Disturbed Flow Induces Reprogramming of Endothelial Cells to Immune-like and Foam Cells under Hypercholesterolemia during Atherogenesis

Disturbed Flow Induces Reprogramming of Endothelial Cells to Immune-like and Foam Cells under Hypercholesterolemia during Atherogenesis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Disturbed Flow Induces Reprogramming of Endothelial Cells to Immune-like and Foam Cells under Hypercholesterolemia during Atherogenesis Hanjoong Jo, Christian Park, Kyung In Baek, Kiyoung Jeong, Paul Kim, and 16 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4397799/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Atherosclerosis 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. Methods Mice 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. Results Atherosclerotic 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 (MΦ), 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 MΦs. 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. Conclusion We 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. Biological sciences/Cell biology/Mechanisms of disease Biological sciences/Physiology/Cardiovascular biology/Cardiovascular diseases/Vascular diseases/Atherosclerosis Endothelial Reprogramming Endothelial-to-immune-cell transition Endothelial-to-foam cell transition Disturbed Flow Atherosclerosis Full Text Additional Declarations Yes there is potential Competing Interest. HJ is the founder of Flowkines Pharma. Supplementary Files NCVR2024050410Crs.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4397799","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":424984936,"identity":"8b58bcf3-aa54-436d-adf7-581eba890e97","order_by":0,"name":"Hanjoong 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Endothelial-to-foam cell transition, Disturbed Flow, Atherosclerosis","lastPublishedDoi":"10.21203/rs.3.rs-4397799/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4397799/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e Atherosclerosis 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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e Mice 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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults \u003c/strong\u003eAtherosclerotic 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 (MΦ), 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 MΦs. 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.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e We 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.\u0026nbsp;\u003c/p\u003e","manuscriptTitle":"Disturbed Flow Induces Reprogramming of Endothelial Cells to Immune-like and Foam Cells under Hypercholesterolemia during Atherogenesis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-07 03:13:52","doi":"10.21203/rs.3.rs-4397799/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"43482eb3-c37b-4086-b2a8-65e57c6a5bdd","owner":[],"postedDate":"March 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":45293330,"name":"Biological sciences/Cell biology/Mechanisms of disease"},{"id":45293331,"name":"Biological sciences/Physiology/Cardiovascular biology/Cardiovascular diseases/Vascular diseases/Atherosclerosis"}],"tags":[],"updatedAt":"2025-04-08T14:16:56+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-07 03:13:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4397799","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4397799","identity":"rs-4397799","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}