Disturbed flow induces reprogramming of endothelial cells to immune-like and foam cells under hypercholesterolaemia during atherogenesis.

aimsAtherosclerosis occurs preferentially in the arteries exposed to disturbed flow (d-flow), while the stable flow (s-flow) regions are protected even under hypercholesterolaemic conditions. We recently showed that d-flow alone initiates flow-induced reprogramming of endothelial cells (FIRE), including the novel concept of partial endothelial-to-immune-cell-like transition (partial EndIT), but it was not validated using a genetic lineage-tracing model. In addition, the combined effect of d-flow and hypercholesterolaemia has not been tested. Here, we tested and validated the two-hit hypothesis that d-flow is an initial instigator of partial FIRE but requires hypercholesterolaemia to induce a full-blown FIRE and atherosclerotic plaque development. METHODS AND

resultsMice were treated with AAV-PCSK9 and a Western diet to induce hypercholesterolaemia and/or partial carotid ligation (PCL) surgery to expose the left common carotid artery (LCA) to d-flow. Single-cell RNA sequencing (scRNA-seq) analysis was performed using single cells obtained from the LCAs and the control right common carotid arteries at 2 and 4 weeks post-PCL. Immunohistochemical staining was performed on EC-specific confetti mice at 4 weeks post-PCL and hypercholesterolaemia to validate endothelial reprogramming. Human aortic endothelial cells (HAECs) exposed to d-flow and hypercholesterolaemic conditions were used to validate FIRE. Atherosclerotic plaques developed by d-flow under hypercholesterolaemia, but not by d-flow or hypercholesterolaemia alone. The scRNA-seq results of 98 553 single cells from 95 mice revealed 25 cell clusters: 5 EC, 3 vascular smooth muscle cell (SMC), 5 macrophage (MΦ), and additional fibroblast, T cell, natural killer cell, dendritic cell, neutrophil, and B-cell clusters. Our scRNA-seq analysis results raised a hypothesis that d-flow under hypercholesterolaemia transitioned healthy ECs to full immune-like (EndIT) and, more surprisingly, foam-like cells (EndFT), in addition to inflammatory and mesenchymal cells (EndMT). Further, ECs with characteristics of foam cells shared remarkably similar transcriptomic profiles with foam cells derived from SMCs and MΦs. Lineage-tracing studies using immunohistochemical staining of canonical protein and lipid markers in the EC-specific confetti mice exposed to d-flow and hypercholesterolaemia demonstrated evidence supporting the novel FIRE hypothesis, including EndIT and EndFT. Moreover, reanalysis of the two publicly available human plaque scRNA-seq datasets and our immunostaining studies suggest that FIRE occurs in human atherosclerotic plaques. Additionally, HAECs exposed to d-flow, high cholesterol, and proinflammatory cytokines (identified in our scRNA-seq data) show the markers of EndIT and EndFT at the mRNA, protein, and functional levels.

conclusionThe scRNA-seq study raised a two-hit hypothesis for FIRE, including EndIT and EndFT, which was validated by the lineage-tracing and in vitro HAEC studies. D-flow induces partial reprogramming, including inflammation, EndMT, and partial EndIT. Under hypercholesterolaemia, d-flow fully reprogrammes arterial ECs, including the novel EndIT and EndFT, in addition to inflammation and EndMT, during atherogenesis. This single-cell atlas and FIRE programs provide a crucial roadmap for novel mechanistic understanding and therapeutics targeting flow-sensitive genes, proteins, and pathways of atherosclerosis.