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

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

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

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

TWIST1 Integrates Endothelial Responses to Flow in Vascular Dysfunction and Atherosclerosis.

RATIONALE: Blood flow-induced shear stress controls endothelial cell (EC) physiology during atherosclerosis via transcriptional mechanisms that are incompletely understood. The mechanosensitive transcription factor TWIST is expressed during embryogenesis but its role in EC responses to shear stress and focal atherosclerosis is unknown. OBJECTIVE: Investigate whether TWIST regulates endothelial responses to shear stress during vascular dysfunction and atherosclerosis, and compare TWIST function in vascular development and disease. METHODS AND RESULTS: The expression and function of TWIST1 was studied in EC in both developing vasculature and during the initiation of atherosclerosis. In zebrafish, twist was expressed in early embryonic vasculature where it promoted angiogenesis by inducing EC proliferation and migration. In adult porcine and murine arteries, TWIST1 was expressed preferentially at low shear stress regions as evidenced by qPCR and en face staining. Moreover, studies of experimental murine carotid arteries and cultured EC revealed that TWIST1 was induced by low shear stress via a GATA4-dependent transcriptional mechanism. Gene silencing in cultured EC and EC-specific genetic deletion in mice demonstrated that TWIST1 promoted atherosclerosis by inducing inflammation and enhancing EC proliferation associated with vascular leakiness. CONCLUSIONS: TWIST expression promotes developmental angiogenesis by inducing EC proliferation and migration. In addition to its role in development, TWIST is expressed preferentially at low shear stress regions of adult arteries where it promotes atherosclerosis by inducing EC proliferation and inflammation. Thus pleiotropic functions of TWIST control vascular disease as well as development

EPAS1 Attenuates Atherosclerosis Initiation at Disturbed Flow Sites Through Endothelial Fatty Acid Uptake.

BackgroundAtherosclerotic plaques form unevenly due to disturbed blood flow, causing localized endothelial cell (EC) dysfunction. Obesity exacerbates this process, but the underlying molecular mechanisms are unclear. The transcription factor EPAS1 (HIF2A) has regulatory roles in endothelium, but its involvement in atherosclerosis remains unexplored. This study investigates the potential interplay between EPAS1, obesity, and atherosclerosis.MethodsResponses to shear stress were analyzed using cultured porcine aortic EC exposed to flow in vitro coupled with metabolic and molecular analyses and by en face immunostaining of murine aortic EC exposed to disturbed flow in vivo. Obesity and dyslipidemia were induced in mice via exposure to a high-fat diet or through Leptin gene deletion. The role of Epas1 in atherosclerosis was evaluated by inducible endothelial Epas1 deletion, followed by hypercholesterolemia induction (adeno-associated virus-PCSK9 [proprotein convertase subtilisin/kexin type 9]; high-fat diet).ResultsEn face staining revealed EPAS1 enrichment at sites of disturbed blood flow that are prone to atherosclerosis initiation. Obese mice exhibited substantial reduction in endothelial EPAS1 expression. Sulforaphane, a compound with known atheroprotective effects, restored EPAS1 expression and concurrently reduced plasma triglyceride levels in obese mice. Consistently, triglyceride derivatives (free fatty acids) suppressed EPAS1 in cultured EC by upregulating the negative regulator PHD2. Clinical observations revealed that reduced serum EPAS1 correlated with increased endothelial PHD2 and PHD3 in obese individuals. Functionally, endothelial EPAS1 deletion increased lesion formation in hypercholesterolemic mice, indicating an atheroprotective function. Mechanistic insights revealed that EPAS1 protects arteries by maintaining endothelial proliferation by positively regulating the expression of the fatty acid-handling molecules CD36 (cluster of differentiation 36) and LIPG (endothelial type lipase G) to increase fatty acid beta-oxidation.ConclusionsEndothelial EPAS1 attenuates atherosclerosis at sites of disturbed flow by maintaining EC proliferation via fatty acid uptake and metabolism. This endothelial repair pathway is inhibited in obesity, suggesting a novel triglyceride-PHD2 modulation pathway suppressing EPAS1 expression. These findings have implications for therapeutic strategies addressing vascular dysfunction in obesity