sensor localized to athero-prone flow areas
Heiden, K. van der
Citation
Heiden, K. van der. (2008, September 11). Primary cilia on endothelial cells : component of the shear stress sensor localized to athero-prone flow areas. Retrieved from
https://hdl.handle.net/1887/13093
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Chapter 3
Does Venous Clip Affect the Distribution Pattern of Endothelial Primary Cilia?
Kim Van der Heiden1; Peter Vennemann2; Christian Poelma2; Jerry Westerweel2; Robert E.
Poelmann1; Beerend P. Hierck1.
1Department of Anatomy and Embryology, Leiden University Medical Center, Leiden, The Netherlands; 2Department of Aero- and Hydrodynamics, Delft University of Technology, Delft, The Netherlands.
Abstract
Shear stress has been shown to drive gene expression in endothelial cells and is therefore considered to play a prominent role in cardiovascular development. Alterations in shear stress during development cause congenital heart malformations. A chicken model was previously developed in which the venous return to the heart and the course of blood flow through the heart are altered by means of extraembryonic venous obstruction (venous clip).
Alterations in shear stress-related gene expression upon venous clip are suggestive for an increase in cardiac shear stress. In the present study, we investigated the immediate effect of venous clip on shear stress in the outflow tract of the heart by means of micro particle image velocimetry (μPIV). In addition, we analyzed the concomitant distribution pattern of endothelial primary cilia, which is shear stress-related. The primary cilium is a constituent of the endothelial shear stress sensor that sensitizes endothelial cells for shear stress in areas of oscillatory shear stress. μPIV measurements are suggestive for an increase in shear stress levels upon venous clip. However, venous clip does not affect endothelial primary cilia patterning.
Introduction
Shear stress is the friction of blood acting on the endothelium in parallel to the direction of the flow. Shear stress drives gene expression in embryonic endothelial cells in vivo1 and in vitro2. Consequently, shear stress is considered to play a substantial role in cardiovascular development3. Several animal models show that disturbing blood flow during development results in congenital heart malformations4-8. Our group developed the chicken venous clip model4 in which the venous return to the heart and the course of blood flow through the heart are altered via ligation of the right lateral vitelline vein. This eventually results in a spectrum of outflow tract (OFT) anomalies4,9. Stekelenburg-de Vos et al.10 demonstrated a direct effect of venous clip on hemodynamics in the dorsal aorta. They reported a decrease in mean and peak blood flow up to 5 hrs after clip, indicating a decrease in shear stress, as shear is directly related to blood flow. Groenendijk et al.11 described alterations in the expression pattern of the shear stress-responsive genes Krüppel-like factor-2 (KLF2), endothelin-1, and endothelial nitric oxide synthase 3 hrs after venous clip, which are suggestive for an increase in cardiac and a decrease in aortic shear stress. The reported changes in the course of blood flow4 and in shear-related gene expression11,12 immediately after clip are most obvious in the OFT of the heart, which is where many malformations eventually occur9. However, the effect of venous clip on hemodynamic parameters and shear stress specifically in the OFT were never measured. Shear stress in the embryonic cardiovascular system can be measured very accurately with micro particle image velocimetry (μPIV), as described by Vennemann et al.13,14. μPIV is a technique that relies on the visualization of blood flow by means of small particles. In general, the velocity of the blood flow is measured by dividing the displacement of the particles in two consecutive images by the time between the images. Shear stress can subsequently be deduced from the flow velocity.
Venous clip-induced alterations in shear stress are sensed by the endothelium. Recently, primary cilia have been shown to function in endothelial shear stress sensing2,15,16. Primary cilia are plasma membrane enclosed, solitary cellular protrusions with 9 microtubule doublets in their core, lacking a central pair of microtubules (9+0), rendering them non- motile17. The primary cilium is in direct contact with the microtubular cytoskeleton, as its
base is formed by the basal body which is the mother centriole of the centrosome18 and part of the microtubule organizing center of the cell19. Not all endothelial cells carry a primary cilium. Their occurrence is restricted to areas of oscillatory shear stress in vivo20,21 and can be induced by exposure to oscillatory shear stress in vitro (Chapter 5). Moreover, in vivo experimentally-induced alterations in shear stress affect the distribution of primary cilia21. We hypothesize that venous clip alters shear stress and concomitantly endothelial ciliation.
To test this hypothesis we analyze shear stress in the OFT of the heart with μPIV and study the distribution pattern of endothelial primary cilia throughout the cardiovascular system before and after venous clip. We show that venous clip appears to increase shear stress levels but does not affect the distribution of primary cilia.
Materials and Methods Venous clip
Fertilised White Leghorn eggs (Gallus domesticus) were incubated at 37°C and 60%
relative humidity for 75 hrs. Only normal embryos that were at Hamburger and Hamilton22 stage 17 (HH17) were selected. Embryos were clipped, essentially as described before4. In short, they were uncovered by creating an opening in the shell and removing the overlying membranes. Adjacent to the right vitelline vein a small incision was made in the vitelline membrane and the yolk sac membrane. Subsequently, the right vitelline vein was ligated by clipping it for several seconds with a pair of tweezers. Cessation of blood flow distal of the clip location was confirmed visually. All experiments were performed according to national and institutional guidelines.
μPIV
After opening the egg and the membranes, 1 μm polyethylene glycol (PEG)-coated polystyrene fluorescent tracer particles (Microparticles, GmbH) were injected into the right vitelline artery. μPIV measurements of blood flow through the OFT of the heart were taken immediately before and after clipping of the right vitelline vein. During the measurements the egg was partially immersed in a constant temperature water bath of 37°C. To prevent dehydration, the egg window was filled up with purified mineral oil (paraffinum subliquidum). To keep the embryo in place a glass coverslip was placed over the window.
Sham operated (n = 2) and clipped (n = 4) embryos were measured in ovo. The application of in vivo μPIV was described by Vennemann et al.13. μPIV uses the displacement of the tracer particles in two sequential digital images to measure the motion of fluid. The wall shear stress is derived with a two-step process. First, the in-plane velocity is determined by μPIV for each step in the cardiac cycle. By stepwise scanning the OFT perpendicular to the mean flow direction, the complete three-dimensional velocity field is obtained. Second, in slices perpendicular to the direction of flow a two-dimensional fit is used to describe the flow velocity. The location of the wall and the local shear rate are determined from this velocity distribution. The former follows from the zero-crossing of the fit, whereas the latter is obtained from the analytical derivative of the fit at the wall locations. The OFT is thus reconstructed from slices perpendicular to the flow direction. To calculate the shear stress (IJ) a typical value of 4x10-3 Pa s is used for the viscosity (ȝ), which links the shear rate (įu/įx) to the shear stress (IJ = ȝ įu/įx). The cardiac cycle is divided in 10 phases. The frame with the highest mean velocity represents systole. The peak systolic velocity is the maximum velocity in the three-dimensional measurement range. Peak and mean blood flow
are determined from the flow through all planes perpendicular to this frame. The heart rate is determined by quantifying the number of maximal velocities per min and the stroke volume is the quotient of the mean blood flow and the heart rate. Data are presented as mean ± standard error of the mean (SEM).
Immunofluorescence
For primary cilia assessment additional clipped embryos were reincubated for 3 hrs, after which they were sacrificed. Sham operated (n = 3) and clipped (n = 3) embryos were perfusion fixed with 4% paraformaldehyde (PFA) in 0.1 mol/L PHEM buffer (60 mmol/L Pipes, 25 mmol/L Hepes, 10 mmol/L EGTA, 2 mmol/L MgCl2 pH 6.97) via the left atrium.
The embryos were subsequently fixed overnight in 4% PFA in 0.1 mol/L PHEM buffer, after which they were dehydrated in graded ethanol and embedded in paraffin. Specimens were sectioned transversely at 5 μm. Primary cilia were detected with a monoclonal antibody directed against acetylated Į-tubulin (clone 6-11B-1, Sigma-Aldrich Chemie)23. Fluorescein isothiocyanate-conjugated rabbit anti-mouse antibody (DAKO, Denmark) was used as secondary antibody. After deparaffination the sections were stained and examined as described previously20.
The number of primary cilia in specific areas of the cardiovascular system was quantified in every fifth tissue section. This number was multiplied by 5 to estimate the absolute number of cilia and an independent-samples t-test was performed (SPSS; SPSS Inc.). For each specific area of the cardiovascular system sham operated embryos were compared with clipped embryos and, as endothelial cells in specific areas of the heart can either protrude a primary cilium from their luminal or abluminal surface, the number of luminal primary cilia was compared to the number of abluminal primary cilia. Data are presented as mean ± SEM P-value < 0.05 and power > 90% was considered significant.
Results
Venous clip appears to increase shear stress levels in the outflow tract
The values (mean ± SEM) for the hemodynamic parameters in the clipped and sham operated embryos before and after clip are summarized in Table 1. To adjust for biological variability the relative changes, i.e., the ratio between the before and after measurements, are given as well. Statistical analyses are not performed because of the sample size.
However, the peak blood flow, mean blood flow, and stroke volume appear to be decreased upon clip.
Table 1. Hemodynamic parameters before (pre) and after (post) venous clip
Pre sham Post sham ǻsham Pre clip Post clip ǻclip Heart Rate
(BPM)
97.63 ± 6.00 105.47 ± 41.01 1.06 ± 0.35 84.27 ± 7.76 92.97 ± 8.36 1.11 ± 0.07
Peak systolic velocity (mm/s)
38.70 ± 3.90 50.45 ± 0.75 1.31 ± 0.11 36.75 ± 6.64 41.80 ± 3.05 1.22 ± 0.17
Peak blood flow (mm3/s)
0.89 ± 0.10 0.97 ± 0.28 1.14 ± 0.44 0.96 ± 0.05 0.81 ± 0.05 0.85 ± 0.05
Mean blood flow (mm3/s)
0.32 ± 0.06 0.40 ± 0.14 1.18 ± 0.21 0.22 ± 0.02 0.20 ± 0.03 0.91 ± 0.15
Stroke Volume (mm3)
0.20 ± 0.02 0.23 ± 0.01 1.19 ± 0.20 0.16 ± 0.01 0.13 ± 0.03 0.82 ± 0.12 Hemodynamic parameters of HH17 sham and clipped embryos derived from ȝPIV measurements and relative changes in hemodynamic parameters upon venous clip. Sham n = 2, Clip n = 4.
Shear stress levels in the OFT before and after clip were analysed for each individual embryo. Fifty percent of all the embryos, sham operated and clipped, show an increase in shear stress levels, whereas in the other embryos the levels are similar before and after clip (not shown). The shear stress distribution in one of the two clipped embryos which is representative for the increase in shear stress is shown in figure 1 and 2. Figure 1 shows the wall shear stress distribution in the midplane of the OFT at peak systole (maximum flow) before (Fig. 1a) and after (Fig. 1b) venous clip. The wall, which is colourcoded with the shear stress level, was visualized by extrapolating the flow velocity to zero. In this way the three-dimensional geometry can be reconstructed without histological sectioning (Fig.
1c,d).
Figure 1. Velocity field at the mid-plane of the outflow tract before (a) and after (b) clip of one of the two clipped embryos that show an increase in shear stress levels upon clip. The wall location is reconstructed and colourcoded with the shear stress level (dyne/cm2). Shown in a and b is a single measurement result from the scanning method, which was used to reconstruct the three-dimensional volumetric data (c). This reconstruction is similar to the morphology of the outflow tract of the chicken embryonic heart, as determined by field emission scanning electron microscopy (d). A, atrium; V, ventricle; OFT, outflow tract.
The measurements before and after clip can not be fitted on top of each other by mere morphological matching, due to the lack of a distinct morphological feature in the high magnification of the interrogation window. Therefore, in Figure 2, the point on the inner OFT wall which is exposed to the highest level of shear stress is designated x=0. The x-axis represents the position along the OFT wall (m). The y-axis represents the level of shear stress (dyne/cm2). There is an increase in shear stress levels upon venous clip, which is highest at x=0 and gradually declines going downstream in the OFT.
Distribution of ciliated endothelial cells in the HH17 chicken embryo
In a previous study we showed that the endothelium is shear responsive at HH171,11, indicating that the mechanosensing machinery is operational. Endothelial primary cilia are indeed present, as is determined by immunofluorescent staining for acetylated Į-tubulin.
Acetylated Į-tubulin is present in the microtubular cytoskeleton, the microtubule organizing center, and in the primary cilium (Fig. 3). Previously, we described the distribution pattern of primary cilia in the cardiovascular system of chicken embryos at HH24, 28, and 3020. At HH17 ciliated endothelial cells are occasionally present in the cardinal veins (Fig. 3a) and in the sinus venosus. In the common atrium (Fig. 3b) the ciliary density increases dramatically. In the atrioventricular (AV) canal, ventricle (Fig. 3c), and OFT (Fig. 3d) many endothelial cells are ciliated, although the prevalence of ciliated endothelial cells is lower than in the atrium. In the aortic sac, pharyngeal arch arteries, and aorta (Fig. 3e) very few ciliated endothelial cells are present. In addition to the primary cilia located on the luminal side of the endothelial cells we observed primary cilia protruding from the abluminal side (Fig. 3b-d). Abluminal endothelial cilia are exclusively present in areas where the endothelium is covering cardiac jelly, i.e., in the atrium (Fig. 3b), the AV canal, the ventricle (Fig. 3c), and the OFT (Fig. 3d). In these areas a difference in the distribution of luminal and abluminal primary cilia is observed. If exposed to high shear stress, as determined by KLF2 expression1, most primary cilia protrude from the abluminal surface.
In all the areas where the endothelium covers cardiac jelly, there appear to be more abluminal than luminal primary cilia. To determine whether this is a significant difference, we quantified the absolute number of luminal and abluminal primary cilia in specific regions of the cardiovascular system. As the previously detected changes in gene
Figure 2. Shear stress along the inner outflow tract wall before (open circles) and after (closed circles) clip of one of the two clipped embryos that show an increase in shear stress level upon clip. The x-axis represents the position on the OFT wall (m). The y-axis shows the corresponding shear stress level (dyne/cm2). X = 0 is the position on the wall exposed to the highest level of shear stress.
expression upon clip were very local11, we subdivided the AV and OFT cushions into separate regions. The AV cushions are subdivided into an upstream (1), middle (2), and downstream (3) part. The junction of the AV and OFT cushion (4) and the upstream (5), middle (6), and downstream (7) part of the OFT cushions are additional subregions. Table 2 shows the absolute number of primary cilia in these regions. However, there are no differences between the absolute number of luminal and abluminal primary cilia.
Figure 3. Confocal laser scanning microscopic (CLSM) images of luminal (arrows) and abluminal (arrowheads) primary cilia on endothelial cells of a HH17 sham operated chicken embryo. (a) Luminal endothelial primary cilium in the cardinal vein. (b) Two ciliated endothelial cells in the atrium. One luminal and one abluminal primary cilium. (c) Luminal and abluminal endothelial primary cilia in the ventricle. (d) Luminal and abluminal primary cilia in the outflow tract. (e) Luminal endothelial primary cilium in the aorta. (f) Ciliated hemopoietic stem cells at the ventral side of the aorta. L, lumen; J, Jelly. Acetylated Į-tubulin (green), nuclei (red; propidium iodide). Scale bar a,b,e,f = 5 ȝm, scale bar c,d = 10ȝm.
Table 2. Absolute number of primary cilia in sham operated and clipped embryos
Sham Clip
Luminal Abluminal Total Luminal Abluminal Total
Atrium 308.3 ± 20.3 450.0 ± 175.0 758.3 ± 174.9 291.7 ± 39.4 425.0 ± 117.6 716.7 ± 156.7 AVC 160.0 ± 25.2 273.3 ± 56.6 433.3 ± 79.3 165.0 ± 13.2 281.7 ± 33.5 446.7 ± 45.9 1 upstream 88.3 ± 30.9 141.7 ± 45.9 230.0 ± 68.3 35.0 ± 5.0 106.7 ± 16.4 141.7 ± 13.3 2 narrow 96.7 ± 21.3 141.7 ± 36.6 238.3 ± 57.8 83.3 ± 19.2 143.3 ± 16.9 226.7 ± 32.4 3 downstream 16.7 ± 3.3 8.3 ± 6.0 25.0 ± 7.6 20.0 ± 5.8 20.0 ± 11.5 40.0 ± 17.3 4 Junction to OFT 28.3 ± 14.5 86.7 ± 46.7 115.0 ± 60.3 21.7 ± 4.4 21.7 ± 10.1 43.3 ± 9.3 Ventricle 133.3 ± 31.8 218.3 ± 21.7 351.7 ± 36.1 76.7 ± 24.9 178.3 ± 54.9 255.0 ± 79.7 OFT 178.3 ± 24.6 343.3 ± 91.1 521.7 ± 103.7 230.0 ± 43.1 360.0 ± 55.7 590.0 ± 97.5 5 upstream 65.0 ± 17.6 143.3 ± 28.0 208.3 ± 17.6 65.0 ± 2.9 123.3±13.0 188.3 ± 14.5 6 middle 90.0 ± 20.2 198.3 ± 85.8 288.3 ± 105.8 128.3 ± 56.0 230.0 ± 68.3 358.3 ± 124.2 7 downstream 35.0 ± 11.5 20.0 ± 7.6 55.0 ± 15.0 43.3 ± 6.0 41.7 ± 3.3 85.0 ± 2.9 Quantification of the absolute number of cilia in regions of the cardiovascular system of sham operated (n = 3) and clipped (n = 3) HH17 chicken embryos. There are no significant differences in the number of luminal and abluminal primary cilia or between sham operated and clipped embryos. The AV and OFT cushions are divided in subregions a described by Groenendijk et al.11. OFT, outflow tract; AVC, atrioventricular canal.
Venous clip does not affect endothelial primary cilia patterning
The distribution of ciliated endothelial cells in the cardiovascular system of a HH17 chicken embryo is not affected by clip. Primary cilia are present in the same areas.
Although the distribution pattern is unaffected the absolute number of primary cilia might be affected. To determine this, we compared the absolute number of luminal, abluminal and the total number of primary cilia in specific regions of the cardiovascular system (Table 2) between sham operated and clipped chicken embryos. There are no significant differences in the number of cilia (luminal, abluminal, or total) between the sham operated and clipped embryos.
Hemopoietic stem cells present primary cilia
In addition to the endothelium, primary cilia are present on several other cell types. To our knowledge, this is the first study to report primary cilia on the intra-aortic hemopoietic stem cells, located at the ventral side of the aorta. These primary cilia protrude in all directions (Fig. 3f). The prevalence of ciliated hemopoietic stem cells is not affected by clip (not shown).
Discussion
Alterations in shear stress-related gene expression upon the ligation of the right lateral vitelline vein (venous clip) of a HH17 chicken embryo are suggestive for an increase in shear stress in the OFT of the chicken embryonic heart11. In this study we determined hemodynamic parameters, such as heart rate, peak systolic velocity, peak blood flow, mean blood flow, stroke volume, and shear stress levels in the OFT of the heart with μPIV immediately before and after venous clip. Although statistical statements can not be made due to the low sample size, peak blood flow, mean blood flow, and stroke volume appear to be slightly decreased upon clip, whereas heart rate and peak systolic velocity are similar to sham operated embryos. This is consistent with data from Stekelenburg-de Vos et al.10 who showed an immediate decrease in the stroke volume and in the peak as well as mean blood flow directly after clip. Venous clip generates an increase in shear stress levels in the OFT (this study), as was previously suggested by Groenendijk et al.11, after they observed an
increased cardiac expression of the high shear stress marker KLF2. We postulate that the local increase in shear stress is not reflected in the overall mean and peak blood flow due to a venous clip-induced shift in flow towards the inner curvature of the heart4,11,24. This shift causes a redistribution of shear stress in the OFT which does not affect the mean and peak blood flow.
Primary cilia function in endothelial shear stress sensing2,15. Moreover, endothelial ciliation is shear-related in the chicken embryonic cardiovascular system at HH24, 28, and 3020. This shear-related distribution of ciliated endothelial cells is evident at HH17 as well. In areas of high shear stress, as determined by KLF2 expression1, no luminal primary cilia are present. This is in line with in vitro data showing that high steady and pulsatile shear stress reduce the prevalence of primary cilia on endothelial cells25, Chapter 5. Strikingly, in high shear stress areas where the endothelium covers cardiac jelly most primary cilia protrude from the abluminal cell surface. The difference in the absolute number of luminal and abluminal primary cilia is not significant, however, which is probably due to the large inter- animal variations in abluminal primary cilia.
During development the cardiac jelly is cellularized via epithelial-mesenchymal transformation (EMT), in which endothelial cells separate from the endothelium, migrate into the cardiac jelly, and transdifferentiate into mesenchymal cells (reviewed by Markwald et al.26). Only a thin layer of cardiac jelly and very few abluminal endothelial cilia remain (Van der Heiden, unpublished data, 2006). Strikingly, the mesenchymal cells filling the cushions are ciliated20. The presence of abluminal cilia on endothelial cells overlying cardiac jelly leads us to speculate that they play a role in the EMT process. We postulate that abluminal cilia have a function in registering relative motions between endothelial cells and the underlying cardiac jelly. The cardiac jelly might induce a mechanical signal, initiating EMT. In that case endothelial cells overlying the cardiac jelly are subjected to forces from two directions and project their cilium either into the lumen or the cardiac jelly.
Although cardiac jelly fluid flow has not been reported, the cushions have been demonstrated to be extremely flexible and to undulate as a wave at HH1727, suggesting fluid-like properties of the cardiac jelly. Besides their well established mechanosensory function on flow-exposed cells2,15,28-31, primary cilia were shown to function in the Hedgehog signaling pathway (reviewed by Singla and Reiter32). A target gene of the Hedgehog signaling pathway is transforming growth factor ȕ (TGFȕ), which plays a major role in EMT (reviewed by Azhar et al.33). However, the presence and function of Hedgehog signaling has not been reported in endothelial primary cilia, to date. In conclusion, EMT is a complex phenomenon, in which different signaling pathways interact and in which the abluminal primary cilium might play a role.
ȝPIV measurements were taken within 30 min after clip. Endothelial ciliation was investigated 3 hrs after clip to compare the ciliation to the previously described gene expression patterns11 and because at least 2 hrs are required to detect changes in ciliation (Chapter 5). Endothelial ciliation is shear-related20,21 and experimentally induced alterations in shear stress result in changes in the distribution and absolute number of endothelial primary cilia21, Chapter 5
. As venous clip induces alterations in flow, concomitant changes in endothelial primary cilia distribution are to be expected. However, in this study we show that the distribution of endothelial primary cilia in the clipped embryos equals that of sham operated embryos. Furthermore, there are no significant differences in the absolute number of primary cilia between clipped and sham operated embryos. Since the inter-animal
variation in luminal primary cilia is small, we can conclude that venous clip does not affect the number of luminal primary cilia. Previously, we found that endothelial ciliation is induced by oscillatory shear stress and independent of the level of shear stress (Chapter 5).
Since venous clip does not affect endothelial ciliation, we can conclude that venous clip does not extend or limit the regions of oscillatory shear stress.
In summary, venous clip appears to increase shear levels in the OFT but does not affect the distribution or absolute number of primary cilia in the cardiovascular system. As endothelial ciliation is induced by oscillatory shear stress and unaffected by an increase in shear stress level, venous clip most likely does not affect the oscillatory shear stress regions. Considering the variations between the embryos in the ȝPIV measurements, sample size should be increased substantially. The mechanism by which albuminal endothelial primary cilia function in mechanosensing and/or intercellular signaling, is a subject of ongoing investigation.
References
1. 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.
2. Hierck BP, Van der Heiden K, Alkemade FE, van de Pas S, van Thienen JV, Groenendijk BCW, Bax WH, Van der Laarse A, DeRuiter MC, Horrevoets AJG, Poelmann RE. Primary cilia sensitize endothelial cells for fluid shear stress. Dev Dyn. 2008;237:725-735.
3. Poelmann RE, Gittenberger-de Groot AC, Hierck BP. The development of the heart and microcirculation:
role of shear stress. Med Biol Eng Comput. 2008;46:479-484.
4. 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.
5. Tobita K, Schroder EA, Tinney JP, Garrison JB, Keller BB. Regional passive ventricular stress-strain relations during development of altered loads in chick embryo. Am J Physiol Heart Circ Physiol.
2002;282:H2386-H2396.
6. Sedmera D, Pexieder T, Rychterova V, Hu N, Clark EB. Remodeling of chick embryonic ventricular myoarchitecture under experimentally changed loading conditions. Anat Rec. 1999;254:238-252.
7. 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.
8. Dealmeida A, McQuinn T, Sedmera D. Increased ventricular preload is compensated by myocyte proliferation in normal and hypoplastic fetal chick left ventricle. Circ Res. 2007;100:1363-1370.
9. 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.
10. 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.
11. Groenendijk BCW, Hierck BP, Vrolijk J, Baiker M, Pourquie MJBM, Gittenberger-de Groot AC, Poelmann RE. Changes in shear stress-related gene expression after experimentally altered venous return in the chicken embryo. Circ Res. 2005;96:1291-1298.
12. Groenendijk BCW, Van der Heiden K, Hierck BP, Poelmann RE. The role of shear stress on ET-1, KLF2, and NOS-3 expression in the developing cardiovascular system of chicken embryos in a venous ligation model. Physiology (Bethesda). 2007;22:380-389.
13. 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. 2006;39:1191-1200.
14. Vennemann P, Lindken R, Westerweel J. In vivo whole-filed blood velocity measurements techniques.
Exp Fluids. 2007;42:495-511.
15. Nauli SM, Kawanabe Y, Kaminski JJ, Pearce WJ, Ingber DE, Zhou J. Endothelial cilia are fluid-shear sensors that regulate calcium signaling and nitric oxide production through polycystin-1. Circulation.
2008;117:1161-1171.
16. Poelmann RE, Van der Heiden K, Gittenberger-de Groot AC, Hierck BP. Deciphering the endothelial shear stress sensor. Circulation. 2008;117:1124-1126.
17. Praetorius HA, Spring KR. A physiological view of the primary cilium. Annu Rev Physiol. 2005;67:515- 529.
18. Vorobjev IA, Chentsov YS. Centrioles in the cell cycle. I. Epithelial cells. J Cell Biol. 1982;93:938-949.
19. Uzbekov R, Prigent C. Clockwise or anticlockwise? Turning the centriole triplets in the right direction!
FEBS Lett. 2007;581:1251-1254.
20. Van der Heiden K, Groenendijk BCW, Hierck BP, Hogers B, Koerten HK, Mommaas AM, Gittenberger- de Groot AC, Poelmann RE. Monocilia on chicken embryonic endocardium in low shear stress areas. Dev Dyn. 2006;235:19-28.
21. Van der Heiden K, Hierck BP, Krams R, de Crom R, Cheng C, Baiker M, Pourquie MJBM, Alkemade FE, DeRuiter MC, Gittenberger-de Groot AC, Poelmann RE. Endothelial primary cilia in areas of disturbed flow are at the base of atherosclerosis. Atherosclerosis. 2008;196:542-550.
22. Hamburger V, Hamilton HL. A series of normal stages in the development of the chick embryo. J Morphol. 1951;88:49-92.
23. Piperno G, Fuller MT. Monoclonal-antibodies specific for an acetylated form of alpha-tubulin recognize the antigen in cilia and flagella from a variety of organisms. J Cell Biol. 1985;101:2085-2094.
24. Hierck BP, Van der Heiden K, Poelmann RE. Fluid shear stress and inner curve remodeling of the embryonic heart. Choosing the right lane! ScientificWorldJournal. 2008;8:212-222.
25. Iomini C, Tejada K, Mo W, Vaananen H, Piperno G. Primary cilia of human endothelial cells disassemble under laminar shear stress. J Cell Biol. 2004;164:811-817.
26. Markwald R, Eisenberg C, Eisenberg L, Trusk T, Sugi Y. Epithelial-mesenchymal transformations in early avian heart development. Acta Anat. 1996;156:173-186.
27. Butcher JT, McQuinn TC, Sedmera D, Turner D, Markwald RR. Transitions in early embryonic atrioventricular valvular function correspond with changes in cushion biomechanics that are predictable by tissue composition. Circ Res. 2007;100:1503-1511.
28. Praetorius HA, Spring KR. Bending the MDCK cell primary cilium increases intracellular calcium. J Membrane Biol. 2001;184:71-79.
29. McGrath J, Somlo S, Makova S, Tian X, Brueckner M. Two populations of node monocilia initiate left- right asymmetry in the mouse. Cell. 2003;114:61-73.
30. Yost HJ. Left-right asymmetry: Nodal cilia make and catch a wave. Curr Biol. 2003;13:R808-R809.
31. Masyuk AI, Masyuk TV, Splinter PL, Huang BQ, Stroope AJ, Larusso NF. Cholangiocyte cilia detect changes in luminal fluid flow and transmit them into intracellular Ca2+ and cAMP signaling.
Gastroenterology. 2006;131:911-920.
32. Singla V, Reiter JF. The primary cilium as the cell's antenna: signaling at a sensory organelle. Science.
2006;313:629-633.
33. Azhar M, Schultz JJ, Grupp I, Dorn GW, Meneton P, Molin DGM, Gittenberger-de Groot AC, Doetschman T. Transforming growth factor beta in cardiovascular development and function. Cytokine Growth Factor Rev. 2003;14:391-407.
