GATA4-Twist1 signalling in disturbed flow-induced atherosclerosis

BACKGROUND: Endothelial cell (EC) dysfunction (enhanced inflammation, proliferation and permeability) is the initial trigger for atherosclerosis. Atherosclerosis shows preferential development near branches and bends exposed to disturbed blood flow. By contrast, sites that are exposed to non-disturbed blood flow are atheroprotected. Disturbed flow promotes atherosclerosis by promoting EC dysfunction. Blood flow controls EC function through transcriptional and post-transcriptional mechanisms that are incompletely understood. METHODS AND RESULTS: We identified the developmental transcription factors Twist1 and GATA4 as being enriched in EC at disturbed flow, atheroprone regions of the porcine aorta in a microarray study. Further work using the porcine and murine aortae demonstrated that Twist1 and GATA4 expression was enhanced at the atheroprone, disturbed flow sites in vivo. Using controlled in vitro flow systems, the expression of Twist1 and GATA4 was enhanced under disturbed compared to non-disturbed flow in cultured cells. Disturbed flow promoted Twist1 expression through a GATA4-mediated transcriptional mechanism as revealed by a series of in vivo and in vitro studies. GATA4-Twist1 signalling promoted EC proliferation, inflammation, permeability and endothelial-to-mesenchymal transition (EndoMT) under disturbed flow, leading to atherosclerosis development, as shown in a combination of in vitro and in vivo studies using GATA4 and Twist1-specific siRNA and EC-specific GATA4 and Twist1 Knock out (KO) mice. CONCLUSIONS: We revealed that GATA4-Twist1-Snail signalling triggers EC dysfunction and atherosclerosis; this work could lead to the development of novel anti-atherosclerosis therapeutics

Aqueous nicotine solutions:PH-measurements and salting-out effects - Analysis of the effective Gibbs energies of hydration and ionic strengths of the solutions

This work is a continuation of previous studies on phase demixing - salting-out effects - in aqueous nicotine solutions. Thus, pH measurements were performed, allowing a brief analysis of the existing hydrogen bond interactions. Salting-out effects - the related experimental cloud point shifts - provoked by the addition of two inorganic salts, potassium nitrate and sodium sulfate, which have not hitherto been studied, were determined. Analysis of the current and previously reported salting-out/or salting-in phenomena in nicotine aqueous solutions was performed. In this respect, five studied salts were included: four inorganic salts (sodium chloride, potassium nitrate, sodium sulfate and trisodium phosphate (Na3PO4)), and ionic the liquid 1-ethyl-3-methylimidazolium ethyl sulfate ([C2mim][EtSO4], commercial name ECOENG212®). Based on pH measurements, the effective Gibbs energies of hydration and the ionic strengths of the respective ternary solutions were calculated and plotted against the related cloud-point shifts caused by the addition of the salts. For the studied salts, the results and diagram obtained within this work may be used to predict the cloud-points shifts, based on the related quantities of the salts added and/or the molar Gibbs energies of hydration and/or ionic strengths requested in each case

Quantifying endothelial cell proliferation in the zebrafish embryo

Introduction: Endothelial cell (EC) proliferation is a fundamental determinant of vascular development and homeostasis, and contributes to cardiovascular disease by increasing vascular permeability to blood-borne lipoproteins. Rodents have been traditionally used to analyse EC proliferation mechanisms in vascular health and disease; however, alternative models such as the zebrafish embryo allow researchers to conduct small scale screening studies in a physiologically relevant vasculature whilst reducing the use of mammals in biomedical research. In vitro models of EC proliferation are valuable but do not fully recapitulate the complexity of the in vivo situation. Several groups have used zebrafish embryos for vascular biology research because they offer the advantages of an in vivo model in terms of complexity but are also genetically manipulable and optically transparent. Methods: Here we investigated whether zebrafish embryos can provide a suitable model for the study of EC proliferation. We explored the use of antibody, DNA labelling, and time-lapse imaging approaches. Results: Antibody and DNA labelling approaches were of limited use in zebrafish due to the low rate of EC proliferation combined with the relatively narrow window of time in which they can label proliferating nuclei. By contrast, time-lapse imaging of fluorescent proteins localised to endothelial nuclei was a sensitive method to quantify EC proliferation in zebrafish embryos. Discussion: We conclude that time-lapse imaging is suitable for analysis of endothelial cell proliferation in zebrafish, and that this method is capable of capturing more instances of EC proliferation than immunostaining or cell labelling alternatives. This approach is relevant to anyone studying endothelial cell proliferation for screening genes or small molecules involved in EC proliferation. It offers greater biological relevance than existing in vitro models such as HUVECs culture, whilst reducing the overall number of animals used for this type of research

The effect of absent blood flow on the zebrafish cerebral and trunk vasculature

The role of blood flow in vascular development is complex and context-dependent. In this study, we quantify the effect of the lack of blood flow on embryonic vascular development on two vascular beds, namely the cerebral and trunk vasculature in zebrafish. We perform this by analysing vascular topology, endothelial cell (EC) number, EC distribution, apoptosis, and inflammatory response in animals with normal blood flow or absent blood flow. We find that absent blood flow reduced vascular area and EC number significantly in both examined vascular beds, but the effect is more severe in the cerebral vasculature, and severity increases over time. Absent blood flow leads to an increase in non-EC-specific apoptosis without increasing tissue inflammation, as quantified by cerebral immune cell numbers and nitric oxide. Similarly, while stereotypic vascular patterning in the trunk is maintained, intra-cerebral vessels show altered patterning, which is likely to be due to vessels failing to initiate effective fusion and anastomosis rather than sprouting or path-seeking. In conclusion, blood flow is essential for cellular survival in both the trunk and cerebral vasculature, but particularly intra-cerebral vessels are affected by the lack of blood flow, suggesting that responses to blood flow differ between these two vascular beds

Zebrafish as a tractable model of human cardiovascular disease

Mammalian models including non-human primates, pigs and rodents have been used extensively to study the mechanisms of cardiovascular disease. However, there is an increasing desire for alternative model systems that provide excellent scientific value while replacing or reducing the use of mammals. Here, we review the use of zebrafish, Danio rerio, to study cardiovascular development and disease. The anatomy and physiology of zebrafish and mammalian cardiovascular systems are compared, and we describe the use of zebrafish models in studying the mechanisms of cardiac (e.g. congenital heart defects, cardiomyopathy, conduction disorders and regeneration) and vascular (endothelial dysfunction and atherosclerosis, lipid metabolism, vascular ageing, neurovascular physiology and stroke) pathologies. We also review the use of zebrafish for studying pharmacological responses to cardiovascular drugs and describe several features of zebrafish that make them a compelling model for in vivo screening of compounds for the treatment cardiovascular disease

A Loss of Function Screen of Identified Genome-Wide Association Study Loci Reveals New Genes Controlling Hematopoiesis

The formation of mature cells by blood stem cells is very well understood at the cellular level and we know many of the key transcription factors that control fate decisions. However, many upstream signalling and downstream effector processes are only partially understood. Genome wide association studies (GWAS) have been particularly useful in providing new directions to dissect these pathways. A GWAS meta-analysis identified 68 genetic loci controlling platelet size and number. Only a quarter of those genes, however, are known regulators of hematopoiesis. To determine function of the remaining genes we performed a medium-throughput genetic screen in zebrafish using antisense morpholino oligonucleotides (MOs) to knock down protein expression, followed by histological analysis of selected genes using a wide panel of different hematopoietic markers. The information generated by the initial knockdown was used to profile phenotypes and to position candidate genes hierarchically in hematopoiesis. Further analysis of brd3a revealed its essential role in differentiation but not maintenance and survival of thrombocytes. Using the from-GWAS-to-function strategy we have not only identified a series of genes that represent novel regulators of thrombopoiesis and hematopoiesis, but this work also represents, to our knowledge, the first example of a functional genetic screening strategy that is a critical step toward obtaining biologically relevant functional data from GWA study for blood cell traits

The effect of absent blood flow on the zebrafish cerebral and trunk vasculature

The role of blood flow in vascular development is complex and context-dependent. In this study, we quantify the effect of the lack of blood flow on embryonic vascular development on two vascular beds, namely the cerebral and trunk vasculature in zebrafish. We perform this by analysing vascular topology, endothelial cell (EC) number, EC distribution, apoptosis, and inflammatory response in animals with normal blood flow or absent blood flow. We find that absent blood flow reduced vascular area and EC number significantly in both examined vascular beds, but the effect is more severe in the cerebral vasculature, and severity increases over time. Absent blood flow leads to an increase in non-EC-specific apoptosis without increasing tissue inflammation, as quantified by cerebral immune cell numbers and nitric oxide. Similarly, while stereotypic vascular patterning in the trunk is maintained, intra-cerebral vessels show altered patterning, which is likely to be due to vessels failing to initiate effective fusion and anastomosis rather than sprouting or path-seeking. In conclusion, blood flow is essential for cellular survival in both the trunk and cerebral vasculature, but particularly intra-cerebral vessels are affected by the lack of blood flow, suggesting that responses to blood flow differ between these two vascular beds

Zebrafish Model for Functional Screening of Flow-Responsive Genes

OBJECTIVE: Atherosclerosis is initiated at branches and bends of arteries exposed to disturbed blood flow that generates low shear stress. This mechanical environment promotes lesions by inducing endothelial cell (EC) apoptosis and dysfunction via mechanisms that are incompletely understood. Although transcriptome-based studies have identified multiple shear-responsive genes, most of them have an unknown function. To address this, we investigated whether zebrafish embryos can be used for functional screening of mechanosensitive genes that regulate EC apoptosis in mammalian arteries. APPROACH AND RESULTS: First, we demonstrated that flow regulates EC apoptosis in developing zebrafish vasculature. Specifically, suppression of blood flow in zebrafish embryos (by targeting cardiac troponin) enhanced that rate of EC apoptosis (≈10%) compared with controls exposed to flow (≈1%). A panel of candidate regulators of apoptosis were identified by transcriptome profiling of ECs from high and low shear stress regions of the porcine aorta. Genes that displayed the greatest differential expression and possessed 1 to 2 zebrafish orthologues were screened for the regulation of apoptosis in zebrafish vasculature exposed to flow or no-flow conditions using a knockdown approach. A phenotypic change was observed in 4 genes; p53-related protein (PERP) and programmed cell death 2-like protein functioned as positive regulators of apoptosis, whereas angiopoietin-like 4 and cadherin 13 were negative regulators. The regulation of perp, cdh13, angptl4, and pdcd2l by shear stress and the effects of perp and cdh13 on EC apoptosis were confirmed by studies of cultured EC exposed to flow. CONCLUSIONS: We conclude that a zebrafish model of flow manipulation coupled to gene knockdown can be used for functional screening of mechanosensitive genes in vascular ECs, thus providing potential therapeutic targets to prevent or treat endothelial injury at atheroprone sites

Understanding mechanobiology in cultured endothelium: A review of the orbital shaker method

A striking feature of atherosclerosis is its highly non-uniform distribution within the arterial tree. This has been attributed to variation in the haemodynamic wall shear stress (WSS) experienced by endothelial cells, but the WSS characteristics that are important and the mechanisms by which they lead to disease remain subjects of intensive investigation despite decades of research. In vivo evidence suggests that multidirectional WSS is highly atherogenic. This possibility is increasingly being studied by culturing endothelial cells in wells that are swirled on an orbital shaker. The method is simple and cost effective, has high throughput and permits chronic exposure, but interpretation of the results can be difficult because the fluid mechanics are complex; hitherto, their description has largely been restricted to the engineering literature. Here we review the findings of such studies, which indicate that putatively atherogenic flow characteristics occur at the centre of the well whilst atheroprotective ones occur towards the edge, and we describe simple mathematical methods for choosing experimental variables that avoid resonance, wave breaking and uncovering of the cells. We additionally summarise a large number of studies showing that endothelium cultured at the centre of the well expresses more pro-inflammatory and fewer homeostatic genes, has higher permeability, proliferation, apoptosis and senescence, and shows more endothelial-to-mesenchymal transition than endothelium at the edge. This simple method, when correctly interpreted, has the potential to greatly increase our understanding of the homeostatic and pathogenic mechanobiology of endothelial cells and may help identify new therapeutic targets in vascular disease