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, 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 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 endothelial cell 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.

BS16 Figure 1 The effect of parasympathetic denervation on acute cardioprotection by SGLT2 inhibitor Ertugliflozin (Ertu). ISinfarct size, AARarea at risk. ** -p<0.01, *** -p<0.001.

The role of blood flow is complex and context-dependent. In this study, we quantify the effect of the lack of blood flow on vascular development and compare its impact in two vascular beds, namely the cerebral and trunk vasculature, using zebrafish as preclinical model. We performed this by analysing vascular topology, endothelial cell number, apoptosis, and inflammatory response in animals with normal blood flow or absent blood flow. We find that absent blood flow reduced vascular area and endothelial cell number significantly in both examined vascular beds, but the effect is more severe in the cerebral vasculature. Similarly, while stereotypic vascular patterning in the trunk is maintained, intra-cerebral vessels show altered patterning. Absent blood flow lead to an increase in non-EC-specific apoptosis without increasing tissue inflammation, as quantified by cerebral immune cell numbers and nitric oxide. 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.Key pointsWe here use zebrafish as a model to quantitatively assess the impact of the lack of blood flow in development and compare its impact in two vascular beds, namely the cerebral to trunk vasculature.In both vascular beds, vascular growth and endothelial cell number are reduced by lack of blood flow, with increasing effect size from 2-5 days post fertilisation.Examination of vascular patterning shows that while stereotypic patterning in the trunk is preserved, the intra-cerebral vasculature patterning is altered.We found non-EC-specific cell death to be increased in both vascular beds, with a larger effect size in the brain, but that this cell death occurs without triggering tissue inflammation.

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.