Atherosclerotic diseases such as myocardial infarction, ischaemic stroke and peripheral artery disease continue to be leading causes of death worldwide despite the success of treatments with cholesterol-lowering drugs and drug-eluting stents, raising the need to identify additional therapeutic targets. Interestingly, atherosclerosis preferentially develops in curved and branching arterial regions, where endothelial cells are exposed to disturbed blood flow with characteristic low-magnitude oscillatory shear stress. By contrast, straight arterial regions exposed to stable flow, which is associated with high-magnitude, unidirectional shear stress, are relatively well protected from the disease through shear-dependent, atheroprotective endothelial cell responses. Flow potently regulates structural, functional, transcriptomic, epigenomic and metabolic changes in endothelial cells through mechanosensors and mechanosignal transduction pathways. A study using single-cell RNA sequencing and chromatin accessibility analysis in a mouse model of flow-induced atherosclerosis demonstrated that disturbed flow reprogrammes arterial endothelial cells in situ from healthy phenotypes to diseased ones characterized by endothelial inflammation, endothelial-to-mesenchymal transition, endothelial-to-immune cell-like transition and metabolic changes. In this Review, we discuss this emerging concept of disturbed-flow-induced reprogramming of endothelial cells (FIRE) as a potential pro-atherogenic mechanism. Defining the flow-induced mechanisms through which endothelial cells are reprogrammed to promote atherosclerosis is a crucial area of research that could lead to the identification of novel therapeutic targets to combat the high prevalence of atherosclerotic disease.

BACKGROUND: Atherosclerosis preferentially occurs in arterial regions of disturbed blood flow, and stable flow (s-flow) protects against atherosclerosis by incompletely understood mechanisms. METHODS: Our single-cell RNA-sequencing data using the mouse partial carotid ligation model was reanalyzed, which identified Heart-of-glass 1 (HEG1) as an s-flow–induced gene. HEG1 expression was studied by immunostaining, quantitive polymerase chain reaction, hybridization chain reaction, and Western blot in mouse arteries, human aortic endothelial cells (HAECs), and human coronary arteries. A small interfering RNA–mediated knockdown of HEG1 was used to study its function and signaling mechanisms in HAECs under various flow conditions using a cone-and-plate shear device. We generated endothelial-targeted, tamoxifen-inducible HEG1 knockout (HEG1 ^iECKO ) mice. To determine the role of HEG1 in atherosclerosis, HEG1 ^iECKO and littermate-control mice were injected with an adeno-associated virus–PCSK9 [proprotein convertase subtilisin/kexin type 9] and fed a Western diet to induce hypercholesterolemia either for 2 weeks with partial carotid ligation or 2 months without the surgery. RESULTS: S-flow induced HEG1 expression at the mRNA and protein levels in vivo and in vitro. S-flow stimulated HEG1 protein translocation to the downstream side of HAECs and release into the media, followed by increased messenger RNA and protein expression. HEG1 knockdown prevented s-flow–induced endothelial responses, including monocyte adhesion, permeability, and migration. Mechanistically, HEG1 knockdown prevented s-flow–induced KLF2/4 (Kruppel-like factor 2/4) expression by regulating its intracellular binding partner KRIT1 (Krev interaction trapped protein 1) and the MEKK3-MEK5-ERK5-MEF2 pathway in HAECs. Compared with littermate controls, HEG1 ^iECKO mice exposed to hypercholesterolemia for 2 weeks and partial carotid ligation developed advanced atherosclerotic plaques, featuring increased necrotic core area, thin-capped fibroatheroma, inflammation, and intraplaque hemorrhage. In a conventional Western diet model for 2 months, HEG1 ^iECKO mice also showed an exacerbated atherosclerosis development in the arterial tree in both sexes and the aortic sinus in males but not in females. Moreover, endothelial HEG1 expression was reduced in human coronary arteries with advanced atherosclerotic plaques. CONCLUSIONS: Our findings indicate that HEG1 is a novel mediator of atheroprotective endothelial responses to flow and a potential therapeutic target.

Microbubble-enhanced ultrasound provides a noninvasive physical method to locally overcome major obstacles to the accumulation of blood-borne therapeutics in the brain, posed by the blood-brain barrier (BBB). However, due to the highly nonlinear and coupled behavior of microbubble dynamics in brain vessels, the impact of microbubble resonant effects on BBB signaling and function remains undefined. Here, combined theoretical and prospective experimental investigations reveal that microbubble resonant effects in brain capillaries can control the enrichment of inflammatory pathways that are sensitive to wall shear stress and promote differential expression of a range of transcripts in the BBB, supporting the notion that microbubble dynamics exerted mechanical stress can be used to establish molecular, in addition to spatial, therapeutic windows to target brain diseases. Consistent with these findings, a robust increase in cytotoxic T-cell accumulation in brain tumors was observed, demonstrating the functional relevance and potential clinical significance of the observed immuno-mechano-biological responses.

Background: Calcific aortic valve disease (CAVD) is a highly prevalent disease, especially in the elderly population, but there are no effective drug therapies other than aortic valve repair or replacement. CAVD develops preferentially on the fibrosa side, while the ventricularis side remains relatively spared through unknown mechanisms. We hypothesized that the fibrosa is prone to the disease due to side-dependent differences in transcriptomic patterns and cell phenotypes. Methods: To test this hypothesis, we performed single-cell RNA sequencing using a new method to collect endothelial-enriched samples independently from the fibrosa and ventricularis sides of freshly obtained human aortic valve leaflets from five donors, ranging from non-diseased to fibrocalcific stages. Results: From the 82,356 aortic valve cells analyzed, we found 27 cell clusters, including seven valvular endothelial cell (VEC), nine valvular interstitial cell (VIC), and seven immune, three transitional, and one stromal cell population. We identified several side-dependent VEC subtypes with unique gene expression patterns. Homeostatic VIC clusters were abundant in non-diseased tissues, while VICs enriched with fibrocalcific genes and pathways were more prevalent in diseased leaflets. Furthermore, homeostatic macrophage (MΦ) clusters decreased while inflammatory MΦ and T-cell clusters increased with disease progression. A foamy MΦ cluster was increased in the fibrosa of mildly diseased tissues. Some side-dependent VEC clusters represented non-diseased, protective phenotypes, while others were CAVD-associated and were characterized by genes enriched in pathways of inflammation, endothelial–mesenchymal transition, apoptosis, proliferation, and fibrosis. Interestingly, we found several activator protein-1 (AP-1)-related transcription factors (FOSB, FOS, JUN, JUNB) and EGR1 to be upregulated in the fibrosa and diseased aortic valve leaflets. Conclusions: Our results showed that VECs are highly heterogeneous in a side- and CAVD-dependent manner. Unique VEC clusters and their differentially regulated genes and pathways found in the fibrosa of diseased tissues may represent novel pathogenic mechanisms and potential therapeutic targets.

Aims: Atherosclerosis 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 results: Mice 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. Conclusion: The 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.