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Research Paper

J Vasc Res 2008;45:54–68 DOI: 10.1159/000109077

The Endothelin-1 Pathway and theDevelopment of Cardiovascular Defectsin the Haemodynamically ChallengedChicken Embryo

Bianca C.W. Groenendijk a, d Sandra Stekelenburg-de Vos b Peter Vennemann c

Juriy W. Wladimiroff b Frans T.M. Nieuwstadt c Ralph Lindken c Jerry Westerweel c

Beerend P. Hierck a Nicolette T.C. Ursem b Robert E. Poelmann a

a Department of Anatomy and Embryology, Leiden University Medical Center, Leiden , b Department of Obstetricsand Gynaecology, Erasmus MC, University Medical Center, Rotterdam , c Department of Aero- and Hydrodynamics, Delft University of Technology, Delft , and d Department of Medical Biochemistry, Academic Medical Center, Amsterdam , The Netherlands

the embryonic heart and extra-embryonic vitelline veins were examined by Doppler and micro-particle image velo-cimetry. Ventricular diastolic filling characteristics were stud-ied at HH24, followed by cardiovascular morphologic inves-tigation (HH35). Results: ET-1 and its receptor antagonists induced haemodynamic effects at HH18. At HH24, a reduced diastolic ventricular passive filling component was demon-strated, which was compensated by an increased active fill-ing component. Thinner ventricular myocardium was shown in 42% of experimental embryos. Conclusion: We conclude that cardiovascular malformations after venous clipping arise from a combination of haemodynamic changes and al-tered gene expression patterns and levels, including those of the endothelin pathway. Copyright © 2007 S. Karger AG, Basel

Introduction

The development of the heart from an almost straight tube to a 4-chambered pump is a complex process in which interactions between genetic and epigenetic influ-

Key Words

Chicken embryo � Atrioventricular function � Blood flow velocity � Endothelin-1 � Endothelin-A receptor � Endothelin-B receptor � Hemodynamics � Cardiac malformation

Abstract

Background/Aims: Ligating the right lateral vitelline vein of chicken embryos (venous clip) results in cardiovascular mal-formations. These abnormalities are similar to malforma-tions observed in knockout mice studies of components of the endothelin-1 (ET-1)/endothelin-converting enzyme-1/endothelin-A receptor pathway. In previous studies we dem-onstrated that cardiac ET-1 expression is decreased 3 h after clipping, and ventricular diastolic filling is disturbed after 2 days. Therefore, we hypothesise that ET-1-related processes are involved in the development of functional and morpho-logical cardiovascular defects after venous clip. Methods: In this study, ET-1 and endothelin receptor antagonists (BQ-123, BQ-788 and PD145065) were infused into the HH18 em-bryonic circulation. Immediate haemodynamic effects on

Received: March 24, 2007 Accepted after revision: June 3, 2007 Published online: September 27, 2007

Prof. Dr. R.E. Poelmann Department of Anatomy and Embryology, Leiden University Medical Center PO Box 9600, Postzone S-1-P NL–2300 RC Leiden (The Netherlands) Tel. +31 71 526 9306, Fax +31 71 526 8289, E-Mail R.E.Poelmann@lumc.nl

© 2007 S. Karger AG, Basel1018–1172/07/0451–0054$23.50/0

Accessible online at:www.karger.com/jvr

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J Vasc Res 2008;45:54–68 55

ences are involved. Haemodynamic forces have been demonstrated to play an important role in cardiac devel-opment [1, 2] . When these forces are impaired or when genes involved in growth and differentiation are not functioning correctly, malformations may arise. Shear stress is one of those haemodynamic forces, and the ex-pression of many genes changes in response to alterations in shear stress [3, 4] . We have shown that the expression of some of these genes is also shear-related in the develop-ing chicken heart [5] .

In the venous clip model, the right lateral vitelline vein is ligated in Hamburger and Hamilton [6] stage 17 (HH17) chicken embryos. This model is expedient to study im-mediate changes in the blood flow patterns through the heart [7] . Furthermore, changes in the dorsal aortic mean and peak blood flow [8] have been demonstrated, as well as alterations in shear-related gene expression [9] . At HH21 the cardiac ventricle is less contractile [10] and at HH24 diastolic ventricular filling is disturbed [11] . In clipped embryos analysed by simultaneous Doppler mea-surements of the dorsal aorta and atrioventricular canal (AVC), we found a reduced diastolic passive filling of the ventricle, which was compensated by an increased active filling component. Over time, the intervention also re-sults in the development of morphological cardiac mal-formations [2] .

Interestingly, knockout mice of components of the en-dothelin-1/endothelin-converting enzyme-1/endothelin-A receptor (ET-1/ECE-1/ETA receptor) pathway demon-strate similar cardiovascular malformations to those en-countered in chicken embryos after venous clip [12–14] . We have previously shown that 3 h after ligation, ET-1 mRNA is down-regulated in the heart [9] . This suggests that components of the ET-1 cascade might be involved in the development of malformations induced by venous clipping.

To test the hypothesis that disturbed ET-1 expression is responsible for functional cardiac impairment and car-diovascular malformations, we infused ET-1 and endo-thelin receptor antagonists into the extra-embryonic vas-culature at HH18, which is the same stage of alterations in gene expression after venous clip. Pulsed Doppler and micro-particle image velocimetry ( � PIV) were used to determine changes in blood flow and velocity. Further-more, we examined the cardiovascular morphology at HH35. The localisation of ETA receptor and endothelin-B (ETB) receptor mRNA in the heart was investigated as well as the differences in presence and localisation of the mRNA of ET-1 , ECE-1 , and the ETA and ETB receptors in the vessel walls of the yolk sac.

Materials and Methods

Animals and Intravenous Infusions Fertilised White Leghorn (Gallus domesticus) eggs were incu-

bated at 37–38 ° C and 60–70% relative humidity. Embryos that were dysmorphic, or exhibited arrhythmias or overt bleeding were excluded. At HH18, the embryos were exposed by window-ing the shell followed by removal of the overlying membranes. A glass micropipette was inserted into one of the third-order branches of the vitelline vein.

One microlitre of the following substances (see below), diluted in phosphate-buffered saline (PBS) stained with indigo carmine blue (0.25 g/ml) for visualisation, was slowly infused without us-ing any additional pressure to keep changes in haemodynamics to a minimum. The final dilutions were determined after range finding, using at least 3 concentrations (n = 5–10) differing in 1 and 2 orders of magnitude. The highest concentrations with no immediate deleterious effects were used. The temperature of the egg was regulated with a thermo element at 37/38 ° C.

For the immediate (HH18) and HH24 ultrasound Doppler procedures, we used 10 –7 M ET-1 (Bachem; n = 16 for HH18,n = 16 for HH24), 10 –5 M BQ-123 (Bachem; n = 22 for HH18,n = 17 for HH24) and BQ-788 (Sigma-Aldrich; n = 24 for HH18, n = 18 for HH24), and 10 –4 M PD145065 (Sigma-Aldrich; n = 24 for HH18, n = 17 for HH24). The embryos were compared with sham-operated animals (n = 6 for HH18, n = 13 for HH24) using PBS with indigo carmine. For the group of embryos of the HH24 analyses, the eggs were reincubated and the blood flow velocity was measured at HH24 (embryonic day 4.5), followed by histo-logical evaluation at HH35 (embryonic day 9). Prior to fixation, the embryos and the hearts were macroscopically evaluated for overt malformations.

For immediate � PIV measurements, 1 � l of the same sub-stances (n = 3 for each) as for the ultrasound Doppler measure-ments was infused and experimental embryos were compared with sham-operated embryos (n = 3).

Measurements and Statistical Analysis Doppler Ultrasound Dorsal aortic and atrioventricular (AV) blood flow velocity

were recorded using a 20-MHz pulsed Doppler velocity meter (model 545C-4; Iowa Doppler Products, Iowa City, Iowa, USA). Dorsal aortic blood velocity was measured with a 750- � m piezo-electric crystal positioned at a 45° angle towards the dorsal aorta. Internal aortic diameter was calculated from a magnified video image [15] . For the HH24 measurements, AV blood flow velocity was measured with a second crystal positioned at the apex of the heart towards the AV orifice. Using complex fast Fourier trans-form analysis, the maximum velocity waveform was reconstruct-ed [15] . Passive filling was defined in the AV flow velocity wave-form from the end of the isovolumic relaxation period to the onset of the A-wave, and active filling from the onset of the A-wave to the onset of the isovolumic contraction period ( fig. 1 ). Portions of passive and active filling overlapped each other at faster heart rates. The demarcation between the passive and active velocities was dependent on heart rate, but was most conspicuous as heart rate slowed. Therefore, heart rate was decreased to approximately 110 bpm by cooling the embryo to discriminate between the pas-sive and active filling phase and to study both groups under sim-ilar conditions, taking into account the influence on haemody-

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J Vasc Res 2008;45:54–6856

namics [16] . Cycle length was defined as the time between con-secutive beats obtained from the dorsal aortic velocity waveform. Dorsal aortic and both passive and active AV velocity profiles were integrated over time. Dorsal aortic blood flow, an estimate of cardiac output, was calculated as the product of the integrated velocity curve and the cross-sectional area of the dorsal aorta. The passive component of ventricular blood flow was defined as mean aortic blood flow multiplied by the fraction of the passive filling area. The active component was defined as mean aortic blood flow multiplied by the fraction of the active filling area. Passive and active ventricular filling volumes equalled dorsal aortic stroke volume multiplied by the fraction of the passive or active filling areas, respectively [17] .

For the immediate analyses, 3 dorsal aortic blood flow veloc-ity measurements were performed per embryo. The first blood flow velocity (baseline) was recorded immediately before infu-sion. The second and third were performed 5 and 30 min after infusion, respectively. We determined peak and mean systolic ve-locity, heart rate, peak and mean blood flow, peak acceleration, cardiac output and stroke volume for each cardiac cycle. We ana-lysed 5 consecutive cycles and the data are presented as means 8 standard error of the mean. The values were standardised by tak-ing the percentage change from baseline level in order to adjust for biological variability. Haemodynamic parameters were com-pared within and between groups with a paired t test. From the

81 chicken embryos that were measured at HH24, the same pa-rameters were determined and an unpaired t test was used. When data was not normally distributed according to the Shapiro-Wilk test, a logarithmic transformation was performed prior to estab-lishing difference between the study groups and sham embryos. Statistical significance was reached at p ! 0.05. Calculations were performed with SPSS 11.5 (SPSS Inc., Chicago, Ill., USA).

Micro-Particle Image Velocimetry � PIV measurements were performed at 3 time points per em-

bryo: before infusion (baseline), as well as 5 and 30 min after in-fusion. At each time point, an optically accessible vitelline vein (35–105 � m diameter) was analysed [18] .

� PIV resolves the spatial velocity distribution (magnitude and direction) in a two-dimensional measurement plane. This allows the derivation of the wall shear stress without the assumption of a certain velocity profile. The velocity gradient perpendicular to the wall, F u / F n , is readily extracted from the velocity field. Mul-tiplication with the viscosity, � , gives the wall shear stress, � w :

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Volume flow rate, peak, minimum and mean velocity, and heart rate can be derived from sequential � PIV measurements at suf-ficient sampling rate. The raw PIV images allow the direct mea-

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Fig. 1. Simultaneously recorded tracings of AV flow velocity (line) and dorsal aortic blood flow (interrupted line) in a sham-operated embryo ( a ) and an embryo treat-ed with the selective ETA receptor antago-nist BQ-123 ( b ) at HH24. Diastole consists of the passive phase (P) and the active phase (A). After BQ-123 infusion, the pas-sive component is decreased, whereas the active component is increased.

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surement of the vessel diameter. Embryonic erythrocytes were used as tracer particles providing an adequate spatial resolution.

Each measurement was based on 500 sequential PIV measure-ments in 688 ! 520 pixel images at 10-Hz repetition rate. This cor-responds to 50 s measurement duration. The blood vessel is located in the middle of the image and has a diameter of approximately 130 � m (corresponding to 200 pixels). The individual evaluation of the measurements with a coarse interrogation window of 64 ! 64 pix-els (40 ! 40 � m 2 ) was used to resolve the flow pulsation. The mag-nitude of the mean displacement of red blood cells for each mea-surement of the time series was determined. The frequency analy-sis of this data resolves the heart rate. Afterwards, the individual measurements were sorted according to their cardiac phase angle. Therefore, the temporal position of the measurements between the 2 closest systolic peaks was determined. The cardiac pulse was then resolved by a dense order of individual measurements. The mea-surements were combined into groups of comparable flow condi-tions by separating the pulse into 12 segments of 50 ms in length. Each group of individual measurements was evaluated by means of an ensemble correlation. The ensemble correlation method enabled the combined evaluation of PIV images to enhance resolution and accuracy in comparison to the individual evaluation of single PIV images [19] . With this method, interrogation windows of 32 ! 32 pixels (20 ! 20 � m 2 ) were used ( fig. 2 ; online suppl. video 1, www.karger.com/doi/10.1159/000109077).

The heart rate, vessel diameter, volume flow rate and mean wall shear stress in the vitelline veins were analysed statistically. As the parameters varied between the differently sized veins of the different embryos, the changes relative to their baseline values per parameter per embryo were calculated and analysed between groups using the non-parametric Mann-Whitney test. Values were considered significant at p ̂ 0.05. Data are presented as the median of the relative change and the 25th and 75th percentiles, showing the variation within the groups.

Advantages of Doppler and � PIV In the present study we used 2 different techniques to investi-

gate the immediate haemodynamic effects, Doppler ultrasound

for measurements in the dorsal aorta and AV canal of the embryo, and � PIV in vitelline vessels, taking advantage of the character-istics of both techniques. It is evident that differences in the out-come exist due to differences in the accuracy of both approaches. The increase in heart rate 5 min after ET-1 infusion, measured with either technique, is only 5%. Using the Doppler technique, however, the variation is much larger than with the � PIV tech-nique, resulting in a statistically significant change only in the latter. This indicates that � PIV measurements are much more ac-curate than Doppler measurements. On the other hand, � PIV measurements are more time consuming than Doppler measure-ments and rely on optical access, which can result in cooling of the embryo and subsequently in a possible decrease in heart rate. This may explain why the heart rate is not observed to increase 30 min after infusion compared with baseline values, as was ob-served with Doppler measurements.

Morphological and Histological Examination After the Doppler measurements at HH24 the embryos were

reincubated until HH35. Embryos that displayed abnormal func-tions at HH24 (n = 6 per substance) were selected for morpho-logical and histological examination. Of HH18 embryos, the vi-telline vessels were examined for the presence of muscle actin.

Embryos were fixed in 4% paraformaldehyde in 0.1 M phos-phate buffer for 24 h, followed by dehydration in graded ethanol and embedding in paraffin. The embryos were sectioned trans-versely to the arterial pole at 5 � m and mounted on glass slides. Routine immunohistochemical staining was performed using an overnight incubation with the primary antibody HHF35 (DAKO, Denmark) against muscle actin [20] diluted 1: 500 in PBS with 0.05% Tween-20 and 1% bovine serum albumin. After rinsing in PBS, the sections were incubated with horseradish peroxidase-conjugated rabbit anti-mouse antibody (1: 250; DAKO) for 75 min. Following thorough rinsing with PBS, the sections were treated with 0.04% diaminobenzidine tetrahydrochloride/0.06‰ H 2 O 2 in 0.05 M Tris-maleic acid (pH 7.6) for 10 min at room tempera-ture. The sections were counter-stained with Mayer’s haematoxy-lin, dehydrated, and mounted in Entellan.

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Fig. 2. Velocity distribution in a vitelline vessel at 3 out of 12 suc-cessive time points. Each measurement is based on a 50-ms time-averaged ensemble evaluation. The diameter of the vessel is about 150 � m. The peak velocity (red vector) is 1.6 mm/s. Note that there is some back flow at t 10 . Assuming a circular vessel cross-

section, the velocity profile (indicated in yellow) can be used to determine the volume flow rate and shear stress. For all 12 suc-cessive time points, see online supplement video 1 (www.karger.com/doi/10.1159/000109077).

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Radioactive in situ Hybridisation In a separate set of embryos, that were fixed in 4% paraformal-

dehyde in 0.1 M phosphate buffer and embedded in paraffin, we determined the localisation of mRNA for the endothelin-1 recep-tors ETA and ETB in the chicken heart at HH18 (n = 3; stage of infusion), HH20 (n = 2), HH22 (n = 2) and HH24 (n = 2; stage of measurements). The presence of ET-1 , ECE-1 , ETA and ETB mRNA in the wall of the vitelline vessels was detected using in situ hybridisation (ISH) on HH18 chicken embryos. The ISHs were performed essentially as described before [21] .

35 S-labelled chicken-specific riboprobes were produced as de-scribed previously [5, 9] . In short, a 619-bp (nucleotides 177–796) ET-1 fragment, a 1,283-bp (nucleotides 325–1608) ECE-1 frag-ment, a 845-bp (nucleotides 2256–3101) ETA fragment and a 440-bp (nucleotides 339–778) ETB fragment were cloned in pCRII (In-vitrogen; ET-1 , ECE-1 , ETB ) or pBSK ( ETA ). After linearisation, sense and antisense cRNA was transcribed in transcription buf-fer, 0.01 M dithiothreitol, 0.25 mM G/A/CTP mix, 1.4 U/ � l RNase inhibitor and 1.5 U/ � l of the appropriate RNA polymerase (T7 for ET-1 , T3 for ETA , and SP6 for ECE-1 and ETB ) in the presence of 2.31 Mb 35 S-UTP. Concentration of the probes was normalised to 1 ! 10 5 cpm/ � l. All sense probes showed negative hybridisation results (not shown).

Hybridised sections were dehydrated in graded ethanol and air dried before being coated with Ilford G5 emulsion (Ilford Ltd., Mobberley, UK), and then exposed for 7–14 days at 4 ° C. Slides were developed in Kodak D19 (Kodak, France) for 4 min at room temperature, rinsed, and fixed for 4 min in Ilford fixative (Ilford Ltd.). Finally, the sections were counterstained with Mayer’s hae-matoxylin, dehydrated, and embedded in Pertex (Histolab, Göte-borg, Sweden).

In vitro Experiments In the venous clip model, ET-1 and KLF2 expression are altered

in a shear-dependent manner [9] . Changes in gene expression upon shear stress have also been described in vitro [3, 4] . ET-1 it-self or its antagonists may also have an effect on gene expression. To investigate whether they induce a similar response as the mechanism of shear-dependent gene expression, we added ET-1, BQ-123 or BQ-788 to primary cultures of chicken endocardial cells. Expression of ET-1 , ECE-1 , ETA , ETB , KLF2 , and CD31 as negative control was quantified by quantitative real-time ampli-fication (QRT-PCR). To obtain endocardial cells, fertilised White Leghorn (G. domesticus) eggs were incubated to stage HH40 (14 days) at 37 ° C and 60–70% relative humidity, and were isolated. The cardiac ventricles of the embryos were used for isolation pro-cedures. After opening along their longitudinal axes, the ventri-cles were incubated with the endocardial side down for 10 min at 37 ° C in 0.1% (w/v in PBS) collagenase A (Roche). The separated cell clusters were flushed with medium, and by filtration on a 30- � m filter (Miltenyi Biotec), sheets of predominantly endocardial cells were isolated. After this, the cells were seeded in fibronectin-coated (20 � g/ml, Sigma) dishes in quail endocardial cell medium consisting of M199/HEPES (Gibco) supplemented with 10% (v/v) fetal calf serum (Gibco), 2% (v/v) chicken serum (Gibco), 2 mM L -glutamine (Gibco), 250 � g/ml endothelial cell growth supple-ment (Sigma), 1 ! antibiotic/antimyotic solution (Gibco) and 50 � g/ml gentamycin (Sigma). The cells were grown to confluency and passed once a week. They were used at p3, at which they were exposed for 5 h to medium containing 10 –7 M ET-1 (n = 4), 10 –5 M

BQ-123 (n = 4) or 10 –5 M BQ-788 (n = 4). Control cells were ex-posed to medium only (n = 4). With our isolation procedure, we obtained cultures that consisted 70–90% of endocardial cells and 10–30% of cells that express � -smooth muscle actin. We took ad-vantage of the presence of a low percentage of muscle cells, since ETA receptors are only present on these cells, and not on endo-cardial cells. ETB receptors, besides their presence on the endo-cardium, are also expressed on muscle cells. For a response of these receptors, muscle cells have to be present.

RNA Isolation and QRT-PCR From each culture, total RNA was isolated (RNeasy; Qiagen)

and prepared for QRT-PCR. RNA was treated with DNAse-I, and 500 ng was reverse transcribed into cDNA with M-MuLV Reverse Transcriptase (Amersham). Equal amounts of cDNA were sub-jected in duplicate to QRT-PCR, using Brilliant SYBR Green QPCR Master Mix (Stratagene) on an MX3000P PCR machine (Stratagene) [22] . The reactions contained 1 ! PCR Master Mix, 2 � l cDNA template and 10 pmol of each primer. As negative con-trols, no-template samples were used. The PCR program consist-ed of a hot start activation step of 10 min at 95 ° C, followed by50 cycles of 15 s at 95 ° C, 30 s at 58 ° C and 30 s at 72 ° C. Dissoci-ation analysis confirmed the amplification of unique targetsand excluded the presence of primer dimers. Specific primersfor chicken � -actin (GenBank L08165), KLF2 (BM490221), ET-1 (XM418943), ECE-1 (AF230274), ETA (AF040634), ETB (AF472616) and CD31 (BX935338) were designed using the Primer3 engine (http://frodo.wi.mit.edu/cgi-bin/primer3/prim-er3_www.cgi) [23] . Equal amounts of RNA from control and ex-perimental samples were analysed for � -actin expression. Cycle threshold values did not vary among these groups (data not shown), therefore, this gene was used as normaliser. Relative ex-pression levels were calculated [24] , corrected for gene-specific variations in amplification efficiencies, derived from serial dilu-tions of cDNA and standard curve analysis. An unpaired t test was used to compare the means between control and experimen-tal samples. p ! 0.05 were considered significant.

Results

HH24 Doppler Ultrasound Measurements Heart rates and cycle lengths were similar in the study

groups and the sham embryos (p 1 0.05). The mean heart rate in the sham embryos was 109 8 1 bpm, correspond-ing to a cycle length of 548 8 4 ms. Figure 1 demonstrates that at this heart rate the passive and active filling phases of the AV velocity waveform during diastole are well de-fined. It is also clear that the AV velocity waveform dur-ing ventricular systole coincides with the dorsal aortic flow velocity. Figure 3 shows all parameters.

ET-1. Embryos infused with ET-1 demonstrated an in-creased dorsal aortic blood flow (0.87 8 0.01 vs. 0.83 8 0.01 � l/s, p ! 0.02). Stroke volume was increased by 3.0% (not significant). The passive ventricular filling volume was decreased by 26.9% (p ! 0.01) and the active filling

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volume was increased by 16.3% (p ! 0.01). This resulted in a decreased ratio of passive to active ventricular filling (0.30 8 0.04 vs. 0.45 8 0.03, p ! 0.01) after ET-1 infu-sion.

BQ-123. Chicken embryos treated with the selective ETA receptor antagonist BQ-123 had a significantly high-er dorsal aortic blood flow compared with sham embryos (0.88 8 0.02 vs. 0.83 8 0.01 � l/s, p ! 0.05). Dorsal aortic stroke volume was elevated by 4.4% (not significant). The passive component of AV blood flow was lower (0.19 8 0.02 vs. 0.25 8 0.01 � l/s, p ! 0.01) in treated embryos and the active component higher (0.69 8 0.03 vs. 0.57 8 0.01 � l/s, p ! 0.01). The ratio of passive to active ventricular filling was therefore decreased by 36% (p ! 0.01) after BQ-123 infusion.

BQ-788. The use of the selective ETB receptor antago-nist BQ-788 displayed a significantly increased dorsal aortic blood flow (10.3%) and stroke volume (12.1%) com-pared with sham embryos (p ! 0.001). The passive ven-tricular filling volume was not different from sham em-bryos (0.14 8 0.01 vs. 0.14 8 0.01 � l, p 1 0.05). The active ventricular filling volume was increased by 18.6% (p ! 0.001). This increase, however, did not change the ratio of passive to active ventricular filling (0.39 8 0.05 vs. 0.45 8 0.03, p 1 0.05).

PD145065. The non-selective endothelin receptor an-tagonist PD145065 caused an increase of 6.3% (p ! 0.001) in dorsal aortic blood flow and 5.2% (p ! 0.01) in stroke volume. The passive ventricular filling flow was de-creased (0.19 8 0.01 vs. 0.25 8 0.01 � l/s, p ! 0.01) and the active flow component was increased (0.69 8 0.01 vs. 0.57 8 0.01 � l/s, p ! 0.001), resulting in a decreased ratio of passive to active ventricular filling of 36% (p ! 0.001).

Morphology of Malformations Macroscopically, no major malformations of the

hearts and pharyngeal arch arteries could be observed after infusion of either substance. Microscopically, small malformations such as (pinpoint) ventricular septum defects, hypoplastic pharyngeal arch arteries and thinned ventricular myocardium were encountered ( fig. 4 ). The total number of malformations was higher in the exper-imental group (68%) than in the shams (1/6) with the highest prevalence in the BQ-123 group (100%), com-pared with the BQ-788 group (40%). Decreased size of the compact layer of the ventricular myocardium was the most common malformation occurring in 42% of the ex-perimental embryos (50% in ET-1; 75% in BQ-123; 40% in BQ-788; 33% in PD145065) compared to 17% in sham embryos.

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Fig. 3. Effects of ET-1, selective ETA an-tagonist (BQ-123), ETB antagonist (BQ-788) and non-selective endothelin recep-tor antagonist (PD145065) infusion on dorsal aortic blood flow ( a ), stroke volume ( b ), the passive component of AV blood flow ( c ) and the active component of AV blood flow ( d ). The black horizontal line in all panels represents the sham values. * p ! 0.05 vs. shams.

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Immediate Haemodynamics Intra-Embryonic Measurements with Doppler Ultrasound Thirty minutes after infusion of either ET-1 or the re-

ceptor antagonists, the heart rate was increased com-pared with the measurements before infusion. This was observed in all cases, including the shams. This increase can be ascribed to growth of the embryo [25, 26] , to the increase in circulating volume with 1 � l [27] , or to the temperature changes that were inevitable in the experi-mental procedures. In contrast to the Doppler measure-ments at HH24, none of the parameters was changed compared with shams immediately after infusion. Fig-ure 5 a and b show the heart rate and mean systolic dorsal aortic velocity with Doppler measurements. The param-eters showed no differences with either concentration of ET-1 or receptor antagonists (not shown).

Extra-Embryonic Measurements Using � PIV The baseline value range in vitelline veins, that is be-

fore infusion, was 83–122 bpm (median 96 bpm) for the heart rate, 35–105 � m (median 55 � m) for the diameter, 0.16–1.68 � l/s (median 0.48 � l/s) for the volume flow rate and 7.42 � 10 –7 to 2.94 � 10 –6 Pa (median 1.80 � 10 –6 Pa) for the mean wall shear stress. To adjust for biological variability, the values were standardised by taking the percentage change from baseline level. These data are summarised in figure 5 c–f; the values of this figure are presented in online supplement table 1 (www.karger.com/doi/10.1159/000109077). The heart rate was increased 5 min after ET-1 infusion, but was back to sham levels after 30 min ( fig. 5 c). With blockade of ETA (BQ-123), the heart rate was in a trend-like manner decreased after 30 min ( fig. 5 c). Blocking both receptors (PD145065) resulted in a sig ni-ficant decrease in heart rate 30 min after infusion ( fig. 5 c).

Five minutes after infusion of the non-selective recep-tor antagonist, a trend-like increase in the diameter of

vitelline veins ( fig. 5 d) in the category of 35–105 � m was shown, whereas after 30 min no difference was measured compared with shams ( fig. 5 d).

The volume flow rate showed a trend in increase 30 min after infusion of ET-1 ( fig. 5 e). According to the data shown in figure 5 c, BQ-123 would also increase the vol-ume flow rate, however, the variation was much larger than that of ET-1, resulting in a p value of 0.513, indicat-ing that there was no detectable change.

The shear stress in veins was decreased 30 min after infusion of the non-selective antagonist PD145065 ( fig. 5 f). Infusion of the ETB receptor antagonist (BQ-788) did not result in any changes of heart rate, diameter, shear stress or volume flow rate in the vitelline veins ( fig. 5 c–f).

Gene Expression To analyse the capacity of embryonic heart tissue to

respond to ET-1 or its antagonists, we determined the lo-calisation of ETA and ETB mRNA during the early stages of development (HH18 to HH24). ETA at stage HH18 was present in all the myocardium of the common atrium, the AVC, the ventricle and the outflow tract (OFT; fig. 6 a–c). During development up to HH24 this was the same, all myocardium showed ETA expression ( fig. 6 g–i). At stage HH18, ETB expression ( fig. 6 d–f) was present in the en-docardium of the atrium, but predominantly on the lat-eral sides and in the narrow part of the AVC ( fig. 6 d and e). Going downstream in the AVC, the endocardium be-comes negative for ETB ( fig. 6 f) . The endocardium lining the ventricular trabeculations showed strong expression, as did the OFT endocardium. In the myocardium, ETB was expressed on the pericardial side in the atrium and AVC ( fig. 6 d–f). This may appear as the epicardium, how-ever, at HH18 the epicardium is not yet present [28] . At the downstream side of the AVC, ETB was also present in the myocardium on the lateral side ( fig. 6 f). The myocar-dium of the ventricle was also positive (data not shown). At HH24 ( fig. 6 j–l), the complete endocardial lining of the atria had become positive, whereas that of the AVC was negative ( fig. 6 j and k). The endocardium along the ventricular trabeculations showed strong expression. That of the OFT showed a patchy expression pattern ( fig. 6 j).

In the vitelline vessels of the yolk sac, ET-1 mRNA ex-pression was shown in the endothelium ( fig. 7 a). ECE-1 mRNA was expressed in the endothelium and slightly in the media ( fig. 7 b). ETB showed the same distribution, but with stronger expression in the media ( fig. 7 d). This expression pattern, and to a lesser degree that of ECE-1 ,

Fig. 4. Sections of anti-actin(HHF35)-stained chicken embryonic ventricles at stage HH35. a , b Sham embryo. c , d Embryo infused with ET-1. e , f Embryo infused with PD145065 (non-selective en-dothelin receptor antagonist) with similar results compared to embryos treated with BQ-123 (selective ETA antagonist) and BQ-788 (selective ETB antagonist; not shown). The thickness of the compact myocardium is indicated with a line in b , d and f . Note that the ventricular myocardium is thinner in embryos treated with either ET-1 or ET receptor antagonists compared to shams. a , c , e Scale bars = 300 � m. b , d , f Scale bars = 200 � m

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Fig. 5. Mean relative change in heart rate ( a ) and mean dorsal aor-tic velocity ( b ) derived from Doppler ultrasound measurements on the dorsal aorta at HH18. The changes at 5 and 30 min after infusion of PBS (sham), ET-1, ETA antagonist (BQ-123), ETB an-tagonist (BQ-788) or the non-selective antagonist (PD145065), are relative to their values before infusion. Note that none of the sub-stances induced a significant change in heart rate or mean dorsal aortic velocity after 5 or 30 min compared to sham. Error bars

represent the standard error of the mean. c–f Median relative changes in heart rate ( c ), vessel diameter ( d ), volume flow ( e ) and shear stress ( f ), obtained from � PIV measurements in vitelline veins. The changes at 5 and 30 min after infusion of PBS, ET-1, BQ-123, BQ-788 or PD145065 are relative to their values before infusion. Bars show the 25th and 75th percentiles. * p ̂ 0.05; ‡ trend (p 1 0.05 vs. shams).

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overlaps clearly with the muscle actin (HHF35) staining (insert in fig. 7 a). Surprisingly, ETA mRNA was unde-tectable ( fig. 7 c) both in the endothelium and media of vitelline arteries and veins, whereas it was strongly pres-ent in the myocardium of the embryo in the same section (data not shown).

In vitro Experiments We added ET-1, BQ-123 or BQ-788 to cultured chick-

en heart endothelial cells for 5 h. The results are sum-marised in figure 8 . Application of the substances did not result in changes in the expression of KLF2 com-pared to control cells, showing a distinct difference in

Fig. 6. ISH sections of an HH18 ( a–f ) and HH24 ( g–l ) chicken embryonic heart showing ETA ( a–c , g–i ) and ETB ( d–f , j–l ) mRNA expression in adjacent sections. a–c , g–i Note that ETA is ex-pressed in the myocardium, and not in the endocardium. d–f , j–l ETB , however, is predominantly expressed in the endocardium, but also shows local myocardial expression. d Endocardial ex-pression of ETB in the common atrium (arrows), whereas the myocardium is slightly positive; the OFT shows little endocardial expression. e ETB in the endocardium of the narrow part of the AVC at the lateral sides and in the OFT, which shows stronger ex-pression than distally (compare with d ). f The downstream part of the AVC shows minor endocardial expression (brackets), ETB expression is strongly present in the ventricle (trabecular part) as

well as in the proximal OFT. The ventricular myocardium (tra-becular part) shows stronger expression than the atrial myocar-dium (compare with d ). Note the positive myocardium in the atri-um and AVC on the pericardial side (arrowheads in d–f ). j Note the positive endocardium in the right and left atria (RA, LA), the distal part of the OFT is slightly positive. The myocardium of the atria also shows ETB expression, but that of the OFT is negative. k The endocardium of the complete AVC is negative, the ventric-ular (V) endocardium is positive. The myocardium of the AVC is negative; in the ventricular part, the myocardium is positive, but the compact myocardium shows less mRNA than the trabecula-tions. l Ventricular myocardium and endocardium are strongly positive. a–f Scale bars = 200 � m g–l Scale bars = 300 � m.

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working mechanism compared to a shear stress trigger. Exposure to the ETA antagonist, BQ-123, showed an in-crease in ETA mRNA (35.5%; p ! 0.02) and a trend in decrease in ECE-1 (–22.6%; p = 0.178). Addition of the ETB antagonist, BQ-788, also resulted in an increase in ETA mRNA (60.9%; p ! 0.02) and ETB mRNA (33.9%;p ! 0.01). Complementary to its receptor antagonists, ET-1 addition showed a trend in decrease in ETA (–27.6%; p = 0.158).

Discussion

The venous clip model was devised to examine the re-lation between disturbed blood flow and cardiac malde-velopment. Because genes of the ET-1/ECE-1/ETA path-way have been shown to be responsive to changes in blood flow and because ET-1 expression is changed in the mod-el [9] , we investigated whether disturbances in ET-1 could be responsible for both functional cardiac impairment and cardiovascular malformations present after venous clipping.

a b

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Fig. 7. Consecutive sections of vitelline vessels of an HH18 em-bryo, showing expression of ET-1 ( a ), ECE-1 ( b ), ETA ( c ) and ETB ( d ) mRNA by means of radioactive ISH, and an antibody staining against muscle actin (HHF35; insert in a ). Note that ET-1 is ex-pressed in the endothelium (arrows). ECE-1 is primarily expressed

in the endothelium (arrows), but also slightly in the media of the vessel wall (arrow heads). ETB is strongly present in both the en-dothelium (arrow) and media of the vessel wall (arrow heads), whereas ETA expression is absent. VV = Vitelline vessels; YSc = yolk sac. Scale bars = 100 � m.

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Directly upon infusion of ET-1 or its receptor antago-nists, the Doppler data did not demonstrate changes compared with shams. This may indicate that there are no immediate embryonic dorsal aortic haemodynamic effects at this developmental stage related to the ET-1 pathway. Decreased expression of KLF-2 and NOS-3 , and increased expression of ET-1 in the dorsal aorta of venous clip embryos, however, indicate diminished shear stress levels in this model [9] . Our new data indicate that ET-1 alone is not able to completely reiterate this phenotype. At HH24, ET-mediated embryonic haemodynamics were significantly changed.

Surprisingly, ETA mRNA is undetectable in the wall of the vitelline vessels at HH18, whereas ETB is expressed in abundance. This implies that ET-1 can only function through the ETB receptors in these vessels. In the embryo this is different, as both ETA and ETB mRNA are present in the heart. We propose, therefore, that in the vitelline or yolk sac vasculature ET-1 exerts its influence through a different mechanism than in the embryo proper.

The Two-Receptor Pathway Activation of the ET-1 pathway might lead to appar-

ently conflicting results. This is due to the involvement

of the 2 receptors. ETB receptor mRNA is expressed in endothelial and smooth muscle cells, whereas ETA recep-tor mRNA is only present in muscle cells [29] . These dif-ferent localisations also involve different actions, as ETA and ETB on smooth muscle cells account for vasocon-striction [30] , and the ETB receptor located on the endo-thelium is responsible for vasodilation [31, 32] .

Heterozygous Etb +/– mice, lacking 40–50% of the total ETB receptor protein, show a significantly higher mean arterial blood pressure, which is attributed to the in-creased influence of ET-1 on the ETA receptors located on resistance vessels [33] . An increase in resistance would lead to a decrease in venous flow. In addition, Verhaar et al. [32] showed that local administration of the selective ETB receptor antagonist triggers a reduction in blood flow through the brachial artery in healthy subjects due to the shift toward more activated ETA receptors. The endothelial ETB receptors are more sensitive to selective agonists compared with those of vascular smooth muscle cells [34] , indicating that ET-1 will mainly bind to the ETB receptors on the endothelium, which will result in vasodilation [35] . As ETA is not present in the vitelline vessels, ET-1 cannot bind locally. Therefore, by blocking ETB, a shift toward the ETA-mediated vasoconstriction,

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Fig. 8. Relative quantity of ET-1 , ECE-1 , ETA , ETB , KLF2 and CD31 mRNA (normalised for � -actin) from primary chicken en-docardial cell cultures after a 5-hour administration of medium only, ET-1, selective ETA receptor antagonist (BQ-123) or selec-tive ETB receptor antagonist (BQ-788). Note that ET-1 addition leads to a trend in decrease in ETA mRNA levels. Blockade of ETA,

however, causes an increase in ETA mRNA and a trend in de-crease in ECE-1 levels. Blocking the ETB receptor demonstrates an increase in both ETA and ETB mRNA. KLF2 and CD31 mRNA is not affected by any of the administered substances. * p ! 0.05; ‡ trend (p 1 0.05).

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is unlikely. Our results demonstrate that all measured and calculated parameters remain constant after block-ing the ETB receptors. By blocking both receptors, a more general systemic effect is observed because both ETA and ETB are expressed in the heart, exerting their inotropic effects [36, 37] .

With blockade of the ETA receptor we now expect haemodynamic changes in the vitelline vessels. Indeed, we show a trend in decrease in heart rate. This may also be due to blockade of ETA receptors in the heart or the vasculature of the embryo, as explained with the non-se-lective antagonist.

When ET-1 is infused, it will activate the endothelial ETB receptors resulting in a release of nitric oxide, and vasodilation and concomitant increased volume flow [35] . Our � PIV data do not show changes in veins small-er than 100 � m. Larger veins between 100 and 200 � m were reported [38] to show a response to ET-1 in the HH21 chicken embryo, although we have not been able to con-firm this, as veins with a diameter larger than 100 � m are not optically accessible.

The shear stress in vitelline veins is decreased after blocking both receptors, due to the decreased heart rate that is not compensated by regulation of the vitelline ves-sel diameter. We suppose that this decrease in shear stress will have an effect on gene expression with ensuing down-stream effects on the vascular bed and circulation.

Long-Term Effects Ventricular Function ET-1 has a negative inotropic and vasodilative effect

through the ETB receptor, and a positive inotropic and vasoconstrictive effect through the ETA receptor [35–37, 39] . During embryogenesis we have shown myocardial expression of ETA (HH18–HH24), and myocardial but even more prominent endocardial expression of ETB , in-dicating that ET-1 could exert its inotropic effects already during these early stages. The effects of ET-1 are immedi-ate but last for only a few hours, partly due to the instabil-ity of the protein [40, 41] . Therefore, 2 days after treat-ment with ET-1, we encounter long-term consequences. Our in vitro data show that when ETA is blocked, ETA mRNA is up-regulated, which indicates a counteracting and probably a more long-lasting mechanism. These in vitro data may even represent an underestimation as ETA mRNA is expressed in muscle cells, whereas the cultures are highly enriched for endocardial cells. Since ETA mRNA is up-regulated after single ETA and single ETB receptor blockade, the results will be similar after infu-sion of the non-selective antagonist. The increased blood

flow induced by both agonist and antagonists of ET-1 re-sults in a higher shear stress. In the venous clip model, changes in flow and shear were observed, resulting in al-tered gene expression [9] . This implies that changes in blood flow through infusion of ET-1 or its antagonists can result in altered gene expression with a subsequent effect on cardiac development.

Ventricular Structure The reduction in the passive component of diastolic

filling after ET-1 infusion, indicating a stiffer and less compliant ventricle, might be due to increased collagen synthesis in the heart through activation of both the ETA and ETB receptor [42] . Furthermore, ET-1 stimulates proliferation of cardiac fibroblasts via ETA receptor acti-vation [43] . We hypothesise that blockade of the ETA re-ceptor will inhibit proliferation, resulting in the demon-strated decreased ventricular wall thickness. This sug-gests that ventricular function during development is hampered, affecting passive filling. Decreased thickness of the compact layer of the myocardium has been de-scribed in several other models, including the venous clip [44] and in a model where antisense inhibition of Ets transcription down-regulates its protein expression [45] . Blockade of the ETB receptor resulted likewise in a re-duced size of the ventricular wall.

In response to the decreased passive filling, cardiac output is maintained by an increased active filling com-ponent. The passive filling is dependent on the ventricu-lar compliance, the active filling on the contraction force, or inotropy, of the atria. Our ISH data show that ETA and ETB receptors are both present in the atrium. It is likely that they counterbalance each other’s inotropic function similar to what we have described for the ventricle result-ing in increased dorsal aortic blood flow. With increased active filling, the blood flow through the AVC must be enhanced, resulting in an increased shear stress. In-creased flow and concomitant shear stress result in the described changes in gene expression [9] followed by car-diac malformations, such as the decreased thickness of the ventricular wall.

Conclusion

The present data show that the mechanism of ET-1-mediated vasoregulation in the vitelline system is differ-ent from embryonic circulation. We postulate that ET-1 pathway-induced changes in haemodynamics influence gene expression and flow patterns in the yolk sac, result-

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Acknowledgements

Part of this research was initiated by Frans T.M. Nieuwstadt, who unfortunately deceased on May 18, 2005. This research was supported by a grant from the Netherlands Heart Foundation, NHF2000.016 (B.C.W.G., S.S., B.P.H.), and a grant from the Dutch Technology Foundation STW, DSF.5695 (P.V.).

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