S1PR1 improves Cardiac Repair by Promoting monocyte/ macrophage infiltration and aggregation via ERK Signaling after myocardial infarction

S1PR1 improves Cardiac Repair by Promoting monocyte/ macrophage infiltration and aggregation via ERK Signaling after myocardial infarction | 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 S1PR1 improves Cardiac Repair by Promoting monocyte/ macrophage infiltration and aggregation via ERK Signaling after myocardial infarction JunpingOu #, ZhongwenLiu #, Qingyi Wang, Yuping Wang, Jie Zeng, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7135671/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 This study aimed investigate the impact and mechanism of S1PR1 on monocyte/macrophage function following myocardial Infarction. Methods We performed cells profiling of Circulating, liver, kidney, and cardiac immune cells in mice challenged with AMI, and the monocyte/macrophage landscape of the infarcted heart were quantified via flow cytometry at 0, 5, and 28 days post-MI. Fluorescence microscopy evaluated monocyte/macrophage fluorescence expression in cardiac tissue. Lentiviral vectors (pCMV.DR8 and pMD2.G) were constructed to infect RAW264.7 macrophages, with optimal multiplicity of infection (MOI) determined. Experimental groups included S1PR1-knockdown, S1PR1-overexpression, U0126 (ERK pathway inhibitor)-treated, and blank control groups. Transwell assays assessed cell migration, while RAW264.7-HUVEC co-cultures evaluated adhesion. Results The experimental group exhibited a significantly higher mononuclear/macrophage count in myocardial tissue compared to controls (P < 0.05). In vitro experiments demonstrated that SEW2871 enhanced both adhesion and migration capacities of RAW264.7 macrophages. These effects were significantly attenuated following S1PR1 gene knockdown. Furthermore, U0126 pretreatment substantially diminished SEW2871-mediated promotion of cellular adhesion and migration. Conclusion S1PR1 promotes monocyte/macrophage adhesion and migration via ERK signaling pathway and thus ameliorates post-MI cardiac repair. Health sciences/Cardiology Health sciences/Diseases Myocardial infarction Sphingosine 1-phosphate receptor 1 Monocytes/macrophages Cardiac repair Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Myocardial infarction (MI), a critical cardiovascular emergency, represents a dynamic continuum encompassing both tissue injury and repair mechanisms [ 1 , 2 ] . During the initial phase, ischemic cardiomyocyte necrosis triggers pronounced inflammatory responses. Subsequently, mononuclear/macrophage populations undergo chemotactic infiltration into myocardial tissue, facilitating phagocytic clearance of necrotic/apoptotic cellular debris and extracellular matrix fragments. This coordinated process promotes neovascularization and fibrous scar formation essential for myocardial restoration [ 2 – 4 ] . Current investigations have demonstrated that targeted modulation of monocyte chemotactic activity or macrophage homeostasis within infarct territories can significantly enhance post-infarction cardiac repair outcomes [ 5 ] . Sphingosine 1-phosphate (S1P), a bioactive lipid mediator, exerts extracellular effects through G protein-coupled receptors (GPCRs) [ 6 ] . Five S1P receptor subtypes (S1PR1-S1PR5) are widely distributed across multiple tissues and cell types, including immune, cardiovascular, respiratory, hepatic, and nervous systems [ 7 – 9 ] . Experimental evidence indicates that S1PR1 facilitates immune cell adhesion to vascular endothelium and subsequent migration to injured tissues [ 10 , 11 ] . This receptor also promotes post-MI angiogenesis, thereby ameliorating cardiac remodeling [ 12 ] . As an intracellular messenger, S1PR1 activates extracellular regulated protein kinases (ERK) [ 13 ] . Phosphorylated ERK translocates from cytoplasm to nucleus, initiating transcriptional activation of Elk-1 and NF-κB [ 14 ] . These molecular events regulate critical cellular processes including proliferation, migration, and apoptosis [ 15 , 16 ] . However, the regulatory role of S1PR1 in monocyte/macrophage functionality during post-MI myocardial repair remains uncharacterized. This study investigated S1PR1-mediated mechanisms in monocyte/macrophage regulation using murine MI models induced by left anterior descending coronary artery ligation, combining in vivo and in vitro experimental approaches. 2. Materials and Methods 2.1 Experimental Animals This study approved by the Medical Ethics Committee of Jinggangshan University.The study protocol was in accordance with The ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments) 2.0. Specific pathogen-free (SPF) female C57BL/6 mice (aged 8–12 weeks, body weight 20–25 g were obtained from Shanghai SLAC Laboratory Animal Co., Ltd. The RAW264.7 macrophage cell line, HUVEC endothelial cell line, and 293T cells were kindly provided by the Oriental Translational Medicine Research Center of Tongji University and the Shanghai Heart Failure Institute. Primary murine monocytes/ macrophages were isolated from peripheral blood of experimental mice, while cardiac endothelial cells were purified via magnetic bead sorting. 2.2Reagents The S1PR1 agonist SEW2871 was purchased from Cayman Chemical. DMEM medium, fetal bovine serum (FBS), and trypsin were acquired from Gibco. RPMI-1640 medium was sourced from Hyclone, and Percoll cell separation solution from GE Healthcare. TRIzol reagent and PCR kits were obtained from Qiagen. Sodium pentobarbital anesthetic, collagenase I, and collagenase XI were procured from Sigma-Aldrich. Transwell chambers were supplied by Corning. S1PR1 gene-silencing and overexpression plasmids were custom-designed by Gima Biotechnology (Shanghai) Co., Ltd. 2.3 Animals and myocardial infarction model This study involving animals and experimental protocol was approved by the Animal Ethics Committee of Jinggangshan University. All experiments of the study were conducted in accordance with relevant guidelines and regulations. The mice aged 8 to 10-week old were housed at 24°C ± 2°C, humidity of 40% ± 5%,. Acute MI models were surgically induced via left anterior descending coronary artery ligation, following established protocols [ 13 ] . The blind method was used for the operation and subsequent echocardiographic evaluation in this study. Mice were euthanized with isoflurane/oxygen and sacrificed on the corresponding days post-MI surgery to obtain their heart samples for corresponding analyses. The experimental group received daily intraperitoneal injections of the S1PR1 agonist SEW2871 (5 mg/kg,Cayman Chemical) until euthanasia, while controls received equivalent volumes of DMSO. 2.4 Echocardiography Cardiac function of mice was evaluated by echocardiography with a high-resolution imaging system (Vevo2100, Visual Sonic Inc., CA) 0, 5, and 28 days after MI In vivo [ 17 , 18 ] .The mice were anesthetized using a mixture of isoflurane and oxygen followed by removal of chest hair and placed in the supine position. The two-dimensional and M-mode echocardiographic views of the mid‐ventricular short axis were acquired at the level of the papillary muscle tips below the mitral valve.Variables including left ventricular internal diameter at the end of diastolic (LVIDd) and systolic (LVIDs) were measured on M‐mode echocardiography. Left ventricular ejection fraction (LVEF), fractional shortening (LVFS) and left ventricular end-diastolic volume (LVEDV) and end-systolic volume (LVESV) were calculated as reported previously [ 18 ] . 2.5 Immunohistochemical Staining Protocol To assess the infarct size, the Immunohistochemical Staining assays were performed as previously described in detail [ 19 ] . In brief, the heart was harvested and fixed in 4% paraformaldehyde (24h).The 5µm -thick paraffin-embedded sections were stained with haematoxylin and eosin (H&E) and Masson’s trichrome. ImageJ software (1.52 V) was used to determine the infarcted area and cardiac fibrosis. Immunostaining was performed on cryostat 8 lm-thick sections by using various antibodies, including antisarcomeric alpha-actinin (Abcam, USA), Anti-F4/80 (Abcam, USA), Anti-Wheat Germ Agglutinin (WGA)-alexa488(Invitrogen, USA), Biotinylated-isolectin B4 antibody (IB4,Vector Laboratories, B-1205, USA) and their corresponding secondary antibodies. Nuclei were stained with 40,6-Diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma-Aldrich, Germany). To evaluate cardiac hypertrophy, we stained heart section with WGA and calculated the average cardiomyocyte cross-sectional area in 10 randomly selected fields of each slide from 5 serial cardiac sections with 500 lm interval distances. 2.6 Single-Cell Suspension Preparation and Flow Cytometry Mice were anesthetized with sodium pentobarbital (50 mg/kg). Peripheral blood, cardiac tissue, bone marrow, liver, and spleen were harvested, homogenized in PBS, and centrifuged at 1000 ×g for 5 min to obtain single-cell suspensions. For temporal analysis of monocyte/macrophage dynamics post-MI, tissues were collected at 0, 5, 7, and 21 days post-infarction. Cell suspensions were stained with anti-CD11b, -F4/80, and -Ly6C antibodies for flow cytometric quantification of monocyte subsets (Ly6Chi/Ly6Clow) and macrophages using FlowJo software. 2.7 Cell Culture and Transfection RAW264.7 macrophages were maintained in RPMI-1640 medium supplemented with 10% FBS, while HUVECs were cultured in EGM2 medium (10% FBS) under 5% CO 2 at 37℃. At 70% confluence, cells were transfected with S1PR1-targeting shRNA, overexpression plasmids, or empty vectors using standard protocols. Transfection efficiency was validated by Western blotting 48 h post-transfection. 2.8 Transwell Migration Assay S1PR1-modulated RAW264.7 migration was assessed using Transwell chambers. Cells were serum-starved overnight in RPMI-1640 with 1% FBS, then seeded in the upper chamber (1×10 5 cells/well in 150µl 1% FBS medium). The lower chamber contained 700µl ~ 800µl 10% FBS medium. After 4 hours incubation, migrated cells were fixed, stained with crystal violet, and quantified microscopically. Triplicate wells were used across three independent experiments. 2.9 Co-Culture Adhesion Assay RAW264.7-HUVEC co-cultures were divided into four groups: control, S1PR1-overexpression, S1PR1-silenced, and S1PR1-overexpression + U0126 (50µmol /L ERK inhibitor). Cells were pretreated with 5 µmol/L S1P for 6 h (with U0126 co-treatment in the inhibitor group). PKH26-labeled RAW264.7 adhesion rates were quantified 30 minutes post-co-culture using fluorescence microscopy. Triplicate measurements were performed in three experimental repeats. 2.10 Statistical Analysis Experimental data were analyzed using SPSS 19.0 (IBM) and visualized with GraphPad Prism 9.0. Intergroup differences were assessed via independent Student’s t-test for two-group comparisons and one-way ANOVA for multi-group analyses (≥ 3 groups). Flow cytometry data were processed using FlowJo v10.8. Results are expressed as mean ± SEM, with statistical significance defined at p < 0.05. 3. Results 3.1 S1PR1 Promotes Monocyte/Macrophage Accumulation in Infarcted Myocardium Inflammatory cells isolated via Percoll gradient centrifugation were analyzed for monocyte/macrophage subpopulations using flow cytometry (Figure 1A). At day 5 post-MI, SEW2871-treated mice exhibited significantly higher counts of CD11b + cells (Figure 1B), Ly6c Low monocytes, and Ly6c high /Ly6c Low macrophages in infarcted myocardium compared to WT controls (Figure 1C). Immunofluorescence staining further revealed increased F4/80 + cells per field in SEW2871-treated mice at 4 weeks post-MI ( p < 0.05), though CD11b + cell counts showed no intergroup difference (Figure 1D–F). 3.2 S1PR1 Enhances Peripheral Blood Monocyte Chemotaxis to Inflamed Tissue FACS analysis demonstrated markedly reduced total monocytes and subtype counts in p < 0.05,peripheral blood of SEW2871-treated mice at day 5 post-MI ( p < 0.05, Figure 2A–C), while liver, spleen, and bone marrow exhibited no significant intergroup differences. 3.3 S1PR1 Facilitates Monocyte Migration Transwell chamber assays revealed a significant increase in migratory cells in the S1PR1-overexpression group versus controls ( p < 0.05, Figure 3A–B). ERK inhibition with U0126 substantially attenuated S1PR1-driven migration, achieving statistical significance compared to controls ( p < 0.05). 3. 4 S1PR1 Augments Monocyte Adhesion In co-cultures of HUVECs and RAW264.7 macrophages, S1PR1-overexpression significantly enhanced adhesion rates versus controls ( p < 0.05). Conversely, U0126 treatment markedly reduced adhesion in the S1PR1-overexpression group ( p < 0.05, Figure 4A–B). 3.5 S1PR1 Upregulates Endothelial Adhesion Molecules and Chemokines RT-qPCR analysis showed significantly elevated E-selectin expression in S1PR1-overexpressing endothelial cells compared to control and knockdown groups ( p < 0.001). SEW2871-treated endothelial cells also exhibited upregulated MCP-1 expression versus controls ( p < 0.05, Figure 5A–B). 4. Discussion Our study demonstrates that: (1) Following myocardial infarction, S1PR1 facilitates monocyte/macrophage infiltration and recruitment within infarcted cardiac tissue. (2) S1PR1 enhances monocyte/ macrophage adhesion and migration. (3) The ERK signaling pathway inhibitor U0126 abolishes S1PR1-induced enhancement of monocyte/macrophage adhesion and migration. These findings advance our understanding of post-MI cardiac repair and potential therapeutic strategies. Previous studies have confirmed that monocytes/macrophages play a pivotal role in myocardial repair following myocardial infarction [20–22] . During the intermediate and late stages of infarction, macrophage accumulation increases in the infarcted myocardium. The elevated population of reparative Ly6C low macrophages stimulates the expression of factors such as TGF-β and VEGF, thereby promoting angiogenesis [23, 24] . Furthermore, prior research indicates that macrophages phagocytose necrotic cardiomyocyte debris and apoptotic immune cells to facilitate cellular regeneration and tissue remodeling in the infarcted area [25] . Our results demonstrate that monocyte/ macrophage infiltration and recruitment in the infarcted myocardium begins to increase on day 3 post-myocardial infarction and peaks on day 5. Notably, the increase in Ly6C low macrophages is particularly pronounced, consistent with previous reports. These findings suggest the initiation of the myocardial repair phase at this stage. Under normal conditions, monocytes/macrophages in myocardial tissue primarily originate from local proliferation. However, under stress conditions such as inflammation, extracardiac sources become predominant [26] . To determine the origin of monocytes/macrophages infiltrating and aggregating in infarcted myocardium and the potential role of S1PR1, we collected tissues including peripheral blood, liver, spleen, and bone marrow from post-myocardial infarction mice and prepared single-cell suspensions. Flow cytometry analysis revealed that, compared with the control group, the S1PR1 agonist group exhibited significantly reduced monocyte counts in peripheral blood, with no significant differences observed in the liver, spleen, or bone marrow. This indicates that S1PR1 primarily promotes chemotaxis, migration, and aggregation of peripheral blood monocytes toward the infarcted area. During inflammation, various cell adhesion molecules, including vascular cell adhesion molecule-1 (VCAM-1) and E-Selectin, play critical roles in inflammatory cell migration [27] . After adhering to vascular endothelial cells, monocytes subsequently transmigrate across the endothelium and migrate toward inflamed tissues under chemokine gradients. Studies reported that S1PR1 upregulates E-selectin expression and enhancing monocyte adhesion by inducing phosphorylation and nuclear translocation of the NF-κB subunit p65 [28, 29] . Both our in vivo and in vitro experiments confirmed that S1PR1 increases E-selectin mRNA levels, an effect inhibited by the ERK pathway inhibitor U0126. This indicates that S1PR1 regulates E-selectin expression via the ERK signaling pathway. As E-selectin is a key adhesion molecule on vascular endothelial cells that mediates leukocyte adhesion, this mechanism partially explains S1PR1's role in promoting monocyte/macrophage adhesion. Furthermore, S1PR1 has been shown to increase MCP-1 expression, thereby enhancing monocyte chemotaxis and adhesion to endothelial cells [30] . Consistent with these reports, our study demonstrated elevated MCP-1 expression in the infarcted myocardium of the experimental group. This suggests that S1PR1 upregulates MCP-1, a major monocyte chemoattractant, thus enhancing monocyte/macrophage migration toward ischemic myocardial tissue. As a component of the MAPK signaling pathway, ERK (extracellular signal-regulated kinase) activation is crucial for transducing signals from cell surface receptors to the nucleus. It plays an essential role in regulating cellular functions such as metabolism, migration, and proliferation [31, 32] . In our study, pretreatment with the ERK pathway inhibitor U0126 significantly attenuated SEW2871-induced monocyte/macrophage adhesion and migration. This confirms the critical involvement of ERK signaling in regulating monocyte/macrophage adhesion and migration. Taken together, our studies demonstrate that S1PR1 agonists play a critical role in the inflammatory response and myocardial repair process following myocardial infarction. We also investigated the role and mechanism of S1PR1 in promoting monocyte/macrophage migration post-myocardial infarction. These investigations revealed that S1PR1 upregulates the expression of E-Selectin and MCP-1 by modulating the ERK signaling pathway. This facilitates the migration and aggregation of peripheral blood monocytes into the infarcted area, thereby increasing monocyte/macrophage accumulation in the infarcted myocardium and promoting cardiac repair. Declarations Authors’ contributions Xiaolin Li put forward the concept and design. Junping Ou made the definition of intellectual content and literature search., Zhongwen Liu made data acquisition, Yuping Wang made data and statistical analysis. Jie Zeng made the manuscript preparation, editing and review. Qingyi Wang quantify experimental data. Funding : This study has been supported by Jiangxi Provincial Natural Science Foundation(20224BAB206014), and the Science and Technology Plan Project of the Jiangxi Provincial Health Commission (202312372；202312384). Data availability statement The data collected in the study is available from the corresponding author on reasonable request, E-mail: [email protected] :+86-13979609896. References Jiahui, Zhang,Yishan, Guo,Yu, Bai et al. 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Park Jong-In, MAPK-ERK Pathway.[J] .Int J Mol Sci, 2023, 24(11):1-3. Additional Declarations No competing interests reported. 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-7135671","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":493413677,"identity":"e7f54ca3-c7b8-4ac8-b6af-d93b15c8498a","order_by":0,"name":"JunpingOu #","email":"","orcid":"","institution":"Ji’an People’s Hospital Affiliated to Jinggangshan University","correspondingAuthor":false,"prefix":"","firstName":"JunpingOu","middleName":"","lastName":"#","suffix":""},{"id":493413678,"identity":"92bfe202-591b-41ea-bcac-8ed478a30fe9","order_by":1,"name":"ZhongwenLiu #","email":"","orcid":"","institution":"Ji’an People’s Hospital Affiliated to Jinggangshan University","correspondingAuthor":false,"prefix":"","firstName":"ZhongwenLiu","middleName":"","lastName":"#","suffix":""},{"id":493413679,"identity":"873e77af-ecbb-4e14-b983-3139a8e04721","order_by":2,"name":"Qingyi Wang","email":"","orcid":"","institution":"Jiangxi Province Key Laboratory of Organ Development and Epigenetics","correspondingAuthor":false,"prefix":"","firstName":"Qingyi","middleName":"","lastName":"Wang","suffix":""},{"id":493413680,"identity":"38f8de20-5a60-456a-89e3-d9caff6b61d4","order_by":3,"name":"Yuping Wang","email":"","orcid":"","institution":"Jiangxi Province Key Laboratory of Organ Development and Epigenetics","correspondingAuthor":false,"prefix":"","firstName":"Yuping","middleName":"","lastName":"Wang","suffix":""},{"id":493413683,"identity":"6867bad3-5af1-4a78-b0e1-b1b76a506297","order_by":4,"name":"Jie Zeng","email":"","orcid":"","institution":"Jiangxi Province Key Laboratory of Organ Development and Epigenetics","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Zeng","suffix":""},{"id":493413684,"identity":"03665cd0-64ea-413d-b896-e57b3df0a3b4","order_by":5,"name":"Xiaolin Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7UlEQVRIie3PMYoCMRSA4SeBTJNx2jeseIZIYFRY2KsogpUHmELcLIKVYGshnsHKOiFglQPYSXbbLbSzUHDUVibaWeQjkBT5SR5AELyhKgFQAC0G0Y9U+xzriS+h9wQZMKPd3LZEKn3JfcNi9XsinuTdlTeJWEMdc6w1YZBhvMTKCoj73ZZ+jHb01CJrS5thukbSBCrEoDQhSsUTZFxPM2yskbYlox/lSUXq8zUxLMPuojgob0KUub2yoT2uJeITCe2YWjELt0Q7uUGejj2zJIkVh//884vvnDSn4eh7Fo3dX1nyAHntehAEQfDABdrqRSfGqry5AAAAAElFTkSuQmCC","orcid":"","institution":"Ji’an People’s Hospital Affiliated to Jinggangshan University","correspondingAuthor":true,"prefix":"","firstName":"Xiaolin","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2025-07-16 04:38:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7135671/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7135671/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":88253640,"identity":"9b58a6e5-11dc-4c79-b4dd-b6bf47ab05f4","added_by":"auto","created_at":"2025-08-04 14:01:38","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":71082,"visible":true,"origin":"","legend":"\u003cp\u003eSEW2871 promotes monocyte/macrophage aggregation in the infarcted myocardium. Myocardial infarction(MI) mice were treated with SEW2871（5 mg/kg/day）, control group (DMSO, 50μl/200μl PBS) until the mice were euthanized. Number of monocytes/macrophages and subtypes by fluorescence activated cell sorting assay(A). Comparison of the number of monocytes/macrophages in myocardial tissue, 5 days after MI(B). Comparison of the number of cell subtypes in myocardial tissue, 5 days after MI(C). Fluorescence staining of myocardial tissue by CD11b and F4/80 (400×) (D). Comparison of the number of monocytes and macrophages in myocardial tissue by Fluorescence staining at 4 weeks after MI(E,F). Scale bar, 20 µm. Mean ± SEM, n = 5, *P \u0026lt; 0.05 vs control group. Scale bar, 50 µm.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7135671/v1/239f0fa942226f07463f3364.jpg"},{"id":88253629,"identity":"924500c1-bc00-491e-b705-1e8121af9211","added_by":"auto","created_at":"2025-08-04 14:01:38","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":44052,"visible":true,"origin":"","legend":"\u003cp\u003eSEW2871 \u0026nbsp;promotes the chemotaxis of monocyte to inflammatory tissues in peripheral blood. MI mice were treated as described in Fig. 1. The number of cells were measured by fluorescence activated cell sorting assay. Mean ± SEM, \u003cem\u003en \u003c/em\u003e= 5. \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01 \u003cem\u003evs\u003c/em\u003e control group;\u003csup\u003e *\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; \u003cem\u003e0.05 vs\u003c/em\u003e control group.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7135671/v1/0ab2f98fea942cb9ba078037.jpg"},{"id":88253647,"identity":"87eb48fe-0105-4cda-ac47-7c946b001711","added_by":"auto","created_at":"2025-08-04 14:01:38","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":150460,"visible":true,"origin":"","legend":"\u003cp\u003eSEW2871 \u0026nbsp;promotes monocyte/macrophage migration.Cells were cultured for 24 h, in 1640 culture medium(containing 1% FBS) by lentiviral infection with S1PR1 and S1PR1 shRNA. The number of cells were measured by transwell transmigration assay. Scale bar, 200 µm. Mean ± SEM, \u003cem\u003e*P \u0026lt; 0.05 vs \u003c/em\u003econtrol group.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7135671/v1/a0861638f394b9b766ba346a.jpg"},{"id":88254520,"identity":"9a7d7734-c826-4e75-af61-22775322725b","added_by":"auto","created_at":"2025-08-04 14:09:38","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":75489,"visible":true,"origin":"","legend":"\u003cp\u003eS1PR1 \u0026nbsp;promotes monocyte/macrophage adhesion.\u003c/p\u003e\n\u003cp\u003eRaw246.7 and HUVECs by lentiviral infection with S1PR1 and S1PR1 shRNA were co-cultured, and each group of cells were pretreated with SEW2871 at 5 mol/L for 6 hours, and then labeled with PKH26 fluorescent dye. Scale bar, 20 µm. Mean ± SEM, \u003cem\u003e*P \u0026lt; 0.05 vs\u003c/em\u003e control group.\u003c/p\u003e\n\u003cp\u003eS1PR1=S1PR1 overexpression; shRNA= shRNA-S1PR1.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7135671/v1/f4f8d1509ff28d8562281f6b.jpg"},{"id":88253645,"identity":"fb22f752-aa9e-47b1-b5d1-b8cfe2d41166","added_by":"auto","created_at":"2025-08-04 14:01:38","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":44831,"visible":true,"origin":"","legend":"\u003cp\u003eS1PR1 \u0026nbsp;upregulates of expression Adhesion molecule and chemokine of vascular endothelial cells. MI mice were treated as described in Fig. 1, and the mRNA levels of E-Selectin and MCP-1 were examined by PCR. Mean ± SEM, \u003cem\u003en \u003c/em\u003e= 3.\u003csup\u003e ***\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001 \u003cem\u003evs\u003c/em\u003e control group;\u003csup\u003e *\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05 \u003cem\u003evs\u003c/em\u003e control group.\u003c/p\u003e\n\u003cp\u003eS1PR1=S1PR1 \u003ca href=\"javascript:;\"\u003eoverexpression\u003c/a\u003e; shRNA= shRNA-S1PR1.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7135671/v1/e3b8a5bb2414c86703da46d2.jpg"},{"id":103205357,"identity":"ce7df950-97eb-41ac-b8d5-cd2bb4babec8","added_by":"auto","created_at":"2026-02-23 07:12:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":936174,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7135671/v1/0d6015ff-8013-4def-b540-9451920caf3b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"S1PR1 improves Cardiac Repair by Promoting monocyte/ macrophage infiltration and aggregation via ERK Signaling after myocardial infarction","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eMyocardial infarction (MI), a critical cardiovascular emergency, represents a dynamic continuum encompassing both tissue injury and repair mechanisms \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. During the initial phase, ischemic cardiomyocyte necrosis triggers pronounced inflammatory responses. Subsequently, mononuclear/macrophage populations undergo chemotactic infiltration into myocardial tissue, facilitating phagocytic clearance of necrotic/apoptotic cellular debris and extracellular matrix fragments. This coordinated process promotes neovascularization and fibrous scar formation essential for myocardial restoration\u003csup\u003e[\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Current investigations have demonstrated that targeted modulation of monocyte chemotactic activity or macrophage homeostasis within infarct territories can significantly enhance post-infarction cardiac repair outcomes\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eSphingosine 1-phosphate (S1P), a bioactive lipid mediator, exerts extracellular effects through G protein-coupled receptors (GPCRs)\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. Five S1P receptor subtypes (S1PR1-S1PR5) are widely distributed across multiple tissues and cell types, including immune, cardiovascular, respiratory, hepatic, and nervous systems\u003csup\u003e[\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. Experimental evidence indicates that S1PR1 facilitates immune cell adhesion to vascular endothelium and subsequent migration to injured tissues\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. This receptor also promotes post-MI angiogenesis, thereby ameliorating cardiac remodeling\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. As an intracellular messenger, S1PR1 activates extracellular regulated protein kinases (ERK)\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Phosphorylated ERK translocates from cytoplasm to nucleus, initiating transcriptional activation of Elk-1 and NF-κB\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. These molecular events regulate critical cellular processes including proliferation, migration, and apoptosis\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. However, the regulatory role of S1PR1 in monocyte/macrophage functionality during post-MI myocardial repair remains uncharacterized. This study investigated S1PR1-mediated mechanisms in monocyte/macrophage regulation using murine MI models induced by left anterior descending coronary artery ligation, combining in vivo and in vitro experimental approaches.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Experimental Animals\u003c/h2\u003e\u003cp\u003eThis study approved by the Medical Ethics Committee of Jinggangshan University.The study protocol was in accordance with The ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments) 2.0. Specific pathogen-free (SPF) female C57BL/6 mice (aged 8\u0026ndash;12 weeks, body weight 20\u0026ndash;25 g were obtained from Shanghai SLAC Laboratory Animal Co., Ltd. The RAW264.7 macrophage cell line, HUVEC endothelial cell line, and 293T cells were kindly provided by the Oriental Translational Medicine Research Center of Tongji University and the Shanghai Heart Failure Institute. Primary murine monocytes/ macrophages were isolated from peripheral blood of experimental mice, while cardiac endothelial cells were purified via magnetic bead sorting.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2Reagents\u003c/h2\u003e\u003cp\u003eThe S1PR1 agonist SEW2871 was purchased from Cayman Chemical. DMEM medium, fetal bovine serum (FBS), and trypsin were acquired from Gibco. RPMI-1640 medium was sourced from Hyclone, and Percoll cell separation solution from GE Healthcare. TRIzol reagent and PCR kits were obtained from Qiagen. Sodium pentobarbital anesthetic, collagenase I, and collagenase XI were procured from Sigma-Aldrich. Transwell chambers were supplied by Corning. S1PR1 gene-silencing and overexpression plasmids were custom-designed by Gima Biotechnology (Shanghai) Co., Ltd.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Animals and myocardial infarction model\u003c/h2\u003e\u003cp\u003e This study involving animals and experimental protocol was approved by the Animal Ethics Committee of Jinggangshan University. All experiments of the study were conducted in accordance with relevant guidelines and regulations. The mice aged 8 to 10-week old were housed at 24\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, humidity of 40% \u0026plusmn; 5%,. Acute MI models were surgically induced via left anterior descending coronary artery ligation, following established protocols\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. The blind method was used for the operation and subsequent echocardiographic evaluation in this study. Mice were euthanized with isoflurane/oxygen and sacrificed on the corresponding days post-MI surgery to obtain their heart samples for corresponding analyses. The experimental group received daily intraperitoneal injections of the S1PR1 agonist SEW2871 (5 mg/kg,Cayman Chemical) until euthanasia, while controls received equivalent volumes of DMSO.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Echocardiography\u003c/h2\u003e\u003cp\u003eCardiac function of mice was evaluated by echocardiography with a high-resolution imaging system (Vevo2100, Visual Sonic Inc., CA) 0, 5, and 28 days after MI In vivo\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e.The mice were anesthetized using a mixture of isoflurane and oxygen followed by removal of chest hair and placed in the supine position. The two-dimensional and M-mode echocardiographic views of the mid‐ventricular short axis were acquired at the level of the papillary muscle tips below the mitral valve.Variables including left ventricular internal diameter at the end of diastolic (LVIDd) and systolic (LVIDs) were measured on M‐mode echocardiography. Left ventricular ejection fraction (LVEF), fractional shortening (LVFS) and left ventricular end-diastolic volume (LVEDV) and end-systolic volume (LVESV) were calculated as reported previously\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Immunohistochemical Staining Protocol\u003c/h2\u003e\u003cp\u003eTo assess the infarct size, the Immunohistochemical Staining assays were performed as previously described in detail\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. In brief, the heart was harvested and fixed in 4% paraformaldehyde (24h).The 5\u0026micro;m -thick paraffin-embedded sections were stained with haematoxylin and eosin (H\u0026amp;E) and Masson\u0026rsquo;s trichrome. ImageJ software (1.52 V) was used to determine the infarcted area and cardiac fibrosis. Immunostaining was performed on cryostat 8 lm-thick sections by using various antibodies, including antisarcomeric alpha-actinin (Abcam, USA), Anti-F4/80 (Abcam, USA), Anti-Wheat Germ Agglutinin (WGA)-alexa488(Invitrogen, USA), Biotinylated-isolectin B4 antibody (IB4,Vector Laboratories, B-1205, USA) and their corresponding secondary antibodies. Nuclei were stained with 40,6-Diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma-Aldrich, Germany). To evaluate cardiac hypertrophy, we stained heart section with WGA and calculated the average cardiomyocyte cross-sectional area in 10 randomly selected fields of each slide from 5 serial cardiac sections with 500 lm interval distances.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Single-Cell Suspension Preparation and Flow Cytometry\u003c/h2\u003e\u003cp\u003eMice were anesthetized with sodium pentobarbital (50 mg/kg). Peripheral blood, cardiac tissue, bone marrow, liver, and spleen were harvested, homogenized in PBS, and centrifuged at 1000 \u0026times;g for 5 min to obtain single-cell suspensions. For temporal analysis of monocyte/macrophage dynamics post-MI, tissues were collected at 0, 5, 7, and 21 days post-infarction. Cell suspensions were stained with anti-CD11b, -F4/80, and -Ly6C antibodies for flow cytometric quantification of monocyte subsets (Ly6Chi/Ly6Clow) and macrophages using FlowJo software.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Cell Culture and Transfection\u003c/h2\u003e\u003cp\u003eRAW264.7 macrophages were maintained in RPMI-1640 medium supplemented with 10% FBS, while HUVECs were cultured in EGM2 medium (10% FBS) under 5% CO\u003csub\u003e2\u003c/sub\u003e at 37℃. At 70% confluence, cells were transfected with S1PR1-targeting shRNA, overexpression plasmids, or empty vectors using standard protocols. Transfection efficiency was validated by Western blotting 48 h post-transfection.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Transwell Migration Assay\u003c/h2\u003e\u003cp\u003eS1PR1-modulated RAW264.7 migration was assessed using Transwell chambers. Cells were serum-starved overnight in RPMI-1640 with 1% FBS, then seeded in the upper chamber (1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well in 150\u0026micro;l 1% FBS medium). The lower chamber contained 700\u0026micro;l\u0026thinsp;~\u0026thinsp;800\u0026micro;l 10% FBS medium. After 4 hours incubation, migrated cells were fixed, stained with crystal violet, and quantified microscopically. Triplicate wells were used across three independent experiments.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9 Co-Culture Adhesion Assay\u003c/h2\u003e\u003cp\u003eRAW264.7-HUVEC co-cultures were divided into four groups: control, S1PR1-overexpression, S1PR1-silenced, and S1PR1-overexpression\u0026thinsp;+\u0026thinsp;U0126 (50\u0026micro;mol /L ERK inhibitor). Cells were pretreated with 5 \u0026micro;mol/L S1P for 6 h (with U0126 co-treatment in the inhibitor group). PKH26-labeled RAW264.7 adhesion rates were quantified 30 minutes post-co-culture using fluorescence microscopy. Triplicate measurements were performed in three experimental repeats.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10 Statistical Analysis\u003c/h2\u003e\u003cp\u003eExperimental data were analyzed using SPSS 19.0 (IBM) and visualized with GraphPad Prism 9.0. Intergroup differences were assessed via independent Student\u0026rsquo;s t-test for two-group comparisons and one-way ANOVA for multi-group analyses (\u0026ge;\u0026thinsp;3 groups). Flow cytometry data were processed using FlowJo v10.8. Results are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM, with statistical significance defined at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e3.1 S1PR1 Promotes Monocyte/Macrophage Accumulation in Infarcted Myocardium\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eInflammatory cells isolated via Percoll gradient centrifugation were analyzed for monocyte/macrophage subpopulations using flow cytometry (Figure 1A). At day 5 post-MI, SEW2871-treated mice exhibited significantly higher counts of CD11b\u003csup\u003e+\u003c/sup\u003e cells (Figure 1B), Ly6c\u003csup\u003eLow\u003c/sup\u003e monocytes, and Ly6c\u003csup\u003ehigh\u003c/sup\u003e/Ly6c\u003csup\u003eLow\u003c/sup\u003e macrophages in infarcted myocardium compared to WT controls (Figure 1C). Immunofluorescence staining further revealed increased F4/80\u003csup\u003e+\u003c/sup\u003e cells per field in SEW2871-treated mice at 4 weeks post-MI (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05), though CD11b\u003csup\u003e+\u003c/sup\u003e cell counts showed no intergroup difference (Figure 1D\u0026ndash;F). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e3.2 S1PR1 Enhances Peripheral Blood Monocyte Chemotaxis to\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eInflamed Tissue\u003c/p\u003e\n\u003cp\u003eFACS analysis demonstrated markedly reduced total monocytes and subtype counts in \u0026nbsp;\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05,peripheral blood of SEW2871-treated mice at day 5 post-MI (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, Figure 2A\u0026ndash;C), while liver, spleen, and bone marrow exhibited no significant intergroup differences.\u003c/p\u003e\n\u003cp\u003e3.3 S1PR1 Facilitates Monocyte Migration \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTranswell chamber assays revealed a significant increase in migratory cells in the S1PR1-overexpression group versus controls (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, Figure 3A\u0026ndash;B). ERK inhibition with U0126 substantially attenuated S1PR1-driven migration, achieving statistical significance compared to controls (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e3. 4 S1PR1 Augments Monocyte Adhesion\u003c/p\u003e\n\u003cp\u003eIn co-cultures of HUVECs and RAW264.7 macrophages, S1PR1-overexpression significantly enhanced adhesion rates versus controls (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). Conversely, U0126 treatment markedly reduced adhesion in the S1PR1-overexpression group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, Figure 4A\u0026ndash;B). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e3.5 \u0026nbsp;S1PR1 Upregulates Endothelial Adhesion Molecules and Chemokines \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRT-qPCR analysis showed significantly elevated E-selectin expression in S1PR1-overexpressing endothelial cells compared to control and knockdown groups (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001). SEW2871-treated endothelial cells also exhibited upregulated MCP-1 expression versus controls (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, Figure 5A\u0026ndash;B).\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eOur study demonstrates that: (1) Following myocardial infarction, S1PR1 facilitates monocyte/macrophage infiltration and recruitment within infarcted cardiac tissue. (2) S1PR1 enhances monocyte/ macrophage adhesion and migration. (3) The ERK signaling pathway inhibitor U0126 abolishes S1PR1-induced enhancement of monocyte/macrophage adhesion and migration.\u003c/p\u003e\n\u003cp\u003eThese findings advance our understanding of post-MI cardiac repair and potential therapeutic strategies.\u003c/p\u003e\n\u003cp\u003ePrevious studies have confirmed that monocytes/macrophages play a pivotal role in myocardial repair following myocardial infarction\u003csup\u003e[20\u0026ndash;22]\u003c/sup\u003e. During the intermediate and late stages of infarction, macrophage accumulation increases in the infarcted myocardium. The elevated population of reparative Ly6C\u003csup\u003elow\u003c/sup\u003e macrophages stimulates the expression of factors such as TGF-\u0026beta; and VEGF, thereby promoting angiogenesis\u003csup\u003e[23, 24]\u003c/sup\u003e. Furthermore, prior research indicates that macrophages phagocytose necrotic cardiomyocyte debris and apoptotic immune cells to facilitate cellular regeneration and tissue remodeling in the infarcted area\u003csup\u003e[25]\u003c/sup\u003e. Our results demonstrate that monocyte/ macrophage infiltration and recruitment in the infarcted myocardium begins to increase on day 3 post-myocardial infarction and peaks on day 5. Notably, the increase in Ly6C\u003csup\u003elow\u003c/sup\u003e macrophages is particularly pronounced, consistent with previous reports. These findings suggest the initiation of the myocardial repair phase at this stage.\u003c/p\u003e\n\u003cp\u003eUnder normal conditions, monocytes/macrophages in myocardial tissue primarily originate from local proliferation. However, under stress conditions such as inflammation, extracardiac sources become predominant\u003csup\u003e[26]\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eTo determine the origin of monocytes/macrophages infiltrating and aggregating in infarcted myocardium and the potential role of S1PR1, we collected tissues including peripheral blood, liver, spleen, and bone marrow from post-myocardial infarction mice and prepared single-cell suspensions. Flow cytometry analysis revealed that, compared with the control group, the S1PR1 agonist group exhibited significantly reduced monocyte counts in peripheral blood, with no significant differences observed in the liver, spleen, or bone marrow. This indicates that S1PR1 primarily promotes chemotaxis, migration, and aggregation of peripheral blood monocytes toward the infarcted area.\u003c/p\u003e\n\u003cp\u003eDuring inflammation, various cell adhesion molecules, including vascular cell adhesion molecule-1 (VCAM-1) and E-Selectin, play critical roles in inflammatory cell migration \u003csup\u003e[27]\u003c/sup\u003e. After adhering to vascular endothelial cells, monocytes subsequently transmigrate across the endothelium and migrate toward inflamed tissues under chemokine gradients. Studies reported that S1PR1 upregulates E-selectin expression and enhancing monocyte adhesion by inducing phosphorylation and nuclear translocation of the NF-\u0026kappa;B subunit p65 \u003csup\u003e[28, 29]\u003c/sup\u003e. Both our in vivo and in vitro experiments confirmed that S1PR1 increases E-selectin mRNA levels, an effect inhibited by the ERK pathway inhibitor U0126. This indicates that S1PR1 regulates E-selectin expression via the ERK signaling pathway. As E-selectin is a key adhesion molecule on vascular endothelial cells that mediates leukocyte adhesion, this mechanism partially explains S1PR1\u0026apos;s role in promoting monocyte/macrophage adhesion. Furthermore, S1PR1 has been shown to increase MCP-1 expression, thereby enhancing monocyte chemotaxis and adhesion to endothelial cells \u003csup\u003e[30]\u003c/sup\u003e. Consistent with these reports, our study demonstrated elevated MCP-1 expression in the infarcted myocardium of the experimental group. This suggests that S1PR1 upregulates MCP-1, a major monocyte chemoattractant, thus enhancing monocyte/macrophage migration toward ischemic myocardial tissue.\u003c/p\u003e\n\u003cp\u003eAs a component of the MAPK signaling pathway, ERK (extracellular signal-regulated kinase) activation is crucial for transducing signals from cell surface receptors to the nucleus. It plays an essential role in regulating cellular functions such as metabolism, migration, and proliferation \u003csup\u003e[31, 32]\u003c/sup\u003e. In our study, pretreatment with the ERK pathway inhibitor U0126 significantly attenuated SEW2871-induced monocyte/macrophage adhesion and migration. This confirms the critical involvement of ERK signaling in regulating monocyte/macrophage adhesion and migration.\u003c/p\u003e\n\u003cp\u003eTaken together, our studies demonstrate that S1PR1 agonists play a critical role in the inflammatory response and myocardial repair process following myocardial infarction. We also investigated the role and mechanism of S1PR1 in promoting monocyte/macrophage migration post-myocardial infarction. These investigations revealed that S1PR1 upregulates the expression of E-Selectin and MCP-1 by modulating the ERK signaling pathway. This facilitates the migration and aggregation of peripheral blood monocytes into the infarcted area, thereby increasing monocyte/macrophage accumulation in the infarcted myocardium and promoting cardiac repair.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors’ contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXiaolin Li put forward the concept and design. Junping Ou made the definition of intellectual content and literature search., Zhongwen Liu made data acquisition, Yuping Wang made data and statistical analysis. Jie Zeng made the manuscript preparation, editing and review. Qingyi Wang quantify experimental data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;:\u003c/strong\u003eThis study has been supported by Jiangxi Provincial Natural Science Foundation(20224BAB206014), and the Science and Technology Plan Project of the Jiangxi Provincial Health Commission (202312372；202312384).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data collected in the study is available from the corresponding author on reasonable request, E-mail: [email protected]:+86-13979609896.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eJiahui, Zhang,Yishan, Guo,Yu, Bai et al. Application of biomedical materials in the diagnosis and treatment of myocardial infarction.[J] .J Nanobiotechnology, 2023, 21(1):298.\u003c/li\u003e\n\u003cli\u003e\u003cem\u003eNikolaos G. 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Sci, 2019, 20(3): 1-16.\u003c/li\u003e\n\u003cli\u003ePark Jong-In, MAPK-ERK Pathway.[J] .Int J Mol Sci, 2023, 24(11):1-3.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"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},"keywords":"Myocardial infarction, Sphingosine 1-phosphate receptor 1, Monocytes/macrophages, Cardiac repair","lastPublishedDoi":"10.21203/rs.3.rs-7135671/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7135671/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eThis study aimed investigate the impact and mechanism of S1PR1 on monocyte/macrophage function following myocardial Infarction.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eWe performed cells profiling of Circulating, liver, kidney, and cardiac immune cells in mice challenged with AMI, and the monocyte/macrophage landscape of the infarcted heart were quantified via flow cytometry at 0, 5, and 28 days post-MI. Fluorescence microscopy evaluated monocyte/macrophage fluorescence expression in cardiac tissue. Lentiviral vectors (pCMV.DR8 and pMD2.G) were constructed to infect RAW264.7 macrophages, with optimal multiplicity of infection (MOI) determined. Experimental groups included S1PR1-knockdown, S1PR1-overexpression, U0126 (ERK pathway inhibitor)-treated, and blank control groups. Transwell assays assessed cell migration, while RAW264.7-HUVEC co-cultures evaluated adhesion.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eThe experimental group exhibited a significantly higher mononuclear/macrophage count in myocardial tissue compared to controls (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In vitro experiments demonstrated that SEW2871 enhanced both adhesion and migration capacities of RAW264.7 macrophages. These effects were significantly attenuated following S1PR1 gene knockdown. Furthermore, U0126 pretreatment substantially diminished SEW2871-mediated promotion of cellular adhesion and migration.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eS1PR1 promotes monocyte/macrophage adhesion and migration via ERK signaling pathway and thus ameliorates post-MI cardiac repair.\u003c/p\u003e","manuscriptTitle":"S1PR1 improves Cardiac Repair by Promoting monocyte/ macrophage infiltration and aggregation via ERK Signaling after myocardial infarction","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-04 14:01:34","doi":"10.21203/rs.3.rs-7135671/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":"031b83f7-bf60-43e8-abd4-f409600f2e7b","owner":[],"postedDate":"August 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":52398708,"name":"Health sciences/Cardiology"},{"id":52398709,"name":"Health sciences/Diseases"}],"tags":[],"updatedAt":"2026-02-23T07:11:28+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-04 14:01:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7135671","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7135671","identity":"rs-7135671","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}