bioRxiv2025
BackgroundAtherosclerosis occurs preferentially in the arteries exposed to disturbed flow (d-flow), while the stable flow (s-flow) regions are protected even under hypercholesterolemic 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 was not validated using a genetic lineage-tracing model. Here, we tested and validated the two-hit hypothesis that d-flow is an initial instigator of partial FIRE but requires hypercholesterolemia to induce a full-blown FIRE and atherosclerotic plaque development. MethodsMice were treated with adeno-associated virus expressing proprotein convertase subtilisin/kexin type 9 and a Western diet to induce hypercholesterolemia 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 cells obtained from the intima and leftover LCAs and the control right common carotid arteries at 2 and 4 weeks post-PCL. Comprehensive immunohistochemical staining was performed on EC-specific confetti mice treated with PCL and hypercholesterolemic conditions at 4 weeks post-PCL to validate endothelial reprogramming. ResultsAtherosclerotic plaques developed by d-flow under hypercholesterolemia at 2 and 4 weeks post-PCL, but not by d-flow or hypercholesterolemia alone, as expected. 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 (M{Phi}), and additional fibroblast, T cell, natural killer cell, dendritic cell, neutrophil, and B cell clusters. Our scRNA-seq analyses showed that d-flow under hypercholesterolemia transitioned healthy ECs to full immune-like (EndIT) and, more surprisingly, foam cells (EndFT), in addition to inflammatory and mesenchymal cells (EndMT). Further, EC-derived foam cells shared remarkably similar transcriptomic profiles with foam cells derived from SMCs and M{Phi}s. Comprehensive lineage-tracing studies using immunohistochemical staining of canonical protein and lipid markers in the EC-specific confetti mice clearly demonstrated direct evidence supporting the novel FIRE hypothesis, including EndIT and EndFT, when d-flow was combined with hypercholesterolemia. Further, reanalysis of the publicly available human carotid plaque scRNA-seq and Perturb-seq datasets supported the FIRE hypothesis and a potential mechanistic link between the genes and FIRE. ConclusionWe provide evidence supporting the two-hit hypothesis: ECs in d-flow regions, such as the branching points, are partially reprogrammed, while hypercholesterolemia alone has minimal endothelial reprogramming effects. Under hypercholesterolemia, d-flow fully reprograms arterial ECs, including the novel EndIT and EndFT, in addition to inflammation and EndMT, during atherogenesis. This single-cell atlas provides a crucial roadmap for developing novel mechanistic understanding and therapeutics targeting flow-sensitive genes, proteins, and pathways of atherosclerosis.