USING ZEBRAFISH EMBRYOS TO IDENTIFY GENES THAT REGULATE ENDOTHELIAL PROLIFERATION

IntroductionAtherosclerosis describes the formation of plaques in the arteries which can lead to myocardial infarction and stroke. Atherosclerosis preferentially occurs in regions of the arteries where the endothelium is exposed to low shear stress, in part, because endothelium in these regions undergoes excessive proliferation. Understanding the mechanisms for this could allow identification of therapeutic targets for patients at risk of cardiovascular disease. We hypothesise that zebrafish embryos can be used to analyse endothelial proliferative responses to flow. If substantiated, this model has the potential to replace rodent models of endothelial pathology.MethodsCandidate regulators of endothelial cell (EC) proliferation in response to flow were identified in prior work (Serbanovic Canic et al, 2017). EC proliferation was quantified by visualising nuclei of Tg fli1a:nls-mCherry embryos in the intersegmental vessels (ISVs) and dorsal longitudinal anastomotic vessel (DLAV) between 54 and 72 hours post fertilisation using a Zeiss LSM880 microscope. Microinjection of morpholino antisense oligonucleotides into single-cell embryos was used to knock-down genes of interest, non-targeting control morpholino and uninjected embryos were used as controls. Flow was prevented by morpholino knockdown of tnnt2a.ResultsGenes that are putative regulators of EC proliferation in response to flow were identified by mining microarray data generated from low and high shear stress regions of the aorta (Serbanovic-Canic et al, 2017). Expression of these genes in zebrafish was validated by quantitative RT-PCR analysis of zebrafish endothelial cells isolated by FACS of cells expressing fli1-EGFP. Morpholinos were then designed against each of these genes to identify those that regulate EC proliferation in response to flow. Endothelial cell proliferation was quantified in embryos with and without blood flow, proliferation was significantly reduced in no-flow embryos (0.81% vs 0.26%; P=0.43). Knockdown of wnk1a significantly increases the rate of EC proliferation in no-flow embryos (0.26% vs 0.;69%; P=0.014), whereas knockdown of gsk3b and fzd5 significantly decreases the rate of EC proliferation in embryos with blood flow (gsk3b 0.51% vs 0.81%: P=0.013. fzd5 0.46% vs 0.81%: P=0.002).ConclusionsEndothelial cell proliferation is reduced in the absence of flow in zebrafish. Putative regulators of EC proliferation in response to flow were identified. Future work will validate the role of these genes in endothelial proliferation and atherosclerosis using mammalian models, and human cells and tissues. The study was funded by the British Heart Foundation and the National Centre for the 3RsReferenceSerbanovic-Canic J, De Luca A, Warboys C, Ferreira PF, Luong LA, Hsiao S, Gauci I, Mahmoud M, Feng S, Souilhol C, Bowden N, Ashton J, Walczak H, Firmin D, Krams R, Mason JC, Haskard DO, Sherwin S, Ridger V, Evans PC. Zebrafish Model for Functional Screening of Flow-Responsive Genes. ATVB 2017;37:130–143.Conflict of Interestnone