43 endothelin-1 knockout phenotype,68 excessive retinoic acid,44 45 and thoracic wall closure defects.24 46 However, the high incidence of semilunar valve abnormalities and the high subaortic VSDs with a fibrous outlet septum are up until now unique in our venous clip model. Further studies will be conducted to determine the underlying morphogenetic mechanism, with endothelial signaling as a likely candidate.
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Acknowledgments |
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This study was supported by grant 900-516-096 of the Netherlands Heart Foundation and the Netherlands Organization for Scientific Research (NWO). The authors are greatly indebted to Mmes A.M.J. Baasten, D. Vermeij, and M.M.T. Mentink for their technical assistance, B. Blankevoort for preparing the illustrations, and J.R. Tooms for computer assistance.
Received June 10, 1996; accepted December 31, 1996.
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