Novel Biomarkers for Ankylosing Spondylitis through Proteomic Profiling of Serum-Derived Extracellular Vesicles

Novel Biomarkers for Ankylosing Spondylitis through Proteomic Profiling of Serum-Derived Extracellular Vesicles | 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 Novel Biomarkers for Ankylosing Spondylitis through Proteomic Profiling of Serum-Derived Extracellular Vesicles Soo-Eun Sung, Wook-Tae Park, Joo-Hee Choi, Young-In Kim, Min-Jung Ma, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5921683/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Jul, 2025 Read the published version in Clinical and Experimental Medicine → Version 1 posted 13 You are reading this latest preprint version Abstract The objective of this study is to analyze the protein composition of extracellular vesicles (EVs) isolated from the serum of ankylosing spondylitis (AS) patients to identify potential biomarkers that could enable the early diagnosis and intervention of this condition. Serum samples were collected from AS patients and healthy controls. EVs were isolated from these samples using ExoQuick® ULTRA solution, and their morphology, size, and concentration were analyzed using transmission electron microscopy and nanoparticle tracking analysis. Proteins within the EVs were identified and quantified through liquid chromatography-mass spectrometry (LC-MS/MS), followed by validation of key proteins using enzyme-linked immunosorbent assay (ELISA). Data were analyzed to identify proteins significantly upregulated in AS patients compared with the levels in controls. Here, through LC-MS/MS analysis, we demonstrated that FBLN1, VWF, CFHR2, and LYZ expression were significantly upregulated in serum-derived EVs from AS patients compared with the levels in healthy controls. These findings were further validated by ELISA, confirming the potential utility of serum-derived EVs as specific biomarkers for AS. The elevated levels of FBLN1, VWF, CFHR2, and LYZ in the EVs of AS patients represent promising candidates for biomarkers in the early diagnosis and treatment of AS. Further research should be performed to validate these findings and explore their clinical applicability. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Ankylosing spondylitis (AS) is a chronic inflammatory disease primarily affecting the axial skeleton, leading to pain and progressive stiffness of the spine [ 1 ]. It is characterized by inflammation of the entheses, the sites where tendons and ligaments attach to bones, and often progresses to vertebral fusion, resulting in loss of spinal mobility and chronic pain [ 2 ]. AS onset typically occurs in young adults, and the condition is much more prevalent in males. Owing to the subtlety and nonspecific nature of early symptoms, AS diagnosis is often delayed until several years after symptom onset, during which irreversible structural damage and disability can occur [ 1 ]. Late diagnosis increases the risk of severe complications, including spinal fractures and cardiovascular diseases, which highlights the need for early detection and intervention [ 3 ]. Currently, AS diagnosis relies on a combination of clinical criteria, imaging studies, and laboratory tests. The modified New York criteria, which include radiographic evidence of sacroiliitis, are commonly used in clinical practice [ 4 ]. Magnetic resonance imaging can detect inflammatory changes at an early stage of AS before radiographic abnormalities become apparent. Laboratory markers such as elevated C-reactive protein and erythrocyte sedimentation rate are supportive of the diagnosis, but not specific to AS [ 5 ]. In cases involving spinal fractures, surgical intervention may be necessary, in addition to pharmacological treatments such as nonsteroidal anti-inflammatory drugs (NSAIDs), biologics, and physical therapy aimed at managing symptoms and maintaining function. AS treatment focuses on managing symptoms, reducing inflammation, and maintaining spinal mobility and function [ 5 ]. NSAIDs are often the first line of treatment due to their effectiveness in reducing pain and stiffness [ 2 ]. For patients who do not respond adequately to NSAIDs, biologic agents such as tumor necrosis factor (TNF) inhibitors (e.g., etanercept, adalimumab) and interleukin-17 (IL-17) inhibitors (e.g., secukinumab) are commonly prescribed, as they target specific pathways in the inflammatory process. Physical therapy and regular exercise are crucial components of AS management, helping to maintain flexibility and posture while reducing pain. In cases of severe disease or complications such as spinal fractures, surgical interventions may be necessary to correct deformities and stabilize the spine [ 5 ]. Patient education and support, along with lifestyle modifications, are also significant in effectively managing AS [ 2 ]. Early diagnosis of AS is vital as it allows the prompt initiation of appropriate treatments, which can slow disease progression, minimize structural damage, and improve long-term outcomes [ 1 ]. Overall, a multidisciplinary approach tailored to the needs of individual patients is essential for optimal outcomes [ 5 ]. Extracellular vesicles (EVs) are membrane-bound particles released by cells into the extracellular environment [ 6 ]. These vesicles, which include exosomes, microvesicles, and apoptotic bodies, carry a diverse cargo of proteins, lipids, RNA, and DNA that reflects the characteristics of their parent cells [ 7 ]. This unique property of EVs makes them a promising source of biomarkers for various diseases, including AS. Recent studies have demonstrated that EVs can provide insights into the pathological processes of diseases and serve as noninvasive diagnostic and prognostic tools [ 6 ]. For example, it has been shown that EVs derived from patients with rheumatoid arthritis carry specific microRNAs and proteins associated with disease activity, suggesting their potential role as biomarkers [ 7 ]. These issues have also been actively investigated in cancer, where EVs derived from certain cancer cells have been shown to trigger angiogenesis and metastasis, among others, through the cargo that they contain. In light of this, it has been suggested that EVs could be used as biomarkers of disease progression and metastasis [ 8 , 9 ]. Here, EVs derived from the serum of AS patients were subjected to proteomic profiling and compared with EVs of normal controls to select candidate biomarkers. The results suggest the possibility of diagnosing and treating AS at an early stage to slow its progression and improve the quality of life of patients. Methods Serum sample collection The study protocol was approved by the Institutional Review Board of Yeungnam University Hospital (IRB no. 2021-08-033). All study participants provided written informed consent before their enrollment. Human serum samples were collected from patients undergoing AS surgery at Yeungnam University Hospital or receiving medical treatment there. AS was diagnosed based on the modified New York criteria for this condition. The control serum samples were collected from patients with conditions other than AS who were free from other orthopedic diseases. Approximately 2.5 mL of blood was collected from each donor using a serum-separating tube (BD Vacutainer, UK). Serum samples were collected in sterile tubes stored at − 80℃ until further study. Extracellular vesicle (EV) isolation and characterization Extracellular vesicles were isolated using the ExoQuick® ULTRA solution (System Biosciences Inc., USA), in accordance with the provided procedure. Briefly, serum was centrifuged at 3,000 × g for 10 min to remove debris. Then, 250 µL of supernatant sample and 67 µL of ExoQuick reagent were incubated for 30 min at 4℃. The EV pellets were obtained by centrifugation at 3,000 × g for 10 min at 4℃. Then, the EV pellets were suspended in 500 µL of phosphate-buffered saline (Gibco, USA). The morphology of vesicles was observed by bio-TEM (Hitachi, Japan). EV size distribution and concentration were measured using NanoSight NS300 (Malvern Panalytical, UK). The surface markers of EV were detected by Novocytes (Agilent Technologies, USA). Liquid chromatography-mass spectrometry (LC-MS/MS) analysis EV samples were lysed by radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific, USA) for protein extraction. The protein concentration was measured by bicinchoninic acid assay (Thermo Fisher Scientific, USA). Filter-aided sample preparation digestion and desalting were conducted for LC-MS/MS analysis. The trapping column was C18, 3 µm, 100 Å, 75 µm × 2 cm. The analytical column was PepMap™ RSLC C18, 2 µm, 100 Å, 75 µm × 50 cm (Thermo Fisher Scientific). Mobile phase A was water with 0.1% formic acid and phase B was 80% acetonitrile (ACN) with 0.1% formic acid. Column flow rate was 300 nL/min and mass range was 400–2,000 m/z. Raw data were analyzed by Proteome Discoverer™ software (Thermo Fisher Scientific) and the human database was downloaded from Uniport. Proteomic data analysis Search parameters were set as follows: 10 ppm tolerance for precursor ion masses, 0.02 Da fragment ion mass, and a maximum of two missed cleavages with trypsin. After the search, peptide lengths of a least 6 were filtered by selecting data results with FDR of less than 1%. Sample normalization was performed using the total peptide amount method with Proteome Discoverer™ software 2.5 [ 10 ]. Fold change was calculated using the protein abundance-based ratio. Differentially expressed proteins were selected based on p-value 1. Proteins with p-value 2.0 were regarded as upregulated, while those with p-value < 0.05 and fold change < 1/2 were regarded as downregulated by ExDEGA Graphic Plus software (e-biogen Inc., Republic of Korea). Protein validation by ELISA Proteomic results were validated using enzyme-linked immunosorbent assay (ELISA) following the manufacturer’s protocol (MyBioSource, USA). Fibulin 1 (FBLN1), von Willebrand factor (VWF), lysozyme C (LYZ), and complement factor H-related protein 2 (CFHR2) were analyzed by ELISA. Statistical analyses Data are expressed as the mean ± standard deviation. Exosomal protein concentration data were analyzed using unpaired sample t-test. All data analyses were performed using GraphPad Prism 8.4.3 (GraphPad Software, USA). Results Isolation of EVs Serum was obtained from AS patients and controls, and EVs were isolated and compared for protein expression ( Fig. 1 ). The demographics and underlying diseases of the control group and AS patients are summarized in Table 1 . The mean age of the patients was 58.9 years, and 97% were male. The diagnosis of AS was based on the modified New York criteria for this condition, with reference to pelvic X-ray and, in some cases, computed tomography and magnetic resonance imaging ( Fig. 2 ). Characterization of EVs Upon analyzing the shape of isolated EVs using bio-TEM equipment, we were able to observe EVs of approximately 100 nm in size, which is their typical size ( Fig. 3a and 3b ). Nanoparticle tracking analysis (NTA) revealed that the mean size of healthy serum-derived EVs was approximately 150.2 nm and the concentration was 8.83e + 10 particles/mL; meanwhile, the corresponding values for AS serum-derived EVs were 126.6 nm and 13.2e + 10 particles/mL, respectively ( Fig. 3c and 3d ). Positivity for the EV surface markers CD9, CD63, and CD81 was detected using flow cytometry. Healthy control serum-derived EVs were largely positive for CD9 (92.08%), CD63 (95.26%), and CD81 (91.81%) ( Fig. 3e ). Similar results were obtained for AS serum-derived EVs as follows: CD9 (90.53%), CD63 (98.15%), and CD81 (97.89%) ( Fig. 3f ). LC-MS analysis of EV proteins in the healthy and AS groups We performed LC-MS/MS for proteomic analysis of the isolated serum-derived EVs. This identified four proteins (FBLN1, VWF, CFHR2, and LYZ) with a fold change of 2 or more and a p-value of 0.05 or less in the AS group, when compared with the levels in the healthy control group ( Table 2 ). Based on the LC-MS/MS results, the normalized values for the four proteins were presented as a significant chart ( Fig. 4a ). In addition, the normalization values of each sample can be used to show differences in protein expression as a clustering heatmap ( Fig. 4b ). The volcano plot shows the distribution of differentially expressed proteins. The four proteins are marked separately ( Fig. 5 ). Validation of EV proteins in the control and AS groups To validate the LC-MS/MS analysis results, ELISA was performed on more patients and healthy control samples. The results showed that the protein with the most significant difference was VWF, with an average of 2,222 ng/mL in the healthy control and 11,193 ng/mL in the AS group. The next most significant protein was Lysozyme, with an average of 77.28 ng/mL in the healthy control and 86.75 ng/mL in the AS group. CFHR2 had an average of 44.77 ng/mL in the healthy control and 100.42 ng/mL in the AS group, while FBLN1 had an average of 20.22 ng/mL in the healthy control and 30.35 ng/mL in the AS group ( Fig. 6 ). Discussion AS is a chronic progressive systemic disease that usually presents with typical spinal stiffness and is characterized by chronic inflammation of the sacroiliac joints and spine and the involvement of peripheral joints, eyes, heart, and intestines [ 11 , 12 ]. Bone diseases are generally caused degeneratively via inflammatory reactions due to aging (e.g., degenerative arthritis), or by specific hormonal disorders due to congenital or acquired problems (e.g., osteoporosis, bone disease) [ 1 , 13 ]. Meanwhile, it is assumed that genetic abnormalities, infection, and immune-inflammatory responses are involved in AS development, but the specific cause of the disease has not yet been revealed [ 14 – 16 ]. One of the main symptoms of AS is inflammatory back pain, which gradually begins in the lumbar or lumbosacral region and, in some cases, is intermittently accompanied by arthritis in the knees and ankles [ 17 – 19 ]. Because this pain is generally vague and undergoes repeated cycles of worsening and improving, it is difficult to distinguish from general back pain [ 20 , 21 ]. Additionally, AS may feature extra-articular-related symptoms such as uveitis, psoriasis, and inflammatory bowel disease, and may in rare cases be accompanied by cardiomyopathy, aortic valve failure, and interstitial pneumonia [ 22 – 26 ]. Although the HLA-B27 gene is known to be associated with AS development, there is a need for further research to elucidate the precise etiology of the disease and to identify reliable biomarkers for its early diagnosis [ 27 – 29 ]. Recently, as new knowledge has been accumulated about this disease’s course and treatment, the issue of early diagnosis has become important as it has been shown that early treatment is effective at preventing AS progression. However, the diagnosis of AS is generally based on the patient’s symptoms, medical history, and radiological findings of the sacroiliac joint and spine. Radiological abnormalities are typically detected only in patients with advanced disease, complicating early diagnosis [ 30 ]. AS, a chronic inflammatory condition primarily affecting the spine and sacroiliac joints, presents significant diagnostic challenges [ 30 , 31 ]. EVs have been reported as potential biomarkers for the diagnosis of specific diseases. In the case of AS, some studies have aimed to develop diagnostic markers based on the expression profiles of genes within EVs [ 32 , 33 ]. However, considering clinical applicability, it is also valuable to investigate biomarkers based on protein expression profiles in EVs. Against this background, this study was implemented to identify novel biomarkers in EVs derived from the serum samples of AS patients, with the goal of improving the utility and timeliness of AS diagnosis. In the present study, serum-derived EVs were isolated and characterized in both normal controls and AS patients. The isolated particles were confirmed to be EVs by TEM, NTA, and flow cytometry. MicroRNAs or proteins in EVs have been studied using bioinformatic methods. Therefore, in this study, we sought to identify potential AS diagnostic biomarkers by analyzing the expression of specific proteins in EVs derived from AS patient serum compared with those from normal control serum. The proteomic analysis results indicated that CFHR2 (Complement factor H-related protein 2), VWF (von Willebrand factor), FBLN1 (Fibulin-1), and LYZ (Lysozyme C) were highly expressed in the exosomes derived from AS patient serum compared with those from the normal control group. The CFHR protein family comprises CFHR1 to CFHR5, each of which binds to the central complement component C3b. Diseases related to this family have been reported, such as atypical hemolytic uremic syndrome (aHUS), C3 glomerulopathies [C3 glomerulonephritis (C3GN), dense deposit disease, and CFHR5 nephropathy], IgA nephropathy, age-related macular degeneration, and systemic lupus erythematosus [ 34 ]. To the best of our knowledge, no studies on disease-specific biomarkers related to the CFHR protein family and AS have been reported. von Willebrand factor (VWF) is a crucial multimeric glycoprotein synthesized exclusively in endothelial cells and megakaryocytes, which plays a vital role in primary hemostasis. VWF biosynthesis involves complex post-translational modifications across different cell organelles, starting in the endoplasmic reticulum and continuing in the Golgi apparatus. This process results in the formation of multimers with over 300 glycan structures, which are essential for the recruitment of platelets at sites of vascular injury [ 35 ]. Recent studies have expanded the understanding of VWF’s role beyond hemostasis. VWF is implicated in various physiological processes, including vascular permeability, inflammation, and angiogenesis. Notably, the absence of VWF, as observed in type 3 von Willebrand disease (VWD), correlates with enhanced vascularization and severe clinical manifestations such as gastrointestinal bleeding due to vascular malformations [ 36 ]. VWD is recognized as the most common inherited bleeding disorder, presenting significant diagnostic and subclassification challenges due to the wide range of plasma VWF levels in the normal population and VWF’s diverse physiological roles [ 37 ]. A study on biomarkers related to VWF and AS reported that the expression of VWF was upregulated in AS patient serum [ 38 ]. However, studies on VWF protein in EVs and AS disease have not been reported. Fibulins are ECM-secreted glycoproteins, notable for their two distinct structural features: a series of calcium-binding epidermal growth factor (cbEGF)-like modules and a unique domain in the C-terminal region. To date, eight family members have been identified, categorized into long (fibulin-1, -2, -6, and − 8) and short types (fibulin-3, -4, -5, and − 7) [ 39 ]. The role of fibulin-1 has attracted increasing attention in a variety of human cancers. However, studies performed to date show contradictory findings regarding fibulin-1’s function across different tumor tissues. While the majority of research supports fibulin-1’s tumor-suppressive capabilities[ 40 ], further investigations are necessary to elucidate its potential association with AS. Lysozyme, a protein produced and released by monocytes and macrophages, is prevalent in various body tissues and secretions, notably saliva [ 41 ]. It endows saliva with antibacterial properties and plays a role in antiviral defense. Studies have identified a negative correlation between the concentration of lysozyme and stress exposure. Additionally, lysozyme expression varies significantly across different diseases; it is notably present in macrophages and giant cells in sarcoidosis and Crohn’s disease granuloma, but is expressed minimally in foreign body granulomas. This variation suggests a link between lysozyme level and disease activity. In the human airways, lysozyme is a major antimicrobial factor, with its activity being reported to positively correlate with neutrophil count in bronchoalveolar lavage fluid in cystic fibrosis patients, but not with bacterial colony count. These observations indicate that certain types of cell-mediated immunity may influence lysozyme activity [ 42 ]. However, the relationship between lysozyme and AS remains unclear. Here, we observed that FBLN1, VWF, CFHR2, and LYZ were expressed at low levels in controls, but showed significantly elevated expression in patients with AS. Utilizing liquid chromatography-mass spectrometry (LC-MS), we confirmed the upregulation of these proteins in the AS patient group compared with the level in the control group through enzyme-linked immunosorbent assay (ELISA). The expression levels of FBLN1, VWF, CFHR2, and LYZ were significantly elevated in the AS group. These findings suggest that FBLN1, VWF, CFHR2, and LYZ could serve as novel biomarkers for the diagnosis of AS. We also successfully isolated EVs from human serum and analyzed and compared the protein expression patterns in EVs from AS patients and controls. This analysis facilitated the identification of specific protein biomarkers potentially linked to AS. However, this study has several limitations. It represents the initial investigation into exosomal biomarkers for AS, focusing exclusively on patients with a confirmed diagnosis, specifically those in advanced stages, to ensure a clear difference between diseased and healthy individuals. Future research will explore the variation in exosomal biomarkers across different stages of AS. Moreover, the small sample size of 10 patients in this study makes further investigation with a larger cohort necessary. In future studies, we aim to include over 100 AS patients to validate and extend the findings on the exosomal biomarkers identified here. Despite these limitations, this research marks a pioneering step in developing exosomal biomarkers for orthopedic diseases and lays the groundwork for subsequent studies, including verification of the accuracy of these biomarkers and their potential application in clinical trials. Conclusion AS is a disease that causes spinal pain, significantly reducing the quality of life and, in severe cases, potentially necessitating surgical intervention due to fractures. Diagnosis is typically based on symptoms and imaging studies. Here, the authors isolated EVs from AS patient serum and subjected them to proteomic analysis, revealing that FBLN1, VWF, CFHR2, and LYZ were significantly upregulated in the AS patient group compared with the levels in healthy controls. These findings suggest that these proteins may serve as novel biomarkers for AS, supporting their potential role in enhancing the clinical diagnosis and management of this disease. Abbreviations AS Ankylosing spondylitis EVs Extracellular vesicles ELISA Enzyme-linked immunosorbent assay FBLN1 Fibulin-1 VWF von Willebrand factor CFHR2 Complement factor H-related protein 2 LYZ Lysozyme C NSAIDs nonsteroidal anti-inflammatory drugs TNF Tumor necrosis factor TEM Transmission electron microscopy NTA Nanoparticle tracking analysis Declarations Data Availability Data is available at the corresponding author upon request. Author Contribution Soo-Eun Sung and Wook-Tae Park contributed as co-first authors and were responsible for drafting the manuscript. Joo-Hee Choi, Young-In Kim, Min-Jung Ma, and Wan-Suk Son led the experiments and organized the data. Sangbum Park reviewed the experimental design. Gun Woo Lee and Min-Soo Seo, as corresponding authors, contributed to the overall study design, manuscript writing, and review. Acknowledgements This study was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (No. 2022R1C1C1005410), grants from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), Ministry of Health & Welfare, Republic of Korea (RS-2023-00305198), and the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (No.00219725). References Braun J, Sieper J. Ankylosing spondylitis. Lancet. 2007;369:1379–90. van der Heijde D, Ramiro S, Landewe R, et al. 2016 update of the ASAS-EULAR management recommendations for axial spondyloarthritis. Ann Rheum Dis. 2017;76:978–91. Sieper J, Poddubnyy D. Axial spondyloarthritis. Lancet. 2017;390:73–84. Rudwaleit M, van der Heijde D, Landewe R, et al. 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Supplementary Files Table1.docx Table2.docx Cite Share Download PDF Status: Published Journal Publication published 01 Jul, 2025 Read the published version in Clinical and Experimental Medicine → Version 1 posted Editorial decision: Revision requested 04 Mar, 2025 Reviews received at journal 03 Mar, 2025 Reviews received at journal 03 Mar, 2025 Reviews received at journal 26 Feb, 2025 Reviewers agreed at journal 11 Feb, 2025 Reviewers agreed at journal 11 Feb, 2025 Reviewers agreed at journal 09 Feb, 2025 Reviewers agreed at journal 09 Feb, 2025 Reviewers agreed at journal 09 Feb, 2025 Reviewers invited by journal 09 Feb, 2025 Editor assigned by journal 29 Jan, 2025 Submission checks completed at journal 29 Jan, 2025 First submitted to journal 28 Jan, 2025 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-5921683","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":408614870,"identity":"88c5c053-bcaf-48fb-a772-d5e293be5a03","order_by":0,"name":"Soo-Eun Sung","email":"","orcid":"","institution":"Daegu-Gyeongbuk Medical Innovation Foundation (K-MEDI hub)","correspondingAuthor":false,"prefix":"","firstName":"Soo-Eun","middleName":"","lastName":"Sung","suffix":""},{"id":408614871,"identity":"ca645f30-2a3b-4c0a-b5d8-142f4d93e073","order_by":1,"name":"Wook-Tae Park","email":"","orcid":"","institution":"Yeungnam University College of Medicine, Yeungnam University Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Wook-Tae","middleName":"","lastName":"Park","suffix":""},{"id":408614872,"identity":"caa48840-e0e4-4602-8c7c-edd23a612253","order_by":2,"name":"Joo-Hee Choi","email":"","orcid":"","institution":"Daegu-Gyeongbuk Medical Innovation Foundation (K-MEDI hub)","correspondingAuthor":false,"prefix":"","firstName":"Joo-Hee","middleName":"","lastName":"Choi","suffix":""},{"id":408614873,"identity":"f1f7ed18-d6b6-4431-b350-7062ac6bb6b4","order_by":3,"name":"Young-In Kim","email":"","orcid":"","institution":"Cellexobio., Ltd","correspondingAuthor":false,"prefix":"","firstName":"Young-In","middleName":"","lastName":"Kim","suffix":""},{"id":408614874,"identity":"c5efad9c-2943-4d1a-a1b6-0e36a6163528","order_by":4,"name":"Min-Jung Ma","email":"","orcid":"","institution":"Kyungpook National University","correspondingAuthor":false,"prefix":"","firstName":"Min-Jung","middleName":"","lastName":"Ma","suffix":""},{"id":408614875,"identity":"f34c78c6-df8b-4b39-a444-2e9acf05db5f","order_by":5,"name":"Wan-Suk Son","email":"","orcid":"","institution":"Kyungpook National University","correspondingAuthor":false,"prefix":"","firstName":"Wan-Suk","middleName":"","lastName":"Son","suffix":""},{"id":408614876,"identity":"ff390016-42d9-43da-b9d9-666d40aaa6e6","order_by":6,"name":"Sangbum Park","email":"","orcid":"","institution":"Michigan State University","correspondingAuthor":false,"prefix":"","firstName":"Sangbum","middleName":"","lastName":"Park","suffix":""},{"id":408614877,"identity":"b9c2ead2-c023-4d8d-92ed-d2ba0c70bee6","order_by":7,"name":"Ju-Hyeon Lim","email":"","orcid":"","institution":"Kolmar Korea Holdings","correspondingAuthor":false,"prefix":"","firstName":"Ju-Hyeon","middleName":"","lastName":"Lim","suffix":""},{"id":408614878,"identity":"c67afa53-605b-4779-ab0c-79bd19e456af","order_by":8,"name":"Min-Soo Seo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAq0lEQVRIiWNgGAWjYNCCigQo4wDRWs6QrIWxjRQtuv3HHz74OC8tz+AA88MPDGfuEdZidiPH2HDmtpxigwNsxhIMN4qJ0cLDJs27rSJxwwEGMwaGDwkEdTCYnT/+/DfvHJAW9m9EajmQYMbM25AD1MIDtOUGMVqAfpGccSwtceZhnmKJBHho43fYww8fapIT+463b/zw4RgRWhCAGYhJ0jAKRsEoGAWjADcAAKlfPZNcEXg7AAAAAElFTkSuQmCC","orcid":"","institution":"Kyungpook National University","correspondingAuthor":true,"prefix":"","firstName":"Min-Soo","middleName":"","lastName":"Seo","suffix":""},{"id":408614879,"identity":"dccefe20-c6f7-4311-a2a6-62a57b21519e","order_by":9,"name":"Gun Woo Lee","email":"","orcid":"","institution":"Yeungnam University College of Medicine, Yeungnam University Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Gun","middleName":"Woo","lastName":"Lee","suffix":""}],"badges":[],"createdAt":"2025-01-29 04:08:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5921683/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5921683/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10238-025-01718-8","type":"published","date":"2025-07-01T15:56:52+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":75191181,"identity":"38fccca9-251b-4466-86f2-97b676ac5d09","added_by":"auto","created_at":"2025-01-31 18:18:59","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":343522,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical abstract. The study was designed to compare protein expression in isolated EVs from AS patients and controls\u003c/p\u003e","description":"","filename":"Fig.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5921683/v1/893811afac8f2d224629b04f.jpg"},{"id":75188884,"identity":"fc609455-3925-4f7c-a69a-661f97da38ce","added_by":"auto","created_at":"2025-01-31 18:02:59","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1220818,"visible":true,"origin":"","legend":"\u003cp\u003eTypical images of AS patients. (a) A pelvic AP X-ray is necessary for diagnosing AS and can show a fused sacroiliac joint (red arrow). (b) X-ray of a thoracolumbar spine fusion in an AS patient. Multiple vertebrae are observed to be fused together (red arrow). (c) X-ray of a fracture of the lumbar spine in an AS patient after surgery to fix the fracture.\u003c/p\u003e","description":"","filename":"Fig.2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5921683/v1/b7b79ad13a3ee51d4bf29563.jpg"},{"id":75190744,"identity":"f4500bee-d4fb-4a6f-87e0-a47e41e3a94e","added_by":"auto","created_at":"2025-01-31 18:10:59","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":685780,"visible":true,"origin":"","legend":"\u003cp\u003eEVs were isolated from the control and ankylosing spondylitis (AS) serum. (a) EV morphology of the healthy control by bio-TEM. (b) EV morphology of the AS patient. (c) Mean size of isolated healthy control EVs, and EV concentration by NTA. (d) Mean size of isolated AS EVs, and EV concentration by NTA. (e) There was positivity for the EV surface markers CD9, CD63, and CD81 in the isolated healthy control EVs. (f) There was positivity for the EV surface markers CD9, CD63, and CD81 in the isolated AS patient serum-derived EVs.\u003c/p\u003e","description":"","filename":"Fig.3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5921683/v1/5e5840cf22ac7c2cd87f0b6f.jpg"},{"id":75188878,"identity":"8683b61a-c842-4838-b920-d55af73c5417","added_by":"auto","created_at":"2025-01-31 18:02:59","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":355676,"visible":true,"origin":"","legend":"\u003cp\u003eProteomic analysis of EVs. (a) Significant chart of LC-MS/MS data. (b) Clustering heatmap for each protein and sample.\u003c/p\u003e","description":"","filename":"Fig.4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5921683/v1/e4a43e8db328bfaa31f8d359.jpg"},{"id":75188880,"identity":"7ae3faed-4925-4142-b974-988556c465f3","added_by":"auto","created_at":"2025-01-31 18:02:59","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":290906,"visible":true,"origin":"","legend":"\u003cp\u003eVolcano plot illustrating the distribution of differentially expressed proteins.\u003c/p\u003e","description":"","filename":"Fig.5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5921683/v1/d890a9f390427638d4f7ab55.jpg"},{"id":75188887,"identity":"383c33b3-9f97-4893-8f04-57811988b867","added_by":"auto","created_at":"2025-01-31 18:02:59","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":342346,"visible":true,"origin":"","legend":"\u003cp\u003eValidation of upregulated proteins in AS extracellular vesicles by ELISA (*p-value \u0026lt; 0.05, **p-value \u0026lt; 0.01, ***p-value \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"Fig.6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5921683/v1/3ebcc63f29b6c0835f9ea336.jpg"},{"id":86179140,"identity":"74cd1555-074c-4f8c-8237-1a1e589ad22f","added_by":"auto","created_at":"2025-07-07 16:16:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3698834,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5921683/v1/dea14991-b7c4-4ef1-bc15-27ce64fc9730.pdf"},{"id":75191180,"identity":"08eeb7a2-4388-4f0d-afc0-c55e2bc593d9","added_by":"auto","created_at":"2025-01-31 18:18:59","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":33383,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-5921683/v1/7ca901106aad2664e7ad3aa6.docx"},{"id":75188883,"identity":"44e5d33d-a90b-4f39-9ef1-35f1813c32fa","added_by":"auto","created_at":"2025-01-31 18:02:59","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":29808,"visible":true,"origin":"","legend":"","description":"","filename":"Table2.docx","url":"https://assets-eu.researchsquare.com/files/rs-5921683/v1/ebda81af28990b0b33ef4386.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Novel Biomarkers for Ankylosing Spondylitis through Proteomic Profiling of Serum-Derived Extracellular Vesicles","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAnkylosing spondylitis (AS) is a chronic inflammatory disease primarily affecting the axial skeleton, leading to pain and progressive stiffness of the spine [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. It is characterized by inflammation of the entheses, the sites where tendons and ligaments attach to bones, and often progresses to vertebral fusion, resulting in loss of spinal mobility and chronic pain [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. AS onset typically occurs in young adults, and the condition is much more prevalent in males. Owing to the subtlety and nonspecific nature of early symptoms, AS diagnosis is often delayed until several years after symptom onset, during which irreversible structural damage and disability can occur [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Late diagnosis increases the risk of severe complications, including spinal fractures and cardiovascular diseases, which highlights the need for early detection and intervention [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCurrently, AS diagnosis relies on a combination of clinical criteria, imaging studies, and laboratory tests. The modified New York criteria, which include radiographic evidence of sacroiliitis, are commonly used in clinical practice [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Magnetic resonance imaging can detect inflammatory changes at an early stage of AS before radiographic abnormalities become apparent. Laboratory markers such as elevated C-reactive protein and erythrocyte sedimentation rate are supportive of the diagnosis, but not specific to AS [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In cases involving spinal fractures, surgical intervention may be necessary, in addition to pharmacological treatments such as nonsteroidal anti-inflammatory drugs (NSAIDs), biologics, and physical therapy aimed at managing symptoms and maintaining function.\u003c/p\u003e \u003cp\u003eAS treatment focuses on managing symptoms, reducing inflammation, and maintaining spinal mobility and function [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. NSAIDs are often the first line of treatment due to their effectiveness in reducing pain and stiffness [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. For patients who do not respond adequately to NSAIDs, biologic agents such as tumor necrosis factor (TNF) inhibitors (e.g., etanercept, adalimumab) and interleukin-17 (IL-17) inhibitors (e.g., secukinumab) are commonly prescribed, as they target specific pathways in the inflammatory process. Physical therapy and regular exercise are crucial components of AS management, helping to maintain flexibility and posture while reducing pain. In cases of severe disease or complications such as spinal fractures, surgical interventions may be necessary to correct deformities and stabilize the spine [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Patient education and support, along with lifestyle modifications, are also significant in effectively managing AS [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Early diagnosis of AS is vital as it allows the prompt initiation of appropriate treatments, which can slow disease progression, minimize structural damage, and improve long-term outcomes [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Overall, a multidisciplinary approach tailored to the needs of individual patients is essential for optimal outcomes [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eExtracellular vesicles (EVs) are membrane-bound particles released by cells into the extracellular environment [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. These vesicles, which include exosomes, microvesicles, and apoptotic bodies, carry a diverse cargo of proteins, lipids, RNA, and DNA that reflects the characteristics of their parent cells [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. This unique property of EVs makes them a promising source of biomarkers for various diseases, including AS. Recent studies have demonstrated that EVs can provide insights into the pathological processes of diseases and serve as noninvasive diagnostic and prognostic tools [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. For example, it has been shown that EVs derived from patients with rheumatoid arthritis carry specific microRNAs and proteins associated with disease activity, suggesting their potential role as biomarkers [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. These issues have also been actively investigated in cancer, where EVs derived from certain cancer cells have been shown to trigger angiogenesis and metastasis, among others, through the cargo that they contain. In light of this, it has been suggested that EVs could be used as biomarkers of disease progression and metastasis [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHere, EVs derived from the serum of AS patients were subjected to proteomic profiling and compared with EVs of normal controls to select candidate biomarkers. The results suggest the possibility of diagnosing and treating AS at an early stage to slow its progression and improve the quality of life of patients.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eSerum sample collection\u003c/p\u003e \u003cp\u003eThe study protocol was approved by the Institutional Review Board of Yeungnam University Hospital (IRB no. 2021-08-033). All study participants provided written informed consent before their enrollment. Human serum samples were collected from patients undergoing AS surgery at Yeungnam University Hospital or receiving medical treatment there. AS was diagnosed based on the modified New York criteria for this condition. The control serum samples were collected from patients with conditions other than AS who were free from other orthopedic diseases. Approximately 2.5 mL of blood was collected from each donor using a serum-separating tube (BD Vacutainer, UK). Serum samples were collected in sterile tubes stored at \u0026minus;\u0026thinsp;80℃ until further study.\u003c/p\u003e \u003cp\u003eExtracellular vesicle (EV) isolation and characterization\u003c/p\u003e \u003cp\u003eExtracellular vesicles were isolated using the ExoQuick\u0026reg; ULTRA solution (System Biosciences Inc., USA), in accordance with the provided procedure. Briefly, serum was centrifuged at 3,000 \u0026times; g for 10 min to remove debris. Then, 250 \u0026micro;L of supernatant sample and 67 \u0026micro;L of ExoQuick reagent were incubated for 30 min at 4℃. The EV pellets were obtained by centrifugation at 3,000 \u0026times; g for 10 min at 4℃. Then, the EV pellets were suspended in 500 \u0026micro;L of phosphate-buffered saline (Gibco, USA). The morphology of vesicles was observed by bio-TEM (Hitachi, Japan). EV size distribution and concentration were measured using NanoSight NS300 (Malvern Panalytical, UK). The surface markers of EV were detected by Novocytes (Agilent Technologies, USA).\u003c/p\u003e \u003cp\u003eLiquid chromatography-mass spectrometry (LC-MS/MS) analysis\u003c/p\u003e \u003cp\u003eEV samples were lysed by radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific, USA) for protein extraction. The protein concentration was measured by bicinchoninic acid assay (Thermo Fisher Scientific, USA). Filter-aided sample preparation digestion and desalting were conducted for LC-MS/MS analysis. The trapping column was C18, 3 \u0026micro;m, 100 \u0026Aring;, 75 \u0026micro;m \u0026times; 2 cm. The analytical column was PepMap\u0026trade; RSLC C18, 2 \u0026micro;m, 100 \u0026Aring;, 75 \u0026micro;m \u0026times; 50 cm (Thermo Fisher Scientific). Mobile phase A was water with 0.1% formic acid and phase B was 80% acetonitrile (ACN) with 0.1% formic acid. Column flow rate was 300 nL/min and mass range was 400\u0026ndash;2,000 m/z. Raw data were analyzed by Proteome Discoverer\u0026trade; software (Thermo Fisher Scientific) and the human database was downloaded from Uniport.\u003c/p\u003e \u003cp\u003eProteomic data analysis\u003c/p\u003e \u003cp\u003eSearch parameters were set as follows: 10 ppm tolerance for precursor ion masses, 0.02 Da fragment ion mass, and a maximum of two missed cleavages with trypsin. After the search, peptide lengths of a least 6 were filtered by selecting data results with FDR of less than 1%. Sample normalization was performed using the total peptide amount method with Proteome Discoverer\u0026trade; software 2.5 [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Fold change was calculated using the protein abundance-based ratio. Differentially expressed proteins were selected based on p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and log2 fold change\u0026thinsp;\u0026gt;\u0026thinsp;1. Proteins with p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and fold change\u0026thinsp;\u0026gt;\u0026thinsp;2.0 were regarded as upregulated, while those with p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and fold change\u0026thinsp;\u0026lt;\u0026thinsp;1/2 were regarded as downregulated by ExDEGA Graphic Plus software (e-biogen Inc., Republic of Korea).\u003c/p\u003e \u003cp\u003eProtein validation by ELISA\u003c/p\u003e \u003cp\u003eProteomic results were validated using enzyme-linked immunosorbent assay (ELISA) following the manufacturer\u0026rsquo;s protocol (MyBioSource, USA). Fibulin 1 (FBLN1), von Willebrand factor (VWF), lysozyme C (LYZ), and complement factor H-related protein 2 (CFHR2) were analyzed by ELISA.\u003c/p\u003e \u003cp\u003eStatistical analyses\u003c/p\u003e \u003cp\u003eData are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Exosomal protein concentration data were analyzed using unpaired sample t-test. All data analyses were performed using GraphPad Prism 8.4.3 (GraphPad Software, USA).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eIsolation of EVs\u003c/p\u003e\n\u003cp\u003eSerum was obtained from AS patients and controls, and EVs were isolated and compared for protein expression (\u003cstrong\u003eFig. 1\u003c/strong\u003e). The demographics and underlying diseases of the control group and AS patients are summarized in \u003cstrong\u003eTable 1\u003c/strong\u003e. The mean age of the patients was 58.9 years, and 97% were male. The diagnosis of AS was based on the modified New York criteria for this condition, with reference to pelvic X-ray and, in some cases, computed tomography and magnetic resonance imaging (\u003cstrong\u003eFig. 2\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eCharacterization of EVs\u003c/p\u003e\n\u003cp\u003eUpon analyzing the shape of isolated EVs using bio-TEM equipment, we were able to observe EVs of approximately 100 nm in size, which is their typical size (\u003cstrong\u003eFig. 3a and 3b\u003c/strong\u003e). Nanoparticle tracking analysis (NTA) revealed that the mean size of healthy serum-derived EVs was approximately 150.2 nm and the concentration was 8.83e + 10 particles/mL; meanwhile, the corresponding values for AS serum-derived EVs were 126.6 nm and 13.2e + 10 particles/mL, respectively (\u003cstrong\u003eFig. 3c and 3d\u003c/strong\u003e). Positivity for the EV surface markers CD9, CD63, and CD81 was detected using flow cytometry. Healthy control serum-derived EVs were largely positive for CD9 (92.08%), CD63 (95.26%), and CD81 (91.81%) (\u003cstrong\u003eFig. 3e\u003c/strong\u003e). Similar results were obtained for AS serum-derived EVs as follows: CD9 (90.53%), CD63 (98.15%), and CD81 (97.89%) (\u003cstrong\u003eFig. 3f\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eLC-MS analysis of EV proteins in the healthy and AS groups\u003c/p\u003e\n\u003cp\u003eWe performed LC-MS/MS for proteomic analysis of the isolated serum-derived EVs. This identified four proteins (FBLN1, VWF, CFHR2, and LYZ) with a fold change of 2 or more and a p-value of 0.05 or less in the AS group, when compared with the levels in the healthy control group (\u003cstrong\u003eTable 2\u003c/strong\u003e). Based on the LC-MS/MS results, the normalized values for the four proteins were presented as a significant chart (\u003cstrong\u003eFig. 4a\u003c/strong\u003e). In addition, the normalization values of each sample can be used to show differences in protein expression as a clustering heatmap (\u003cstrong\u003eFig. 4b\u003c/strong\u003e). The volcano plot shows the distribution of differentially expressed proteins. The four proteins are marked separately (\u003cstrong\u003eFig. 5\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003eValidation of EV proteins in the control and AS groups\u003c/p\u003e\n\u003cp\u003eTo validate the LC-MS/MS analysis results, ELISA was performed on more patients and healthy control samples. The results showed that the protein with the most significant difference was VWF, with an average of 2,222 ng/mL in the healthy control and 11,193 ng/mL in the AS group. The next most significant protein was Lysozyme, with an average of 77.28 ng/mL in the healthy control and 86.75 ng/mL in the AS group. CFHR2 had an average of 44.77 ng/mL in the healthy control and 100.42 ng/mL in the AS group, while FBLN1 had an average of 20.22 ng/mL in the healthy control and 30.35 ng/mL in the AS group (\u003cstrong\u003eFig. 6\u003c/strong\u003e).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAS is a chronic progressive systemic disease that usually presents with typical spinal stiffness and is characterized by chronic inflammation of the sacroiliac joints and spine and the involvement of peripheral joints, eyes, heart, and intestines [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Bone diseases are generally caused degeneratively via inflammatory reactions due to aging (e.g., degenerative arthritis), or by specific hormonal disorders due to congenital or acquired problems (e.g., osteoporosis, bone disease) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Meanwhile, it is assumed that genetic abnormalities, infection, and immune-inflammatory responses are involved in AS development, but the specific cause of the disease has not yet been revealed [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. One of the main symptoms of AS is inflammatory back pain, which gradually begins in the lumbar or lumbosacral region and, in some cases, is intermittently accompanied by arthritis in the knees and ankles [\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Because this pain is generally vague and undergoes repeated cycles of worsening and improving, it is difficult to distinguish from general back pain [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Additionally, AS may feature extra-articular-related symptoms such as uveitis, psoriasis, and inflammatory bowel disease, and may in rare cases be accompanied by cardiomyopathy, aortic valve failure, and interstitial pneumonia [\u003cspan additionalcitationids=\"CR23 CR24 CR25\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Although the HLA-B27 gene is known to be associated with AS development, there is a need for further research to elucidate the precise etiology of the disease and to identify reliable biomarkers for its early diagnosis [\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Recently, as new knowledge has been accumulated about this disease\u0026rsquo;s course and treatment, the issue of early diagnosis has become important as it has been shown that early treatment is effective at preventing AS progression. However, the diagnosis of AS is generally based on the patient\u0026rsquo;s symptoms, medical history, and radiological findings of the sacroiliac joint and spine. Radiological abnormalities are typically detected only in patients with advanced disease, complicating early diagnosis [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. AS, a chronic inflammatory condition primarily affecting the spine and sacroiliac joints, presents significant diagnostic challenges [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. EVs have been reported as potential biomarkers for the diagnosis of specific diseases. In the case of AS, some studies have aimed to develop diagnostic markers based on the expression profiles of genes within EVs [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. However, considering clinical applicability, it is also valuable to investigate biomarkers based on protein expression profiles in EVs. Against this background, this study was implemented to identify novel biomarkers in EVs derived from the serum samples of AS patients, with the goal of improving the utility and timeliness of AS diagnosis.\u003c/p\u003e \u003cp\u003eIn the present study, serum-derived EVs were isolated and characterized in both normal controls and AS patients. The isolated particles were confirmed to be EVs by TEM, NTA, and flow cytometry. MicroRNAs or proteins in EVs have been studied using bioinformatic methods. Therefore, in this study, we sought to identify potential AS diagnostic biomarkers by analyzing the expression of specific proteins in EVs derived from AS patient serum compared with those from normal control serum. The proteomic analysis results indicated that CFHR2 (Complement factor H-related protein 2), VWF (von Willebrand factor), FBLN1 (Fibulin-1), and LYZ (Lysozyme C) were highly expressed in the exosomes derived from AS patient serum compared with those from the normal control group.\u003c/p\u003e \u003cp\u003eThe CFHR protein family comprises CFHR1 to CFHR5, each of which binds to the central complement component C3b. Diseases related to this family have been reported, such as atypical hemolytic uremic syndrome (aHUS), C3 glomerulopathies [C3 glomerulonephritis (C3GN), dense deposit disease, and CFHR5 nephropathy], IgA nephropathy, age-related macular degeneration, and systemic lupus erythematosus [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. To the best of our knowledge, no studies on disease-specific biomarkers related to the CFHR protein family and AS have been reported.\u003c/p\u003e \u003cp\u003evon Willebrand factor (VWF) is a crucial multimeric glycoprotein synthesized exclusively in endothelial cells and megakaryocytes, which plays a vital role in primary hemostasis. VWF biosynthesis involves complex post-translational modifications across different cell organelles, starting in the endoplasmic reticulum and continuing in the Golgi apparatus. This process results in the formation of multimers with over 300 glycan structures, which are essential for the recruitment of platelets at sites of vascular injury [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Recent studies have expanded the understanding of VWF\u0026rsquo;s role beyond hemostasis. VWF is implicated in various physiological processes, including vascular permeability, inflammation, and angiogenesis. Notably, the absence of VWF, as observed in type 3 von Willebrand disease (VWD), correlates with enhanced vascularization and severe clinical manifestations such as gastrointestinal bleeding due to vascular malformations [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. VWD is recognized as the most common inherited bleeding disorder, presenting significant diagnostic and subclassification challenges due to the wide range of plasma VWF levels in the normal population and VWF\u0026rsquo;s diverse physiological roles [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. A study on biomarkers related to VWF and AS reported that the expression of VWF was upregulated in AS patient serum [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. However, studies on VWF protein in EVs and AS disease have not been reported.\u003c/p\u003e \u003cp\u003eFibulins are ECM-secreted glycoproteins, notable for their two distinct structural features: a series of calcium-binding epidermal growth factor (cbEGF)-like modules and a unique domain in the C-terminal region. To date, eight family members have been identified, categorized into long (fibulin-1, -2, -6, and \u0026minus;\u0026thinsp;8) and short types (fibulin-3, -4, -5, and \u0026minus;\u0026thinsp;7) [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The role of fibulin-1 has attracted increasing attention in a variety of human cancers. However, studies performed to date show contradictory findings regarding fibulin-1\u0026rsquo;s function across different tumor tissues. While the majority of research supports fibulin-1\u0026rsquo;s tumor-suppressive capabilities[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], further investigations are necessary to elucidate its potential association with AS.\u003c/p\u003e \u003cp\u003eLysozyme, a protein produced and released by monocytes and macrophages, is prevalent in various body tissues and secretions, notably saliva [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. It endows saliva with antibacterial properties and plays a role in antiviral defense. Studies have identified a negative correlation between the concentration of lysozyme and stress exposure. Additionally, lysozyme expression varies significantly across different diseases; it is notably present in macrophages and giant cells in sarcoidosis and Crohn\u0026rsquo;s disease granuloma, but is expressed minimally in foreign body granulomas. This variation suggests a link between lysozyme level and disease activity. In the human airways, lysozyme is a major antimicrobial factor, with its activity being reported to positively correlate with neutrophil count in bronchoalveolar lavage fluid in cystic fibrosis patients, but not with bacterial colony count. These observations indicate that certain types of cell-mediated immunity may influence lysozyme activity [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. However, the relationship between lysozyme and AS remains unclear.\u003c/p\u003e \u003cp\u003eHere, we observed that FBLN1, VWF, CFHR2, and LYZ were expressed at low levels in controls, but showed significantly elevated expression in patients with AS. Utilizing liquid chromatography-mass spectrometry (LC-MS), we confirmed the upregulation of these proteins in the AS patient group compared with the level in the control group through enzyme-linked immunosorbent assay (ELISA). The expression levels of FBLN1, VWF, CFHR2, and LYZ were significantly elevated in the AS group. These findings suggest that FBLN1, VWF, CFHR2, and LYZ could serve as novel biomarkers for the diagnosis of AS. We also successfully isolated EVs from human serum and analyzed and compared the protein expression patterns in EVs from AS patients and controls. This analysis facilitated the identification of specific protein biomarkers potentially linked to AS. However, this study has several limitations. It represents the initial investigation into exosomal biomarkers for AS, focusing exclusively on patients with a confirmed diagnosis, specifically those in advanced stages, to ensure a clear difference between diseased and healthy individuals. Future research will explore the variation in exosomal biomarkers across different stages of AS. Moreover, the small sample size of 10 patients in this study makes further investigation with a larger cohort necessary. In future studies, we aim to include over 100 AS patients to validate and extend the findings on the exosomal biomarkers identified here. Despite these limitations, this research marks a pioneering step in developing exosomal biomarkers for orthopedic diseases and lays the groundwork for subsequent studies, including verification of the accuracy of these biomarkers and their potential application in clinical trials.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eAS is a disease that causes spinal pain, significantly reducing the quality of life and, in severe cases, potentially necessitating surgical intervention due to fractures. Diagnosis is typically based on symptoms and imaging studies. Here, the authors isolated EVs from AS patient serum and subjected them to proteomic analysis, revealing that FBLN1, VWF, CFHR2, and LYZ were significantly upregulated in the AS patient group compared with the levels in healthy controls. These findings suggest that these proteins may serve as novel biomarkers for AS, supporting their potential role in enhancing the clinical diagnosis and management of this disease.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAnkylosing spondylitis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEVs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eExtracellular vesicles\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eELISA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEnzyme-linked immunosorbent assay\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFBLN1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFibulin-1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eVWF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003evon Willebrand factor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCFHR2\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eComplement factor H-related protein 2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLYZ\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLysozyme C\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNSAIDs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003enonsteroidal anti-inflammatory drugs\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTNF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTumor necrosis factor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTEM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTransmission electron microscopy\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNTA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNanoparticle tracking analysis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData is available at the corresponding author upon request.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eSoo-Eun Sung and Wook-Tae Park contributed as co-first authors and were responsible for drafting the manuscript. Joo-Hee Choi, Young-In Kim, Min-Jung Ma, and Wan-Suk Son led the experiments and organized the data. Sangbum Park reviewed the experimental design. Gun Woo Lee and Min-Soo Seo, as corresponding authors, contributed to the overall study design, manuscript writing, and review.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThis study was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (No. 2022R1C1C1005410), grants from the Korea Health Technology R\u0026amp;D Project through the Korea Health Industry Development Institute (KHIDI), Ministry of Health \u0026amp; Welfare, Republic of Korea (RS-2023-00305198), and the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (No.00219725).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBraun J, Sieper J. Ankylosing spondylitis. 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Br J Haematol. 2020;191:329\u0026ndash;39.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTaylan A, Sari I, Kozaci DL, et al. Evaluation of various endothelial biomarkers in ankylosing spondylitis. Clin Rheumatol. 2012;31:23\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTimpl R, Sasaki T, Kostka G, Chu ML. Fibulins: a versatile family of extracellular matrix proteins. Nat Rev Mol Cell Biol. 2003;4:479\u0026ndash;89.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHedayati M, Abooshahab R, Razavi SA, Salehipour P, Ahmadikia K, Boroomand S. Low level of plasma fibulin-1 in patients with thyroid lesions: a case-control study and literature review. Mol Biol Rep. 2020;47:8859\u0026ndash;66.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLos K, Waszkiewicz N. Biological Markers in Anxiety Disorders. J Clin Med. 2021;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIshii H, Iwata A, Oka H, Sakamoto N, Ishimatsu Y, Kadota J. Elevated serum levels of lysozyme in desquamative interstitial pneumonia. Intern Med. 2010;49:847\u0026ndash;51.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 and 2 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"clinical-and-experimental-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"clem","sideBox":"Learn more about [Clinical and Experimental Medicine](https://www.springer.com/journal/10238)","snPcode":"10238","submissionUrl":"https://submission.nature.com/new-submission/10238/3","title":"Clinical and Experimental Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5921683/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5921683/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe objective of this study is to analyze the protein composition of extracellular vesicles (EVs) isolated from the serum of ankylosing spondylitis (AS) patients to identify potential biomarkers that could enable the early diagnosis and intervention of this condition. Serum samples were collected from AS patients and healthy controls. EVs were isolated from these samples using ExoQuick\u0026reg; ULTRA solution, and their morphology, size, and concentration were analyzed using transmission electron microscopy and nanoparticle tracking analysis. Proteins within the EVs were identified and quantified through liquid chromatography-mass spectrometry (LC-MS/MS), followed by validation of key proteins using enzyme-linked immunosorbent assay (ELISA). Data were analyzed to identify proteins significantly upregulated in AS patients compared with the levels in controls. Here, through LC-MS/MS analysis, we demonstrated that FBLN1, VWF, CFHR2, and LYZ expression were significantly upregulated in serum-derived EVs from AS patients compared with the levels in healthy controls. These findings were further validated by ELISA, confirming the potential utility of serum-derived EVs as specific biomarkers for AS. The elevated levels of FBLN1, VWF, CFHR2, and LYZ in the EVs of AS patients represent promising candidates for biomarkers in the early diagnosis and treatment of AS. Further research should be performed to validate these findings and explore their clinical applicability.\u003c/p\u003e","manuscriptTitle":"Novel Biomarkers for Ankylosing Spondylitis through Proteomic Profiling of Serum-Derived Extracellular Vesicles","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-31 18:02:54","doi":"10.21203/rs.3.rs-5921683/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-03-04T08:01:38+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-03T21:09:51+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-03T14:14:04+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-02-27T04:23:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"21231816114857591738256954038315748781","date":"2025-02-11T12:38:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"233009072750704192737466067994735497947","date":"2025-02-11T07:58:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"28575968794308665458773433089178111995","date":"2025-02-10T00:46:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"3798679221939282823065582027826211550","date":"2025-02-09T22:05:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"305552971262479509602827559931381726187","date":"2025-02-09T13:23:01+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-02-09T12:15:39+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-01-29T11:45:07+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-01-29T11:43:10+00:00","index":"","fulltext":""},{"type":"submitted","content":"Clinical and Experimental Medicine","date":"2025-01-29T04:01:30+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"clinical-and-experimental-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"clem","sideBox":"Learn more about [Clinical and Experimental Medicine](https://www.springer.com/journal/10238)","snPcode":"10238","submissionUrl":"https://submission.nature.com/new-submission/10238/3","title":"Clinical and Experimental Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"8b3cb1ae-c6b5-4120-9da5-00d489afed32","owner":[],"postedDate":"January 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-07-07T16:05:25+00:00","versionOfRecord":{"articleIdentity":"rs-5921683","link":"https://doi.org/10.1007/s10238-025-01718-8","journal":{"identity":"clinical-and-experimental-medicine","isVorOnly":false,"title":"Clinical and Experimental Medicine"},"publishedOn":"2025-07-01 15:56:52","publishedOnDateReadable":"July 1st, 2025"},"versionCreatedAt":"2025-01-31 18:02:54","video":"","vorDoi":"10.1007/s10238-025-01718-8","vorDoiUrl":"https://doi.org/10.1007/s10238-025-01718-8","workflowStages":[]},"version":"v1","identity":"rs-5921683","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5921683","identity":"rs-5921683","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}