Atherosclerosis, a disease of arteries leading to angina, heart attack and stroke, is the leading cause of death in the UK. It is initiated at branches and bends of arteries that display enhanced rates of endothelial cell (EC) proliferation associated with vascular leakiness and influx of cholesterol-rich lipoproteins. These sites are exposed to disturbed blood flow which generates low wall shear stress (WSS; mechanical drag) which promotes EC proliferation and atherogenesis. By contrast, high WSS protects arteries by inducing EC quiescence. Elucidating the molecular mechanisms linking WSS to EC proliferation could lead to the development of novel therapies to treat atherosclerosis. Studying EC responses to flow using in vitro flow bioreactors has had limited success because they do not generate physiological shear stress waveforms and EC lack cross-talk with smooth muscle and other vascular cell types. Because of these considerations, we have developed zebrafish embryo models to investigate vascular responses to flow. We recently used a zebrafish embryo model for functional screening of flow-responsive genes that regulate EC apoptosis, and found that shear stress regulation of EC proliferation was also conserved between murine and fish models (Serbanovic-Canic et al. 2017 ATVB;37:130-143 and Evans, unpublished). The proposed NC3Rs/BHF PhD Studentship will build on these observations by developing a zebrafish embryo model to identify genes that control EC proliferation under shear, thus enhancing the applicability and value of zebrafish models in studies of atherosclerosis.
Thus we hypothesize that zebrafish can be used to identify flow-responsive genes that regulate disease-promoting EC proliferation in mammalian arteries
The objectives of the study are:
(1) Establish a reporter system in zebrafish embryos for dynamic measurement of EC proliferative responses to flow.
We will use fluorescence ubiquitin cell cycle indicator (FUCCI) technology in which cell cycle stage is reported via the expression fluorescent fusion proteins (Bouldin CM and Kimelman D. Zebrafish. 2014;11:182-183). Thus cells in G0/G1 phase (expressing cherry-Cdt; red) and those in S/G2/M (Cerulean-Geminin; cyan) phase can be distinguished. Signal from EC will be identified by crossing reporter lines onto a fli1-EGFP (green) background. Flow will be reduced using genetic (e.g. troponin T morphant) or pharmacological (e.g. tricaine) approaches. The effects of flow cessation on EC proliferation will be quantified at varying time points using fucci reporters and compared to conventional measures of proliferation (Ki67, PCNA staining).
(2) Use the zebrafish model to perform functional screening.
A microarray study from our group identified 12 putative regulators of proliferation that were enriched at a low shear region in the porcine aorta (Serbananovic-Canic et al 2017 ATVB;37:130-143). Their function will be determined by silencing (using CRISPRi-knockdown; with Dr Rob Wilkinson, UoS; see letter) prior to measurement of EC proliferation as above.
(3) Validate screening studies using mouse arteries and human plaques.
We anticipate that functional screening will identify several genes that control EC proliferation under shear stress. The 5 genes that display the greatest influence on EC proliferation in zebrafish will be validated by: (i) measuring their expression in murine arteries exposed to low or high WSS by en face staining, (ii) quantifying expression in EC in human coronary artery plaques.