BS21 Endothelial cell profile in responses to high shear stress is different in healthy arteries and plaques

ntroduction Shear stress is a parallel force generated by blood flow on the endothelial surface of blood vessel. It controls endothelial physiology and plaque biology. It is well established that low shear stress promotes the atherogenesis by increasing endothelial apoptosis, inflammation and vascular permeability. However, the role of shear stress in plaque growth and rupture is still controversial. It is hypothesized that endothelial cell (EC) responses to shear stress may be different in plaques compared to EC responses in healthy arteries. One interesting possibility is that plaque endothelium may have altered mechanosensing compared to healthy ECs. To test this hypothesis, we perform a transcriptome analysis of high shear stress endothelium in health versus diseased tissue.

Methods ApoE-/-mice were exposed to a high-fat diet for 16 weeks to induce atherosclerotic plaques or were given normal chow as a control. Wild-type (WT) mice (C57BL/6) were also studied. Aortic arches were optically cleared using the CUBIC protocol and eNOS was analysed in 3D by immunofluorescent staining coupled to light-sheet imaging (Zeiss Light-sheet Z.1). Shear stress maps were generated by OPT (optical projection tomography) imaging and computational fluid dynamics, registered against maps of eNOS expression using ITK snap software. Aortic ECs were analysed by single-cell RNA sequencing (scRNAseq) by enzymatic digestion, sorting of CD31+ CD45- cells and analysed using Bioturing software.

Results We established that eNOS is a high shear stress marker both in healthy and diseased aorta and used this to compare the transcriptional profiles of EC exposed to high shear stress in health and disease. We performed scRNAseq analysis of aorta from Apoe-/- normal diet (ND; intermediate cholesterol) mice and Apoe-/- high fat diet (HFD; high cholesterol) mice. eNOShigh cells were selected for transcriptome analysis (Figure 1A). We observed in t-distributed stochastic neighbour embedding (t-SNE) plot that eNOShigh cells in Apoe-/- HFD had a strikingly different transcriptional profile compared to eNOShigh cells from WT and Apoe-/- ND (Figure 1A). Some shear stress related genes were differently expressed between healthy arteries and plaques, including Klk10 which was enriched in high shear regions of healthy arteries and significantly reduced in plaque (Figure 1B).