Single-cell RNA-seq reveals cellular heterogeneity of mouse carotid artery under disturbed flow

Abstract

Disturbed blood flow (d-flow) has been known to induce changes of the cells in the arterial wall, increasing the risk of atherosclerosis. However, the heterogeneity of the vascular cell populations under d-flow remains less understood. To generate d-flow in vivo, partial carotid artery ligation (PCL) was performed. Seven days after ligation, single-cell RNA sequencing of nine left carotid arteries (LCA) from the PCL group (10,262 cells) or control group (14,580 cells) was applied and a single-cell atlas of gene expression was constructed. The integrated analysis identified 15 distinct carotid cell clusters, including 10 d-flow-relevant subpopulations. Among endothelial cells, at least four subpopulations were identified, including Klk8hi ECs, Lrp1hi ECs, Dkk2hi ECs, and Cd36hi ECs. Analysis of GSVA and single-cell trajectories indicated that the previously undescribed Dkk2hi ECs subpopulation was mechanosensitive and potentially transformed from Klk8hi ECs under d-flow. D-flow-induced Spp1hi VSMCs subpopulation that appeared to be endowed with osteoblast differentiation, suggesting a role in arterial stiffness. Among the infiltrating cell subpopulations, Trem2hi Mφ, Birc5hi Mφ, DCs, CD4+ T cells, CXCR6+ T cells, NK cells, and granulocytes were identified under d-flow. Of note, the novel Birc5hi Mφ was identified as a potential contributor to the accumulation of macrophages in atherosclerosis. Finally, Dkk2hi ECs, and Cd36hi ECs were also found in the proatherosclerotic area of the aorta where the d-flow occurs. In conclusion, we presented a comprehensive single-cell atlas of all cells in the carotid artery under d-flow, identified previously unrecognized cell subpopulations and their gene expression signatures, and suggested their specialized functions.

Next-Generation and Single-Cell Sequencing Approaches to Study Atherosclerosis and Vascular Inflammation Pathophysiology: A Systematic Review

“…Two articles ( 43 , 44 ) specifically explore the cellular and molecular constituents of cardiac tissue using single-cell sequencing, with the first ( 43 ) investigating the heterogeneity of aortic tissue in healthy vs. disease states, and the second ( 44 ) addressing the impact of hemodynamic factors, specifically disturbed flow, on the composition of carotid arteries.…”

“…Li et al ( 44 ) addressed the effects of disturbed blood flow at the single-cell level using mice which underwent partial carotid artery ligation. Clustering analysis showed ten distinct cell clusters specific to disturbed flow compared to control: two EC clusters, one SMC, three macrophage, and four immune cell clusters.…”

The Applications of Single-Cell RNA Sequencing in Atherosclerotic Disease

“…A disturbed flow EC population was found to be more enriched in processes such as epithelial to mesenchymal transition and TGF-β signaling. Disturbed flow-specific SMC and Mφs were also reported (44). Current studies rarely take into account the influence of flow on the outcome of scRNA-seq experiments.…”

“…Endothelial cells can become mesenchymal cells by a process called EndoMT, and SMCs can undergo phenotypic modulation and differentiate into Mϕ-like, fibroblast-like, osteoblast-like, or chondrocyte-like cells. EndoMT can occur under the influence of disturbed blood flow ( 43 , 44 ) and strongly affect the number of SMCs that stabilize a fibrous cap ( 45 ). However, the role and existence of EndoMT in disease is not without controversy ( 46 ).…”

“…Similarly, Li et al ( 44 ) performed scRNA-seq on mice lesions after partial carotid ligation. Compared to the laminar flow condition, they found cell populations specific for both models.…”