CARM1 inhibitor TP064 attenuates endothelial cell dysfunction via inhibits inflammatory response in vitro model of subarachnoid hemorrhage

CARM1 inhibitor TP064 attenuates endothelial cell dysfunction via inhibits inflammatory response in vitro model of subarachnoid hemorrhage | 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 Research Article CARM1 inhibitor TP064 attenuates endothelial cell dysfunction via inhibits inflammatory response in vitro model of subarachnoid hemorrhage Qingtao Zhang, Ping Zhang, Yidan Liang, Qiang Yang, Lei Xu, Yongbing Deng, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4432703/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 Cerebral endothelial cell dysfunction plays a critical role in the pathophysiology of vascular injury subsequent to subarachnoid hemorrhage (SAH), yet the precise molecular mechanism remains largely speculative. Inflammation stands out as a pivotal contributor to an unfavorable prognosis post-SAH, with nuclear factor-κB (NF-κB) pathways being initiated and ultimately leading to inflammation activation and pro-inflammatory cytokine release following SAH. In this study, we explored the impact of the Coactivator-associated arginine methyltransferase 1 (CARM1) inhibitor TP-064 on inflammation using an in vitro SAH model. Exposure of endothelial cells to TP-064 resulted in a significant reduction in CAMR1 and NF-κB expression upon hemoglobin exposure. Similarly, endothelial cells treated with TP-064 following hemoglobin incubation exhibited decreased expression levels of intercellular adhesion molecule-1 (ICAM1), myeloperoxidase (MPO), and cytokine production including interleukin-1β (IL-1β), interleukin-12 (IL-12), tumor necrosis factor-α (TNF-α) in response to hemoglobin exposure. Moreover, subsequent investigations demonstrated that CARM1 transcriptionally regulates NF-κB via methylation. Additionally, TP-064 notably mitigated endothelial dysfunction. Collectively, our findings identify TP-064 as a CARM1 inhibitor targeting inflammation and neutrophil infiltration, offering new insights into therapeutic strategies for addressing endothelial cell dysfunction following SAH. CARM1 inflammation endothelial dysfunction Methylation subarachnoid hemorrhage Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1.Introduction Subarachnoid hemorrhage (SAH) is a cerebrovascular ailment characterized by elevated morbidity and mortality rates[ 1 ]. Impaired cerebral autoregulation emerges as a significant contributor to unfavorable prognoses in SAH patients, encompassing vasospasm and delayed cerebral ischemia (DCI)[ 2 ]. Endothelial cells (ECs), integral components of cerebral vasculature, play pivotal roles in upholding the blood-brain barrier (BBB) and modulating vascular tone and hemodynamics, thereby exerting influence on vascular function and homeostasis[ 3 , 4 ]. Previous research demonstrates the presence of inflammatory cytokines within the endothelium of spastic arteries, indicating the potential contribution of inflammatory factors to ECs damage[ 5 , 6 ]. Nuclear factor-κB(NF-κB) is a key factor in the regulation of the expression of pro-inflammatory cytokines, adhesion molecules and enzymes[ 7 ]. NF-κB-mediated pro-inflammatory signaling in endothelial cells and leukocytes induces the chronic inflammatory pathology atherosclerosis[ 8 ].Coactivator-associated arginine methyltransferase1(CARM1) is an arginine methyltransferase that controls gene transcription, which is know known to enhance transcriptional activation by nuclear receptors[ 9 ]. Recently, accumulative evidence showed that CARM1 can act as a coactivator for the transcription factor NF-κB and enhance its activity[ 10 , 11 ]. Increased expression of CARM1 is associated with elevated levels of pro-inflammatory mediators in atherosclerosis-related cardiovascular disease[ 10 ]. Thus, inhibition of CARM1 activity might be a promising strategy to reduce the pro-inflammatory response. However, it remains unclear whether CARM1 participate in the endothelial cell dysfunction after SAH. In this study, we established cell models of SAH to observe the effects of CARM1 on the endothelial cell dysfunction, we also apply TP-064 as CARM1 inhibitor to study the effects of TP-064 treatment on inflammation to explore the potential mechanisms in this pathophysiological process. 2.Materials and Methods 2.1 Cell culture. ECs were cultured in dulbecco modified eagle medium and ham’s F-12 medium (DMEM/DF12, Gibco, USA, 1:1) supplemented with 10% fetal bovine serum (FBS, Gibco, USA) in 5% CO2 at 37°C,with medium renewal every 48 hours. To simulate the pathophysiological conditions of subarachnoid hemorrhage (SAH) and determine the optimal stimulating concentration, cells were exposed to hemoglobin (Sigma, USA) at concentrations ranging from 5 µM to 20 µM for 24 hours prior to subsequent assays. Following hemoglobin exposure, western blot analysis was employed to assess the expression of CARM1 across different concentration groups. Cells were treated with 10 µM TP-064 dissolved in an equivalent concentration of dimethyl sulfoxide (DMSO) for 24 hours. Subsequently, cells were stimulated with hemoglobin for an additional 24 hours. Following stimulation, cells and supernatants were harvested and stored at − 20°C until further analysis of gene expression or cytokine levels. 2.3 Western blotting. Endothelial cells (ECs) cultured in T-25 flasks following the aforementioned treatments were subjected to triple washing, and the proteins were extracted using RIPA buffer supplemented with protease inhibitors (Biotime, China). The total protein concentration was determined using BCA Protein Assay Reagent (Beyotime, China). Subsequently, the protein samples were denatured by boiling and separated (20 µg) via 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer onto polyvinylidene difluoride membranes (PVDF, Bio-Rad, USA). The membranes were then blocked with 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 hour, and incubated overnight at 4°C with specific antibodies: CARM1 (Abcam, United Kingdom, 1:200), MPO (Abcam, United Kingdom, 1:500), ICAM1 (Abcam, United Kingdom, 1:200), NF-κB (Abcam, United Kingdom, 1:400), GAPDH (ProteinTech Group, China, 1:1000), and Histone (1:1000, Proteintech). Blots were subsequently incubated with the appropriate horseradish peroxidase-conjugated IgG for 1 hour at 37°C and visualized using the Chemiluminescence Kit (Beyotime, China) with X-ray film. The optical densities of these bands were quantified using Quantity One software 4.6.2. GAPDH and Histone were employed as loading controls for whole cell and nuclear proteins. 2.4 ELISA. The concentration of pro-inflammatory cytokines were analyzed in the medium of endothelial cells using the ELISA protocols provided by BD Biosciences and Biolegend (San Jose and San Diego, CA, USA). Absorbance measurements were conducted at 450 nm and 570 nm. 2.5 Immunofluorescence. ECs were cultured on glass coverslips placed in 6-well plates at 65% confluence and pre-incubated TP-064. For immunostaining, cells were washed three times with PBS, fixed with 4% paraformaldehyde,, permeabilized with 0.01% Triton X-100 in PBS, and blocked with 10% goat serum for 30 minutes at 37°C. The cells were then incubated overnight at 4°C with the following primary antibodies: rabbit anti–CARM1 (Abcam, United Kingdom, 1:200), mouse-CD31(Abcam, United Kingdom, 1:200), rabbit anti–MPO (Abcam, United Kingdom, 1:200) followed by appropriate fluorescein isothiocyanate-conjugated and tetramethyl rhodamine isothiocyanate-conjugated secondary antibodies (Abbkine, USA, 1:200) for 1hours at room temperature. Subsequently, all cells were incubated with DAPI for 15minutes. Coverslips were mounted in antifade regent (Beyotime, China) and visualized by a fluorescence microscopy timely and effectively (Leica, Germany). 2.6 Gene expression analysis. Total RNA was extracted from cells following the standard protocol outlined by Chomczynski and Sacchi[ 11 ]. RNA was reverse transcribed into cDNA using Maxima H Minus Reverse Transcriptase. The PCR cycle threshold (Ct) values were then determined after incorporation of SensiMix SYBR low-ROX mix, utilizing the ABI 7500 Real Time PCR system (Applied Biosystems, Foster City, CA, USA). The average Ct values of acidic ribosomal phosphoprotein P0 (36b4), hypoxanthine guanine phosphoribosyl transferase (Hprt), and ribosomal protein L27 (Rpl27) were employed as housekeeping genes. 2.7 Chromatin immunoprecipitation assay (ChIP) The ChIP assay was conducted using a commercially available kit (Millipore) following the manufacturer's instructions. Briefly, cells were crosslinked with 1% formaldehyde for 10 minutes, followed by quenching with glycine (0.125 M). Subsequently, the cell pellets were lysed in lysis buffer and sonicated for 5 minutes. The lysates were centrifuged, and the supernatants were incubated with specified antibodies overnight at 4°C. Immunocomplexes were captured using 30 µl of protein A/G sepharose on a rotator at 4°C for 2 hours. Following four times of washing, protein complex attached to the beads was dissolved in the SDS sample buffer and subsequently resolved in 7% SDSPAGE gel. All experiments were performed in three biological replicates. 2.8 Statistical Analysis. Statistical analysis was carried out using SPSS 20.0 and GraphPad Prism 9.5. All values were expressed as mean ± standard deviation. Multi-group comparisons were conducted through one-way analysis of variance (ANOVA) followed by Bonferroni post hoc analysis. Each experiment was repeated a minimum of three times. A significance level of P < 0.05 was deemed statistically significant. 3.Results 3.1 Hemoglobin Induced increased CARM1 in Cultured Cerebral ECs. The cells were treated with varying concentrations of hemoglobin, and the expression of CARM1 and NF-κB were examined by western blot analysis after 24 hours. As depicted in Fig. 1 A, elevated levels of CARM1 and NF-κB were detected within cultured ECs following hemoglobin treatment. The expression of both proteins reached maximal levels with a hemoglobin concentration of 10 µM. However, excessive hemoglobin dosage led to a decrease in the expression of both proteins(Fig. 1 a,b,c). Based on the preliminary data, a concentration of 10 µM (optimal stimulating concentration) was chosen for subsequent experiments. Following a 24-hour incubation with hemoglobin, a significant increase in the expression of CARM1 and NF-κB was observed compared to the control group. Similarly, an enhanced immunoreactivity of CARM1 was observed within cultured ECs after hemoglobin treatment (Fig. 1 d). 3.2 TP-064 treatment inhibited the expression of CARM1 and attenuated the hemoglobin-induced pro-inflammatory response of endothelial cells (ECs) in vitro. To investigate the effects of CARM1 on the pro-inflammatory response of ECs, we sought to inhibit CARM1 expression using TP-064. As illustrated in Fig. 2 a,b, immunoblot results demonstrated a significant increase in CARM1 expression following hemoglobin incubation, which was reversed by TP-064 treatment. Additionally, TP-064 markedly reduced the expression of NF-κB, ICAM1, and MPO (Fig. 2 a, c, d, e). Similarly, ELISA results revealed an elevation in the expression of inflammatory cytokines (TNF-α, IL-1β, and IL-12) induced by hemoglobin, which was mitigated by TP-064 treatment (Fig. 3 d, e, f). Immunofluorescence analysis showed colocalization of the neutrophil marker (MPO) with a specific marker of endothelial cells (CD31) in ECs. These findings indicate that TP-064 treatment significantly suppressed neutrophil infiltration and the inflammatory response (Fig. 2 f). 3.3 TP-064 treatment downregulates inflammation-related gene expression. Consistently, the inhibition of CARM1 activity by TP-064 was associated with a decrease in NF-κB mRNA expression levels. Our current in vitro study demonstrated that TP-064-mediated inhibition of CARM1 resulted in decreased expression levels of ICAM1 and MPO in endothelial cells (ECs) (Fig. 3 a, b, c). 3.4 CARM1 enhances NF-κB activation through Methylation. As confirmed previously, CARM1 upregulated NF-κB expression in endothelial cells (ECs), suggesting a potential modulation of NF-κB's transcriptional activity by CARM1. As expected, our co-immunoprecipitation (co-IP) assays demonstrated the interaction between CARM1 and NF-κB in ECs (Fig. 4 a). Subsequently, we investigated whether NF-κB served as a substrate for CARM1. ECs underwent co-IP with immunoglobulin G (IgG) or anti-NF-κB antibodies, followed by immunoblotting with an asymmetric dimethylarginine (ADMA)-specific antibody (Fig. 4 b). The presence of methylated NF-κB in the immunoprecipitation (IP) lysates indicated the methylation of NF-κB by CARM1 in vitro. 3.5 TP-064 treatment alleviated hemoglobin induced endothelial dysfunction. To assess the impact of TP-064 treatment on endothelial dysfunction levels of endothelin-1 (ET-1) and nitric oxide (NO) were measured using enzyme-linked immunosorbent assay (ELISA). Following hemoglobin treatment, ET-1 levels increased while NO levels decreased in endothelial cells (ECs). We demonstrated that the CARM1 inhibitor TP-064 effectively reduced ET-1 levels and increased NO levels (Fig. 5 a, b), indicating a beneficial effect of CARM1 inhibition on endothelial dysfunction. 4.Discussion In the current study, we explored the potential impact of the CARM1 inhibitor TP-064 on endothelial cell dysfunction following SAH in vitro. The primary findings from our study are as follows: 1) CARM1-mediated NF-κB activation through methylation contributes to inflammatory response and endothelial cell dysfunction; 2) Regulation of CARM1 expression by TP-064 in cultured cerebral ECs effectively alleviates inflammation and endothelial cell dysfunction after hemoglobin incubation. In the cerebral vasculature, healthy endothelial cells regulate blood viscosity, control blood flow, and maintain the integrity of the blood-brain barrier (BBB). [ 12 ]. Previous research has described inflammation in blood vessels as involving leukocyte-endothelial cell interactions and the release of inflammatory mediators, resulting in endothelial cell integrity disruption and subsequent dysfunction [ 6 , 13 ]. Hemoglobin, a product of blood hemolysis after SAH, oxidizes into various forms, becoming a potent pro-inflammatory and cytotoxic molecule known to activate NF-κB.[ 14 , 15 ]. Hemoglobin-induced endothelial damage and apoptosis, neuroinflammation, and BBB disruption have been reported[ 16 ]. Following hemoglobin incubation with ECs to mimic SAH neuropathology, our results revealed heightened NF-κB expression. Coactivator-associated arginine methyltransferase 1 (CARM1) acts as a coactivator and a novel transcriptional regulator of NF-κB-mediated inflammatory gene expression[ 17 , 18 ].However, the expression level and role of CARM1 in ECs following hemoglobin treatment have yet to be determined. Our study demonstrated a significant increase in both mRNA and protein levels of CARM1 in ECs after hemoglobin treatment compared to controls, with CARM1 also colocalizing with ECs. Our co-IP assay confirmed the interaction between CARM1 and NF-κB, suggesting that CARM1 interacts with NF-κB to catalyze its methylation and restrict its nuclear localization, thereby promoting inflammation. Elevated levels of inflammatory cytokines (TNF-α, IL-1β, and IL-12) correlated with increased expression of CARM1 and NF-κB. Additionally, CARM1 mRNA levels positively correlated with mRNA expression levels of NF-κB, MPO, and ICAM1 in ECs following hemoglobin treatment. To our knowledge, this study is the first to report elevated CARM1 levels in ECs following hemoglobin treatment, further supporting a role for CARM1 in the inflammatory response. Recent studies have identified TP-064 as a selective and potent inhibitor of CARM1 function [ 19 , 20 ]. Consistent with previous observations that CARM1 acts as an NF-κB coactivator, our research showed that TP-064 reduced pro-inflammatory cytokine secretion[ 17 , 21 ]. Western blot analysis revealed increased expression of MPO and ICAM-1 following hemoglobin treatment, which was significantly reduced by TP-064 administration. The administration of TP-064 significantly decreased the expression of MPO and ICAM-1. Furthermore, MPO is a marker of leukocyte activity[ 22 ], and fluorescence microscopy demonstrated MPO colocalization with CD31, indicating leukocyte infiltration into ECs. We also observed that TP-064 suppressed neutrophil infiltration in ECs following hemoglobin treatment. These findings collectively suggest that TP-064 inhibits neutrophil infiltration, possibly through decreased ICAM-1 and MPO expression. Endothelial cells regulate vascular tone and blood flow through a delicate balance of vasoconstrictors such as endothelin-1 (ET-1) and vasodilators such as nitric oxide (NO)[ 23 ]. Animal studies have shown that SAH can induce functional changes in vascular endothelium, resulting in reduced NO levels and increased ET-1 levels[ 24 , 25 ]. In our investigation, TP-064 treatment significantly reduced ET-1 levels and increased NO levels, resulting in significant improvement in endothelial dysfunction following hemoglobin incubation in the TP-064 treatment group. In conclusion, these findings imply that inhibition of CARM1 function by TP-064 may represent a promising novel therapeutic approach for NF-κB-driven pathologies, a matter of considerable importance in elucidating the molecular mechanisms underlying endothelial dysfunction after SAH. Further validation will be conducted in subsequent animal experiments. Declarations Ethical Statements: It is not applicable. Conflicts of interest The authors declare no competing financial interests. Acknowledgements We acknowledge the service provided by the Key Laboratory of Chongqing University Central Hospital. Funding This work was supported by the Post-Doctoral Science Fund of Chongqing Natural Science Foundation(cstc2021jcyj-bshX0051), National Natural Science Foundation of China(82160222), Guizhou Provincial Science and Technology Projects (Qiankehe Foundation ZK (2022) General 261), Guizhou Provincial People’s Hospital National Science Foundation(GPPH-NSFC-2021-14). Author contributions: Liu Liu and Min Wu conceived and designed the project, Qingtao Zhang and Ping Zhang executing all experiments and contributing to manuscript preparation. Yidan Liang, Qiang Yang, Min Cui, and Chao Sun assisted in completing specific experiments. Yongbing Deng and Weiduo Zhou conducted data analysis, Lei Xu participated in manuscript revision, particularly focusing on language refinement. All authors critically reviewed and approved the final version of the manuscript. Data availability statement: The data that support the findings of this study are available from the corresponding author upon reasonable request. References Lawton MT and Vates GE (2017) Subarachnoid Hemorrhage. N Engl J Med 377:257-266. doi: 10.1056/NEJMcp1605827 Otite F, Mink S, Tan CO, Puri A, Zamani AA, Mehregan A, Chou S, Orzell S, Purkayastha S, Du R and Sorond FA (2014) Impaired cerebral autoregulation is associated with vasospasm and delayed cerebral ischemia in subarachnoid hemorrhage. Stroke 45:677-82. doi: 10.1161/STROKEAHA.113.002630 Komarova YA, Kruse K, Mehta D and Malik AB (2017) Protein Interactions at Endothelial Junctions and Signaling Mechanisms Regulating Endothelial Permeability. 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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-4432703","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":307250741,"identity":"c025d1dc-47ea-4ccb-b59e-9aad429b921f","order_by":0,"name":"Qingtao Zhang","email":"","orcid":"","institution":"Chongqing University Central Hospital, Chongqing University","correspondingAuthor":false,"prefix":"","firstName":"Qingtao","middleName":"","lastName":"Zhang","suffix":""},{"id":307250743,"identity":"131e3edd-0e31-4819-92f0-f9874fd1b014","order_by":1,"name":"Ping Zhang","email":"","orcid":"","institution":"The First Affiliated Hospital of 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Central Hospital, Chongqing University","correspondingAuthor":true,"prefix":"","firstName":"Liu","middleName":"","lastName":"Liu","suffix":""},{"id":307250752,"identity":"44eec0b6-36a4-4b85-bf4d-02f1078e8836","order_by":10,"name":"Min Wu","email":"","orcid":"","institution":"Guizhou Provincial People’s Hospital","correspondingAuthor":false,"prefix":"","firstName":"Min","middleName":"","lastName":"Wu","suffix":""}],"badges":[],"createdAt":"2024-05-16 18:02:50","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4432703/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4432703/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":57412290,"identity":"b03827a3-ef71-4835-ad70-71a9db80bcf9","added_by":"auto","created_at":"2024-05-30 10:52:44","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":756831,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of CARM1 and NF-κB in cerebral endothelial cell after treatment with Hb.\u003c/strong\u003e \u003cstrong\u003ea-c\u003c/strong\u003e Representative image and quantitative analysis of CAMR1 and NF-κB expression in cerebral ECs at 24hours after incubation with various concentrations of hemoglobin respectively (n=6 per group).\u003cstrong\u003e d\u003c/strong\u003e Immunohistochemical staining for CARM1 (red), CD31 (green), and DAPI (blue) in cerebral endothelial cells at 24 hours following hemoglobin incubation. All quantitative data were presented as mean±standard deviation. Scale Bar=100μm. *P\u0026lt;0.05 vs Control group.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-4432703/v1/7f35939fadc792bfe6021b56.png"},{"id":57413017,"identity":"9908a5ec-9010-4a0c-a4c3-b067c812efe0","added_by":"auto","created_at":"2024-05-30 11:00:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":813306,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTP-064 inhibited CAMR1 expression and suppressed pro-inflammatory response of ECs. a-e \u003c/strong\u003eRepresentative image and quantitative analysis of CAMR1,NF-κB ,ICAM1 and MPO in cerebral ECs at 24hours post-incubation with 10μM Hb, with or without TP-064 pretreatment, respectively. \u003cstrong\u003ef\u003c/strong\u003e Immunohistochemistry for mpo(green), CD31 (red), and DAPI (blue) in cerebral ECs at 24hours post-Hb incubation with or without TP064 pretreatment, respectively. All quantitative data were presented as mean±standard deviation. Scale Bar=100μm. *P\u0026lt;0.05 vs Control group; # P\u0026lt;0.05 vs. Hb group.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-4432703/v1/a3db755095ad8453ac7835e3.png"},{"id":57412292,"identity":"8ac21077-0d55-4c66-8b7b-6da35769c6d4","added_by":"auto","created_at":"2024-05-30 10:52:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":121392,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTP-064 treatment decreases pro-inflammatory gene expression and cytokine production. a-c\u003c/strong\u003e Relative mRNA expression levels of pro-inflammatory trigger genes (a-c) in ECs at 24hours after 10μM Hb incubation with or without TP-064 pretreated respectively Pro-inflammatory cytokine genes in ECs at 24hours after 10μM Hb incubation with or without TP064 pretreated respectively.\u003cstrong\u003e d-f \u003c/strong\u003ePro-inflammatory cytokines in ECs at 24hours post-incubation with 10μM Hb, with or without TP064 pretreatment, respectively. *P\u0026lt;0.05 vs Control group; # P\u0026lt;0.05 vs. Hb group.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-4432703/v1/fa25f54afac646f652fd63fc.png"},{"id":57412288,"identity":"aacf9cde-b38f-417a-b820-12b510115792","added_by":"auto","created_at":"2024-05-30 10:52:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":107958,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCARM1 transcriptionally controls NF-κB through Methylation.\u003c/strong\u003e \u003cstrong\u003ea \u003c/strong\u003eco-IP assay demonstrated the interaction between CARM1 and NF-κB in endothelial cells (ECs); \u003cstrong\u003eb \u003c/strong\u003eCell lysates from ECs were incubated with anti-IgG or NF-κB antibody, and followed by WB with anti-ADMA.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-4432703/v1/5cf5ad31a28636247ea1ec22.png"},{"id":57412289,"identity":"86f4939b-0cef-4e6d-8ebe-7249785a4eed","added_by":"auto","created_at":"2024-05-30 10:52:44","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":61029,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTP-064 treatment alleviated endothelial dysfunction\u003c/strong\u003e. Representative analysis of effects of TP064 on the expression of ET-1 and NO in cerebral ECs after 10μM Hb incubation. *P\u0026lt;0.05 vs Control group; # P\u0026lt;0.05 vs. Hb group.\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-4432703/v1/e99b8654e02cd1ab74339092.png"},{"id":60808524,"identity":"9dc8d6f1-b7e3-4f7e-a158-d2a981a383d4","added_by":"auto","created_at":"2024-07-22 10:34:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2459244,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4432703/v1/7e761405-b981-4b2b-98f0-bbffd257d6e5.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"CARM1 inhibitor TP064 attenuates endothelial cell dysfunction via inhibits inflammatory response in vitro model of subarachnoid hemorrhage","fulltext":[{"header":"1.Introduction","content":"\u003cp\u003eSubarachnoid hemorrhage (SAH) is a cerebrovascular ailment characterized by elevated morbidity and mortality rates[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Impaired cerebral autoregulation emerges as a significant contributor to unfavorable prognoses in SAH patients, encompassing vasospasm and delayed cerebral ischemia (DCI)[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Endothelial cells (ECs), integral components of cerebral vasculature, play pivotal roles in upholding the blood-brain barrier (BBB) and modulating vascular tone and hemodynamics, thereby exerting influence on vascular function and homeostasis[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Previous research demonstrates the presence of inflammatory cytokines within the endothelium of spastic arteries, indicating the potential contribution of inflammatory factors to ECs damage[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNuclear factor-κB(NF-κB) is a key factor in the regulation of the expression of pro-inflammatory cytokines, adhesion molecules and enzymes[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. NF-κB-mediated pro-inflammatory signaling in endothelial cells and leukocytes induces the chronic inflammatory pathology atherosclerosis[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].Coactivator-associated arginine methyltransferase1(CARM1) is an arginine methyltransferase that controls gene transcription, which is know known to enhance transcriptional activation by nuclear receptors[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Recently, accumulative evidence showed that CARM1 can act as a coactivator for the transcription factor NF-κB and enhance its activity[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Increased expression of CARM1 is associated with elevated levels of pro-inflammatory mediators in atherosclerosis-related cardiovascular disease[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Thus, inhibition of CARM1 activity might be a promising strategy to reduce the pro-inflammatory response. However, it remains unclear whether CARM1 participate in the endothelial cell dysfunction after SAH. In this study, we established cell models of SAH to observe the effects of CARM1 on the endothelial cell dysfunction, we also apply TP-064 as CARM1 inhibitor to study the effects of TP-064 treatment on inflammation to explore the potential mechanisms in this pathophysiological process.\u003c/p\u003e"},{"header":"2.Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e2.1 Cell culture.\u003c/strong\u003e ECs were cultured in dulbecco modified eagle medium and ham\u0026rsquo;s F-12 medium (DMEM/DF12, Gibco, USA, 1:1) supplemented with 10% fetal bovine serum (FBS, Gibco, USA) in 5% CO2 at 37\u0026deg;C,with medium renewal every 48 hours. To simulate the pathophysiological conditions of subarachnoid hemorrhage (SAH) and determine the optimal stimulating concentration, cells were exposed to hemoglobin (Sigma, USA) at concentrations ranging from 5 \u0026micro;M to 20 \u0026micro;M for 24 hours prior to subsequent assays. Following hemoglobin exposure, western blot analysis was employed to assess the expression of CARM1 across different concentration groups.\u003c/p\u003e\n\u003cp\u003eCells were treated with 10 \u0026micro;M TP-064 dissolved in an equivalent concentration of dimethyl sulfoxide (DMSO) for 24 hours. Subsequently, cells were stimulated with hemoglobin for an additional 24 hours. Following stimulation, cells and supernatants were harvested and stored at \u0026minus;\u0026thinsp;20\u0026deg;C until further analysis of gene expression or cytokine levels.\u003c/p\u003e\n\u003cp\u003e\u003cspan\u003e\u003cstrong\u003e2.3 Western blotting.\u003c/strong\u003e Endothelial cells (ECs) cultured in T-25 flasks following the aforementioned treatments were subjected to triple washing, and the proteins were extracted using RIPA buffer supplemented with protease inhibitors (Biotime, China). The total protein concentration was determined using BCA Protein Assay Reagent (Beyotime, China). Subsequently, the protein samples were denatured by boiling and separated (20 \u0026micro;g) via 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer onto polyvinylidene difluoride membranes (PVDF, Bio-Rad, USA). The membranes were then blocked with 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 hour, and incubated overnight at 4\u0026deg;C with specific antibodies: CARM1 (Abcam, United Kingdom, 1:200), MPO (Abcam, United Kingdom, 1:500), ICAM1 (Abcam, United Kingdom, 1:200), NF-\u0026kappa;B (Abcam, United Kingdom, 1:400), GAPDH (ProteinTech Group, China, 1:1000), and Histone (1:1000, Proteintech). Blots were subsequently incubated with the appropriate horseradish peroxidase-conjugated IgG for 1 hour at 37\u0026deg;C and visualized using the Chemiluminescence Kit (Beyotime, China) with X-ray film. The optical densities of these bands were quantified using Quantity One software 4.6.2. GAPDH and Histone were employed as loading controls for whole cell and nuclear proteins.\u003cbr\u003e\u003c/span\u003e\u003cspan\u003e\u003cstrong\u003e2.4 ELISA.\u003c/strong\u003e The concentration of pro-inflammatory cytokines were analyzed in the medium of endothelial cells using the ELISA protocols provided by BD Biosciences and Biolegend (San Jose and San Diego, CA, USA). Absorbance measurements were conducted at 450 nm and 570 nm.\u003cbr\u003e\u003c/span\u003e\u003cspan\u003e\u003cstrong\u003e2.5 Immunofluorescence.\u003c/strong\u003e ECs were cultured on glass coverslips placed in 6-well plates at 65% confluence and pre-incubated TP-064. For immunostaining, cells were washed three times with PBS, fixed with 4% paraformaldehyde,, permeabilized with 0.01% Triton X-100 in PBS, and blocked with 10% goat serum for 30 minutes at 37\u0026deg;C. The cells were then incubated overnight at 4\u0026deg;C with the following primary antibodies: rabbit anti\u0026ndash;CARM1 (Abcam, United Kingdom, 1:200), mouse-CD31(Abcam, United Kingdom, 1:200), rabbit anti\u0026ndash;MPO (Abcam, United Kingdom, 1:200) followed by appropriate fluorescein isothiocyanate-conjugated and tetramethyl rhodamine isothiocyanate-conjugated secondary antibodies (Abbkine, USA, 1:200) for 1hours at room temperature. Subsequently, all cells were incubated with DAPI for 15minutes. Coverslips were mounted in antifade regent (Beyotime, China) and visualized by a fluorescence microscopy timely and effectively (Leica, Germany).\u003cbr\u003e\u003c/span\u003e\u003cspan\u003e\u003cstrong\u003e2.6 Gene expression analysis.\u003c/strong\u003e Total RNA was extracted from cells following the standard protocol outlined by Chomczynski and Sacchi[\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e]. RNA was reverse transcribed into cDNA using Maxima H Minus Reverse Transcriptase. The PCR cycle threshold (Ct) values were then determined after incorporation of SensiMix SYBR low-ROX mix, utilizing the ABI 7500 Real Time PCR system (Applied Biosystems, Foster City, CA, USA). The average Ct values of acidic ribosomal phosphoprotein P0 (36b4), hypoxanthine guanine phosphoribosyl transferase (Hprt), and ribosomal protein L27 (Rpl27) were employed as housekeeping genes.\u003cbr\u003e\u003c/span\u003e\u003cspan\u003e\u003cstrong\u003e2.7 Chromatin immunoprecipitation assay (ChIP)\u003c/strong\u003e The ChIP assay was conducted using a commercially available kit (Millipore) following the manufacturer\u0026apos;s instructions. Briefly, cells were crosslinked with 1% formaldehyde for 10 minutes, followed by quenching with glycine (0.125 M). Subsequently, the cell pellets were lysed in lysis buffer and sonicated for 5 minutes. The lysates were centrifuged, and the supernatants were incubated with specified antibodies overnight at 4\u0026deg;C. Immunocomplexes were captured using 30 \u0026micro;l of protein A/G sepharose on a rotator at 4\u0026deg;C for 2 hours. Following four times of washing, protein complex attached to the beads was dissolved in the SDS sample buffer and subsequently resolved in 7% SDSPAGE gel. All experiments were performed in three biological replicates.\u003cbr\u003e\u003c/span\u003e\u003cspan\u003e\u003cstrong\u003e2.8 Statistical Analysis.\u003c/strong\u003e Statistical analysis was carried out using SPSS 20.0 and GraphPad Prism 9.5. All values were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Multi-group comparisons were conducted through one-way analysis of variance (ANOVA) followed by Bonferroni post hoc analysis. Each experiment was repeated a minimum of three times. A significance level of P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was deemed statistically significant.\u003cbr\u003e\u003c/span\u003e\u003c/p\u003e\n"},{"header":"3.Results","content":"\u003cp\u003e\u003cstrong\u003e3.1 Hemoglobin Induced increased CARM1 in Cultured Cerebral ECs.\u003c/strong\u003e The cells were treated with varying concentrations of hemoglobin, and the expression of CARM1 and NF-\u0026kappa;B were examined by western blot analysis after 24 hours. As depicted in Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA, elevated levels of CARM1 and NF-\u0026kappa;B were detected within cultured ECs following hemoglobin treatment. The expression of both proteins reached maximal levels with a hemoglobin concentration of 10 \u0026micro;M. However, excessive hemoglobin dosage led to a decrease in the expression of both proteins(Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea,b,c). Based on the preliminary data, a concentration of 10 \u0026micro;M (optimal stimulating concentration) was chosen for subsequent experiments. Following a 24-hour incubation with hemoglobin, a significant increase in the expression of CARM1 and NF-\u0026kappa;B was observed compared to the control group. Similarly, an enhanced immunoreactivity of CARM1 was observed within cultured ECs after hemoglobin treatment (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ed).\u003c/p\u003e\n\u003cp\u003e3.2 TP-064 treatment inhibited the expression of CARM1 and attenuated the hemoglobin-induced pro-inflammatory response of endothelial cells (ECs) in vitro. To investigate the effects of CARM1 on the pro-inflammatory response of ECs, we sought to inhibit CARM1 expression using TP-064. As illustrated in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea,b, immunoblot results demonstrated a significant increase in CARM1 expression following hemoglobin incubation, which was reversed by TP-064 treatment. Additionally, TP-064 markedly reduced the expression of NF-\u0026kappa;B, ICAM1, and MPO (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea, c, d, e). Similarly, ELISA results revealed an elevation in the expression of inflammatory cytokines (TNF-\u0026alpha;, IL-1\u0026beta;, and IL-12) induced by hemoglobin, which was mitigated by TP-064 treatment (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ed, e, f). Immunofluorescence analysis showed colocalization of the neutrophil marker (MPO) with a specific marker of endothelial cells (CD31) in ECs. These findings indicate that TP-064 treatment significantly suppressed neutrophil infiltration and the inflammatory response (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ef).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 TP-064 treatment downregulates inflammation-related gene expression.\u003c/strong\u003e Consistently, the inhibition of CARM1 activity by TP-064 was associated with a decrease in NF-\u0026kappa;B mRNA expression levels. Our current in vitro study demonstrated that TP-064-mediated inhibition of CARM1 resulted in decreased expression levels of ICAM1 and MPO in endothelial cells (ECs) (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea, b, c).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 CARM1 enhances NF-\u0026kappa;B activation through Methylation.\u003c/strong\u003e As confirmed previously, CARM1 upregulated NF-\u0026kappa;B expression in endothelial cells (ECs), suggesting a potential modulation of NF-\u0026kappa;B\u0026apos;s transcriptional activity by CARM1. As expected, our co-immunoprecipitation (co-IP) assays demonstrated the interaction between CARM1 and NF-\u0026kappa;B in ECs (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea). Subsequently, we investigated whether NF-\u0026kappa;B served as a substrate for CARM1. ECs underwent co-IP with immunoglobulin G (IgG) or anti-NF-\u0026kappa;B antibodies, followed by immunoblotting with an asymmetric dimethylarginine (ADMA)-specific antibody (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eb). The presence of methylated NF-\u0026kappa;B in the immunoprecipitation (IP) lysates indicated the methylation of NF-\u0026kappa;B by CARM1 in vitro.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5 TP-064 treatment alleviated hemoglobin induced endothelial dysfunction.\u003c/strong\u003e To assess the impact of TP-064 treatment on endothelial dysfunction levels of endothelin-1 (ET-1) and nitric oxide (NO) were measured using enzyme-linked immunosorbent assay (ELISA). Following hemoglobin treatment, ET-1 levels increased while NO levels decreased in endothelial cells (ECs). We demonstrated that the CARM1 inhibitor TP-064 effectively reduced ET-1 levels and increased NO levels (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea, b), indicating a beneficial effect of CARM1 inhibition on endothelial dysfunction.\u003c/p\u003e"},{"header":"4.Discussion","content":"\u003cp\u003eIn the current study, we explored the potential impact of the CARM1 inhibitor TP-064 on endothelial cell dysfunction following SAH in vitro. The primary findings from our study are as follows: 1) CARM1-mediated NF-κB activation through methylation contributes to inflammatory response and endothelial cell dysfunction; 2) Regulation of CARM1 expression by TP-064 in cultured cerebral ECs effectively alleviates inflammation and endothelial cell dysfunction after hemoglobin incubation.\u003c/p\u003e \u003cp\u003eIn the cerebral vasculature, healthy endothelial cells regulate blood viscosity, control blood flow, and maintain the integrity of the blood-brain barrier (BBB). [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Previous research has described inflammation in blood vessels as involving leukocyte-endothelial cell interactions and the release of inflammatory mediators, resulting in endothelial cell integrity disruption and subsequent dysfunction [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Hemoglobin, a product of blood hemolysis after SAH, oxidizes into various forms, becoming a potent pro-inflammatory and cytotoxic molecule known to activate NF-κB.[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Hemoglobin-induced endothelial damage and apoptosis, neuroinflammation, and BBB disruption have been reported[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Following hemoglobin incubation with ECs to mimic SAH neuropathology, our results revealed heightened NF-κB expression.\u003c/p\u003e \u003cp\u003eCoactivator-associated arginine methyltransferase 1 (CARM1) acts as a coactivator and a novel transcriptional regulator of NF-κB-mediated inflammatory gene expression[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].However, the expression level and role of CARM1 in ECs following hemoglobin treatment have yet to be determined. Our study demonstrated a significant increase in both mRNA and protein levels of CARM1 in ECs after hemoglobin treatment compared to controls, with CARM1 also colocalizing with ECs. Our co-IP assay confirmed the interaction between CARM1 and NF-κB, suggesting that CARM1 interacts with NF-κB to catalyze its methylation and restrict its nuclear localization, thereby promoting inflammation. Elevated levels of inflammatory cytokines (TNF-α, IL-1β, and IL-12) correlated with increased expression of CARM1 and NF-κB. Additionally, CARM1 mRNA levels positively correlated with mRNA expression levels of NF-κB, MPO, and ICAM1 in ECs following hemoglobin treatment. To our knowledge, this study is the first to report elevated CARM1 levels in ECs following hemoglobin treatment, further supporting a role for CARM1 in the inflammatory response.\u003c/p\u003e \u003cp\u003eRecent studies have identified TP-064 as a selective and potent inhibitor of CARM1 function [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Consistent with previous observations that CARM1 acts as an NF-κB coactivator, our research showed that TP-064 reduced pro-inflammatory cytokine secretion[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Western blot analysis revealed increased expression of MPO and ICAM-1 following hemoglobin treatment, which was significantly reduced by TP-064 administration. The administration of TP-064 significantly decreased the expression of MPO and ICAM-1. Furthermore, MPO is a marker of leukocyte activity[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], and fluorescence microscopy demonstrated MPO colocalization with CD31, indicating leukocyte infiltration into ECs. We also observed that TP-064 suppressed neutrophil infiltration in ECs following hemoglobin treatment. These findings collectively suggest that TP-064 inhibits neutrophil infiltration, possibly through decreased ICAM-1 and MPO expression.\u003c/p\u003e \u003cp\u003eEndothelial cells regulate vascular tone and blood flow through a delicate balance of vasoconstrictors such as endothelin-1 (ET-1) and vasodilators such as nitric oxide (NO)[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Animal studies have shown that SAH can induce functional changes in vascular endothelium, resulting in reduced NO levels and increased ET-1 levels[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In our investigation, TP-064 treatment significantly reduced ET-1 levels and increased NO levels, resulting in significant improvement in endothelial dysfunction following hemoglobin incubation in the TP-064 treatment group.\u003c/p\u003e \u003cp\u003eIn conclusion, these findings imply that inhibition of CARM1 function by TP-064 may represent a promising novel therapeutic approach for NF-κB-driven pathologies, a matter of considerable importance in elucidating the molecular mechanisms underlying endothelial dysfunction after SAH. Further validation will be conducted in subsequent animal experiments.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical Statements:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIt is not applicable.\u003c/p\u003e\u003cp\u003eConflicts of interest\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing financial interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Acknowledgements\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe acknowledge the service provided by the Key Laboratory of Chongqing University Central Hospital.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Post-Doctoral Science Fund of Chongqing Natural Science Foundation(cstc2021jcyj-bshX0051), National Natural Science Foundation of China(82160222), Guizhou Provincial Science and Technology Projects (Qiankehe Foundation ZK (2022) General 261), Guizhou Provincial People\u0026rsquo;s Hospital National Science Foundation(GPPH-NSFC-2021-14).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u003c/strong\u003eLiu Liu and Min Wu conceived and designed the project, Qingtao Zhang and Ping Zhang executing all experiments and contributing to manuscript preparation. Yidan Liang, Qiang Yang, Min Cui, and Chao Sun assisted in completing specific experiments.\u0026nbsp;Yongbing Deng\u0026nbsp;and Weiduo Zhou conducted data analysis, Lei Xu participated in manuscript revision, particularly focusing on language refinement. All authors critically reviewed and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement:\u0026nbsp;\u003c/strong\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLawton MT and Vates GE (2017) Subarachnoid Hemorrhage. 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Biochem Biophys Res Commun 280:824-30. doi: 10.1006/bbrc.2000.4167\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":"CARM1, inflammation, endothelial dysfunction, Methylation, subarachnoid hemorrhage","lastPublishedDoi":"10.21203/rs.3.rs-4432703/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4432703/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCerebral endothelial cell dysfunction plays a critical role in the pathophysiology of vascular injury subsequent to subarachnoid hemorrhage (SAH), yet the precise molecular mechanism remains largely speculative. Inflammation stands out as a pivotal contributor to an unfavorable prognosis post-SAH, with nuclear factor-κB (NF-κB) pathways being initiated and ultimately leading to inflammation activation and pro-inflammatory cytokine release following SAH. In this study, we explored the impact of the Coactivator-associated arginine methyltransferase 1 (CARM1) inhibitor TP-064 on inflammation using an in vitro SAH model. Exposure of endothelial cells to TP-064 resulted in a significant reduction in CAMR1 and NF-κB expression upon hemoglobin exposure. Similarly, endothelial cells treated with TP-064 following hemoglobin incubation exhibited decreased expression levels of intercellular adhesion molecule-1 (ICAM1), myeloperoxidase (MPO), and cytokine production including interleukin-1β (IL-1β), interleukin-12 (IL-12), tumor necrosis factor-α (TNF-α) in response to hemoglobin exposure. Moreover, subsequent investigations demonstrated that CARM1 transcriptionally regulates NF-κB via methylation. Additionally, TP-064 notably mitigated endothelial dysfunction. Collectively, our findings identify TP-064 as a CARM1 inhibitor targeting inflammation and neutrophil infiltration, offering new insights into therapeutic strategies for addressing endothelial cell dysfunction following SAH.\u003c/p\u003e","manuscriptTitle":"CARM1 inhibitor TP064 attenuates endothelial cell dysfunction via inhibits inflammatory response in vitro model of subarachnoid hemorrhage","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-30 10:52:40","doi":"10.21203/rs.3.rs-4432703/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":"361024bf-03f0-42fc-a58d-130ffa322df9","owner":[],"postedDate":"May 30th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-08-12T12:42:55+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-30 10:52:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4432703","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4432703","identity":"rs-4432703","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}