Altered Wall Shear Stresses in Embryonic Chicken Outflow
Tract Due to Homocysteine Exposure
Annelien M. Oosterbaan
1,*Christian Poelma
2Els Bon
1Regine P. M. Steegers-Theunissen
1,3,4Eric A. P. Steegers
1Nicolette T. C. Ursem
11Department of Obstetrics and Gynecology, Division of Obstetrics and Prenatal Medicine, Erasmus MC, University Medical Center, 3015 GE Rotterdam, The Netherlands
2Laboratory for Aero and Hydrodynamics, Delft University of Technology, 2628 CN Delft, The Netherlands 3Department of Clinical Genetics, Erasmus MC, University Medical Center, 3015 GE Rotterdam, The Netherlands
Received 8 Jan 2013; Accepted 22 May 2013;doi: 10.5405/jmbe.1417
Abstract
The embryonic heart is already beating before cardiac morphogenesis is complete. Therefore, the effect of blood flow on cardiogenesis has been subject of many studies. Shear stress, acting on the endocardial cells, has been shown to alter the expression of shear-responsive genes implicated in heart development. The congenital heart defects (CHDs) induced in the chicken embryo after altering blood flow and intracardiac shear stress are comparable to the type of defects observed after homocysteine (Hcy) exposure. This may suggest that altered blood flow and shear stress have a role in the etiology of Hcy-induced CHDs. The aim of this study is to quantitate wall shear stress (WSS) in the outflow tract (OFT) of Hcy-exposed chicken embryos. WSS was derived from the velocity field obtained with microscopic particle image velocimetry in the OFT of the embryonic-day-3 Hcy- and sham-treated chicken embryos. Hcy treatment consisted of L-Hcy-thiolactone 30 µM solution injected into the neural tube of the embryos. The results suggest that Hcy has an inhibiting effect on WSS in the early embryonic chicken heart. These alterations in shear stress may cause altered gene expression and behaviour of endothelial cells, eventually contributing to the development of CHDs.
Keywords: Wall shear stress, Animals, Homocysteine, Microscopic particle image velocimetry, Cardiac outflow tract
1. Introduction
During embryonic development, the heart is already beating before cardiac development is complete. Cardiac morphology has thus often been linked to cardiac function. Studies on the effects of blood flow on cardiac morphogenesis have shown that the formation of the functional heart is regulated by the interaction between blood flow and the vessel wall [1,2] and the response to shear stress [3]. Endocardial cells are endothelial cells that line the inner vessel wall of the heart and are subjected to blood flow. Therefore, they are also the cells that sense shear stress, which is the frictional force (per unit area) acting on the cells parallel to blood flow. Because of its low Reynolds number, embryonic blood flow is laminar flow despite irregularities such as trabeculations and endocardial cushions. From the definition of the wall shear stress (WSS), τ = η du/dn, where η represents the dynamic viscosity and du/dn is the wall-normal fluid velocity gradient
* Corresponding author: Annelien M. Oosterbaan Tel: +31-6-14390685; Fax: +31-10-7043532 E-mail: annelienoosterbaan@gmail.com
evaluated at the wall, one would expect an increase in WSS with increased blood flow velocity. Shear stress was first shown to influence gene expression in vitro [4,5]. Subsequently, increasing in vivo evidence has underlined the importance of the effects of shear stress on the expression of genes implicated in heart development [6,7]. Therefore, early cardiac functioning and, specifically, shear stress are considered to play a substantial role in cardiovascular development. Consequently, shear stress is seen as an important epigenetic factor in embryonic cardiogenesis [8,9]. Methods to accurately quantitate WSS in vivo, in a beating heart, are limited. Recently, WSSs have been analyzed by Liu et al. [10] using synchronized in vivo optical
coherence tomography (OCT) images and pressure
measurements. To better understand the relationships between hemodynamics and cardiac development, Goenezen et al. reviewed the recent literature, including studies that have applied new technologies such as high-resolution imaging modalities and computational modeling [11].
To study early cardiovascular development in vivo, the chicken embryo model has proven to be a suitable animal experimental model [12,13]. In this model, successful alteration of intracardiac blood flow in order to explore the role of shear stress was achieved by clipping of the right lateral vitelline vein
[14-16]. Such venous clipping studies have demonstrated that altered hemodynamics in the developing heart can result in cardiovascular malformations, mostly malformations of the outflow tract (OFT) [9,14].
Maternal hyperhomocysteinemia, an increased level of the methionine derivative homocysteine (Hcy) in maternal blood, has been associated with an approximately three-fold increased risk of congenital cardiovascular malformations [17]. This association has been substantiated in the chicken embryo, with specific cardiac malformations such as ventricular septal defects and conotruncal heart defects reported after Hcy treatment [18-20]. These specific defects are comparable to the type of defects observed in the venous clipping model, suggesting that Hcy may induce its effects, among others, by altering blood flow and shear stress levels, resulting in heart defects. We have previously shown that Hcy alters the hemodynamic parameters of early embryonic chicken heart function [21]. To further test this hypothesis, the present study quantifies WSS in the OFT of an Hcy-exposed chicken embryo. WSS was derived from the velocity field obtained with the use of microscopic particle image velocimetry (μPIV) since it cannot be measured directly. The OFT of chicken embryos was studied at embryonic day (ED) 3, Hamburger and Hammilton (HH) stage 18 [22]. At this stage, the heart is under constant development, and the OFT of the developing heart has been described to be most sensitive to alterations in blood flow dynamics [14,23].
2. Materials and methods
2.1 Injection of homocysteine
Fertilized White Leghorn chicken eggs (Gallus Gallus) (Drost Loosdrecht BV, Loosdrecht, The Netherlands) were incubated horizontally at 37 °C. After approximately 30 h (HH 9-10), the embryos were taken out of the incubator. An egg was placed in a metal holder on a heated plate to maintain temperature at 37 °C. A small window of approximately 1.5 cm × 1.5 cm was sawn in the eggshell to allow embryo treatment. Either an Hcy (n = 30) or a sham solution (n = 30), with M199 medium (Gibco, Invitrogen) and Indigo Carmine Bleu, was injected into the lumen of the neural tube of the embryos at somite level 4-6, filling the neural tube in the anterior direction until the solution reached the otic placode level. The Hcy treatment consisted of 30 µM L-Hcy- thiolactone solution in MilliQ water, and was performed according to the protocol reported by Boot et al. [24]. This concentration has been demonstrated to result in OFT defects [18]. Sham treatment consisted of the physiological MilliQ solution with M199 medium and Indigo Carmine Blue only. After injection, the eggs were sealed with Scotch tape and placed back into the incubator for further development. 2.2 Microscopic particle image velocimetry
At ED 3, the eggs were taken out of the incubator and the window was reopened. Embryos were staged macroscopically with the use of a microscope. HH stage 18 embryos were
included for further examination. An egg was placed under an epifluorescent microscope (Leica MZ 16 FA), while partially submerged in a temperature-controlled water bath at 37 °C. To prevent dehydration and allow undistorted imaging, a glass coverslip was placed on top of the window. Visualization of blood flow was achieved by injection of a total injection volume of < 1 μl of 1 µm polyethylene glycol (PEG)-coated polystyrene fluorescent tracer particles (Microparticles GmbH, Berlin, Germany) into the right vitelline artery. Due to the small size of the particles, they accurately follow blood flow. The improved accuracy of blood flow measurements when using tracer particles has recently been reported by Poelma et al. [25]. The tracer particles were illuminated using a dual- cavity Nd:YLF laser (Pegasus, New Wave, ESI, Fremont CA, USA). Flow velocity was determined by imaging the particles with a CCD camera (Imager Intense QE, LaVision, Goettingen, Germany) that records image pairs at a frame rate of 9.9 Hz. The measurements were taken at the midplane of the OFT (Fig. 1). The delay between the two images of each of these pairs was set to 500 ms. At this interval between frames, the maximum tracer displacement at peak systole is 20-25 pixels, which is equivalent to 26-32 µ m. The local displacement was determined by cross-correlating the images and the velocity and then dividing the result by the delay time [26]. A series of 500 image pairs was recorded for each measurement, with a duration of approximately 50 s. The cardiac cycle was divided into 10 phase steps and the data were grouped and averaged within a group to improve accuracy. A vector field at one phase step is based on typically 50 image pairs. Phase 0 was assigned to systole. The result of this approach is a stack of vector fields (i.e., x, y-velocity components in a series of x, y-planes). As the flow is incompressible (i.e., divergence-free), the third velocity component can also be reconstructed, so that in principle all velocity components in a three-dimensional volume as a function of time are available. In previous studies, we found that the in-plane gradients of the streamwise velocity component (i.e., parallel to the walls) are dominant over all other terms in the determination of the WSS tensor [27,28]. This means
Figure 1. Schematic presentation of the anatomy of an HH 18 chicken heart. Blood flows from the primitive atrium (PA) into the primitive left ventricle (PLV). In between, the atrio- ventricular cushions (AVC) are situated. Between the primitive right ventricle (PRV) and the outflow tract (OFT), the outflow cushions (OC) are situated. The blue square containing an X axis and a Y axis represents the midplane of the OFT in which μPIV measurements were recorded.
that the WSS in the OFT midplane can be estimated from a single measurement plane of two components, greatly simplifying the analysis from which the three-dimensional, time-dependent geometry can be determined. We here report only the velocity at the midplane of the OFT.
Figures 2 and 3 show images of the velocity distribution at the midplane of the OFT for 5 successive time points of one cardiac cycle. Detailed descriptions of the in vivo µPIV procedure were given by Vennemann et al. [26] and Poelma et al. [28]. Because of the complexity of the experiments,
successful μPIV measurements of only 3 Hcy and 4 control embryos were obtained. Mostly practical issues prevented successful measurements, such as insufficient number of tracers or movement of the embryo out of the region of interest. Some embryos did not survive the experiments.
2.3 Determination of wall shear stress
The WSS distribution was derived from the velocity fields as determined by μPIV using τ = η du/dn, where η is the
(a) (b) (c)
(d) (e) (f)
Figure 2. Velocity distribution at the midplane of the OFT in an HH 18 homocysteine embryo at 5 successive time points of one cardiac cycle, starting from (a) (phase -0.4 (Ф)) until (e) (Ф = 0.4). Phase 0 represents systole during the cardiac cycle. The wall location is reconstructed and color-coded with the shear stress level. Shear stress is presented in τ (Pa). The arrows indicate flow direction. (f) Schematic representation of mean blood flow velocity (mm/s) present in the OFT at 2 consecutive cardiac cycles. One complete cardiac cycle starts from Ф = -0.4 until Ф = 0.6. No wall location is presented in image (d) because there is no blood flow during this phase of the cardiac cycle. The velocity profile is indicated in yellow (forward flow).
(a) (b) (c)
(d) (e) (f)
Figure 3. Velocity distribution at the midplane of the OFT in an HH 18 control embryo at 5successive time points of one cardiac cycle, starting from (a) (phase -0.4 (Ф)) until (e) (Ф = 0.4). Phase 0 represents systole during the cardiac cycle. The wall location is reconstructed and color-coded with the shear stress level. Shear stress is presented in τ (Pa). The arrows indicate flow direction. (f) Schematic representation of mean blood flow velocity (mm/s) present in the OFT at 2 consecutive cardiac cycles. One complete cardiac cycle starts from Ф = -0.4 until Ф = 0.6. No wall location is presented in image d because there is no blood flow during this phase of the cardiac cycle. The velocity profile is indicated in yellow (forward flow) and red (backward flow).
dynamic viscosity of blood and du/dn is the gradient of the velocity in the wall-normal direction. A value of 0.003 Pa·s ( = 3 cP) was used as a representative value for the dynamic viscosity of embryonic chicken blood. As stated before, the total gradient tensor at the wall is approximated by only considering the in-plane (x,y) velocity components and gradients, which dominate over the other terms. The gradient at the wall (du/dn) is determined by fitting a polynomial function to the velocity profile along a cross-section of the vessel. The location of the wall can be obtained by finding the roots of the polynomial and the WSS follows from the gradient of the polynomial at the wall locations. This process is repeated in the next vessel segment to reconstruct the vessel geometry and WSS distribution. For an extensive explanation of this technique, we refer to a study by Poelma et al. [28]. This method is applicable to any flow pattern and is thus more accurate than conventional approaches that rely on Poiseuille’s law, which assumes a parabolic flow profile. For the complex, curved geometry of the embryonic heart and OFT, the latter assumption cannot be relied on.
2.4 Statistical analysis
Statistical analyses for small sample sizes were performed. Student’s t-test was applied to compare the hemodynamic parameters of sham- and Hcy-treated embryos. Data were corrected for heart rate. A Pearson correlation matrix was calculated to test correlations between parameters. A p value of <0.05 was considered statistically significant.
3. Results and discussion
3.1 Wall shear stress
With the use of velocity fields, obtained by μPIV, the WSS was calculated. Additionally, the location of the vessel wall was extracted from the flow data. Figures 2 and 3 show WSS measurements in Hcy-treated and control embryos at 5 successive time points of one cardiac cycle. Figures 2 and 3 show that, in general, the WSS is lower in Hcy-treated embryos compared to control embryos, most evidently visible during systole (Ф = 0). The mean WSS in Hcy-treated embryos was 1.12 (0.25) Pa, compared to 1.24 (0.22) Pa in control embryos. During some phases of the cardiac cycle, the OFT cushions (Fig. 1) were closed and no blood flow was detected. Consequently, no vessel wall could be extracted in these plains (Figs. 2(d) and 3(d)). Figures 2(f) and 3(f) display the mean blood flow velocity (mm/s) measured in the OFT at 5 successive time points of 2 consecutive cardiac cycles.
3.2 Flow characteristics in OFT
The hemodynamic parameters obtained with μPIV are presented in Table 1. Although no significance was reached, the mean heart rate was slightly higher in Hcy-treated embryos, with a difference of 14%. Other parameters tended to be lower in Hcy-treated embryos compared to control embryos, with a reduction of 10 to 24%. For all parameters, no level of significance was reached when comparing data of Hcy-treated embryos with control embryos. As multiple planes were recorded, the midplane of the OFT was selected for analysis (Fig. 1).
Table 1. Hemodynamic parameters of homocysteine-treated and control embryos Hemodynamic parameters of HH 18 homocysteine- and sham-treated (control) chicken embryos obtained with μPIV measurements presented as mean (standard deviation). The difference between homocysteine-treated and control embryos is presented in percentages. Hcy: homocysteine; HR: heart rate; OFT: outflow tract.
Hcy-treated (n = 3) Control (n = 4) Difference Hcy vs. Control (%) Heart rate (1/min) 134.6 (6.9) 117.9 (20.3) +14 Maximum
velocity (mm/s) 29.3 (4.6) 34.5 (7.9) -15 OFT diameter (mm) 0.31 (0.03) 0.32 (0.04) -3 Mean wall shear
stress (Pa) 1.12 (0.25) 1.24 (0.22) -10 Shear rate (1/s) 373.1 (82.0) 414.6 (74.9) -10 Peak flow (mm3/s) 1.13 (0.18) 1.48 (0.56) -24 Mean flow (mm3/s) 0.26 (0.06) 0.30 (0.08) -13
3.3 Correlation matrix
Data from all embryos were tested for correlations between the different parameters (Table 2). Maximum velocity was correlated to shear rate, mean WSS, and peak flow. Shear rate was correlated to mean WSS. OFT diameter was correlated to peak flow and mean flow. Peak flow was correlated to mean flow.
This study shows that Hcy treatment during early embryonic development tends to reduce the WSS in the OFT of the chicken embryonic heart. Additionally, Hcy treatment causes a reduction of peak flow and peak velocity. This coincides with the results from a previous Doppler study performed by our research group, which found reduced hemodynamic parameters of chicken embryonic heart function, such as peak velocities and velocity time integrals, after Hcy treatment [21]. The decrease in shear stress found in this study may result from a reduction of hemodynamic parameters induced by Hcy. From the definition of the WSS, τ = η du/dn, one can expect this decrease:
Table 2. Correlation matrix for all embryos Correlation matrix of parameters obtained from all embryos (n = 7). Values are given for Pearson’s correlation. * indicates values that have reached statistical significance (p < 0.05). OFT: outflow tract.
Heart rate Maximum velocity OFT diameter Mean wall shear stress Shear rate Peak flow Mean flow
(1/min) (mm/s) (mm) (Pa) (1/s) (mm3/s) (mm3/s)
Heart rate (1/min) - - - -
Maximum velocity (mm/s) -0.40 - - - -
OFT diameter (mm) -0.68 0.39 - - - - -
Mean wall shear stress (Pa) -0.02 0.83* -0.19 - - - -
Shear rate (1/s) -0.02 0.83* -0.19 1.00* - - -
Peak flow (mm3/s) -0.63 0.87* 0.79* 0.45 0.45 - -
when the flow rate, and consequently the associated velocity profile, decreases, the resulting WSS will decrease proportionally. Since the correlation matrix shows no correlation between heart rate and the other parameters, the effect of Hcy on WSS appears to be independent of heart rate. Still, because of the low number of embryos, a correlation cannot really be excluded.
The effects of Hcy on hemodynamic parameters and shear stress presented in this study may influence cardiogenesis. It has been previously shown that altered intracardiac blood flow may cause altered cardiac morphogenesis [15]. After venous clipping, decreased heart rate and peak velocities have been reported [16]. Hcy treatment, such as venous clipping, results in disturbance of normal intracardiac blood flow patterns [21]. Shear stress is dependent on volume flow and is therefore affected in a situation of altered flow. Shear stress exerted on the endothelial cells that line the inner walls of the OFT drives gene expression [29,30]. Endothelial cells respond to the magnitude, frequency, and direction of the shear stress, and alterations from normal shear, either an increase or decrease, will lead to signalling events [29]. Altered expression of the shear-stress-responsive genes Krüppel-like factor-2 (KLF-2), endothelin-1 (ET-1), and endothelial nitric oxide synthase (NOS-3) have been reported after venous clipping [31]. Altered gene expression in reaction to lowered shear stress may cause changes in cardiac morphology. This is supported by the venous clipping studies that have shown that alterations in blood flow and consequently shear stress levels can lead to the development of specific heart defects [15,31]. Therefore, we conclude that altered hemodynamics and the WSS induced by Hcy treatment may influence cardiac morphology.
In our experiments, heart rates obtained by µPIV were lower than those previously reported [32,33]. This may be explained by the fact that these previous data were obtained by Doppler ultrasonography. This method does not require manipulation of the embryo preceding the recording of flow velocity profiles, and therefore less influence on embryo hemodynamics is expected.
It has been shown by others that hypothermia directly decreases heart rate [34]. In our experiments, an egg was partly placed in a temperature-controlled water bath to maintain temperature at 37 °C, and the heart rate and hemodynamic parameters were not affected. Heart rates were probably reduced because of the complexity of the performed technique. This is supported by the fact that successful µPIV measurements were obtained for only approximately 10% of the treated embryos. Even though the injection of fluorescent tracer particles is needed to be able to perform µPIV measurements and quantify the WSS, it is plausible that this affects the embryo as well as its hemodynamic functioning.
The 30 µmol/l concentration of Hcy solution applied to the embryos in this study has been shown to result in OFT defects in the chicken embryo [18] and is comparable to the mild to moderate hyperhomocysteinemia (15-150 μmol/l) in humans associated with neural tube defects and congenital heart defects [17,35,36].
The reported shear stress in control embryos, 1.24 Pa,
coincides well with levels of 1-3 Pa previously reported for HH 18 chicken embryos [28]. It also matches well with the shear stress of 3.1 Pa found by Liu et al. [23], and of 5 Pa reported by Vennemann et al. [26] and Hierck et al. [2], since this concerns the maximum shear stress at the inner curvature of the OFT and we have presented the mean shear stress levels in the OFT. Also, Liu et al. studied older embryos (HH stage 21). During development, as the chicken embryo grows, hemodynamic parameters of chicken embryonic heart function increase [32], consequently leading to higher shear stress levels. According to Poelma et al., the shear stress levels in Hcy-exposed embryos of 1.12 (0.25) Pa, also fall in the normal range [28]. This is most probably due to low sample size. Greater differences in hemodynamic parameters and WSS between control and Hcy- treated embryos are expected with additional experiments since we have previously reported lower mean heart rates in Hcy- exposed embryos [21]. With a lower heart rate, reduced WSS is expected, as has recently been shown by Lee et al. [34].
A limitation of our study is the low sample size. Also, with techniques such as µPIV, it is not possible to directly measure shear stress. However, the WSS can in theory be much more accurately determined from data obtained by µPIV compared to studies using (Hagen-) Poisseuille’s law, requiring less information. Still, shear stress levels are estimated from the velocity profile, which is difficult to measure near the wall of a beating heart.
4. Conclusion
Reduced hemodynamic parameters were observed after Hcy treatment in the early embryonic chicken heart. Disturbance of normal intracardiac blood flow patterns affects WSS. These alterations in WSS may cause different behaviour and gene expression of the endothelial cells in the embryonic chicken heart, eventually leading to the development of congenital heart defects. These findings warrant further investigation.
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