Beerend P. Hierck

Changes in Shear Stress-Related Gene Expression After Experimentally Altered Venous Return in the Chicken Embryo

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.

Primary Cilia Sensitize Endothelial Cells for Fluid Shear Stress

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. Non‐ciliated cells that were subjected to fluid shear stress showed significantly less induction of the shear marker Krüppel‐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.

Lack of primary cilia primes shear-induced Endothelial-to-Mesenchymal Transition

Rationale: Primary cilia are cellular protrusions that serve as mechanosensors for fluid flow. In endothelial cells (ECs), they function by transducing local blood flow information into functional responses, such as nitric oxide production and initiation of gene expression. Cilia are present on ECs in areas of low or disturbed flow and absent in areas of high flow. In the embryonic heart, high-flow regime applies to the endocardial cushion area, and the absence of cilia here coincides with the process of endothelial-to-mesenchymal transition (EndoMT). Objective: In this study, we investigated the role of the primary cilium in defining the responses of ECs to fluid shear stress and in EndoMT. Methods and Results: Nonciliated mouse embryonic ECs with a mutation in Tg737/Ift88 were used to compare the response to fluid shear stress to that of ciliated ECs. In vitro, nonciliated ECs undergo shear-induced EndoMT, which is accompanied by downregulation of Klf4. This Tgf&bgr;/Alk5-dependent transformation is prevented by blocking Tgf&bgr; signaling, overexpression of Klf4, or rescue of the primary cilium. In the hearts of Tg737orpk/orpk embryos, Tgf&bgr;/Alk5 signaling was activated in areas in which ECs would normally be ciliated but now lack cilia because of the mutation. In these areas, ECs show increased Smad2 phosphorylation and expression of &agr;-smooth muscle actin. Conclusions: This study demonstrates the central role of primary cilia in rendering ECs prone to shear-induced activation of Tgf&bgr;/Alk5 signaling and EndoMT and thereby provides a functional link between primary cilia and flow-related endothelial performance.

MONOCILIA ON CHICKEN EMBRYONIC ENDOCARDIUM IN LOW SHEAR STRESS AREAS

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 Hensens node in the embryo where they transduce mechanical signals into an intracellular Ca2+ 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 alpha‐tubulin. The distribution pattern of the monocilia was compared to the chicken embryonic expression pattern of the high shear stress marker Krüppel‐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. Developmental Dynamics 235:19–28, 2006.

Variants of the α6 β1 Laminin Receptor in Early Murine Development: Distribution, Molecular Cloning and Chromosomal Localization of the Mouse Integrin α6 Subunit

Laminin (A:B1:B2) is a major component of the first basement membrane to appear in the developing mouse embryo. Its effects on morphogenesis and differentiation are mediated by interaction with cell surface receptors that are members of the integrin family. We have studied the expression of the alpha 6 subunit of murine alpha 6 beta 1 and its ligand, laminin, in preimplantation mouse embryos, embryo outgrowths and in embryonic stem (ES) cells and embryonal carcinoma (EC) cells. The alpha 6 subunit is present in the oocyte and throughout preimplantation development. Laminin A chain appears later than alpha 6 and has a more restricted distribution until the late blastocyst stage. alpha 6 beta 1 is strongly expressed in ES and EC cells; the levels of mRNA expression are not altered by differentiation. Molecular cloning of cDNA for the murine integrin alpha 6 subunit from a mammary gland lambda gt11 library showed, as in man, an open reading frame encoding two variants of alpha 6, alpha 6A and alpha 6B. The identity of the alpha 6 amino acid sequence to that in man and chicken is 93% and 73%, respectively. The gene for murine alpha 6 was mapped to chromosome 2. While undifferentiated ES and EC cells express only alpha 6B, alpha 6A is co-expressed in ES cells after differentiation is induced by retinoic acid. alpha 6B is also the only variant expressed in blastocyst stage embryos, but when blastocysts have grown out in culture both alpha 6A and alpha 6B are expressed reflecting the results in the cell lines. We suggest that the deposition of laminin in the embryo is a receptor-mediated process and that the shift in the expression of the variants, as the inner cell mass forms its first differentiated progeny, reflects a change in functional properties.

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

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.

Expression patterns of Tgfβ1–3 associate with myocardialisation of the outflow tract and the development of the epicardium and the fibrous heart skeleton

Transforming growth factor‐beta (Tgfβ) is essential for normal embryogenesis. The cardiac phenotypes obtained after knockout of each of the three mammalian isoforms suggest different roles during morphogenesis. We studied cardiovascular expression of Tgfβ1–3 in parallel tissue sections of normal mouse embryos from 9.5 to 15.5 days post coitum (dpc) by using radioactive in situ hybridisation. The Tgfβ isoforms are differentially expressed in unique and in overlapping patterns during cardiovascular development. In the vessels, Tgfβ1 is found in the intima, whereas Tgfβ2 and ‐β3 are mainly present in the media and adventitia of the great arteries. Tgfβ1 is present in the endocardium at all stages examined. The Tgfβ2 signal in the endocardium of the atrioventricular canal and outflow tract (9.5 dpc) shifts during epithelial–mesenchymal transformation toward the mesenchymal cushions (10.5–11.5 dpc) after which it exhibits a marked spatiotemporal expression pattern as the cushion differentiation progresses (11.5–15.5 dpc). The myocardium underlying the endocardial cushions and the atrial muscular septum are intensely positive for Tgfβ2 at early stages (9.5–11.5 dpc) and expression decreases at 12.5 days. In contrast to earlier reports, we find marked overlap of Tgfβ2 and ‐β3 expression. Tgfβ3 expression shows a characteristic distribution in the mesenchymal cushions, suggesting a role in cushion differentiation, possibly additional to Tgfβ2. From 14.5 dpc onward, a strong Tgfβ3 signal is found in the fibrous septum primum of the atrium and in the fibrous skeleton of the heart. Special attention was paid to the proepicardial organ and its derivatives. The proepicardial organ strongly expresses Tgfβ2 as early as 9.5 days, and all isoforms are present in the epicardium from 12.5 dpc onward. The spatiotemporal cardiovascular expression of Tgfβ1–3 supports both specific and complementary functions during cardiovascular development that might explain functional redundancy between the Tgfβ‐isoforms. The information provided favors novel roles of Tgfβ1–3 in epicardial development, of Tgfβ2 in myocardialisation, and of Tgfβ3 in differentiation of the fibrous structures of the heart. Developmental Dynamics 227:431–444, 2003.

Measurements of the wall shear stress distribution in the outflow tract of an embryonic chicken heart

In order to study the role of blood–tissue interaction in the developing chicken embryo heart, detailed information about the haemodynamic forces is needed. In this study, we present the first in vivo measurements of the three-dimensional distribution of wall shear stress (WSS) in the outflow tract (OFT) of an embryonic chicken heart. The data are obtained in a two-step process: first, the three-dimensional flow fields are measured during the cardiac cycle using scanning microscopic particle image velocimetry; second, the location of the wall and the WSS are determined by post-processing flow velocity data (finding velocity gradients at locations where the flow approaches zero). The results are a three-dimensional reconstruction of the geometry, with a spatial resolution of 15–20 µm, and provides detailed information about the WSS in the OFT. The most significant error is the location of the wall, which results in an estimate of the uncertainty in the WSS values of 20 per cent.

Prenatal Exposure to apoE Deficiency and Postnatal Hypercholesterolemia Are Associated with Altered Cell-Specific Lysine Methyltransferase and Histone Methylation Patterns in the Vasculature

We recently demonstrated that neointima formation of adult heterozygous apolipoprotein E (apoE(+/-)) offspring from hypercholesterolemic apoE(-/-) mothers was significantly increased as compared with genetically identical apoE(+/-) offspring from normocholesterolemic wild-type mothers. Since atherosclerosis is the consequence of a complex microenvironment and local cellular interactions, the effects of in utero programming and type of postnatal diet on epigenetic histone modifications in the vasculature were studied in both groups of offspring. An immunohistochemical approach was used to detect cell-specific histone methylation modifications and expression of accompanying lysine methyltransferases in the carotid arteries. Differences in histone triple-methylation modifications in vascular endothelial and smooth muscle cells revealed that the offspring from apoE(-/-) mothers had significantly different responses to a high cholesterol diet when compared with offspring from wild-type mothers. Our results suggest that both in utero programming and postnatal hypercholesterolemia affect epigenetic patterning in the vasculature, thereby providing novel insights regarding initiation and progression of vascular disease in adults.

Fluid Shear Stress and Inner Curvature Remodeling of the Embryonic Heart. Choosing the Right Lane

Cardiovascular development is directed or modulated by genetic and epigenetic factors. The latter include blood flow-related shear stress and blood pressure-related circumferential strain. This review focuses on shear stress and its effects on endothelial cells lining the inner surfaces of the heart and blood vessels. Flow characteristics of the embryonic blood, like velocity, viscosity and periodicity, are taken into account to describe the responses of endothelial cells to shear stress and the sensors for this friction force. The primary cilium, which is an integral part of the shear sensor, connects to the cytoskeletal microtubules and transmits information about the level and direction of blood flow into the endothelial cell. When the heart remodels from a more or less straight into a c-shaped tube the sharp curvature, in combination with the small vessel dimensions and high relative viscosity, directs the highest shear stress to the inner curvature of this pump. This proves to be an important epigenetic modulator of cardiac morphogenesis because when shear stress is experimentally altered inner curvature remodeling is affected which leads to the development of congenital cardiovascular anomalies. The best of both worlds, mechanics and biology, are used here to describe early cardiogenesis.