EC proliferation is significantly reduced in embryos without blood flow. Transgenic Tg(fli1a:LifeAct-mClover; fli1a:nls-mCherry) embryos were analysed. The ISVs of four embryos with flow, five tnnt2a-MO (No-Flow), and six tricaine-treated (No-Flow) embryos were imaged from 54 to 70 hpf. For each embryo, data were pooled from 7 ISVs. (A) Representative images showing a dividing EC (arrows) in an ISV exposed to flow. (B) The rate of EC proliferation in the ISVs was quantified as a percentage of nuclei dividing per hour. Mean values + /− standard deviations are shown. Differences between means were analysed using an one-way ANOVA with multiple comparisons.

Sorting of EC from fli1a:EGFP; gata1adsRed zebrafish. Approximately 40 Tg(fli1a:EGFP; gata1a:dsRed) embryos at 48 hpf were dissociated into single cells using trypsin. Using FACS, cells were gated using forward scatter (FSC) and side scatter (SSC) and then GFP + and GFP-single cells were sorted. N = 3 independent experiments were performed. (A) Typical flow cytometry data showing FSC and SSC profiles (upper panel; gating indicated as red box) and delineation of GFP + and GFP- single cells (lower panel; gating indicated as red boxes). (B) cdh5 expression was quantified in GFP + cells by qRT-PCR using actin as a control for total RNA levels. Mean 1/dCt values and individual data points are shown. Differences between means were analysed using a t-test. cdh5 expression was enriched in GFP + cells. (C) Quantification of expression of genes of interest in zebrafish endothelium. GFP + cells were analyzed by qRT-PCR. Expression of genes of interest was quantified relative to the expression of actin. Mean values + /− standard deviations and individual data points are shown. Genes which were expressed at detectable levels in one or more experiment were selected for study.

Knock-down of candidate genes does not affect morphology or blood flow in the ISVs. Zebrafish embryos were injected with a gene-specific or non-targeting control MO. (A) Morphology of Tg(fli1a:LifeAct-mClover) embryos was observed during development and is shown here at 54 hpf. Lateral view, anterior to the left, dorsal up. Scale bar: 100 μm. (B) Blood flow was quantified in the ISVs of Tg(gata1a:dsRed) morphants at 72 hpf. n = 5 embryos per group.Mean values were compared using a Kruskal–Wallis test, none of the morphants had significantly different blood flow to the control.

Knock-down of candidate genes influenced EC proliferation in ISVs with and without blood flow. Embryos expressing Tg(fli1a:nls-mCherry) with a gene-specific or non-targeting control MO. Some embryos were co-injected with tnnt2a-MO to prevent blood flow (no flow), whereas controls did not receive tnnt2a-MO (flow). (A) Representative images showing a dividing EC (arrows) in a wnk1a MO-treated embryo under no flow conditions. (B) Proliferation was quantified from 54 to 70 hpf in a minimum of eight embryos per condition. The rate of EC proliferation in the ISVs was quantified as a percentage of nuclei dividing per hour. Data are presented as violin plots. Differences between means were analysed by two-way ANOVA. For the no-flow condition, only significant differences are shown, with non-significant differences omitted. In the flow condition, no statistically significant differences were detected.

WNK1 is enriched at a low WSS region of the mouse endothelium. WNK1 was analysed by en face staining of the murine aorta using anti-WNK1 antibodies and Alexafluor568-conjugated secondary antibodies (red) (n = 3 mice). EC were co-stained using anti-CDH5 (green) and nuclei were detected with TO-PRO-3 (blue). WNK1 expression was quantified in high and low WSS regions. Mean values + /− standard error of mean are shown. Differences between means were analysed using a paired t-test.

WNK1 was expressed in the endothelium of human atherosclerotic plaques. Endothelial WNK1 expression was determined by immunofluorescent staining of sections of human carotid artery plaques (symptomatic (N = 5); asymptomatic (N = 3) with anti-WNK1 (red) and anti-VWF antibodies (green) to identify EC. Nuclei were counterstained using DAPI (blue). Staining was analysed by fluorescence microscopy and the proportion of WNK1-positive endothelial cells calculated.Representative images are shown. WNK1—positive EC are indicated with arrows. Differences between means were calculated using an unpaired t-test.

Schematic summary.