development of cardiac malformations : The involvement of the
endothelin-1 pathway
Groenendijk, B.C.W.
Citation
Groenendijk, B. C. W. (2006, March 23). Influence of blood flow on shear stress responsive
genes in the development of cardiac malformations : The involvement of the endothelin-1
pathway. Retrieved from https://hdl.handle.net/1887/4346
Version:
Corrected Publisher’s Version
License:
Licence agreement concerning inclusion of doctoral thesis in the
Institutional Repository of the University of Leiden
Changes in Shear Stress-related Gene
Expression After Experimentally Altered
Venous Return in the Chicken Embryo
Bianca CW Groenendijk, Beerend P Hierck, Johannes Vrolijk, Martin
Baiker, Mathieu JBM Pourquie, Adriana C Gittenberger-de Groot, Robert
E Poelmann
Abstract
Hemodynamics play an important role in cardiovascular development, and changes in blood flow can cause congenital heart malformations. The endothelium and endocardium are subjected to mechanical forces, of which fluid shear stress is correlated to blood flow velocity. The shear stress responsive genes lung Krüppel-like factor (KLF2), endothelin-1 (ET-1), and endothelial nitric oxide synthase (NOS-3) display specific expression patterns in
vivo during chicken cardiovascular development. Non-overlapping patterns of these genes
were demonstrated in the endocardium at structural lumen constrictions that are subjected to high blood flow velocities. Previously, we described in chicken embryos a dynamic flow model (the venous clip) in which the venous return to the heart is altered and cardiac blood flow patterns are disturbed, causing the formation of congenital cardiac malformations. In the present study, we test the hypothesis that disturbed blood flow can induce altered gene expression. In situ hybridisations indeed show a change in gene expression after venous clip. The level of expression of ET-1 in the heart is locally decreased, whereas KLF2 and
NOS-3 are both up-regulated. We conclude that venous obstruction results in altered
expression patterns of KLF2, ET-1 and NOS-3, suggestive for increased cardiac shear stress.
Introduction
Hemodynamic forces generated by blood flow modulate the structure and function of both fetal and adult endothelial cells (reviewed by Gimbrone et al1). In pathogenesis, shear stress
is important as atherosclerosis develops in low and unsteady shear stress areas2. During
embryogenesis, blood flow plays an important role in cardiac development3;4. We developed
the chicken venous clip model3 in which the right lateral vitelline vein is ligated. This results
in immediate changes in blood flow patterns through the heart, and eventually to cardiovascular malformations, including ventricular septal defects, semilunar valve anomalies, and several types of pharyngeal arch artery abnormalities5. Additionally,
Stekelenburg-de Vos et al.6 have shown that up to 5 hours after venous clip the dorsal aortic
mean and peak blood flow is decreased, demonstrating a change in hemodynamics. Shear stress is directly related to blood flow, therefore it is likely that this is also altered in the venous clip model, and involved in the development of abnormalities in the cardiovascular system.
(NOS-3/eNOS) are shear-dependent in their expression in vitro7-9. Previously, we suggested that
these genes are also shear-related in vivo10. Endothelin-1 is a growth hormone and
vasoconstrictor. NOS-3 catalyses the conversion of L-arginine to L-citrullin, generating nitric oxide (NO). NO is involved in, e.g., vasodilation. KLF2 is a member of the SP/XKLF family of transcription factors11, and is expressed in the endothelium of the adult human aorta at
sites of high shear stress9. During normal chicken cardiovascular development
(HH16-HH3012), we demonstrated that KLF2 and NOS-3 were expressed in the endocardium of
structural lumen constrictions of the heart, such as the atrioventricular canal and the outflow tract, where shear stress is higher than in the adjacent areas. These patterns become mutually exclusive during development with ET-1 positive regions located in low shear areas. This implies that there is differential shear dependent gene expression in cardiovascular development10.
Our in vivo findings confirm the results from in vitro studies, which have shown that both
KLF2 and NOS-3 are up-regulated9;13, and that ET-1 is down-regulated by increased shear
stress14. The regulation of ET-1 and NOS-3 is probably a multi-step process as it is not only
directly shear-related, but also under the regulation of KLF215. It has been shown that these
genes are involved in embryonic development, particularly in cardiovascular development. Knockout mice for Klf2 die at approximately embryonic day 12.5 because of massive hemorrhaging in the outflow tract region, and KLF2 is found to be important for the formation of the media of the vessel wall16. Nos-3-deficient mice show atrial and ventricular
septal defects, heart failure17, and bicuspid aortic valves18. Knockout mice for Edn-119,
endothelin converting enzyme-120 (Ece-1), and the endothelin-A receptor21 (Eta) display a
spectrum of craniofacial, pharyngeal arch artery and cardiac malformations. Interestingly, the cardiovascular anomalies are similar to the ones that result from venous clip5.
Materials and Methods
Ligation Procedures
Fertilised White Leghorn eggs (Gallus domesticus) were incubated at 37°C and 60-70% relative humidity for 70 hours to obtain embryos of stage HH17. Eggs were windowed, and the right lateral vitelline vein was clipped as described before6. The cessation of blood flow
downstream, and the re-routing of blood flow upstream of the microclip were confirmed visually. Eggs were resealed, reincubated for 3 hours, and the embryos were euthanised. Control (n=5) and clipped (n=8) embryos were fixed overnight in 4% paraformaldehyde in 0,1mol/L phosphate buffer at 4°C, dehydrated in graded ethanol, and embedded in paraffin. The embryos were sectioned at 5μm and mounted serially for in situ hybridisation. All experiments were performed according to institutional guidelines.
Radioactive In Situ Hybridisation
Probes
35S-labelled chicken-specific riboprobes were produced as described previously10. In short, a
607-bp (nucleotides 339-946) KLF2 fragment, a 619-bp (nucleotides 177-796) ET-1 fragment, and a 521-bp (nucleotides 701-1222) NOS-3 fragment were cloned in PCRII (Invitrogen; KLF2 and ET-1) or pBSKP (Stratagene; NOS-3). After linearisation, sense and antisense cRNA was transcribed in transcription buffer, 0.01mol/L dithiothreitol, 0.25mmol/L G/A/CTP mix, 1.4U/μL RNase-inhibitor, and 1.5U/μL of the appropriate RNA polymerase (T7 for KLF2 and
ET-1, and T3 for NOS-3) in the presence of 2.31MB 35S-UTP. Concentration of the probes was
normalised to 1 x 105cpm/μL. All sense probes showed negative hybridisation results (not
shown).
In Situ Hybridisation
In situ hybridisation (ISH) was performed as described before22. Control and clipped
Three-Dimensional Reconstruction
A three-dimensional (3D)-reconstruction of the endothelial lining of heart and vessels of a stage HH18 chicken embryo was created, using the Amira software package (TGS, San Diego, CA), as described before10.
CFD Model
The patterning of shear stress distribution was analysed in a CFD model of a HH14 chicken embryonic heart that was acquired by 3D-reconstruction of the endocardium upon in vivo confocal imaging. The model represents the heart in maximal dilatation, and input parameters were based on in vivo hemodynamic measurements6;23. The colour distribution
represents the shear stress distribution throughout the heart.
(Semi-)Quantification
Real Time RT-PCR
To quantify the changes in endocardial expression via real time RT-PCR, hearts of HH18 control (n=8) and clipped (n=10) embryos were used. Two hearts per sample were pooled to obtain enough RNA for downstream analysis. From the narrow part of the atrioventricular canal (AVC) to the upstream part of the distal outflow tract (OFT) total RNA was isolated, and treated with DNAse-I (RNeasy-micro, Qiagen). To obtain cDNA, M-MuLV Reverse Transcriptase (Amersham) was used with 500ng of RNA. Quantitative real-time amplification (qRT-PCR) was performed on equal amounts of cDNA, in duplicate, using SYBR green I Master Mix (Applied Biosystems) on an MX3000P PCR machine (Stratagene)24.
Reactions contained 1xPCR Master Mix, 2μL cDNA template, and 10pmol of each primer. As negative controls no-template samples were used. The PCR program commenced with a hot start activation step of 10 min 95°C, followed by 50 cycles of 15 sec 95°C, 30 sec 58°C, and 30 sec 70°C. Dissociation analysis confirmed the amplification of only one target and excluded the presence of primer-dimers. Gene specific primers for chicken β-actin (GenBank: L08165), KLF2 (BM490221), ET-1 (XM418943), and NOS-3 (BI064564) were designed using the Primer3 engine (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi)25. β-Actin expression was analysed for equal amounts of
RNA from control and experimental samples. This gene was used as normaliser, as the Ct values did not vary among the groups. Relative expression levels were calculated26,
cDNA and standard curve analysis. An independent samples t-test was used to compare the means between clipped samples and controls. p-values <0.05 were considered significant.
ColourProc
It has been demonstrated that radioactive ISH is a quantifiable technique27. Therefore, the
ColourProc program, originally developed for the acquisition and analysis of COBRA MFISH28, was adapted for the semi-quantification and analysis of the radioactive ISH of
mRNA in sections. Utmost care was taken to standardise the steps for histology and staining procedures. First, dark-field photomicrographs were taken of the in situ hybridisation sections of the same areas in the different embryos, taking into consideration to use the same exposure time for each section hybridised with the same probe. These images were imported in ColourProc, and areas of interest were marked. For the different areas (Fig. 3.1) the endothelium/endocardium on the dorsal and ventral side of the lumen were analysed separately. Five control embryos and 8 clipped embryos were used for quantification, leading to a maximum of 10 control values, and 16 experimental values per area. Normalisation on tissue area was obtained by measuring the number of pixels above an arbitrary but fixed grey-scale threshold per specified area, which represents a percentage of expression. Each section was standardised by measuring and subtracting the expression of a small area in the adjacent lumen for background correction. In addition, expression of ET-1 in non-endothelial cells was quantified with the same technique to confirm that changes in gene expression are solely endocardially related.
All data represent ‘the percentage change in means ± percentage of the standard error of the difference (%SED)’. Values of expression of control and clipped embryos were first compared with an unpaired Student’s t-test. If this showed differences in the variation within the two groups, a nonparametric Mann Whitney U-test was applied to the data. Differences with p<0.05 were considered significant.
Results
In General
(Fig. 3.2) were demonstrated. ISH showed clear differences in regional gene patterns. This means that the standard quantification technique was too general for the regional differences. Therefore, we semi-quantified the gene signal from ISH at the various areas represented in Figure 3.1a. The AVC was subdivided in three levels: an upstream part (area 1), the narrowest part (2), and the downstream slope (3). Area 4 represents the junction between the AVC to the OFT cushions in the inner curvature. The OFT is also divided in three regions: the proximal, upstream slope (5), the narrow part (6), and the downstream part (7). Area 8 represents the descending dorsal aorta. Figure 3.1b shows the shear stress-independent expression of ET-1 in the endoderm of the pharyngeal arches of a clipped embryo. This expression is unaltered compared with control embryos (the difference is 0.7 ± 4.8% SED, histogram not shown). After 3 hours of ligation the cardiovascular morphology does not show malformations (not shown).
In Situ Hybridisation
KLF2 Expression Pattern
KLF2 mRNA is present in the endothelial/endocardial cells of the developing cardiovascular
system. In control embryos of stage HH18, KLF2 expression is located predominantly in the sinus venosus, the AVC, in scattered cells at the top of the ventricular trabeculations, in the OFT, the aortic sac, and the pharyngeal arch arteries. The dorsal aorta shows a very weak patchy expression. Obstructing the venous return at stage HH17 does not lead to a change in KLF2 mRNA pattern in the sinus venosus, atrium, and upstream slope of the AVC (area 1 in Fig. 3.1a; not shown). However, expression in the narrow part of the AVC (area 2) is extended in clipped embryos (Fig. 3.1c,d). The expression is not restricted to the endocardium covering the inner curvature of the heart tube, but it also spreads toward the remaining endocardium. KLF2 in the downstream slope of the AVC (area 3) appears unchanged (Fig. 3.3a,b). In the ventricle, expression in the endocardium lining the junction between the AVC to the OFT cushions (area 4) is augmented (Fig. 3.3a,b), as is the case in the upstream slope of the outflow tract (area 5; Fig. 3.3a,b). In the distal part (area 6 and 7) no difference has been observed (Fig. 3.1c,d).
ET-1 Expression Pattern
The mRNA of endothelin-1 is not only confined to the endothelium/endocardium. It is also present in, e.g., the endoderm of the pharyngeal pouches, and the ectoderm and mesodermal core of the pharyngeal arches. The normal endothelial/endocardial expression (HH18) is located in the sinus venosus, the AV canal, in a few single cells in the ventricle, in the OFT, and in the dorsal aorta. After clipping, expression of ET-1 in the atrium, ventricle, aortic sac, pharyngeal arch arteries, and the ascending dorsal aorta does not change (not shown). In contrast, in the sinus venosus the ET-1 pattern shifts. At the site where the cardinal veins enter the sinus venosus ET-1 is present in the ventral and right lateral wall of control embryos (Fig. 3.3g). After the 3-hour clip, expression is only present at the right lateral side (Fig. 3.3h). Further downstream it is evident that in the upstream slope and the narrow part of the AVC (areas 1 and 2) the ET-1 pattern is unaltered (Fig. 3.1e,f). On the downstream slope (area 3), however, expression is down-regulated (Fig. 3.3c,d). The junction between AVC to OFT cushions (area 4) shows a decrease in ET-1 (Fig. 3.3c,d). In addition, in the upstream part of the OFT (area 5), ET-1 is down-regulated (Fig. 3.3c,d) as it is in the distal part (area 6 and 7; Fig. 3.1e,f).
In the endothelium of the dorsal aorta (area 8), ET-1 is weakly present at the lateral sides. In clipped embryos, however, the signal is stronger, suggesting increased mRNA levels. This was confirmed by semi-quantitative analysis (see below).
NOS-3 Expression Pattern
NOS-3 mRNA is present in endocardial and endothelial cells. In control embryos (HH18),
NOS-3 expression is observed in the sinus venosus, AVC, ventricle, OFT, pharyngeal arch
arteries, and dorsal aorta. After ligation, no changes in expression pattern have been observed in the sinus venosus, atrium, and the upstream slope of the AVC (area 1 in Fig. 3.1a; not shown). The narrow part of the AVC (area 2), on the other hand, shows an increase in expression. It is not only expressed in the endocardium of the inner curvature as in control embryos, but it is extended to all of the endocardium in that area (Fig. 3.1g,h).
The downstream part of the AVC (area 3) shows no differences (Fig. 3.3e,f). In the ventricle,
NOS-3 expression in the endocardium lining the junction between the AVC-OFT cushions
(area 4), and the upstream slope of the OFT (area 5) is increased (Fig. 3.3e,f). The OFT further downstream (areas 6 and 7) shows no alterations (Fig. 3.1g,h).
The aortic sac, pharyngeal arch arteries and ascending aorta do not show differences between control and clipped embryos (not shown). The descending aorta (area 8) shows lower NOS-3 expression levels in clipped embryos (see below).
Figure 3.2. Level of expression of KLF2, ET-1 and NOS-3 in control versus clipped embryos, in the ventricular segment of the heart, using real time RT-PCR. Note the trend in decrease of ET-1 and increase of both KLF2 and NOS-3 after venous clip. Error bars represent SEM.
Semi-Quantification
Semi-quantification is performed by measuring the number of pixels exceeding a fixed threshold of grey level in 8 different regions from the AVC (3 levels), ventricle, OFT (3 levels), and descending aorta as described. In Figure 3.4, the percentages in change are shown, and the numbers are presented in Table 3.1.
KLF2
endothelium of the dorsal aorta (area 8) a significant decrease of 24.5% in KLF2 is shown in clipped embryos (Fig. 3.4a8).
ET-1
The ET-1 expression levels in the proximal and narrow part of the AVC (areas 1 and 2) are not altered after ligation (Fig. 3.4b1,2). From the downstream slope of the AVC to the distal OFT (areas 3 to 7) expression is down-regulated (38.4% to 58.2%, Fig. 3.4b3-7). In contrast, the dorsal aorta (area 8) shows an increase of 21.5% (Fig. 3.4b8).
Table 3.1. Percentage change in gene expression after venous clipa.
*significantly different, using p<0.05.
aPercentage change in gene expression levels upon semi-quantification of gene expression in ISH sections of KLF2, ET-1 and NOS-3 from control versus clipped embryos. The numbers ET-1 to 8 refer to the different areas from Figure 3.ET-1a. Note that in the heart (areas 1 to 7) KLF2 and NOS-3 are mainly increased, whereas ET-1 is down-regulated. In the dorsal aorta (area 8), this is the opposite: ET-1 is up-regulated, and KLF2 and NOS-3 show a (for NOS-3 a not significant) decrease.
NOS-3
After ligation no changes occur in NOS-3 levels in the upstream slope of the AVC (area 1; Fig. 3.4c1). The narrow part of the AVC (area 2) shows a trend in increase of 63.7% (Fig. 3.4c2). The downstream slope of the AVC (area 3) is not altered (Fig. 3.4c3). In the ventricle of clipped embryos, NOS-3 in area 4 is in a trend-like fashion increased by 28.9% (Fig. 3.4c4). The upstream slope of the OFT (area 5) shows a significant up-regulation of 64.3% (Fig. 3.4c5). The distal OFT (areas 6 and 7) shows no differences (Fig. 3.4c6,7), but the descending aorta (area 8) shows a trend in down-regulation by 22.2% (Fig. 3.4c8).
Shear Stress Distribution
A 3D reconstruction of a HH14 embryonic chicken heart was used to demonstrate the shear stress distribution in a CFD model (Fig. 3.5). The model represents the heart in maximal dilatation, and input parameters were based on in vivo hemodynamic measurements23.
Highest levels were detected in the inner curvature and at sites of lumen constrictions, i.e., AVC and OFT. Lowest shear is in the outer curvature and intertrabecular sinuses.
KLF2 ET-1 NOS-3
Area
Figure 3.5.
Shear stress distribution demonstrated in a static CFD model from a
HH14 embryonic
chicken heart. The input of this model is from real and actual in vivo parameters23. The colour code represents the shear stress distribution throughout the heart with the highest shear in white and red, the lowest in blue. Note high shear in the inflow tract (IFT), AVC, OFT, and inner curvature (∗). Low shear is shown in the outer curvature of the ventricle (V) and the intertrabecular sinuses (arrows).
Discussion
In the present study, we show that the gene expression patterns and levels in the heart and dorsal aorta are altered three hours after ligation of the right lateral vitelline vein. Likely, these alterations are caused by shear stress changes, and not by potential changes in cyclic strain, as preliminary in vitro data do not show alterations in KLF2 or ET-1 expression in avian endocardial cells under cyclic strain (Hierck, unpublished data 2005). In a previous study we have already shown that the expression of genes is shear-related in vivo10.
Combining the shear distribution patterns and the KLF2 expression10 confirms this. The
narrow regions are the higher shear stress areas, e.g., the AV canal and outflow tract, but the inner curvature as well. These are also the regions where KLF2 is expressed. In the outer curvature of the ventricle, shear stress is low, and KLF2 mRNA is absent. During development, KLF2 and NOS-3 are expressed at sites of high shear stress, whereas ET-1 is present in areas of low shear10. This has been confirmed by in vitro studies showing KLF2
andNOS-3 up-regulation9;29, and a decrease of ET-130 by high shear stress. Recent data show
ligation of the vitelline vein. This led to our hypothesis that expression patterns and levels of shear stress responsive genes change in the cardiovascular system after venous clip. A time period of 3 hours was used, because this is within the 5-hour window after clip, at which a decrease in dorsal aortic blood flow was shown6.
Gene patterns were evaluated by radioactive ISH, and gene levels were quantified in two ways. The first method was real time qRT-PCR on the ventricular segment of the heart. Trends in up- and down-regulation were demonstrated between control and clipped embryos. These alterations were in accordance with the significant changes observed with ISH. The differences using PCR were not significant because any change in mRNA from the specific sites was most likely masked by the mRNA signal from the complete ventricle, even on analysis of endothelial specific genes like KLF2, ET-1 and NOS-3. As was obvious from the ISH, the alterations in expression were detected in restricted and specific regions. Differences in gene expression levels were semi-quantified and showed important changes. With the adapted ColourProc method the differences in hybridisation signals could be efficiently semi-quantified. In addition, non-endothelial cells, i.e., cells not exposed to shear stress, showed no alterations in ET-1 expression, confirming that altered shear stress is the cause of the endothelial changes in gene expression.
The mRNA levels in the dorsal aorta show that the alterations in flow directly after placement of the clip6 were accompanied by an immediate change in gene expression. Most
in vitro data link increases of shear stress to a decrease of ET-1 expression30. Here, we show
that the opposite is also true. A decrease in blood flow velocity, and a concomitant decrease in shear stress, led to an increase in ET-1 mRNA levels. This confirms our previous observations of high ET-1 expression in the heart at sites of presumed low shear stress. Another finding linking changes in both shear stress and gene expression in vivo is the shift of ET-1 in the sinus venosus reflecting the decrease in blood flow in the right cardinal vein after right lateral vitelline vein ligation.
In sharp contrast to the increase of ET-1 in the dorsal aorta is the response in the heart. Here,
ET-1 is generally down-regulated, whereas both KLF2 and NOS-3 are mostly up-regulated,
albeit in specific regions. No changes in gene expression are observed in the atrium and upstream part of the AVC. Interestingly, in these particular areas no malformations develop in the venous clip model5. This may imply that changes in gene expression are necessary for
downstream slope of the AVC both ET-1 and KLF2 are down-regulated, whereas NOS-3 shows no changes. This concerted response in KLF2 and ET-1 was not expected. In the narrow region of the AVC an increase in shear stress is apparent, but following the downstream slope the lumen widens and the flow patterns will diverge. From the study by Hogers et al.3 can be deduced that ligation of the right lateral vitelline vein results in blood
flow profiles being more widespread in that area, which could lead to locally diminished shear and a concomitant drop in KLF2. How ET-1 responds by a down-regulation is under investigation.
Figure 3.6. Scheme, showing the presumed influences of altered gene expression by changes in shear stress on biological processes leading to congenital heart malformations. For composition of the scheme the references used show, e.g., pharmacological, knockout, and in vitro data16-19;21;31-40. ↑ indicates increase; ↓, decrease. a, described in
The part that showed the most pronounced changes in all three gene levels is the upstream slope of the outflow tract. This is very interesting, because this region is very susceptible for emerging cardiac malformations. Ventricular septal defects and semilunar valve abnormalities may arise during maldevelopment of this region, and OFT malformations are commonly encountered in many animal models and humans5. This also implies that altered
gene expression is involved in the development of these malformations.
Our clip data demonstrate overlapping KLF2 and NOS-3 signals, as we have shown in the normal expression study10, which implies that they change co-ordinately. This has been
confirmed by studies from SenBanerjee et al.15, where it was shown that KLF2 can induce
NOS-3 expression. Although there is much inter-embryonic variation in the NOS-3 signal, it
alters similarly to KLF2.
After venous clip the heart rate is decreased, like other parameters, such as the stroke volume and the dorsal aortic blood flow6. This suggests a lower flow and thus shear stress in
the heart. However, the shift of flow patterns after clip toward the inner curvature5 implies a
local increase in flow and shear stress in this region. In addition, according to the elevated
KLF2 and NOS-3 mRNA, and the down-regulated ET-1 expression, it is suggested that the
endocardium lining the primary heart tube, including the inner curvature of the heart, is subjected to higher shear stress in clipped embryos compared with controls. It has been suggested that shear stress in the outer curvature is higher because of higher ECE-1 expression in this area41. However, high ECE-1 expression should imply a lower shear stress
as this gene, like ET-1, is down-regulated by increased shear stress42. A model study of
curved tubes also demonstrated that shear stress is highest in the inner curvature43. More
importantly, we now also demonstrate using a CFD model that in the inner curvature the shear stress is higher than in the outer curvature. Although the static nature of the model exaggerates shear levels in the inflow and outflow of the heart, the model accurately shows relative changes in shear stress distribution, and produces natural shear values in areas that are within the same phase of the contraction cycle. In the venous clip model cardiac looping is disturbed5. Our expression data suggest that the inner curvature is involved in the
formation of this anomaly. A similar important role for the inner curvature is reflected in the ingrowth of epicardially-derived cells (EPDC) into the myocardium, which is earlier in the inner curvature than in the outer curvature44. This implies that the increase in shear and the
In summary, in the dorsal aorta flow6 and presumably shear stress are decreased, leading to
down-regulation of KLF2 and NOS-3, and up-regulation of ET-1. To our surprise the shear stress in the heart appeared to be increased instead of decreased after venous clip. This was suggested from the changes in gene expression, which are the opposite of the changes in the dorsal aorta: KLF2 and NOS-3 were both augmented, whereas ET-1 was down-regulated. Especially the inner curvature and the upstream slope of the outflow tract cushions, being areas of relatively high shear in normal embryos, showed a prominent change in expression. These are also the regions where most of the cardiac malformations occur after venous clip5.
Thus, by ligating the right lateral vitelline vein blood flow patterns through the heart shift, resulting in a change in shear stress and in alterations of gene expression, which in turn lead to the cardiovascular malformations found in this venous clip model.
Acknowledgements
The authors thank J. Lens for his expert assistance preparing the figures. This research is supported by a grant from the Netherlands Heart Foundation, grant number NHF2000.016.
References
1. Gimbrone MA Jr, Topper JN, Nagel T, Anderson KR, Garcia-Cardena G. Endothelial dysfunction, hemodynamic forces, and atherogenesis. Ann N Y Acad Sci. 2000;902:230-239.
2. Moore JE, Xu C, Glagov S, Zarins CK, Ku DN. Fluid wall shear stress measurements in a model of the human abdominal aorta: oscillatory behavior and relationship to atherosclerosis. Atherosclerosis. 1994;110:225-240. 3. Hogers B, DeRuiter MC, Gittenberger-de Groot AC, Poelmann RE. Unilateral vitelline vein ligation alters
intracardiac blood flow patterns and morphogenesis in the chick embryo. Circ Res. 1997;80:473-481.
4. Hove JR, Koster RW, Forouhar AS, Acevedo-Bolton G, Fraser SE, Gharib M. Intracardiac fluid forces are an essential epigenetic factor for embryonic cardiogenesis. Nature. 2003;421:172-177.
5. Hogers B, DeRuiter MC, Gittenberger-de Groot AC, Poelmann RE. Extraembryonic venous obstructions lead to cardiovascular malformations and can be embryolethal. Cardiovasc Res. 1999;41:87-99.
6. Stekelenburg-de Vos S, Ursem NTC, Hop WCJ, Wladimiroff JW, Gittenberger-de Groot AC, Poelmann RE. Acutely altered hemodynamics following venous obstruction in the early chick embryo. J Exp Biol. 2003;206:1051-1057.
7. Malek AM, Izumo S. Physiological fluid shear stress causes downregulation of endothelin-1 mRNA in bovine aortic endothelium. Am J Physiol. 1992;263 (2Pt1):C389-C396.
9. Dekker RJ, Van Soest S, Fontijn RD, Salamanca S, de Groot PG, VanBavel E, Pannekoek H, Horrevoets AJG. Prolonged fluid shear stress induces a distinct set of endothelial cell genes, most specifically lung Krüppel-like factor (KLF2). Blood. 2002;100:1689-1698.
10. Groenendijk BCW, Hierck BP, Gittenberger-de Groot AC, Poelmann RE. Development-related changes in the expression of shear stress responsive genes KLF-2, ET-1, and NOS-3 in the developing cardiovascular system of chicken embryos. Dev Dyn. 2004;230:57-68.
11. Philipsen S, Suske G. A tale of three fingers: the family of mammalian Sp/XKLF transcription factors. Nucleic Acids Res. 1999;27:2991-3000.
12. Hamburger V, Hamilton HL. A series of normal stages in the development of the chick embryo. J Morphol. 1951;88:49-92.
13. Mattart M, Mazzolai L, Chambaz C, Hayoz D, Brunner HR, Silacci P. ET-1 and NOSIII gene expression regulation by plaque-free and plaque-prone hemodynamic conditions. Biorheology. 2003;40:289-297.
14. Tsukurov OI, Kwolek CJ, L'Italien GJ, Benbrahim A, Milinazzo BB, Conroy NE, Gertler JP, Orkin RW, Abbott WM. The response of adult human saphenous vein endothelial cells to combined pressurized pulsatile flow and cyclic strain, in vitro. Ann Vasc Surg. 2000;14:260-267.
15. SenBanerjee S, Lin Z, Atkins GB, Greif DM, Rao RM, Kumar A, Feinberg MW, Chen Z, Simon DI, Luscinskas FW, Michel TM, Gimbrone MA, Jr., Garcia-Cardena G, Jain MK. KLF2 Is a novel transcriptional regulator of endothelial proinflammatory activation. J Exp Med. 2004;199:1305-1315.
16. Kuo CT, Veselits ML, Barton KP, Lu MM, Clendenin C, Leiden JM. The LKLF transcription factor is required for normal tunica media formation and blood vessel stabilization during murine embryogenesis. Genes Dev. 1997;11:2996-3006.
17. Feng Q, Song W, Lu X, Hamilton JA, Lei M, Peng T, Yee SP. Development of heart failure and congenital septal defects in mice lacking endothelial nitric oxide synthase. Circulation. 2002;106:873-879.
18. Lee TC, Zhao YD, Courtman DW, Stewart DJ. Abnormal aortic valve development in mice lacking endothelial nitric oxide synthase. Circulation. 2000;101:2345-2348.
19. Kurihara Y, Kurihara H, Oda H, Maemura K, Nagai R, Ishikawa T, Yazaki Y. Aortic arch malformations and ventricular septal defect in mice deficient in endothelin-1. J Clin Invest. 1995;96:293-300.
20. Yanagisawa H, Yanagisawa M, Kapur RP, Richardson JA, Williams SC, Clouthier DE, de Wit D, Emoto N, Hammer RE. Dual genetic pathways of endothelin-mediated intercellular signaling revealed by targeted disruption of endothelin converting enzyme-1 gene. Development. 1998;125:825-836.
21. Clouthier DE, Hosoda K, Richardson JA, Williams SC, Yanagisawa H, Kuwaki T, Kumada M, Hammer RE, Yanagisawa M. Cranial and cardiac neural crest defects in endothelin-A receptor-deficient mice. Development. 1998;125:813-824.
22. Hierck BP, Poelmann RE, VanIperen L, Brouwer A, Gittenberger-de Groot AC. Differential expression of α6 and other subunits of laminin binding integrins during development of the murine heart. Dev Dyn. 1996;206:100-111. 23. Vennemann P, Kiger KT, Lindken R, Groenendijk BCW, Stekelenburg-de Vos S, ten Hagen TLM, Ursem NTC,
Poelmann RE, Westerweel J, Hierck BP. In vivo micro particle image velocimetry measurements of blood-plasma in the embryonic avian heart. J Biomech. 2005. In Press. http://dx.doi.org/10.1016/j.jbiomech.2005.03.015
24. Hierck BP, Molin DGM, Boot MJ, Poelmann RE, Gittenberger-de Groot AC. A chicken model for DGCR6 as a modifier gene in the DiGeorge critical region. Pediatr Res. 2004;56:440-448.
25. Rozen S, Skaletsky H. Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol. 2000;132:365-386.
27. Jonker A, de Boer PAJ, van den Hoff MJB, Lamers WH, Moorman AFM. Towards quantitative in situ hybridization. J Histochem Cytochem. 1997;45:413-423.
28. Tanke HJ, Wiegant J, van Gijlswijk RPM, Bezrookove V, Pattenier H, Heetebrij RJ, Talman EG, Raap AK, Vrolijk J. New strategy for multi-colour fluorescence in situ hybridisation: COBRA: COmbined Binary RAtio labelling. Eur J Hum Genet. 1999;7:2-11.
29. Topper JN, Cai J, Falb D, Gimbrone MA, Jr. Identification of vascular endothelial genes differentially responsive to fluid mechanical stimuli: cyclooxygenase-2, manganese superoxide dismutase, and endothelial cell nitric oxide synthase are selectively up-regulated by steady laminar shear stress. Proc Natl Acad Sci U S A. 1996;93:10417-10422.
30. McCormick SM, Eskin SG, McIntire LV, Teng CL, Lu CM, Russell CG, Chittur KK. DNA microarray reveals changes in gene expression of shear stressed human umbilical vein endothelial cells. Proc Natl Acad Sci U S A. 2001;98:8955-8960.
31. Hirata Y, Emori T, Eguchi S, Kanno K, Imai T, Ohta K, Marumo F. Endothelin Receptor Subtype-B Mediates Synthesis of Nitric-Oxide by Cultured Bovine Endothelial-Cells. J Clin Invest. 1993;91:1367-1373.
32. Bucci M, Roviezzo F, Posadas I, Yu J, Parente L, Sessa WC, Ignarro LJ, Cirino G. Endothelial nitric oxide synthase activation is critical for vascular leakage during acute inflammation in vivo. Proc Natl Acad Sci U S A. 2005;102:904-908.
33. Rudic RD, Shesely EG, Maeda N, Smithies O, Segal SS, Sessa WC. Direct evidence for the importance of endothelium-derived nitric oxide in vascular remodeling. J Clin Invest. 1998;101:731-736.
34. Leite-Moreira AF, Bras-Silva C, Pedrosa CA, Rocha-Sousa AA. ET-1 increases distensibility of acutely loaded myocardium: a novel ETA and Na+/H+ exchanger-mediated effect. Am J Physiol Heart Circ Physiol. 2003;284:H1332-H1339.
35. D'Orleans-Juste P, Labonte J, Bkaily G, Choufani S, Plante M, Honore JC. Function of the endothelin(B) receptor in cardiovascular physiology and pathophysiology. Pharmacol Ther. 2002;95:221-238.
36. Guarda E, Katwa LC, Myers PR, Tyagi SC, Weber KT. Effects of endothelins on collagen turnover in cardiac fibroblasts. Cardiovasc Res. 1993;27:2130-2134.
37. Fujisaki H, Ito H, Hirata Y, Tanaka M, Hata M, Lin MH, Adachi S, Akimoto H, Marumo F, Hiroe M. Natriuretic Peptides Inhibit Angiotensin-Ii-Induced Proliferation of Rat Cardiac Fibroblasts by Blocking Endothelin-1 Gene-Expression. J Clin Invest. 1995;96:1059-1065.
38. Marsault R, Feolde E, Frelin C. Receptor Externalization Determines Sustained Contractile Responses to Endothelin-1 in the Rat Aorta. Am J Physiol. 1993;264:C687-C693.
39. Kelso EJ, McDermott BJ, Silke B, Spiers JP. EndothelinA receptor subtype mediates endothelin-induced contractility in left ventricular cardiomyocytes isolated from rabbit myocardium. J Pharmacol Exp Ther. 2000;294:1047-1052.
40. Watanabe T, Kusumoto K, Kitayoshi T, Shimamoto N. Positive Inotropic and Vasoconstrictive Effects of Endothelin-1 in Invivo and Invitro Experiments - Characteristics and the Role of L-Type Calcium Channels. J Cardiovasc Pharmacol. 1989;13:S108-S111.
41. Hall CE, Hurtado R, Hewett KW, Shulimovich M, Poma CP, Reckova M, Justus C, Pennisi DJ, Tobita K, Sedmera D, Gourdie RG, Mikawa T. Hemodynamic-dependent patterning of endothelin converting enzyme 1 expression and differentiation of impulse-conducting Purkinje fibers in the embryonic heart. Development. 2004;131:581-592. 42. Masatsugu K, Itoh H, Chun TH, Ogawa Y, Tamura N, Yamashita J, Doi K, Inoue M, Fukunaga Y, Sawada N,
Saito T, Korenaga R, Ando J, Nakao K. Physiologic shear stress suppresses endothelin-converting enzyme-1 expression in vascular endothelial cells. J Cardiovasc Pharmacol. 1998;31(Suppl 1):S42-S45.
