Changes in Shear StresseCRelated Gene Expression After Experimentally Altered Venous Return in the Chicken Embryo
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循环研究杂志 2005年第6期
The Department of Anatomy and Embryology (B.C.W.G., B.P.H., M.B., A.C.G.-d.G., R.E.P.) and the Laboratory for Cytochemistry and Cytometry, Department of Molecular Cell Biology (J.V.), Leiden University Medical Center, Leiden, The Netherlands
the Department of Aero- and Hydro-dynamics (M.B., M.J.B.M.P.), Delft University of Technology, Delft, The Netherlands.
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 Kre筽pel-like factor (KLF2), endothelin-1 (ET-1), and endothelial nitric oxide synthase (NOS-3) display specific expression patterns in vivo during chicken cardiovascular development. Nonoverlapping 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 hybridizations 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 upregulated. We conclude that venous obstruction results in altered expression patterns of KLF2, ET-1, and NOS-3, suggestive for increased cardiac shear stress.
Key Words: cardiovascular physiology embryonic circulation endothelium gene expression 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 areas.2 During embryogenesis blood flow plays an important role in cardiac development.3,4 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 abnormalities.5 Additionally, Stekelenburg-de Vos et al6 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.
Important genes encoding transcription factors and signaling molecules, eg, lung Kre筽pel-like factor (KLF2/LKLF), endothelin-1 (ET-1), and endothelial nitric oxide synthase (NOS-3/eNOS) are shear-dependent in their expression in vitro.7eC9 Previously, we suggested that these genes are also shear-related in vivo.10 Endothelin-1 is a growth hormone and vasoconstrictor. NOS-3 catalyzes the conversion of L-arginine to L-citrullin, generating nitric oxide (NO). NO is involved in, eg, 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 stress.9 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-1eCpositive regions located in low shear areas. This implies that there is differential shear-dependent gene expression in cardiovascular development.10
Our in vivo findings confirm the results from in vitro studies, which have shown that both KLF2 and NOS-3 are upregulated9,13 and that ET-1 is downregulated by increased shear stress.14 The regulation of ET-1 and NOS-3 is probably a multistep process as it is not only directly shear-related, but also under the regulation of KLF2.15 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 wall.16 Nos-3eCdeficient mice show atrial and ventricular septal defects, heart failure,17 and bicuspid aortic valves.18 Knockout mice for Edn-1,19 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 clip.5
As KLF2, ET-1, and NOS-3 expression is shear related, it is likely that they respond to venous clipeCinduced changes in shear stress by an alteration in their expression patterns and levels. Therefore, we hypothesize that changes in shear stress by venous clip leads to alterations in the expression of these genes, which is likely to be implicated in cardiovascular malformations. To test this hypothesis, we investigated the expression patterns and levels of KLF2, ET-1, and NOS-3 in the chicken cardiovascular system after ligation of the right lateral vitelline vein.
Materials and Methods
Ligation Procedures
Fertilized white Leghorn eggs (Gallus domesticus; Intervet International, Nieuwegein, The Netherlands) were incubated at 37°C and 60% to 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 before.6 The cessation of blood flow downstream and the rerouting of blood flow upstream of the microclip were confirmed visually. Eggs were resealed, reincubated for 3 hours, and the embryos were euthanized. 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 5 e and mounted serially for in situ hybridization. All experiments were performed according to institutional guidelines.
Data Analysis
Radioactive in situ hybridization was performed as described previously.10 For a detailed description, please refer to the online data supplement available at http://circres.ahajournals.org. The hybridization sections were used for 3D reconstructions and semiquantification.
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), as described before.10 Generation of a CFD model, based on 3D-reconstruction, is described in the online data supplement.
ColourProc
It has been demonstrated that radioactive ISH is a quantifiable technique.22 Therefore, the ColourProc program, originally developed for the acquisition and analysis of COBRA MFISH,23 was adapted for the semiquantification and analysis of the radioactive ISH of mRNA in sections. For a detailed description see the online data supplement.
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. Total RNA was isolated from the narrow part of the atrioventricular canal (AVC) to the upstream part of the distal outflow tract (OFT). Please refer to the online data supplement for details.
Results
In General
We focused on the mRNA expression levels and localizations in endothelium and endocardium of the large vessels and heart in control and experimentally challenged embryos. Changes after clip were most obvious in the ventricular part of the heart (caudally from level B in Figure 1a). Therefore, mRNA levels were quantified using real time RT-PCR. Trends in downregulation of ET-1 and upregulation of KLF2 and NOS-3 expression levels (supplemental Figure I) 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 semiquantified the gene signal from ISH at the various areas represented in Figure 1a. The AVC was subdivided in 3 levels: an upstream part (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 3 regions: the proximal upstream slope (5), the narrow part (6), and the downstream part (7). Area 8 represents the descending dorsal aorta. Figure 1b shows the shear stresseCindependent 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 Hybridization
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 Figure 1a; not shown). However, expression in the narrow part of the AVC (area 2) is extended in clipped embryos (Figure 1c and 1d). 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 (Figure 2a and 2b). In the ventricle, expression in the endocardium lining the junction between the AVC to the OFT cushions (area 4) is augmented (Figure 2a and 2b), as is the case in the upstream slope of the outflow tract (area 5; Figure 2a and 2b). In the distal part (area 6 and 7) no difference has been observed (Figure 1c and 1d).
The aortic sac and the pharyngeal arch arteries show no change in pattern (not shown). In the endothelium of the descending dorsal aorta (area 8), however, KLF2 is decreased (see the following).
ET-1 Expression
The mRNA of endothelin-1 is not only confined to the endothelium/endocardium. It is also present in, eg, 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 (Figure 2g). After the 3-hour clip, expression is only present at the right lateral side (Figure 2h). 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 (Figure 1e and 1f). On the downstream slope (area 3), however, expression is downregulated (Figure 2c and 2d). The junction between AVC to OFT cushions (area 4) shows a decrease in ET-1 (Figure 2c and 2d). In addition, in the upstream part of the OFT (area 5), ET-1 is downregulated (Figure 2c and 2d) as it is in the distal part (area 6 and 7; Figure 1e and 1f).
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 semiquantitative analysis (see below).
NOS-3 Expression
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 Figure 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 (Figure 1g and 1h). The downstream part of the AVC (area 3) shows no differences (Figure 2e and 2f). 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 (Figure 2e and 2f). The OFT further downstream (areas 6 and 7) shows no alterations (Figure 1g and 1h).
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).
Semiquantification
Semiquantification is performed by measuring the number of pixels exceeding a fixed threshold of gray level in 8 different regions from the AVC (3 levels), ventricle, OFT (3 levels), and descending aorta as described.
KLF2
After venous obstruction, the KLF2 level in the upstream slope of the AVC (area 1) is not altered (Figure 3A1). Expression in area 2 is increased by 130% (Figure 3A2), but area 3 shows a significant downregulation of 19.0% (Figure 3A3). The level in the endocardium lining the junction between the AVC-OFT cushions (area 4) is augmented by 70.3% (Figure 3A4). Reaching the upstream slope of the OFT (area 5) the KLF2 signal is elevated after clipping (135.1%; Figure 3A5). The distal OFT (area 6 and 7) is not changed (Figure 3A6 and 3A7). In the endothelium of the dorsal aorta (area 8) a significant decrease of 24.5% in KLF2 is shown in clipped embryos (Figure 3A8).
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 (Figure 3B1 and 3B2). From the downstream slope of the AVC to the distal OFT (areas 3 to 7) expression is downregulated (38.4% to 58.2%, Figure 3B3 through 3B7). In contrast, the dorsal aorta (area 8) shows an increase of 21.5% (Figure 3B8).
NOS-3
After ligation no changes occur in NOS-3 levels in the upstream slope of the AVC (area 1; Figure 3C1). The narrow part of the AVC (area 2) shows a trend in increase of 63.7% (Figure 3C2). The downstream slope of the AVC (area 3) is not altered (Figure 3C3). In the ventricle of clipped embryos, NOS-3 in area 4 is in a trend-like fashion increased by 28.9% (Figure 3C4). The upstream slope of the OFT (area 5) shows a significant upregulation of 64.3% (Figure 3C5). The distal OFT (areas 6 and 7) shows no differences (Figure 3C6 and 3C7), but the descending aorta (area 8) shows a trend in downregulation by 22.2% (Figure 3C8).
Shear Stress Distribution
A 3D reconstruction of a HH14 embryonic chicken heart was used to demonstrate the shear stress distribution in a CFD model (Figure 4). The model represents the heart in maximal dilatation, and input parameters were based on in vivo hemodynamic measurements.24 Highest levels were detected in the inner curvature and at sites of lumen constrictions, ie, AVC and OFT. Lowest shear is in the outer curvature and intertrabecular sinuses.
Discussion
In the present study we show that the gene expression patterns and levels in the heart and dorsal aorta are altered 3 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 vivo.10 Combining the shear distribution patterns and the KLF2 expression10 confirms this. The narrow regions are the higher shear stress areas, eg, 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 shear.10 This has been confirmed by in vitro studies showing KLF2 and NOS-3 upregulation,9,25 and a decrease of ET-126 by high shear stress. Recent data show that avian embryonic endocardial cells show a similar response (Hierck, unpublished data, 2005). Shear stress is positively correlated with blood flow, and will also be altered after 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 shown.6
Gene patterns were evaluated by radioactive ISH, and gene levels were quantified in 2 ways. The first method was real time qRT-PCR on the ventricular segment of the heart. Trends in up- and downregulation 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 semiquantified and showed important changes. With the adapted ColourProc method the differences in hybridization signals could be efficiently semiquantified. In addition, nonendothelial cells, ie, 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 expression.26 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 downregulated, whereas both KLF-2 and NOS-3 are mostly upregulated, 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 model.5 This may imply that changes in gene expression are necessary for anomalies to develop. It is not clear whether shear stress is not altered at all or only subliminally, resulting in the absence of alterations in gene expression. In the narrow part of the AVC, KLF2 and NOS-3 are both upregulated, whereas ET-1 shows no alterations. The level of change in shear may not be sufficient enough for ET-1 to alter. Toward the downstream slope of the AVC both ET-1 and KLF2 are downregulated, 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 al3 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 downregulation is under investigation.
The part that showed the most pronounced changes in all 3 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 humans.5 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 study,10 which implies that they change coordinately. 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 interembryonic 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 flow.6 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 downregulated 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 area.27 However, high ECE-1 expression should imply a lower shear stress as this gene, like ET-1, is downregulated by increased shear stress.28 A model study of curved tubes also demonstrated that shear stress is highest in the inner curvature.29 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 disturbed.5 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 into the myocardium, which is earlier in the inner curvature than in the outer curvature.30 This implies that the increase in shear and the resulting change in gene expression in the inner curvature can lead to impaired cardiac development. Figure 5 shows a scheme of how altered shear stress and changed gene expression can modify signaling processes, which may influence heart development and can lead to congenital heart malformations.
In summary, in the dorsal aorta flow6 and presumably shear stress are decreased, leading to downregulation of KLF2 and NOS-3 and upregulation 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 downregulated. 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 clip.5 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.
Acknowledgments
This research is supported by grant number NHF2000.016 from the Netherlands Heart Foundation. The authors thank J. Lens for his expert assistance preparing the figures.
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Guarda E, Katwa LC, Myers PR, Tyagi SC, Weber KT. Effects of endothelins on collagen turnover in cardiac fibroblasts. Cardiovasc Res. 1993; 27: 2130eC2134.
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: 1059eC1065.
Marsault R, Feolde E, Frelin C. Receptor externalization determines sustained contractile responses to endothelin-1 in the rat aorta. Am J Physiol. 1993; 264: C687eCC693.
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: 1047eC1052.
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: S108eCS111.(Bianca C.W. Groenendijk, )
the Department of Aero- and Hydro-dynamics (M.B., M.J.B.M.P.), Delft University of Technology, Delft, The Netherlands.
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 Kre筽pel-like factor (KLF2), endothelin-1 (ET-1), and endothelial nitric oxide synthase (NOS-3) display specific expression patterns in vivo during chicken cardiovascular development. Nonoverlapping 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 hybridizations 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 upregulated. We conclude that venous obstruction results in altered expression patterns of KLF2, ET-1, and NOS-3, suggestive for increased cardiac shear stress.
Key Words: cardiovascular physiology embryonic circulation endothelium gene expression 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 areas.2 During embryogenesis blood flow plays an important role in cardiac development.3,4 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 abnormalities.5 Additionally, Stekelenburg-de Vos et al6 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.
Important genes encoding transcription factors and signaling molecules, eg, lung Kre筽pel-like factor (KLF2/LKLF), endothelin-1 (ET-1), and endothelial nitric oxide synthase (NOS-3/eNOS) are shear-dependent in their expression in vitro.7eC9 Previously, we suggested that these genes are also shear-related in vivo.10 Endothelin-1 is a growth hormone and vasoconstrictor. NOS-3 catalyzes the conversion of L-arginine to L-citrullin, generating nitric oxide (NO). NO is involved in, eg, 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 stress.9 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-1eCpositive regions located in low shear areas. This implies that there is differential shear-dependent gene expression in cardiovascular development.10
Our in vivo findings confirm the results from in vitro studies, which have shown that both KLF2 and NOS-3 are upregulated9,13 and that ET-1 is downregulated by increased shear stress.14 The regulation of ET-1 and NOS-3 is probably a multistep process as it is not only directly shear-related, but also under the regulation of KLF2.15 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 wall.16 Nos-3eCdeficient mice show atrial and ventricular septal defects, heart failure,17 and bicuspid aortic valves.18 Knockout mice for Edn-1,19 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 clip.5
As KLF2, ET-1, and NOS-3 expression is shear related, it is likely that they respond to venous clipeCinduced changes in shear stress by an alteration in their expression patterns and levels. Therefore, we hypothesize that changes in shear stress by venous clip leads to alterations in the expression of these genes, which is likely to be implicated in cardiovascular malformations. To test this hypothesis, we investigated the expression patterns and levels of KLF2, ET-1, and NOS-3 in the chicken cardiovascular system after ligation of the right lateral vitelline vein.
Materials and Methods
Ligation Procedures
Fertilized white Leghorn eggs (Gallus domesticus; Intervet International, Nieuwegein, The Netherlands) were incubated at 37°C and 60% to 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 before.6 The cessation of blood flow downstream and the rerouting of blood flow upstream of the microclip were confirmed visually. Eggs were resealed, reincubated for 3 hours, and the embryos were euthanized. 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 5 e and mounted serially for in situ hybridization. All experiments were performed according to institutional guidelines.
Data Analysis
Radioactive in situ hybridization was performed as described previously.10 For a detailed description, please refer to the online data supplement available at http://circres.ahajournals.org. The hybridization sections were used for 3D reconstructions and semiquantification.
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), as described before.10 Generation of a CFD model, based on 3D-reconstruction, is described in the online data supplement.
ColourProc
It has been demonstrated that radioactive ISH is a quantifiable technique.22 Therefore, the ColourProc program, originally developed for the acquisition and analysis of COBRA MFISH,23 was adapted for the semiquantification and analysis of the radioactive ISH of mRNA in sections. For a detailed description see the online data supplement.
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. Total RNA was isolated from the narrow part of the atrioventricular canal (AVC) to the upstream part of the distal outflow tract (OFT). Please refer to the online data supplement for details.
Results
In General
We focused on the mRNA expression levels and localizations in endothelium and endocardium of the large vessels and heart in control and experimentally challenged embryos. Changes after clip were most obvious in the ventricular part of the heart (caudally from level B in Figure 1a). Therefore, mRNA levels were quantified using real time RT-PCR. Trends in downregulation of ET-1 and upregulation of KLF2 and NOS-3 expression levels (supplemental Figure I) 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 semiquantified the gene signal from ISH at the various areas represented in Figure 1a. The AVC was subdivided in 3 levels: an upstream part (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 3 regions: the proximal upstream slope (5), the narrow part (6), and the downstream part (7). Area 8 represents the descending dorsal aorta. Figure 1b shows the shear stresseCindependent 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 Hybridization
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 Figure 1a; not shown). However, expression in the narrow part of the AVC (area 2) is extended in clipped embryos (Figure 1c and 1d). 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 (Figure 2a and 2b). In the ventricle, expression in the endocardium lining the junction between the AVC to the OFT cushions (area 4) is augmented (Figure 2a and 2b), as is the case in the upstream slope of the outflow tract (area 5; Figure 2a and 2b). In the distal part (area 6 and 7) no difference has been observed (Figure 1c and 1d).
The aortic sac and the pharyngeal arch arteries show no change in pattern (not shown). In the endothelium of the descending dorsal aorta (area 8), however, KLF2 is decreased (see the following).
ET-1 Expression
The mRNA of endothelin-1 is not only confined to the endothelium/endocardium. It is also present in, eg, 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 (Figure 2g). After the 3-hour clip, expression is only present at the right lateral side (Figure 2h). 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 (Figure 1e and 1f). On the downstream slope (area 3), however, expression is downregulated (Figure 2c and 2d). The junction between AVC to OFT cushions (area 4) shows a decrease in ET-1 (Figure 2c and 2d). In addition, in the upstream part of the OFT (area 5), ET-1 is downregulated (Figure 2c and 2d) as it is in the distal part (area 6 and 7; Figure 1e and 1f).
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 semiquantitative analysis (see below).
NOS-3 Expression
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 Figure 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 (Figure 1g and 1h). The downstream part of the AVC (area 3) shows no differences (Figure 2e and 2f). 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 (Figure 2e and 2f). The OFT further downstream (areas 6 and 7) shows no alterations (Figure 1g and 1h).
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).
Semiquantification
Semiquantification is performed by measuring the number of pixels exceeding a fixed threshold of gray level in 8 different regions from the AVC (3 levels), ventricle, OFT (3 levels), and descending aorta as described.
KLF2
After venous obstruction, the KLF2 level in the upstream slope of the AVC (area 1) is not altered (Figure 3A1). Expression in area 2 is increased by 130% (Figure 3A2), but area 3 shows a significant downregulation of 19.0% (Figure 3A3). The level in the endocardium lining the junction between the AVC-OFT cushions (area 4) is augmented by 70.3% (Figure 3A4). Reaching the upstream slope of the OFT (area 5) the KLF2 signal is elevated after clipping (135.1%; Figure 3A5). The distal OFT (area 6 and 7) is not changed (Figure 3A6 and 3A7). In the endothelium of the dorsal aorta (area 8) a significant decrease of 24.5% in KLF2 is shown in clipped embryos (Figure 3A8).
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 (Figure 3B1 and 3B2). From the downstream slope of the AVC to the distal OFT (areas 3 to 7) expression is downregulated (38.4% to 58.2%, Figure 3B3 through 3B7). In contrast, the dorsal aorta (area 8) shows an increase of 21.5% (Figure 3B8).
NOS-3
After ligation no changes occur in NOS-3 levels in the upstream slope of the AVC (area 1; Figure 3C1). The narrow part of the AVC (area 2) shows a trend in increase of 63.7% (Figure 3C2). The downstream slope of the AVC (area 3) is not altered (Figure 3C3). In the ventricle of clipped embryos, NOS-3 in area 4 is in a trend-like fashion increased by 28.9% (Figure 3C4). The upstream slope of the OFT (area 5) shows a significant upregulation of 64.3% (Figure 3C5). The distal OFT (areas 6 and 7) shows no differences (Figure 3C6 and 3C7), but the descending aorta (area 8) shows a trend in downregulation by 22.2% (Figure 3C8).
Shear Stress Distribution
A 3D reconstruction of a HH14 embryonic chicken heart was used to demonstrate the shear stress distribution in a CFD model (Figure 4). The model represents the heart in maximal dilatation, and input parameters were based on in vivo hemodynamic measurements.24 Highest levels were detected in the inner curvature and at sites of lumen constrictions, ie, AVC and OFT. Lowest shear is in the outer curvature and intertrabecular sinuses.
Discussion
In the present study we show that the gene expression patterns and levels in the heart and dorsal aorta are altered 3 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 vivo.10 Combining the shear distribution patterns and the KLF2 expression10 confirms this. The narrow regions are the higher shear stress areas, eg, 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 shear.10 This has been confirmed by in vitro studies showing KLF2 and NOS-3 upregulation,9,25 and a decrease of ET-126 by high shear stress. Recent data show that avian embryonic endocardial cells show a similar response (Hierck, unpublished data, 2005). Shear stress is positively correlated with blood flow, and will also be altered after 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 shown.6
Gene patterns were evaluated by radioactive ISH, and gene levels were quantified in 2 ways. The first method was real time qRT-PCR on the ventricular segment of the heart. Trends in up- and downregulation 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 semiquantified and showed important changes. With the adapted ColourProc method the differences in hybridization signals could be efficiently semiquantified. In addition, nonendothelial cells, ie, 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 expression.26 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 downregulated, whereas both KLF-2 and NOS-3 are mostly upregulated, 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 model.5 This may imply that changes in gene expression are necessary for anomalies to develop. It is not clear whether shear stress is not altered at all or only subliminally, resulting in the absence of alterations in gene expression. In the narrow part of the AVC, KLF2 and NOS-3 are both upregulated, whereas ET-1 shows no alterations. The level of change in shear may not be sufficient enough for ET-1 to alter. Toward the downstream slope of the AVC both ET-1 and KLF2 are downregulated, 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 al3 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 downregulation is under investigation.
The part that showed the most pronounced changes in all 3 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 humans.5 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 study,10 which implies that they change coordinately. 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 interembryonic 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 flow.6 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 downregulated 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 area.27 However, high ECE-1 expression should imply a lower shear stress as this gene, like ET-1, is downregulated by increased shear stress.28 A model study of curved tubes also demonstrated that shear stress is highest in the inner curvature.29 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 disturbed.5 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 into the myocardium, which is earlier in the inner curvature than in the outer curvature.30 This implies that the increase in shear and the resulting change in gene expression in the inner curvature can lead to impaired cardiac development. Figure 5 shows a scheme of how altered shear stress and changed gene expression can modify signaling processes, which may influence heart development and can lead to congenital heart malformations.
In summary, in the dorsal aorta flow6 and presumably shear stress are decreased, leading to downregulation of KLF2 and NOS-3 and upregulation 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 downregulated. 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 clip.5 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.
Acknowledgments
This research is supported by grant number NHF2000.016 from the Netherlands Heart Foundation. The authors thank J. Lens for his expert assistance preparing the figures.
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