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Abstract-Hemodynamics play an important role in cardiovascular development, and changes in blood flow can cause congenital heart malformations. The endothelium and endocardium are subjected to mechanical forces, of which fluid shear stress is correlated to blood flow velocity. The shear stress responsive genes lung Krüppel-like factor (KLF2), endothelin-1 (ET-1), and endothelial nitric oxide synthase (NOS-3) display specific expression patterns in vivo during chicken cardiovascular development. 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 Ⅲ embryonic circulation Ⅲ endothelium Ⅲ gene expression Ⅲ shear stress H emodynamic forces generated by blood flow modulate the structure and function of both fetal and adult endothelial cells (reviewed by Gimbrone et al 1 ). 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 model 3 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 al 6 have shown that up to 5 hours after venous clip the dorsal aortic mean and peak blood flow is decreased, demonstrating a change in hemodynamics. Shear stress is directly related to blood flow, therefore it is likely that this is also altered in the venous clip model and involved in the development of abnormalities in the cardiovascular system.Important genes encoding transcription factors and signaling molecules, eg, lung Krüppel-like factor (KLF2/LKLF), endothelin-1 (ET-1), and endothelial nitric oxide synthase (NOS-3/eNOS) are shear-dependent in their expression in vitro. 7-9 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 t...

During cardiovascular development, fluid shear stress patterns change dramatically due to extensive remodeling. This biomechanical force has been shown to drive gene expression in endothelial cells and, consequently, is considered to play a role in cardiovascular development. The mechanism by which endothelial cells sense shear stress is still unidentified. In this study, we postulate that primary cilia function as fluid shear stress sensors of endothelial cells. Such a function already has been attributed to primary cilia on epithelial cells of the adult kidney and of Hensen's node in the embryo where they transduce mechanical signals into an intracellular Ca 2ϩ signaling response. Recently, primary cilia were observed on human umbilical vein endothelial cells. These primary cilia disassembled when subjected to high shear stress levels. Whereas endocardial-endothelial cells have been reported to be more shear responsive than endothelial cells, cilia are not detected, thus far, on endocardial cells. In the present study, we use field emission scanning electron microscopy to show shear stress-related regional differences in cell protrusions within the cardiovasculature of the developing chicken. Furthermore, we identify one of these cell protrusions as a monocilium with monoclonal antibodies against acetylated and detyrosinated alphatubulin. The distribution pattern of the monocilia was compared to the chicken embryonic expression pattern of the high shear stress marker Krü ppel-like factor-2. We demonstrate the presence of monocilia on endocardial-endothelial cells in areas of low shear stress and postulate that they are immotile primary cilia, which function as fluid shear stress sensors.

Primary cilia are mechanosensors for fluid shear stress, and are involved in a number of syndromes and congenital anomalies. We identified endothelial cilia in areas of low shear stress in the embryonic heart. The objective of the present study was to demonstrate the role of primary cilia in mechanosensing. Ciliated embryonic endothelial cells were cultured from the heart, and non-ciliated cells from the arteries. Nonciliated cells that were subjected to fluid shear stress showed significantly less induction of the shear marker Krü ppel-Like Factor-2, as compared to ciliated cells. In addition, ciliated cells from which the cilia were chemically removed show a similar decrease in flow response. This shows that primary cilia sensitize endothelial cells for fluid shear stress. In addition, we targeted and stabilized the connection of the cilium to the cytoplasm by treatment with Colchicine and Taxol/Paclitaxel, respectively, and show that microtubular integrity is essential to sense shear stress. Developmental Dynamics 237:725-735, 2008.

In this review, the role of wall shear stress in the chicken embryonic heart is analyzed to determine its effect on cardiac development through regulating gene expression. Therefore, background information is provided for fluid dynamics, normal chicken and human heart development, cardiac malformations, cardiac and vitelline blood flow, and a chicken model to induce cardiovascular anomalies. A set of endothelial shear stress-responsive genes coding for endothelin-1 (ET-1), lung Krüppel-like factor (LKLF/KLF2), and endothelial nitric oxide synthase (eNOS/NOS-3) are active in development and are specifically addressed.

Blood flow patterns play an important role in cardiovascular development, as changes can cause congenital heart malformations. Shear stress is positively correlated to blood flow. Therefore, it is likely that shear stress is also involved in cardiac development. In this study, we investigated the expression patterns of ET-1, NOS-3, and KLF-2 mRNA in a series of developmental stages of the chicken embryo. These genes are reported to be shear responsive. It has been demonstrated that KLF-2 is confined to areas of high shear stress in the adult human aorta. From in vitro studies, it is known that ET-1 is down-regulated by shear stress, whereas NOS-3 is up-regulated. Therefore, we expect ET-1 to be low or absent and NOS-3 to be high at sites where KLF-2 expression is high. Our study shows that, in the early stages, expression patterns are mostly not shear stress-related, whereas during development, this correlation becomes stronger. We demonstrate overlapping expression patterns of KLF-2 and NOS-3 in the narrow parts of the cardiovascular system, like the cardiac inflow tract, the atrioventricular canal, outflow tract, and in the early stages in the aortic sac and the pharyngeal arch arteries. In these regions, the expression patterns of KLF-2 and NOS-3 exclude that of ET-1. Our results suggest that, in the embryonic cardiovascular system, KLF-2 is expressed in regions of highest shear stress, and that ET-1 and NOS-3 expression, at least in the later stages, is related to shear stress. Developmental Dynamics 230:57-68, 2004.

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Background/Aims: Ligating the right lateral vitelline vein of chicken embryos (venous clip) results in cardiovascular malformations. These abnormalities are similar to malformations observed in knockout mice studies of components of the endothelin-1 (ET-1)/endothelin-converting enzyme-1/endothelin-A receptor pathway. In previous studies we demonstrated that cardiac ET-1 expression is decreased 3 h after clipping, and ventricular diastolic filling is disturbed after 2 days. Therefore, we hypothesise that ET-1-related processes are involved in the development of functional and morphological cardiovascular defects after venous clip. Methods: In this study, ET-1 and endothelin receptor antagonists (BQ-123, BQ-788 and PD145065) were infused into the HH18 embryonic circulation. Immediate haemodynamic effects on the embryonic heart and extra-embryonic vitelline veins were examined by Doppler and micro-particle image velocimetry. Ventricular diastolic filling characteristics were studied at HH24, followed by cardiovascular morphologic investigation (HH35). Results: ET-1 and its receptor antagonists induced haemodynamic effects at HH18. At HH24, a reduced diastolic ventricular passive filling component was demonstrated, which was compensated by an increased active filling component. Thinner ventricular myocardium was shown in 42% of experimental embryos. Conclusion: We conclude that cardiovascular malformations after venous clipping arise from a combination of haemodynamic changes and altered gene expression patterns and levels, including those of the endothelin pathway.