Flow-induced reprogramming of endothelial cells in atherosclerosis

Abstract

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.

Fig. 1. Atherosclerosis preferentially develops at sites of disturbed flow.

a, Stages of atherosclerotic plaque development: LDL particles infiltrate into the subendothelial space in areas of endothelial dysfunction (1); oxidized LDL promotes inflammation (2); circulating monocytes (3) and vascular smooth muscle cells (VSMCs) from the media (4) migrate towards the region of inflammation; macrophages and VSMCs ingest oxidized LDL particles (5) and, eventually, transform into lipid-laden foam cells (6), contributing to the development of atherosclerotic plaques (7). b, The haemodynamic forces acting on the artery wall are blood pressure, circumferential stress and shear stress. The three layers of artery wall are the intima (which contains endothelial cells), the media (with VSMCs) and the adventitia (which contains fibroblasts). c, Common sites of atherosclerosis development, with the associated prevalence of plaques in middle-aged adults reported in the AWHS and PESA studies and the shear stress level average and ranges, based on available literature^,. d, Time-averaged shear stress levels in the left carotid bifurcation in a healthy individual show that the lateral wall of the internal carotid, a common site of atherosclerosis development, experiences low and oscillating shear stress from disturbed flow. Panel d adapted from ref. , Elsevier.

Fig. 2. Models of atherosclerosis induced by disturbed flow.

a, Schematic representation of the partial carotid artery ligation mouse model of atherosclerosis (left panel). The external carotid artery (ECA), occipital artery (OA) and internal carotid artery (ICA) are surgically ligated (black lines) to induce disturbed flow in the left carotid artery (LCA), which promotes atherosclerosis development in the LCA in hypercholesterolaemic conditions, such as in Apoe^−/− mice fed a high-fat diet (central panel)^, and mice with adeno-associated virus (AAV)-mediated PCSK9 overexpression fed a high-fat diet (right image, middle-right and bottom panels; shown by oil-Red-O staining). By contrast, even in hypercholesterolaemic conditions, the right carotid artery (RCA), which is exposed to stable flow, does not develop atherosclerosis (right image, middle-left panel). Mice without AAV-PCSK9-induced hypercholesterolaemia do not develop atherosclerotic plaques in the RCA or the ligated LCA (right image, top panels). b, Schematic representation of the shear-modifying constrictive cuff model. Implanting a constrictive cuff (white bracket) on the RCA shown in the magnetic resonance imaging angiogram (MRA) exposes endothelial cells to low-magnitude, unidirectional, laminar shear stress (ULS) in the proximal region of the cast, high-magnitude ULS within the cuff and low-magnitude oscillatory shear stress (OSS) in the distal region of the cuff. In hypercholesterolaemic conditions, such as in Apoe^−/− mice fed a Western diet for 8 weeks, the low-magnitude OSS induces atherosclerotic plaque (P) development with a large lipid core (black arrows) in the carotid artery, as shown by haematoxylin and eosin staining. The vessel lumen is indicated by an asterisk. c, Schematic representation of a cone-and-plate viscometer. d, Schematic representation of a parallel-plate flow chamber. Endothelial cells are exposed to differential shear stress with the use of a rotating Teflon cone in the cone-and-plate viscometer and with computer-generated hydrostatic pressure in the parallel-plate system. C1 and C2, cuffs; LSA, left subclavian artery; RSA, right subclavian artery; STA, superior thyroid artery. Panel a left drawing adapted from ref. , APS; central image adapted from ref. , CC0; and right images adapted from ref. , Elsevier. Panel b adapted from ref.  (Kuhlmann, M. T., Cuhlmann, S., Hoppe, I., Krams, R., Evans, P. C., Strijkers, G. J., Nicolay, K., Hermann, S., Schäfers, M. Implantation of a carotid cuff for triggering shear-stress induced atherosclerosis in mice. J. Vis. Exp. (59), e3308, 10.3791/3308 (2012)). Panel c adapted from ref. , Elsevier. Panel d adapted from ref. , Elsevier.

Fig. 3. Mechanosensors and mechanosignal transduction pathways in endothelial cells.

The apical surface of the endothelial cell contains protein mechanosensors, such as plexin D1, NOTCH1, PIEZO1, P2X4 and G protein-coupled receptors (such as GPR68), as well as mechanosensitive cell structures, such as caveolae, primary cilia and the glycocalyx. Cell–cell junctions contain the mechanosensory complex comprising VE-cadherin, platelet endothelial cell adhesion molecule (PECAM1), vascular endothelial growth factor receptor 2 (VEGFR2) and VEGFR3. The basal surface of endothelial cells contains integrin mechanosensors. Mechanosignal transduction pathways include the PI3K–AKT pathway, ERK1–ERK2 pathway, YAP–TAZ pathway and the RHO signalling pathway. Many mechanosignal transduction pathways result in activation of transcription factors including Krüppel-like factor 2 (KLF2) and KLF4, nuclear factor-κB (NF-κB) and hypoxia-inducible factor 1α (HIF1α). EndMT, endothelial-to-mesenchymal transition; eNOS, endothelial nitric oxide synthase; FAK, focal adhesion kinase; JAM, junctional adhesion molecule; NRF2, nuclear factor erythroid 2-related factor 2; ROS, reactive oxygen species; SOX13, transcription factor SOX13.

Fig. 4. Single-cell RNA sequencing reveals disturbed-flow-induced reprogramming of endothelial cells.

a, Disturbed flow stimulates the transition of healthy endothelial cells (ECs) to mesenchymal cells (EndMT; E8) and to an immune cell-like state (EndIT; E8), as determined by a pseudotime trajectory analysis of single-cell RNA sequencing datasets obtained from a mouse model of partial carotid artery ligation. The dots along the trajectory lines represent the status of the cells transitioning towards differentiated cell types. b, Disturbed flow induces epigenomic changes, such as chromatin remodelling, and transcriptomic changes that lead to pro-atherogenic gene expression patterns, which in turn induce flow-induced reprogramming of ECs (which we term as FIRE, an emerging concept that collectively refers to EndMT, EndIT and EC inflammation) and, eventually, atherosclerosis development. VSMC, vascular smooth muscle cell. Panel a adapted with permission from ref. , Elsevier.