Mendelian randomization study on the causal link between neutrophil extracellular traps and cardiovascular diseases

Mendelian randomization study on the causal link between neutrophil extracellular traps and cardiovascular diseases | 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 Mendelian randomization study on the causal link between neutrophil extracellular traps and cardiovascular diseases Siping Peng, Tao Hu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7056612/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 investigates the causal relationship between neutrophil extracellular traps (NETs) and cardiovascular diseases (CVDs) using two-sample Mendelian randomization (MR). Methods We performed a two-sample MR analysis utilizing GWAS summary statistics from the GWAS Catalog and IEU databases for six cardiovascular outcomes and various NET-related exposures. The main analysis employed the inverse variance-weighted (IVW) method, with supplementary analyses using MR-Egger, weighted median, and weighted mode methods. Results Genetic predisposition to higher neutrophil counts was found to increase the risk of coronary artery disease (OR = 1.0829, 95% CI: 1.0317–1.1366, P = 0.001). No causal link between NET measurements and CVDs was detected (OR = 1.0014, 95% CI: 0.9942–1.0087, P = 0.698), nor for other NET-related exposures (All P > 0.05). Sensitivity analyses confirmed the robustness of these findings. Conclusion Our results indicate that elevated neutrophil counts may heighten coronary artery disease risk. Further studies should investigate the mechanisms underlying this association and explore therapeutic interventions targeting neutrophils in CVD prevention and treatment. neutrophil extracellular traps cardiovascular diseases neutrophil count Mendelian randomization causal inference Figures Figure 1 Figure 2 Introduction Cardiovascular diseases (CVDs) represent a group of disorders affecting the heart and blood vessels, including heart failure, myocardial infarction, coronary artery disease (CAD), atrial fibrillation, stroke, and atherosclerosis. These conditions collectively constitute the leading cause of morbidity and mortality worldwide, accounting for an estimated 31% of all global deaths[ 1 ]. The prevalence and incidence of CVDs continue to rise, posing significant challenges to healthcare systems and economies globally. The clinical manifestations of CVDs are multifaceted and can range from subtle symptoms like chest pain and shortness of breath to life-threatening events such as sudden cardiac arrest and stroke[ 2 ]. The known risk factors for cardiovascular disease include age, sex, unhealthy lifestyle, systemic autoimmune diseases and hypertension and diabetes[ 3 , 4 ]. Given the substantial burden of CVDs on public health, understanding the underlying risk factors and mechanisms driving their development is paramount for effective prevention and treatment strategies. Neutrophil Extracellular Traps (NETs) are web-like structures composed of DNA, histones, and antimicrobial proteins released by activated neutrophils as part of the innate immune response[ 5 ]. Interleukin-4 (IL-4), IL-13, IL5 can interact with the IL receptor of neutrophil and promote the production of NETs[ 6 ]. Initially discovered as a defense mechanism against pathogens, NETs have recently emerged as potential contributors to various pathological conditions, including cardiovascular diseases[ 7 ]. Clinical observational studies have reported associations between NETs and several cardiovascular disorders. Elevated circulating levels of NET components, such as cell-free DNA and myeloperoxidase (MPO), accompanying with the accumulation of neutrophil and increased levels of certain cytokines, have been associated with an increased risk of CAD, stroke, and heart failure[ 8 – 10 ]. Circulating NETs have been correlated with CVDs disease severity and poor prognosis[ 11 ]. The presence of NETs in coronary artery plaques suggests their involvement in plaque instability and progression of CAD[ 11 ]. In atrial fibrillation, NETs have been detected in left atrial appendage thrombi, potentially contributing to thrombogenesis[ 12 ]. Ischemic stroke patients have shown a positive correlation between NET levels and infarct size, as well as neurological deficit severity[ 8 ]. Furthermore, NETs have been associated with endothelial dysfunction and foam cell formation in atherosclerosis[ 13 ]. While these observational studies suggest a link between NETs and CVDs, the causal nature of this relationship remains unclear. To address this gap, we employed a Mendelian randomization (MR) approach, which utilizes genetic variants as instrumental variables (IVs) to infer causality[ 14 , 15 ]. It can provide evidence for potential causal associations by leveraging genetic variants that are less prone to confounding and reverse causation, which is useful in informing public health policies and guiding interventions for various diseases and conditions [ 14 , 16 ]. This study aimed to elucidate the causal direction, if any, between NETs-related traits, including NETs measurement, neutrophil count, MPO and several interleukins (IL), and CVDs through a two-sample MR study, providing insights into the etiology and potential therapeutic targets for this condition. Methods Study Design and Data Sources We applied a two-sample MR design to evaluate the causal relationship between NETs (NETs measurement, neutrophil count, IL-4, IL-5, IL-13, MPO and MPO-DNA) and CVDs (heart failure, myocardial infarction, CAD, atrial fibrillation, stroke, and atherosclerosis). The study followed the three key assumptions of MR: the IVs are associated with the exposure, independent of any confounders of the exposure-outcome association, and only affect the outcome through the exposure. The article adheres to the STROBE-MR checklist for reporting MR studies [ 15 ]. GWAS summary statistics for CVDs were obtained from the IEU and GWAS catalog databases. For instance, GWSA dataset for CAD includes 122,733 cases and 424,528 controls from European. GWSA dataset for heart failure includes 47,309 cases and 930,014 controls from European. For exposures related to NETs, we utilized GWAS data from the GWAS Catalog. Detailed sample characteristics and GWAS information are provided in the Table S1 . This study is based on publicly available summary statistics; therefore, no ethical approval is required. Instrumental Variable Selection SNPs were selected based on genome-wide significance (P < 5 * 10 − 8 ) for neutrophil count and MPO, and a more lenient threshold (P 0.01, excluded SNPs with linkage disequilibrium (LD) R 2 > 0.001 within 10,000 kb windows[ 18 ], and replaced unavailable SNPs with proxies having R 2 > 0.8[ 19 ]. To assess IV strength and mitigate potential weak instrument bias between IVs and exposure factors, the F-statistic was calculated for each SNP in the IV set. The calculation formula is as follows: F = R 2 * (N-2) / (1-R 2) where R 2 represents the proportion of exposure variance explained by the SNP in the IV. The F-statistic was required to be > 10[ 18 ]. MR Analysis We employed an inverse variance weighted (IVW) method as the primary analysis, complemented by weighted median, weighted mode, and MR-Egger regression for sensitivity assessments[ 20 ]. MR methods estimate odds ratios (OR) and 95% confidence intervals (CIs) for the causal effect[ 21 ]. The MR-Egger method was employed to investigate the presence of an intercept and provide precise causal effect estimates in the presence of pleiotropic bias[ 22 ]. In contrast, the weighted median approach relies on the assumption that at least 50% of the instrumental variables are valid for evaluating the causal relationship between exposure and outcome [ 23 ]. All analyses were performed using the "TwoSampleMR" package (V 0.5.11) within the R (V 4.0.5). Visual representations were generated through scatter plots and sensitivity analysis diagrams. Statistical significance was defined as an P 0.05 indicating no heterogeneity[ 25 ]. To account for the potential impact of genetic variation pleiotropy on association effect estimates, the MR-Egger regression approach was applied to investigate the presence of horizontal pleiotropy. A null intercept or lack of statistical significance in the MR-Egger regression suggests the absence of pleiotropy. Additionally, the MR-PRESSO method was employed to identify potential outlier single nucleotide polymorphisms (SNPs) with p < 0.05, and the causal association was reassessed following their exclusion [ 26 ]. This procedure corrects for horizontal pleiotropy. Leave-one-out analysis was conducted to remove each SNP step by step, calculate the meta-effect of the remaining SNPs, and observe whether the results change after removing each SNP [ 27 ]. Results Incorporation of instrumental variables We extracted SNPs from GWAS summary statistics of each phenotypic exposure under a well-recognized threshold. In total, we select 12, 6, 12, 10, 17, 429, 5 IVs for IL-4 levels, IL-5 levels, IL-13 levels, blood protein levels of MPO-DNA complexes, NETs measurement, neutrophil count and blood protein levels of MPO. The mean value of the F-statistic of IVs is calculated to be over 20, reflecting strong instrument validity ( Table S2 ). In addition, there were 8 proxy SNPs were used to replace SNPs, when using ‘atherosclerosis’ as the outcome ( Table S3 ). This process ensured a robust set of instruments for MR analysis across different exposures. The casual effect of the NETs-related traits on CVDs As shown in Table 1 , the scatter plot (Fig. 2 A), and the forest plot (Fig. 2 B), our MR analysis indicated a significant association between neutrophil count (OR = 1.0829, 95% CI: 1.0317–1.1366, P = 0.001) and CAD. The results of MR Egger and Weighted median methods verified it. Which suggests that neutrophil count play the protective effect on the development of CAD. No significant causal effects were detected for other NETs-related traits (All P > 0.05, Table S4 ). For example, there was no evidence of causal effect between NETs measurement and CAD (OR = 1.0014, 95% CI: 0.9942–1.0087, P = 0.698). Table 1 MR analyses showed a causal relationship between Neutrophil count and CAD. Exposure Outcome Methods OR (95% CI) P Neutrophil count Coronary artery disease IVW 1.0829 ( 1.0317–1.1366 ) 0.001 MR Egger 1.1155 ( 1.0144–1.2267 ) 0.024 Weighted median 1.0882 ( 1.0124–1.1696 ) 0.022 Weighted mode 1.0490 ( 0.9664–1.1387 ) 0.254 Heterogeneity, directional pleiotropy, and horizontal pleiotropy among the genetic instruments were evaluated using MR-Egger regression analysis, Cochran's Q test, and the MR-PRESSO test. The Q test results showed that there was heterogeneity in several NETs-related traits, such as blood protein levels of myeloperoxidase-DNA complexes (P = 0.025) as exposure and atherosclerosis as outcome ( Table S5 ). Since the IVW method we primarily use is based on random effects, it can accept a certain degree of heterogeneity (Fig. 2 C). MR-Egger regression analysis did not find horizontal pleiotropy. However, the MR-PRESSO results showed that there were outliers in many exposures ( Table S6 ), but the removal of outliers did not affect our results. The analysis results are now presented after outlier correction. The stability of our findings was validated by leave-one-out analyses, which verified that the exclusion of any individual SNP did not substantially affect the observed associations (Fig. 2 D). Discussion Our MR study provides evidence for a causal role of neutrophil count in the development of CAD. In addition, there was no evidence from MR analyses indicating that other types of NETs-related traits, increased or decreased the risk of CVDs. Our results suggest that targeting neutrophil activity could be a promising therapeutic strategy for preventing or treating CAD. However, further mechanistic studies are warranted to understand the specific pathways involved and to identify potential biomarkers for clinical intervention. Our results suggest that circulating neutrophil count is a risk factor for CAD. Over the past 2 decades, numerous studies have described the association between elevated neutrophil counts and increased risk of CAD. High neutrophil counts and low lymphocyte counts within the normal clinical range have demonstrated a strong linear correlation with all stages of CAD[ 28 , 29 ]. A European cohort study indicated that neutrophil counts are associated with the incidence of hypoechoic plaques and disease progression in CAD patients[ 30 ]. Similar findings have been reported in large-scale Asian cohorts; for instance, the Chinese Dongfeng-Tongji cohort found that higher neutrophil and monocyte counts were associated with increased CAD risk[ 31 ]. These findings align with our research results, suggesting a positive correlation between elevated neutrophil counts and increased CAD risk across diverse age groups, genders, and ethnicities. Mechanistically, research over the past decade has elucidated the crucial role of neutrophils in various stages of CAD-related inflammation. At sites of vascular inflammation, activated neutrophils employ several synergistic strategies to fulfill their functions, including the release of reactive oxygen species (ROS), degranulation to release proteolytic enzymes, secretion of pro-inflammatory alarmins (such as S100A8/A9) and proteases (including cathepsin G, neutrophil elastase, and myeloperoxidase), as well as the formation of NETs[ 32 ]. Excessive ROS secretion leads to endothelial cell dysfunction and activation, extracellular matrix degradation, enhanced monocyte adhesion and recruitment, and facilitates the transfer of low-density lipoprotein (LDL) from the lumen to the arterial intima [ 33 ]. ROS mediates the formation of oxidized LDL and may activate matrix metalloproteinases, potentially leading to plaque rupture [ 34 ]. Neutrophils also participate in immune system regulation, interacting with T cells, macrophages, and other immune cells, influencing the overall inflammatory response. In certain circumstances, neutrophil hyperactivation and excessive inflammation may damage the myocardium, leading to cardiomyocyte apoptosis and necrosis [ 35 ]. Although our results indicate an association between neutrophils and CAD risk, we did not find a correlation between NETs and the risk of CAD or other CVDs. However, this does not negate the role of NETs in CVDs. NETs may play an indirect role in CVDs that may not be apparent in MR studies. A study identified the contribution of neutrophils, particularly NETs, in promoting plaque instability by precisely facilitating necrotic core growth and fibrous cap thinning[ 36 ]. Mechanistically, regulatory smooth muscle cells in the fibrous cap physically interact with neutrophils, leading to their activation, ROS production, and NETs release, mediated by smooth muscle cell-derived CCL7[ 37 ]. Thus, the involvement of NETs in CVD progression may depend on activated neutrophils and systemic inflammatory responses. In vitro studies have shown that NETs kill smooth muscle cells. This effect is not dependent on neutrophil proteins derived from granules or cytoplasm, but rather on histones, particularly histone H4, consistent with earlier reports on the cytotoxic activity of NET-borne histones[ 37 , 38 ]. Currently, the lack of GWAS data on histone H4 limits further validation, which needs to be addressed in future research. This study presents several notable strengths. It marks the inaugural use of two-sample MR analysis to investigate potential causal relationships between the NETs related traits and CVD. Traditional observational studies are more susceptible to bias due to confounding variables and the possibility of reverse causation. Furthermore, the epidemiological impact of MR analysis is substantial, and its application is likely to continue increasing in the coming years. Nevertheless, there are certain limitations to the conclusions that can be drawn from this study. Primarily, the use of summary statistics rather than individual-level data precludes additional subgroup analyses, such as distinguishing between different types of CVDs. Moreover, MR studies typically require a large sample size, and the current study sample size for NETs (N = 657) may be insufficient, affecting the reliability of the findings. Conclusion In conclusion, our findings support a causal link between neutrophil count and CAD, highlighting the importance of neutrophil biology in cardiovascular health and disease. Future research should aim to translate these findings into clinical applications for better management of CVDs. Declarations Ethics approval and consent to participate Not applicable. Conflict of interests All authors declare that they have no any conflict of interests. Funding None. Author Contribution Siping Peng carried out the studies, participated in collecting data, and drafted the manuscript. Tao Hu performed the statistical analysis and participated in its design. Siping Peng and Tao Hu participated in acquisition, analysis, or interpretation of data and draft the manuscript. All authors read and approved the final manuscript. Availability of data and materials All data generated or analysed during this study are included in this published article. References Lavie CJ. Cardiovascular statistics 2023. Prog Cardiovasc Dis. 2023;79:112–3. Bułdak Ł. Cardiovascular Diseases-A Focus on Atherosclerosis, Its Prophylaxis, Complications and Recent Advancements in Therapies. Int J Mol Sci. 2022;23. Wong ND, Sattar N. Cardiovascular risk in diabetes mellitus: epidemiology, assessment and prevention. Nat Rev Cardiol. 2023;20:685–95. Funada S, Luo Y, Nishioka N, Yoshioka T. Cardiovascular risk in systemic autoimmune diseases. Lancet. 2023;401:21. Ravindran M, Khan MA, Palaniyar N. Neutrophil Extracellular Trap Formation: Physiology, Pathology, and Pharmacology. Biomolecules. 2019;9. Impellizzieri D, Ridder F, Raeber ME, Egholm C, Woytschak J, Kolios AGA et al. IL-4 receptor engagement in human neutrophils impairs their migration and extracellular trap formation. 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Associations of Baseline and Changes in Leukocyte Counts with Incident Cardiovascular Events: The Dongfeng-Tongji Cohort Study. J Atheroscler Thromb. 2022;29:1040–58. Luo J, Thomassen JQ, Nordestgaard BG, Tybjærg-Hansen A, Frikke-Schmidt R. Neutrophil counts and cardiovascular disease. Eur Heart J. 2023;44:4953–64. Sugamura K, Keaney JF. Jr. Reactive oxygen species in cardiovascular disease. Free Radic Biol Med. 2011;51:978–92. Moris D, Spartalis M, Spartalis E, Karachaliou GS, Karaolanis GI, Tsourouflis G, et al. The role of reactive oxygen species in the pathophysiology of cardiovascular diseases and the clinical significance of myocardial redox. Ann Transl Med. 2017;5:326. Fan Q, Tao R, Zhang H, Xie H, Lu L, Wang T, et al. Dectin-1 Contributes to Myocardial Ischemia/Reperfusion Injury by Regulating Macrophage Polarization and Neutrophil Infiltration. Circulation. 2019;139:663–78. Döring Y, Libby P, Soehnlein O. Neutrophil Extracellular Traps Participate in Cardiovascular Diseases: Recent Experimental and Clinical Insights. Circ Res. 2020;126:1228–41. Silvestre-Roig C, Braster Q, Wichapong K, Lee EY, Teulon JM, Berrebeh N, et al. Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death. Nature. 2019;569:236–40. Saffarzadeh M, Juenemann C, Queisser MA, Lochnit G, Barreto G, Galuska SP, et al. Neutrophil extracellular traps directly induce epithelial and endothelial cell death: a predominant role of histones. PLoS ONE. 2012;7:e32366. Additional Declarations No competing interests reported. Supplementary Files SupplementatyTables.docx TableS2IVsusedinthisstudy.xlsx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7056612","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":502572892,"identity":"f67e6bed-0b8e-4b5c-abb0-cfd16751f9c0","order_by":0,"name":"Siping Peng","email":"","orcid":"","institution":"Jiangxi Pingxiang People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Siping","middleName":"","lastName":"Peng","suffix":""},{"id":502572896,"identity":"c7054b6d-1cb9-4400-8e35-e24251ce10d9","order_by":1,"name":"Tao Hu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzUlEQVRIiWNgGAWjYBAC++P9Hx9IGLDJsbE3EKvnzAFjA4sCPmN+ngPEarnhYCZR8UEuceaMBCJ1MM5gSJO4YWDGuOHm4403GGpsoglqYZZuOGw5wyCN2eB2WrEFw7G03AZCWthkDjbeljA4xmZwO8dMgrHhMGEtPBLJDNJ/DP7zGNw8Q6QWCYk0JglgIEtIzuAhUosBzxlmA6AWA34eoF8SiPGLAXsP4wOJP2z1beyHN974UGNDWAuKdokEUpRDtJCqYxSMglEwCkYGAAAENj0QLeMQUgAAAABJRU5ErkJggg==","orcid":"","institution":"Jiangxi Provincial People's Hospital, The First Affiliated Hospital of Nanchang Medical College","correspondingAuthor":true,"prefix":"","firstName":"Tao","middleName":"","lastName":"Hu","suffix":""}],"badges":[],"createdAt":"2025-07-06 08:23:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7056612/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7056612/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":89696614,"identity":"60066225-5af1-4b68-9987-49a1d25f2fd8","added_by":"auto","created_at":"2025-08-22 18:17:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":533531,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFlowchart of the Mendelian Randomization (MR) study.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7056612/v1/b0ab6544f041ce5e3c1a9eb3.png"},{"id":89697393,"identity":"21ca7918-1512-47f4-8788-b714c48bf9c1","added_by":"auto","created_at":"2025-08-22 18:25:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4013050,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of the casual effect of\u003c/strong\u003e \u003cstrong\u003eneutrophil count on CAD. through MR.\u003c/strong\u003e (A) A scatter plot illustrates the association between neutrophil count and CAD risk. (B) The MR estimate is depicted using a forest plot. (C) A funnel plot assesses consistency and potential heterogeneity. (D) Leave-one-out analysis confirms the robustness of the causal inference.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7056612/v1/1709f1e549f6e08608098042.png"},{"id":93065717,"identity":"44d3e02e-341a-4ab3-84d1-f9497e96cc11","added_by":"auto","created_at":"2025-10-08 16:45:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5403829,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7056612/v1/e6158b72-315b-4867-9400-ae3643a69347.pdf"},{"id":89696611,"identity":"70ccfd1c-e67b-43a7-8432-97444154108c","added_by":"auto","created_at":"2025-08-22 18:17:48","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":34632,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementatyTables.docx","url":"https://assets-eu.researchsquare.com/files/rs-7056612/v1/b076100d73b48aaadc1156f3.docx"},{"id":89696613,"identity":"5e803772-4797-45df-975d-db76c2dbc7c5","added_by":"auto","created_at":"2025-08-22 18:17:48","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":66240,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2IVsusedinthisstudy.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7056612/v1/9fe9781a7975202984f3c60b.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Mendelian randomization study on the causal link between neutrophil extracellular traps and cardiovascular diseases","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCardiovascular diseases (CVDs) represent a group of disorders affecting the heart and blood vessels, including heart failure, myocardial infarction, coronary artery disease (CAD), atrial fibrillation, stroke, and atherosclerosis. These conditions collectively constitute the leading cause of morbidity and mortality worldwide, accounting for an estimated 31% of all global deaths[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The prevalence and incidence of CVDs continue to rise, posing significant challenges to healthcare systems and economies globally. The clinical manifestations of CVDs are multifaceted and can range from subtle symptoms like chest pain and shortness of breath to life-threatening events such as sudden cardiac arrest and stroke[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The known risk factors for cardiovascular disease include age, sex, unhealthy lifestyle, systemic autoimmune diseases and hypertension and diabetes[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Given the substantial burden of CVDs on public health, understanding the underlying risk factors and mechanisms driving their development is paramount for effective prevention and treatment strategies.\u003c/p\u003e\u003cp\u003eNeutrophil Extracellular Traps (NETs) are web-like structures composed of DNA, histones, and antimicrobial proteins released by activated neutrophils as part of the innate immune response[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Interleukin-4 (IL-4), IL-13, IL5 can interact with the IL receptor of neutrophil and promote the production of NETs[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Initially discovered as a defense mechanism against pathogens, NETs have recently emerged as potential contributors to various pathological conditions, including cardiovascular diseases[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Clinical observational studies have reported associations between NETs and several cardiovascular disorders. Elevated circulating levels of NET components, such as cell-free DNA and myeloperoxidase (MPO), accompanying with the accumulation of neutrophil and increased levels of certain cytokines, have been associated with an increased risk of CAD, stroke, and heart failure[\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Circulating NETs have been correlated with CVDs disease severity and poor prognosis[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The presence of NETs in coronary artery plaques suggests their involvement in plaque instability and progression of CAD[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In atrial fibrillation, NETs have been detected in left atrial appendage thrombi, potentially contributing to thrombogenesis[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Ischemic stroke patients have shown a positive correlation between NET levels and infarct size, as well as neurological deficit severity[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Furthermore, NETs have been associated with endothelial dysfunction and foam cell formation in atherosclerosis[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. While these observational studies suggest a link between NETs and CVDs, the causal nature of this relationship remains unclear.\u003c/p\u003e\u003cp\u003eTo address this gap, we employed a Mendelian randomization (MR) approach, which utilizes genetic variants as instrumental variables (IVs) to infer causality[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. It can provide evidence for potential causal associations by leveraging genetic variants that are less prone to confounding and reverse causation, which is useful in informing public health policies and guiding interventions for various diseases and conditions [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. This study aimed to elucidate the causal direction, if any, between NETs-related traits, including NETs measurement, neutrophil count, MPO and several interleukins (IL), and CVDs through a two-sample MR study, providing insights into the etiology and potential therapeutic targets for this condition.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cb\u003eStudy Design and Data Sources\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe applied a two-sample MR design to evaluate the causal relationship between NETs (NETs measurement, neutrophil count, IL-4, IL-5, IL-13, MPO and MPO-DNA) and CVDs (heart failure, myocardial infarction, CAD, atrial fibrillation, stroke, and atherosclerosis). The study followed the three key assumptions of MR: the IVs are associated with the exposure, independent of any confounders of the exposure-outcome association, and only affect the outcome through the exposure. The article adheres to the STROBE-MR checklist for reporting MR studies [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eGWAS summary statistics for CVDs were obtained from the IEU and GWAS catalog databases. For instance, GWSA dataset for CAD includes 122,733 cases and 424,528 controls from European. GWSA dataset for heart failure includes 47,309 cases and 930,014 controls from European. For exposures related to NETs, we utilized GWAS data from the GWAS Catalog. Detailed sample characteristics and GWAS information are provided in the \u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e. This study is based on publicly available summary statistics; therefore, no ethical approval is required.\u003c/p\u003e\u003cp\u003e\u003cb\u003eInstrumental Variable Selection\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSNPs were selected based on genome-wide significance (P\u0026thinsp;\u0026lt;\u0026thinsp;5 * 10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e) for neutrophil count and MPO, and a more lenient threshold (P\u0026thinsp;\u0026lt;\u0026thinsp;5 * 10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e) for other exposures due to limited SNP availability[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. We required minor allele frequencies (MAF)\u0026thinsp;\u0026gt;\u0026thinsp;0.01, excluded SNPs with linkage disequilibrium (LD) R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.001 within 10,000 kb windows[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], and replaced unavailable SNPs with proxies having R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.8[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. To assess IV strength and mitigate potential weak instrument bias between IVs and exposure factors, the F-statistic was calculated for each SNP in the IV set. The calculation formula is as follows: F\u0026thinsp;=\u0026thinsp;R\u003csup\u003e2\u003c/sup\u003e * (N-2) / (1-R\u003csup\u003e2)\u003c/sup\u003e where R\u003csup\u003e2\u003c/sup\u003e represents the proportion of exposure variance explained by the SNP in the IV. The F-statistic was required to be \u0026gt;\u0026thinsp;10[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cb\u003eMR Analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe employed an inverse variance weighted (IVW) method as the primary analysis, complemented by weighted median, weighted mode, and MR-Egger regression for sensitivity assessments[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. MR methods estimate odds ratios (OR) and 95% confidence intervals (CIs) for the causal effect[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The MR-Egger method was employed to investigate the presence of an intercept and provide precise causal effect estimates in the presence of pleiotropic bias[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In contrast, the weighted median approach relies on the assumption that at least 50% of the instrumental variables are valid for evaluating the causal relationship between exposure and outcome [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. All analyses were performed using the \"TwoSampleMR\" package (V 0.5.11) within the R (V 4.0.5). Visual representations were generated through scatter plots and sensitivity analysis diagrams. Statistical significance was defined as an P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cb\u003eSensitivity Analyses\u003c/b\u003e\u003c/p\u003e\u003cp\u003eHeterogeneity among instrumental variables (IVs) was assessed using Cochran's Q test, with p\u0026thinsp;\u0026gt;\u0026thinsp;0.05 indicating no heterogeneity[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. To account for the potential impact of genetic variation pleiotropy on association effect estimates, the MR-Egger regression approach was applied to investigate the presence of horizontal pleiotropy. A null intercept or lack of statistical significance in the MR-Egger regression suggests the absence of pleiotropy. Additionally, the MR-PRESSO method was employed to identify potential outlier single nucleotide polymorphisms (SNPs) with p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, and the causal association was reassessed following their exclusion [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. This procedure corrects for horizontal pleiotropy. Leave-one-out analysis was conducted to remove each SNP step by step, calculate the meta-effect of the remaining SNPs, and observe whether the results change after removing each SNP [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eIncorporation of instrumental variables\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe extracted SNPs from GWAS summary statistics of each phenotypic exposure under a well-recognized threshold. In total, we select 12, 6, 12, 10, 17, 429, 5 IVs for IL-4 levels, IL-5 levels, IL-13 levels, blood protein levels of MPO-DNA complexes, NETs measurement, neutrophil count and blood protein levels of MPO. The mean value of the F-statistic of IVs is calculated to be over 20, reflecting strong instrument validity (\u003cb\u003eTable \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e). In addition, there were 8 proxy SNPs were used to replace SNPs, when using \u0026lsquo;atherosclerosis\u0026rsquo; as the outcome (\u003cb\u003eTable S3\u003c/b\u003e). This process ensured a robust set of instruments for MR analysis across different exposures.\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe casual effect of the NETs-related traits on CVDs\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAs shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the scatter plot (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), and the forest plot (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), our MR analysis indicated a significant association between neutrophil count (OR\u0026thinsp;=\u0026thinsp;1.0829, 95% CI: 1.0317\u0026ndash;1.1366, P\u0026thinsp;=\u0026thinsp;0.001) and CAD. The results of MR Egger and Weighted median methods verified it. Which suggests that neutrophil count play the protective effect on the development of CAD. No significant causal effects were detected for other NETs-related traits (All P\u0026thinsp;\u0026gt;\u0026thinsp;0.05, \u003cb\u003eTable S4\u003c/b\u003e). For example, there was no evidence of causal effect between NETs measurement and CAD (OR\u0026thinsp;=\u0026thinsp;1.0014, 95% CI: 0.9942\u0026ndash;1.0087, P\u0026thinsp;=\u0026thinsp;0.698).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMR analyses showed a causal relationship between Neutrophil count and CAD.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eExposure\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOutcome\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMethods\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eOR (95% CI)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eP\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003eNeutrophil count\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003eCoronary artery disease\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eIVW\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.0829 ( 1.0317\u0026ndash;1.1366 )\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.001\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMR Egger\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.1155 ( 1.0144\u0026ndash;1.2267 )\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.024\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eWeighted median\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.0882 ( 1.0124\u0026ndash;1.1696 )\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.022\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eWeighted mode\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1.0490 ( 0.9664\u0026ndash;1.1387 )\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.254\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eHeterogeneity, directional pleiotropy, and horizontal pleiotropy among the genetic instruments were evaluated using MR-Egger regression analysis, Cochran's Q test, and the MR-PRESSO test. The Q test results showed that there was heterogeneity in several NETs-related traits, such as blood protein levels of myeloperoxidase-DNA complexes (P\u0026thinsp;=\u0026thinsp;0.025) as exposure and atherosclerosis as outcome (\u003cb\u003eTable S5\u003c/b\u003e). Since the IVW method we primarily use is based on random effects, it can accept a certain degree of heterogeneity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). MR-Egger regression analysis did not find horizontal pleiotropy. However, the MR-PRESSO results showed that there were outliers in many exposures (\u003cb\u003eTable S6\u003c/b\u003e), but the removal of outliers did not affect our results. The analysis results are now presented after outlier correction. The stability of our findings was validated by leave-one-out analyses, which verified that the exclusion of any individual SNP did not substantially affect the observed associations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur MR study provides evidence for a causal role of neutrophil count in the development of CAD. In addition, there was no evidence from MR analyses indicating that other types of NETs-related traits, increased or decreased the risk of CVDs. Our results suggest that targeting neutrophil activity could be a promising therapeutic strategy for preventing or treating CAD. However, further mechanistic studies are warranted to understand the specific pathways involved and to identify potential biomarkers for clinical intervention.\u003c/p\u003e\u003cp\u003eOur results suggest that circulating neutrophil count is a risk factor for CAD. Over the past 2 decades, numerous studies have described the association between elevated neutrophil counts and increased risk of CAD. High neutrophil counts and low lymphocyte counts within the normal clinical range have demonstrated a strong linear correlation with all stages of CAD[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. A European cohort study indicated that neutrophil counts are associated with the incidence of hypoechoic plaques and disease progression in CAD patients[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Similar findings have been reported in large-scale Asian cohorts; for instance, the Chinese Dongfeng-Tongji cohort found that higher neutrophil and monocyte counts were associated with increased CAD risk[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. These findings align with our research results, suggesting a positive correlation between elevated neutrophil counts and increased CAD risk across diverse age groups, genders, and ethnicities. Mechanistically, research over the past decade has elucidated the crucial role of neutrophils in various stages of CAD-related inflammation. At sites of vascular inflammation, activated neutrophils employ several synergistic strategies to fulfill their functions, including the release of reactive oxygen species (ROS), degranulation to release proteolytic enzymes, secretion of pro-inflammatory alarmins (such as S100A8/A9) and proteases (including cathepsin G, neutrophil elastase, and myeloperoxidase), as well as the formation of NETs[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Excessive ROS secretion leads to endothelial cell dysfunction and activation, extracellular matrix degradation, enhanced monocyte adhesion and recruitment, and facilitates the transfer of low-density lipoprotein (LDL) from the lumen to the arterial intima [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. ROS mediates the formation of oxidized LDL and may activate matrix metalloproteinases, potentially leading to plaque rupture [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Neutrophils also participate in immune system regulation, interacting with T cells, macrophages, and other immune cells, influencing the overall inflammatory response. In certain circumstances, neutrophil hyperactivation and excessive inflammation may damage the myocardium, leading to cardiomyocyte apoptosis and necrosis [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAlthough our results indicate an association between neutrophils and CAD risk, we did not find a correlation between NETs and the risk of CAD or other CVDs. However, this does not negate the role of NETs in CVDs. NETs may play an indirect role in CVDs that may not be apparent in MR studies. A study identified the contribution of neutrophils, particularly NETs, in promoting plaque instability by precisely facilitating necrotic core growth and fibrous cap thinning[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Mechanistically, regulatory smooth muscle cells in the fibrous cap physically interact with neutrophils, leading to their activation, ROS production, and NETs release, mediated by smooth muscle cell-derived CCL7[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Thus, the involvement of NETs in CVD progression may depend on activated neutrophils and systemic inflammatory responses. In vitro studies have shown that NETs kill smooth muscle cells. This effect is not dependent on neutrophil proteins derived from granules or cytoplasm, but rather on histones, particularly histone H4, consistent with earlier reports on the cytotoxic activity of NET-borne histones[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Currently, the lack of GWAS data on histone H4 limits further validation, which needs to be addressed in future research.\u003c/p\u003e\u003cp\u003eThis study presents several notable strengths. It marks the inaugural use of two-sample MR analysis to investigate potential causal relationships between the NETs related traits and CVD. Traditional observational studies are more susceptible to bias due to confounding variables and the possibility of reverse causation. Furthermore, the epidemiological impact of MR analysis is substantial, and its application is likely to continue increasing in the coming years. Nevertheless, there are certain limitations to the conclusions that can be drawn from this study. Primarily, the use of summary statistics rather than individual-level data precludes additional subgroup analyses, such as distinguishing between different types of CVDs. Moreover, MR studies typically require a large sample size, and the current study sample size for NETs (N\u0026thinsp;=\u0026thinsp;657) may be insufficient, affecting the reliability of the findings.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, our findings support a causal link between neutrophil count and CAD, highlighting the importance of neutrophil biology in cardiovascular health and disease. Future research should aim to translate these findings into clinical applications for better management of CVDs.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eConflict of interests\u003c/h2\u003e\u003cp\u003eAll authors declare that they have no any conflict of interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eNone.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eSiping Peng carried out the studies, participated in collecting data, and drafted the manuscript. Tao Hu performed the statistical analysis and participated in its design. Siping Peng and Tao Hu participated in acquisition, analysis, or interpretation of data and draft the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e\u003cp\u003eAll data generated or analysed during this study are included in this published article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLavie CJ. Cardiovascular statistics 2023. Prog Cardiovasc Dis. 2023;79:112\u0026ndash;3.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBułdak Ł. Cardiovascular Diseases-A Focus on Atherosclerosis, Its Prophylaxis, Complications and Recent Advancements in Therapies. Int J Mol Sci. 2022;23.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWong ND, Sattar N. Cardiovascular risk in diabetes mellitus: epidemiology, assessment and prevention. Nat Rev Cardiol. 2023;20:685\u0026ndash;95.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFunada S, Luo Y, Nishioka N, Yoshioka T. Cardiovascular risk in systemic autoimmune diseases. 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PLoS ONE. 2012;7:e32366.\u003c/span\u003e\u003c/li\u003e\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":"neutrophil extracellular traps, cardiovascular diseases, neutrophil count, Mendelian randomization, causal inference","lastPublishedDoi":"10.21203/rs.3.rs-7056612/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7056612/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eThis study investigates the causal relationship between neutrophil extracellular traps (NETs) and cardiovascular diseases (CVDs) using two-sample Mendelian randomization (MR).\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eWe performed a two-sample MR analysis utilizing GWAS summary statistics from the GWAS Catalog and IEU databases for six cardiovascular outcomes and various NET-related exposures. The main analysis employed the inverse variance-weighted (IVW) method, with supplementary analyses using MR-Egger, weighted median, and weighted mode methods.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eGenetic predisposition to higher neutrophil counts was found to increase the risk of coronary artery disease (OR\u0026thinsp;=\u0026thinsp;1.0829, 95% CI: 1.0317\u0026ndash;1.1366, P\u0026thinsp;=\u0026thinsp;0.001). No causal link between NET measurements and CVDs was detected (OR\u0026thinsp;=\u0026thinsp;1.0014, 95% CI: 0.9942\u0026ndash;1.0087, P\u0026thinsp;=\u0026thinsp;0.698), nor for other NET-related exposures (All P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Sensitivity analyses confirmed the robustness of these findings.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eOur results indicate that elevated neutrophil counts may heighten coronary artery disease risk. Further studies should investigate the mechanisms underlying this association and explore therapeutic interventions targeting neutrophils in CVD prevention and treatment.\u003c/p\u003e","manuscriptTitle":"Mendelian randomization study on the causal link between neutrophil extracellular traps and cardiovascular diseases","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-22 18:17:44","doi":"10.21203/rs.3.rs-7056612/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":"2eccad1a-00a9-4277-a046-a4d786612e10","owner":[],"postedDate":"August 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-08T16:39:33+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-22 18:17:44","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7056612","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7056612","identity":"rs-7056612","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}