Characteristic changes in the mRNA expression profile of plasma exosomes from patients with MPO-ANCA-associated vasculitis and its possible correlations with pathogenesis

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Methods Plasma exosomes were isolated from MPO-AAV patients and healthy controls (HCs) to screen for differential mRNA expression via exosomal mRNA sequencing. The differentially expressed mRNAs in exosomes from the 2 groups were comparatively explored by bioinformatics analysis. The six most differentially expressed mRNAs were selected and validated in larger groups of MPO-AAV patients and HCs by real-time quantitative polymerase chain reaction (RT‒qPCR). The relationships between these selected mRNAs and patient characteristics were statistically analyzed. Results Compared with HCs, a total of 1,077 mRNAs in exosomes from MPO-AAV patients were found to be significantly upregulated, including DEPDC1B and TPST1, while NSUN4 and AK4 were significantly downregulated. Statistical analysis did not reveal any correlation between the six selected mRNAs and clinical indicators, including disease activity. GO enrichment analysis revealed that these differentially expressed genes participate in various enzyme activities, protein synthesis, etc. KEGG pathway analysis revealed that metabolic pathways, cell adhesion molecules, epithelial signaling, and mitogen-activated protein kinase (MAPK) signaling pathways were significantly enriched in the exosomal mRNAs. Conclusions There were significant differences in the expression of exosomal mRNAs between MPO-AAV patients and HCs, which may be related to the occurrence and development of MPO-AAV. These findings provide clues for further investigations of MPO-AAV pathogenesis and the identification of new potential therapeutic targets. MPO-ANCA-associated vasculitis exosomes mRNA pathogenesis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a group of autoimmune diseases characterized by the inflammatory destruction of small blood vessels, mostly resulting in multiple types of systemic damage and frequently affecting the kidney and lung[ 1 , 2 ]. The common ANCAs that have been identified are self-antigens, mainly myeloperoxidase (MPO) and neutral protease 3 (PR3), referred to as MPO-ANCA and PR3-ANCA, which are associated with MPO-AAV and PR3-AAV, respectively[ 3 ]. Clinically, AAV is largely divided into three groups: microscopic polyangitis (MPA), granuloma with polyangitis (GPA), and eosinophilic granuloma with polyangitis (EGPA), in which MPA and EPGA are predominantly related to MPO-ANCA, while PR3-ANCA is mainly related to GPA [ 4 – 7 ] [ 8 ]. However, genetic studies have revealed that MPO-AAV and PR3-AAV are distinct autoimmune syndromes[ 9 , 10 ], implying that their pathogenesis may differ, although they may sometimes share some clinical manifestations and therefore could be classified as MPA, GPA or EGPA. Therefore, to explore their pathogenesis, MPO-AAV and PR3-AAV should be investigated separately. Research has shown an increasing trend in the incidence of AAV[ 11 , 12 ]. PR3-AAV is more common than MPO-AAV in Western countries[ 12 , 13 ], while MPO-AAV is the most common in East Asian countries, including China and Japan[ 14 , 15 ]. ANCAs are thought to be directly involved in the pathogenesis of this disease. They can induce preactivated neutrophils to be hyperactivated, to release superoxides and lyases and to produce neutrophil extracellular traps (NETs), exacerbating vascular inflammation and injury. In addition, the alternative complement pathway and T lymphocytes play important roles in the pathogenesis of MPO-AAV [ 16 ]. Although all of these pathological processes in AAV are caused by immune intolerance, the exact mechanism that leads to the immune intolerance has remained elusive. Exosomes are cystic vesicles secreted by living cells that carry large amounts of proteins, lipids, DNA and different forms of RNA, including messenger RNA (mRNA) and noncoding RNA (ncRNA). They play important roles in multiple biological processes, such as intracellular signaling, coagulation, angiogenesis, inflammation, antigen presentation, apoptosis, and cellular homeostasis[ 17 – 20 ]. In particular, exosomes are also presumably involved in pathogenic pathways by modulating the immune response[ 21 ]. Exosomes derived from T-regulated cells (Tregs) can inhibit Th1 (T helper 1) cell proliferation and cytokine production by transferring their microRNAs (miRNAs)[ 22 ]. Exosomes from B cells can express functional integrins that mediate cell adhesion during inflammatory processes[ 23 ]. In the pathological process of AAV, biological molecules such as autoantibodies (ANCAs), cytokines and signaling pathway molecules are translated from mRNAs; however, to our knowledge, the exosomal mRNAs of AAV have not yet been investigated. Here, we preliminarily explored the potential role of exosomal mRNAs in the pathogenesis of MPO-AAV and their possible influences on disease activity. 2. Patients and Methods 2.1 Participants and heath record collection During the screening phase, six patients with MPO-AAV and six age- and sex-matched healthy controls (HCs) were selected. To exclude the possible impact of therapeutic agents on the experimental data, all 6 patients were recruited at their first onset of illness and had not yet received any treatments. During the validation phase, 22 additional MPO-AAV patients at various disease states and 22 matched HCs were further included in this study. All the patients tested positive for MPO-ANCAs and fulfilled the AAV classification criteria released by the American College of Rheumatology (ACR)/European Alliance of Associations for Rheumatology (EULAR) in 2022[ 5 , 6 ]. The clinical records and health check records of all the selected patients were collected. Patients with acute or chronic infections, neoplasms, other chronic diseases or patient who were pregnant were excluded. This study was performed according to the principles of the Declaration of Helsinki, and each participant provided informed consent before entering the study. The ethical committee of Anhui Medical University approved the study protocol. 2.2 Isolation and identification of peripheral blood exosomes After referencing relevant literature on the isolation and identification of exosomes from peripheral blood, exosomes were isolated from the peripheral blood of patients with MPO-AAV and HCs by ultracentrifugation[ 24 , 25 ]. Briefly, EDTA-anticoagulant venous blood samples (15 ml) from participants were centrifuged at 4000 rpm for 10 min to obtain the plasma. Exosomes were isolated from the plasma by ultracentrifugation at 110,000 × g for 70 minutes. To determine the morphology and size of the exosomes obtained, transmission electron microscopy and nanoparticle tracking analysis (NTA) were employed. In brief, the purified exosomes suspended in phosphate-buffered saline were dripped onto a copper-coated grid, phosphotungstic acid was added dropwise, and the sample was incubated for 5 minutes. Then, the samples were dried at room temperature for a few minutes. The grid was moved to a transmission electron microscope and observed at 100 kV using a Hitachi H-7650 transmission electron microscope. NTA was used to detect the obtained exosomes directly. 2.3 Extraction of exosomal total RNA Total RNA was extracted from purified exosomes using TRIzol. All procedures were carried out according to the manufacturer's instructions. A NanoDrop™ 2000 spectrophotometer (Thermo Scientific, USA) was used to determine the RNA concentration. 2.4 Exosomal mRNA bioinformatics analysis Beijing Genomics Institute (BGI) (China) prepared, sequenced, and conducted the bioinformatics analysis of the RNA library. The BGISEQ-500 platform was used for library sequencing. DESeq2 software was used to analyze mRNAs in the samples. A threshold P value 2 was used to identify the upregulated and downregulated mRNAs. Cluster hierarchy and volcano maps were used to display differences in exosomal mRNA expression patterns between the patient and HC groups. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were subsequently performed to explore the roles of the target mRNAs. 2.5 Validation of differential mRNA expression by qRT‒PCR During the validation stage, total RNA was extracted from the exosomes of 22 patients with MPO-AAV and 22 HCs. The data were used as original template information for real-time quantitative polymerase chain reaction (qRT‒PCR) quantification. The primer information is shown in Supplementary Table S1 online. In brief, cDNA was transcribed using PrimeScript™ RT Master Mix. PCR was performed on the Cobas 4800 PCR platform. The RT‒qPCR thermocycling protocol was as follows: 95°C for 30 s, 95°C for 5 s and 60°C for 30 s for 40 cycles. The qPCR data were quantified using the 2 − ΔΔCt method (ΔCt = Cttarget − Ctreference, −ΔΔCt = sample ΔCT − β-actin ΔCT)[ 26 ]. 2.6 Statistical analysis All the statistical analyses were performed with SPSS version 22.0 (SPSS Inc., Chicago, IL). Categorical variables are presented as frequencies and percentages. Quantitative variables are presented as the median and 25th–75th percentiles (interquartile range) or as the mean ± standard deviation (SD). Differences between two groups were assessed by t tests or nonparametric tests. The chi-square test was used to analyze the count data. Group comparisons were performed using Spearman’s correlation test. Risk factors were analyzed using logistic multiple regression. The term “significant” in this report was used to denote statistical significance ( P < 0.05). 3. Results 3.1 The demographic and clinical characteristics of the participants During the screening phase, 6 patients were matched with 6 HCs in terms of sex and age. All 6 patients were diagnosed with MPO-AAV for the first time and therefore had relatively short disease courses with active disease status. In the validation phase, there was no statistically significant difference in sex or age between the 22 patients and 22 HCs. All the patients had a longer course of disease, and their condition was also considered to be in an active state. In addition, in both the screening and validation phases, the erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) level, and creatinine level were significantly greater in the patients than in the controls. The demographic and disease characteristics of all the included patients and controls are listed in Table 1 . Table 1 Demographic and clinical characteristics of the MPO-AAV patients and healthy controls Phase Demographic and clinical characteristics MPO-AAV HC P value Screening n 6 6 Age (years, mean ± SD) 66.7 ± 10.8 60.2 ± 5.3 0.215 * Sex (male/female) 3/3 3/3 1.000 Disease duration (months, M (P25, P75)) 2.8 (1.9, 5.2) / ESR (mm/h, mean ± SD) 69.3 ± 25.8 3.8 ± 1.9 < 0.001 * CRP level (mg/L, M (P25, P75)) 42.0 (19.3, 57.0) 1.3 (0.9, 1.9) 0.009 Creatinine level (umol/mL, M (P25, P75)) 115.7 (43.3, 161.5) 67.2 (64.3, 70.3) 0.460 BVAS-V3 (M (P25, P75)) 17.5 (16, 20.3) / Validation n 22 22 Age (years, mean ± SD) 63.5 ± 8.7 59.5 ± 14.3 0.268 * Sex (male/female) 1.1:1 1.3:1 0.502 # Disease duration (months, mean ± SD) 1.2 (1.0, 3.25) / ESR (mm/h, M (P25, P75)) 68.1(48.5, 89.3) 3.0 (2.0, 5.0) < 0.001 # CRP level (mg/L, M (P25, P75)) 22.7 (4.3, 58.5) 3.2 (1.3, 5.7) < 0.001 # Creatinine level (umol/mL, M (P25, P75)) 180.0 (64.0, 367.5) 64.0 (45.0, 75.5) < 0.001 # BVAS-V3 (M mean ± SD) 17.7 ± 2.5 / Notes: *: T test result of the comparison between the RA and HC groups. #: result of the nonparametric test. Abbreviations: BVAS-V3, Birmingham vasculitis activity score-version 3; CRP, C-reactive protein; ESR erythrocyte sedimentation rate; HC, healthy control; MPO-ANCA, antineutrophil cytoplasmic antibody against myeloperoxidase; MPO-AAV, MPO-ANCA associated vasculitis; SD, standard deviation. 3.2 Features of the acquired plasma exosomes Transmission electron microscopy clearly revealed that the exocrine bodies were round or oval vesicles with sizes ranging from 30–100 nm. The comparison of the transmission electron microscopy images of the exocrine bodies from different sources between the two groups showed that exosomes were successfully isolated from plasma, and there was no significant difference in the morphology of the exocrine bodies between the AAV patients and the healthy control group (Fig. 1 ). 3.3 Hierarchical clustering analysis of the mRNAs from exosomes Hierarchical clustering analysis was carried out based on the exosomal mRNA results. The heatmap shows differences in the levels of plasma exosomal mRNAs between MPO-AAV patients and healthy controls (Fig. 2 ). The results of the GO enrichment analysis and KEGG pathway analysis of the target genes of the identified exosomal mRNAs are shown in Fig. 3 . A total of 3 GO terms (molecular function, cellular components, and biological processes) were found in the analysis. The results indicate that these differential genes are related to various enzyme activities, protein synthesis, etc., and are involved in various biological processes, such as retinal ganglion cell axon guidance, choline catabolic processes, mitotic sister chromatid segregation, spliceosomal tri-snRNP complex assembly, amyloid-beta metabolic processes, positive regulation of osteoblast differentiation, and cell responses to hepatocyte growth factor stimuli. KEGG pathway analysis revealed that metabolic pathways, cell adhesion molecules, epithelial signaling, and mitogen-activated protein kinase (MAPK) signaling pathways were significantly enriched in the exosomal mRNAs. 3.4 Candidate mRNAs screened by high-throughput sequencing and validated by qRT‒PCR Differentially expressed exosomal mRNAs An analysis of the differentially expressed extracellular mRNAs was conducted, and a total of 1,077 mRNAs in exosomes were screened from plasma samples. The statistical results of the differentially expressed exosomal mRNAs are shown in a volcano plot (Fig. 4 ). Then, the differentially expressed exosomal mRNAs between the AAV group and HC group were screened according to the fold change (log2, multiple expression differences). The selection criteria were as follows: Q value ≤ 0.05 or |Log2FC|≥ 2; 34 exosomal mRNAs showed significantly upregulated expression levels; and 74 exosomal mRNAs had significantly downregulated expression levels (Supplementary Table S2 online). Moreover, we randomly selected six differentially expressed exosomal mRNAs (DEPDC1B, TPST1, LSM2, NSUN4, FBXO34, and AK4) and validated them by qRT–PCR (Table 2). The expression levels of DEPDC1B and TPST1 in the exosomes from MPO-AAV patients were significantly greater than those in the exosomes from normal controls, while those of NSUN4 and AK4 were significantly lower ( P < 0.05, Fig. 5 ) in the patient group. Table 2. The expression differences of 6 candidate exosomal mRNAs in the validation phase ( n =22) Exosomal mRNA MPO-AAV * HC * * P # DEPDC1B 1.73 (0.50,1.97) 0.24 (0.03,0.32) 0.002 TPST1 1.78 (0.37,2.58) 0.64 (0.20,1.47) 0.008 LSM2 1.48 (0.24,2.34) 0.90 (0.65,1.24) 0.088 NSUN4 1.95 (0.63,2.45) 4.77 (1.55,9,53) 0.008 FBXO34 2.78 (0.90,4.38) 2.87 (0.20,5.26) 0.890 AK4 19.91(5.80,19.14) 56.56 (20.80,90.15) 0.013 Abbreviations: HC, healthy control; MPO-AAV, antineutrophil cytoplasmic antibody against myeloperoxidase associated vasculitis. *: data are presented as the median and 25 th and 75 th percentiles. #: the result of the Mann‒Whitney U test between the MPO-AAV and HC groups. 3.5 Relationships between the 6 selected mRNAs and MPO-AAV-related clinical indices The results obtained from the univariate linear regression analysis of the 6 selected mRNAs and clinical or laboratory parameters in MPO-AAV patients are shown in Table 3 . The results indicated that there were no statistically significant correlations between the expression of the 6 selected mRNAs and disease activity parameters (ESR, CRP level, and Birmingham vasculitis activity score version 3 (BVAS-V3)). Table 3. Results of the correlation analysis between the 6 candidate mRNAs and the clinical indices Clinical index DEPDC1B TPST1 LSM2 NSUN4 FBXO34 AK4 r P r P r P r P r P r P Disease duration (months) -0.11 0.96 -0.21 0.38 -0.24 0.38 -0.24 0.28 -0.18 0.43 0.15 0.52 ESR (mm/h) 0.13 0.56 -0.07 0.76 0.09 0.76 0.34 0.13 0.22 0.32 -0.22 0.34 CRP level (mg/L) -0.10 0.66 -0.08 0.74 0.14 0.74 0.28 0.19 0.16 0.47 -0.27 0.24 BVAS-V3 0.12 0.59 0.15 0.53 0.34 0.53 0.03 0.91 0.10 0.68 -0.04 0.87 Abbreviations: BVAS-V3, Birmingham vasculitis activity score-version 3; CRP, C-reactive protein; ESR, erythrocyte sedimentation rate. *: The results of the Spearman rank correlation analysis. Discussion In recent years, researchers have investigated miRNAs in AAV exosomes [ 27 – 29 ], but studies on the mRNAs in AAV exosomes are lacking. The relationship between plasma-derived exosomal mRNA and inflammatory injury in vascular endothelial cells in patients with AAV is not clear. Exosomes enter receptor cells through various mechanisms, such as phagocytosis, fusion and signal transduction[ 30 ]. Vascular endothelial cells can produce and absorb extracellular vesicles. Therefore, exosomes may be captured by vascular endothelial cells throughout the body and cause cell damage, leading to the onset of AAV[ 31 – 33 ]. Exosomes are widely distributed in the human body. They can carry proteins, lipids, and nucleic acids to exchange information between cells and regulate the activity of recipient cells. Studies have shown that exosomes can act as mediators of immune stimulation and regulation and participate in the regulation of a variety of immune processes, including antigen presentation, T-cell activation and polarization, and immunosuppression[ 34 , 35 ]. In the present study, we investigated the characteristics of mRNA expression profiles in the circulating exosomes of AAV patients. We isolated exosomes from peripheral blood for mRNA sequencing analysis and then identified candidate mRNAs by comparing the mRNA expression profiles between AAV patients and healthy controls. The RNA-sequencing data revealed that mRNAs may contribute to AAV disease progression. In this study, we identified three upregulated mRNAs (DEPDC1B, TPST1, and LSM2) and three downregulated mRNAs (NSUN4, FBXO34, and AK4) and validated the differential expression of these mRNAs by qRT‒PCR in 22 samples from AAV patients. The results indicated that the expression levels of DEPDC1B and TPST1 in the exosomes of AAV patients were significantly greater than those in the exosomes of normal controls, whereas those of NSUN4 and AK4 were significantly lower. The enrichment analysis results indicated that metabolic pathways, cell adhesion molecules, epithelial signaling, and MAPK signaling pathways are involved in AAV pathogenesis. Cell adhesion molecules and MAPK signaling pathways are considered to play crucial roles in autoimmune disease[ 28 , 36 , 37 ]. Studies have shown that plasma levels of extracellular vesicles increase when the vascular endothelium is activated or damaged[ 38 , 39 ]. Exosomes may damage endothelium-dependent vasodilation and reduce NO, leading to endothelial dysfunction[ 40 ]. In addition, exosomes can bind to the endothelium and activate it by increasing the production of reactive oxygen species (ROS)[ 41 ]. Neutosis is a special form of neutrophil death in which nuclear DNA is released from the broken nuclear membrane[ 42 ] and the broken plasma membrane forms NETs, which may lead to endothelial dysfunction through the activation of matrix metalloproteinases2[ 43 ], leading to vasculitis. Moreover, studies have confirmed a positive correlation between proinflammatory cytokines and adhesion molecules[ 37 ]. Therefore, we believe that endothelial injury and/or activation are essential features of AAV. Adhesion molecules in endothelial cells are upregulated during metabolism and inflammatory activation, which then mediate the adhesion and migration of white blood cells in blood vessels and promote inflammation and tissue damage. In general, cell adhesion molecules play a role in the pathogenesis of AAV. MAPK signaling pathways are also considered to play a crucial role in autoimmune disease[ 44 , 45 ]. In the vascular wall, macrophages produce proinflammatory cytokines (IL-1β and TNF-α). These proinflammatory cytokines aggravate vascular inflammation through phosphorylation of signaling pathways, such as the MAPK pathway. During inflammation, adhesion molecules, cytokines, and chemokines are regulated by the MAPK signaling pathway. Increasing evidence suggests that the inhibition of signaling cascade phosphorylation is effective in treating inflammatory diseases[ 46 , 47 ]. In AAV, ANCAs activate human neutrophils through the p38MAPK-mediated pathway, and this signaling cascade is responsible for the transfer of ANCA-specific antigens from cytoplasmic particles to the cell surface[ 48 ]. Many studies have confirmed that inflammatory damage to the vascular endothelium is caused by leukocyte infiltration in AAV[ 49 , 50 ]. The patients we selected were all active AAV patients (with elevated BVAS-V3), and compared to those of healthy individuals, their ESR, CRP levels, and creatinine levels were significantly greater, indicating that the patients were experiencing a systemic inflammatory response. Considering the key role of inflammatory injury in endothelial cells in the pathogenesis of AAV, we speculate that the mRNAs from the exosomes of patients with active AAV may be absorbed by vascular endothelial cells and contribute to the induction of endothelial cell inflammation and neutrophil adhesion. An increase in the mRNAs DEPDC1B and TPST1 in AAV exosomes may promote the adhesion of neutrophils. DEPDC1B recognizes G protein-coupled receptors and regulates signaling pathways through effector and regulatory factors[ 51 ]. DEPDC1B interacts with diverse signaling molecules, including splicing regulatory molecules and transmembrane proteins. Studies have shown that DEPDC1B participates in cell adhesion, cell proliferation, and cell cycle regulation[ 52 , 53 ]. Some studies have reported that the expression of the TPST1 gene may affect the tumor microenvironment and may even be related to the immunotherapy response in bladder cancer[ 54 ]. Neutrophils express TPST1[ 55 ]. TPST1 plays a role in the production of proinflammatory cytokines in LPS-induced macrophages, and the downregulation of TPST1 inhibits LPS-induced IL-6 production[ 56 ]. The expression of TPST1, which is related to the immune response, is upregulated in the bone marrow monocytes of patients with rheumatoid arthritis[ 57 ]. Therefore, TPST1 may play an important role in the development of inflammation. Additionally, some studies have shown that the mRNA expression of NSUN4 and AK4 is related to tumors, but its role in autoimmune diseases needs further research[ 58 – 60 ]. The pathophysiological mechanisms of AAV are complex. According to the bioinformatics analysis of the differentially expressed mRNAs, we believe that the disease activity of AAV is related to an activation and imbalance of inflammation-related signaling pathways. Although these mRNAs can partially predict the onset of AAV, further studies are needed to understand the mechanisms underlying the effect of these target mRNAs on disease progression. In conclusion, this study revealed two mRNAs (DEPDC1B and TPST1) that were significantly upregulated in patients with AAV and two mRNAs (NSUN4 and AK4) that were significantly downregulated in patients with AAV. However, through correlation analysis, we found that these indicators were not related to disease activity, and these mRNA interactions may be involved in the development or progression of AAV. These data suggest that DEPDC1B, TPST1, NSUN4 and AK4 may be key regulators of AAV. Targeting these mRNAs may be useful for inhibiting the inflammatory response in AAV. However, the exact role of these mRNAs in the pathogenesis of AAV and their diagnostic and prognostic value for this disease are still unclear. This study detected the serum exosomal mRNAs of patients with AAV by high-throughput sequencing technology and further confirmed that exosomal mRNAs can be used for the early diagnosis and treatment of AAV. However, the sample size of this study was small, and thus should be further experimentally verified with an expanded sample size. Abbreviations ANCA antineutrophil cytoplasmic antibody AAV ANCA-associated vasculitis BVAS-V3 Birmingham vasculitis activity score-version 3 CRP C-reactive protein EGPA eosinophilic granuloma with polyangiitis ESR erythrocyte sedimentation rate GO Gene Ontology HC healthy control KEGG Encyclopedia of Genes and Genomes MAPK mitogen-activated protein kinase miRNA microRNA MPO myeloperoxidase mRNA messenger RNA MPA microscopic polyangiitis,ncRNA,noncoding RNA NETs neutrophil extracellular traps PR3 neutral protease 3 qRT‒PCR real-time quantitative polymerase chain reaction ROS reactive oxygen species SD standard deviation GPA granuloma with polyangitis Treg T-regulated cell Th1 T helper 1. Declarations Data availability Sequence data that support the findings of this study have been deposited in the NCBI with the primary accession code PRJNA1114026. Declarations of interest The authors declare no conflicts of interest. Ethics approval and consent to participate All subjects signed informed consent forms in accordance with the ethical principles of the Declaration of Helsinki. The research protocol was approved by the Ethics Committee of Anhui Medical University (PJ2020-06-11). Funding sources The current study received support from the Basic and Clinical Cooperative Research Promotion Program of Anhui Medical University (2021xkjT034). Acknowledgments We thank all the patients for their enthusiastic participation in the study. Author contributions YF Chen contributed to the study design, experiment conduct, data analysis and drafting of the article. X Qian, Y Wang and SQ Ge contributed to sample collection, experiment conduct and data analysis. YF Chen and ZW Shuai reviewed and edited the article. References Kitching A. R., Anders H. J., Basu N., et al. ANCA-associated vasculitis. Nat Rev Dis Primers. 2020;6(1):71. Almaani S., Fussner L. A., Brodsky S., Meara A. S., Jayne D. 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XY Li, XT Yang, Id Orcid. - Correlation Between the RNA Methylation Genes and Immune Infiltration and. D - 101512684. (- 1178-7031 (Print)):- 3941-56. X Zhang, Y Zhou, Z Shi, et al. - Integrated analysis of genes encoding ATP-dependent chromatin remodellers. D - 101597971. (- 2001-1326 (Electronic)):- e953. Additional Declarations No competing interests reported. Supplementary Files SupplementaryMaterial.docx Cite Share Download PDF Status: Published Journal Publication published 17 Sep, 2024 Read the published version in Clinical and Experimental Medicine → Version 1 posted Editorial decision: Revision requested 07 Jul, 2024 Reviews received at journal 07 Jul, 2024 Reviews received at journal 04 Jul, 2024 Reviewers agreed at journal 26 Jun, 2024 Reviewers agreed at journal 26 Jun, 2024 Reviewers invited by journal 25 Jun, 2024 Editor assigned by journal 24 Jun, 2024 Submission checks completed at journal 24 Jun, 2024 First submitted to journal 22 Jun, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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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-4622546","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":323781082,"identity":"ca50e9cc-3fdd-42e5-964e-3edfa5530ad5","order_by":0,"name":"杨凡 陈","email":"","orcid":"","institution":"The First Affiliated Hospital of Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"杨凡","middleName":"","lastName":"陈","suffix":""},{"id":323781086,"identity":"49c634f0-21e1-4b9b-8e80-3d7a1bedae05","order_by":1,"name":"东清 周","email":"","orcid":"","institution":"The First Affiliated Hospital of Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"东清","middleName":"","lastName":"周","suffix":""},{"id":323781090,"identity":"793f44b2-e73e-45e3-9b9e-2a8fccf1fbe3","order_by":2,"name":"辛 钱","email":"","orcid":"","institution":"The First Affiliated Hospital of Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"辛","middleName":"","lastName":"钱","suffix":""},{"id":323781091,"identity":"90bf0052-b513-4d21-a1df-98b7be759550","order_by":3,"name":"尚庆 格","email":"","orcid":"","institution":"The First Affiliated Hospital of Anhui Medical University","correspondingAuthor":false,"prefix":"","firstName":"尚庆","middleName":"","lastName":"格","suffix":""},{"id":323781092,"identity":"bc7de066-c209-4389-80bc-1dcb5d22ce81","order_by":4,"name":"宗文 帅","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+UlEQVRIiWNgGAWjYDACCSBOgHE+MByACxKnhXEG0VpggJmHGC3ys3sMPzz4Y5MnH5F87LFNzZ1ogwPMB2/zMNjl4dJicOeMsUQCT1qx4Y20dOOcY89yNxxgS7bmYUguxqlFIsdAIkHicOLG2Tlm0jlsh4FaeMykgS5MbMDlsBk5xj8SDEBa8r9JW/wDaeH/hlcLw40cM4mEhMOJ84FWSDO2gW1hw6vF4EZamUXCgbTEDfLPzCR7+w7nzjzMZmw5xyAZj8OSN9/88ccmcX7P4WcSP74dzu073vzwxpsKO9wOg1t3AMZiBnMJqQdZR9DQUTAKRsEoGLEAACWqXO7/1NxDAAAAAElFTkSuQmCC","orcid":"","institution":"The First Affiliated Hospital of Anhui Medical University","correspondingAuthor":true,"prefix":"","firstName":"宗文","middleName":"","lastName":"帅","suffix":""}],"badges":[],"createdAt":"2024-06-22 15:44:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4622546/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4622546/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10238-024-01457-2","type":"published","date":"2024-09-17T15:57:14+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":60624869,"identity":"e1d5feaa-f1fa-4409-b7a8-715cc4bb5c97","added_by":"auto","created_at":"2024-07-18 22:17:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1210569,"visible":true,"origin":"","legend":"\u003cp\u003eTransmission electron microscope image of the exosome morphology in patients with AAV (A) and healthy controls (B) (scale bar: 100 nm) and the size distributions of the exosomes from MPO-AAV patients (C) and healthy controls (D), which indicated that exosomes were successfully obtained in our study.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4622546/v1/d8361c0501365b359db00428.png"},{"id":60624864,"identity":"06878240-d98e-47c7-88b7-2f38b8244781","added_by":"auto","created_at":"2024-07-18 22:17:56","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":204029,"visible":true,"origin":"","legend":"\u003cp\u003eHeatmap showing the comparison of the expression levels of exosomal mRNAs between the MPO-AAV group and the healthy control group. The X axis shows the MPO-AAV patient and HC groups in the cluster analysis, and the Y axis represents the sample differential exosomal mRNAs. The red color represents an increase in expression, while the blue color represents a decrease in expression; the larger the value is, the darker the color.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4622546/v1/c453f93cbbc10b2e22ad1859.png"},{"id":60625635,"identity":"aafcf32c-d339-4ce7-a9a6-0f4e82dde75b","added_by":"auto","created_at":"2024-07-18 22:25:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":574040,"visible":true,"origin":"","legend":"\u003cp\u003eGO enrichment analysis and KEGG pathway analysis of the genes associated with exosomal mRNAs. (A) GO enrichment analysis molecular function terms; (B) GO enrichment analysis cellular components terms; (C) GO enrichment analysis biological processes terms; (D) KEGG pathway analysis results.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4622546/v1/52f1a539cd1ffe1b04a4bc5d.png"},{"id":60624865,"identity":"32d662c0-a280-4819-bc01-7886df320c66","added_by":"auto","created_at":"2024-07-18 22:17:56","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":413749,"visible":true,"origin":"","legend":"\u003cp\u003eVolcano plot revealing the differentially expressed mRNAs between the patient and HC groups. The X axis is the log2 (fold change) values, and the Y axis is −log10 (Q value) values. The gray dots represent the mRNAs with no significant differences, the red dots represent the upregulated mRNAs, and the green dots represent the downregulated mRNAs.\u003c/p\u003e","description":"","filename":"figure4..png","url":"https://assets-eu.researchsquare.com/files/rs-4622546/v1/721c69a3ad184903cc95cdc5.png"},{"id":60624867,"identity":"48af1530-0b87-45f0-b688-0c380fd34dae","added_by":"auto","created_at":"2024-07-18 22:17:56","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":82237,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of the expression levels of exosomal mRNAs (DEPDC1B, TPST1, FBXO34, and AK4) between the patient and HC groups (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4622546/v1/496208086ca8233acd378f18.png"},{"id":65104466,"identity":"086cb5e4-39b8-4557-a621-fc59c31d662e","added_by":"auto","created_at":"2024-09-23 16:13:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3681534,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4622546/v1/f062c664-1e61-49c7-a528-f264534f77ce.pdf"},{"id":60624866,"identity":"0a5f90d1-0a54-4a1c-a06c-38e06ed2028c","added_by":"auto","created_at":"2024-07-18 22:17:56","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":17669,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-4622546/v1/2fb5bde02802be570970131a.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Characteristic changes in the mRNA expression profile of plasma exosomes from patients with MPO-ANCA-associated vasculitis and its possible correlations with pathogenesis","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAntineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a group of autoimmune diseases characterized by the inflammatory destruction of small blood vessels, mostly resulting in multiple types of systemic damage and frequently affecting the kidney and lung[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The common ANCAs that have been identified are self-antigens, mainly myeloperoxidase (MPO) and neutral protease 3 (PR3), referred to as MPO-ANCA and PR3-ANCA, which are associated with MPO-AAV and PR3-AAV, respectively[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Clinically, AAV is largely divided into three groups: microscopic polyangitis (MPA), granuloma with polyangitis (GPA), and eosinophilic granuloma with polyangitis (EGPA), in which MPA and EPGA are predominantly related to MPO-ANCA, while PR3-ANCA is mainly related to GPA [\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. However, genetic studies have revealed that MPO-AAV and PR3-AAV are distinct autoimmune syndromes[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], implying that their pathogenesis may differ, although they may sometimes share some clinical manifestations and therefore could be classified as MPA, GPA or EGPA. Therefore, to explore their pathogenesis, MPO-AAV and PR3-AAV should be investigated separately. Research has shown an increasing trend in the incidence of AAV[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. PR3-AAV is more common than MPO-AAV in Western countries[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], while MPO-AAV is the most common in East Asian countries, including China and Japan[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eANCAs are thought to be directly involved in the pathogenesis of this disease. They can induce preactivated neutrophils to be hyperactivated, to release superoxides and lyases and to produce neutrophil extracellular traps (NETs), exacerbating vascular inflammation and injury. In addition, the alternative complement pathway and T lymphocytes play important roles in the pathogenesis of MPO-AAV [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Although all of these pathological processes in AAV are caused by immune intolerance, the exact mechanism that leads to the immune intolerance has remained elusive.\u003c/p\u003e \u003cp\u003eExosomes are cystic vesicles secreted by living cells that carry large amounts of proteins, lipids, DNA and different forms of RNA, including messenger RNA (mRNA) and noncoding RNA (ncRNA). They play important roles in multiple biological processes, such as intracellular signaling, coagulation, angiogenesis, inflammation, antigen presentation, apoptosis, and cellular homeostasis[\u003cspan additionalcitationids=\"CR18 CR19\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In particular, exosomes are also presumably involved in pathogenic pathways by modulating the immune response[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Exosomes derived from T-regulated cells (Tregs) can inhibit Th1 (T helper 1) cell proliferation and cytokine production by transferring their microRNAs (miRNAs)[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Exosomes from B cells can express functional integrins that mediate cell adhesion during inflammatory processes[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In the pathological process of AAV, biological molecules such as autoantibodies (ANCAs), cytokines and signaling pathway molecules are translated from mRNAs; however, to our knowledge, the exosomal mRNAs of AAV have not yet been investigated. Here, we preliminarily explored the potential role of exosomal mRNAs in the pathogenesis of MPO-AAV and their possible influences on disease activity.\u003c/p\u003e"},{"header":"2. Patients and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Participants and heath record collection\u003c/h2\u003e \u003cp\u003eDuring the screening phase, six patients with MPO-AAV and six age- and sex-matched healthy controls (HCs) were selected. To exclude the possible impact of therapeutic agents on the experimental data, all 6 patients were recruited at their first onset of illness and had not yet received any treatments. During the validation phase, 22 additional MPO-AAV patients at various disease states and 22 matched HCs were further included in this study. All the patients tested positive for MPO-ANCAs and fulfilled the AAV classification criteria released by the American College of Rheumatology (ACR)/European Alliance of Associations for Rheumatology (EULAR) in 2022[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The clinical records and health check records of all the selected patients were collected. Patients with acute or chronic infections, neoplasms, other chronic diseases or patient who were pregnant were excluded. This study was performed according to the principles of the Declaration of Helsinki, and each participant provided informed consent before entering the study. The ethical committee of Anhui Medical University approved the study protocol.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Isolation and identification of peripheral blood exosomes\u003c/h2\u003e \u003cp\u003eAfter referencing relevant literature on the isolation and identification of exosomes from peripheral blood, exosomes were isolated from the peripheral blood of patients with MPO-AAV and HCs by ultracentrifugation[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Briefly, EDTA-anticoagulant venous blood samples (15 ml) from participants were centrifuged at 4000 rpm for 10 min to obtain the plasma. Exosomes were isolated from the plasma by ultracentrifugation at 110,000 \u0026times; g for 70 minutes. To determine the morphology and size of the exosomes obtained, transmission electron microscopy and nanoparticle tracking analysis (NTA) were employed. In brief, the purified exosomes suspended in phosphate-buffered saline were dripped onto a copper-coated grid, phosphotungstic acid was added dropwise, and the sample was incubated for 5 minutes. Then, the samples were dried at room temperature for a few minutes. The grid was moved to a transmission electron microscope and observed at 100 kV using a Hitachi H-7650 transmission electron microscope. NTA was used to detect the obtained exosomes directly.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Extraction of exosomal total RNA\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from purified exosomes using TRIzol. All procedures were carried out according to the manufacturer's instructions. A NanoDrop\u0026trade; 2000 spectrophotometer (Thermo Scientific, USA) was used to determine the RNA concentration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Exosomal mRNA bioinformatics analysis\u003c/h2\u003e \u003cp\u003eBeijing Genomics Institute (BGI) (China) prepared, sequenced, and conducted the bioinformatics analysis of the RNA library. The BGISEQ-500 platform was used for library sequencing. DESeq2 software was used to analyze mRNAs in the samples. A threshold P value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 plus a multiple change\u0026thinsp;\u0026gt;\u0026thinsp;2 was used to identify the upregulated and downregulated mRNAs. Cluster hierarchy and volcano maps were used to display differences in exosomal mRNA expression patterns between the patient and HC groups. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were subsequently performed to explore the roles of the target mRNAs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Validation of differential mRNA expression by qRT‒PCR\u003c/h2\u003e \u003cp\u003eDuring the validation stage, total RNA was extracted from the exosomes of 22 patients with MPO-AAV and 22 HCs. The data were used as original template information for real-time quantitative polymerase chain reaction (qRT‒PCR) quantification. The primer information is shown in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e online. In brief, cDNA was transcribed using PrimeScript\u0026trade; RT Master Mix. PCR was performed on the Cobas 4800 PCR platform. The RT‒qPCR thermocycling protocol was as follows: 95\u0026deg;C for 30 s, 95\u0026deg;C for 5 s and 60\u0026deg;C for 30 s for 40 cycles. The qPCR data were quantified using the 2\u0026thinsp;\u0026minus;\u0026thinsp;ΔΔCt method (ΔCt\u0026thinsp;=\u0026thinsp;Cttarget\u0026thinsp;\u0026minus;\u0026thinsp;Ctreference, \u0026minus;ΔΔCt\u0026thinsp;=\u0026thinsp;sample ΔCT\u0026thinsp;\u0026minus;\u0026thinsp;β-actin ΔCT)[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Statistical analysis\u003c/h2\u003e \u003cp\u003eAll the statistical analyses were performed with SPSS version 22.0 (SPSS Inc., Chicago, IL). Categorical variables are presented as frequencies and percentages. Quantitative variables are presented as the median and 25th\u0026ndash;75th percentiles (interquartile range) or as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Differences between two groups were assessed by \u003cem\u003et\u003c/em\u003e tests or nonparametric tests. The chi-square test was used to analyze the count data. Group comparisons were performed using Spearman\u0026rsquo;s correlation test. Risk factors were analyzed using logistic multiple regression. The term \u0026ldquo;significant\u0026rdquo; in this report was used to denote statistical significance (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.1 The demographic and clinical characteristics of the participants\u003c/h2\u003e \u003cp\u003eDuring the screening phase, 6 patients were matched with 6 HCs in terms of sex and age. All 6 patients were diagnosed with MPO-AAV for the first time and therefore had relatively short disease courses with active disease status. In the validation phase, there was no statistically significant difference in sex or age between the 22 patients and 22 HCs. All the patients had a longer course of disease, and their condition was also considered to be in an active state. In addition, in both the screening and validation phases, the erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) level, and creatinine level were significantly greater in the patients than in the controls. The demographic and disease characteristics of all the included patients and controls are listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\u003cdiv class=\"gridtable\"\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=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\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\u003eDemographic and clinical characteristics of the MPO-AAV patients and healthy controls\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePhase\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDemographic and clinical characteristics\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMPO-AAV\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHC\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eP value\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eScreening\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003en\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAge (years, mean ± SD)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e66.7 ± 10.8\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e60.2 ± 5.3\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.215\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSex (male/female)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3/3\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3/3\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.000\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDisease duration (months, M (P25, P75))\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.8 (1.9, 5.2)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eESR (mm/h, mean ± SD)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e69.3 ± 25.8\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.8 ± 1.9\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt; 0.001\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCRP level (mg/L, M (P25, P75))\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e42.0 (19.3, 57.0)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.3 (0.9, 1.9)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.009\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCreatinine level (umol/mL, M (P25, P75))\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e115.7 (43.3, 161.5)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e67.2 (64.3, 70.3)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.460\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBVAS-V3 (M (P25, P75))\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17.5 (16, 20.3)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eValidation\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003en\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAge (years, mean ± SD)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e63.5 ± 8.7\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e59.5 ± 14.3\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.268\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSex (male/female)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.1:1\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.3:1\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.502\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDisease duration (months, mean ± SD)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.2 (1.0, 3.25)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eESR (mm/h, M (P25, P75))\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e68.1(48.5, 89.3)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.0 (2.0, 5.0)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt; 0.001\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCRP level (mg/L, M (P25, P75))\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22.7 (4.3, 58.5)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.2 (1.3, 5.7)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt; 0.001\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCreatinine level (umol/mL, M (P25, P75))\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e180.0 (64.0, 367.5)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e64.0 (45.0, 75.5)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt; 0.001\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBVAS-V3 (M mean ± SD)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17.7 ± 2.5\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e/\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003eNotes: *: T test result of the comparison between the RA and HC groups. #: result of the nonparametric test. Abbreviations: BVAS-V3, Birmingham vasculitis activity score-version 3; CRP, C-reactive protein; ESR erythrocyte sedimentation rate; HC, healthy control; MPO-ANCA, antineutrophil cytoplasmic antibody against myeloperoxidase; MPO-AAV, MPO-ANCA associated vasculitis; SD, standard deviation.\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e \u003cp\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Features of the acquired plasma exosomes\u003c/h2\u003e \u003cp\u003eTransmission electron microscopy clearly revealed that the exocrine bodies were round or oval vesicles with sizes ranging from 30–100 nm. The comparison of the transmission electron microscopy images of the exocrine bodies from different sources between the two groups showed that exosomes were successfully isolated from plasma, and there was no significant difference in the morphology of the exocrine bodies between the AAV patients and the healthy control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Hierarchical clustering analysis of the mRNAs from exosomes\u003c/h2\u003e \u003cp\u003eHierarchical clustering analysis was carried out based on the exosomal mRNA results. The heatmap shows differences in the levels of plasma exosomal mRNAs between MPO-AAV patients and healthy controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe results of the GO enrichment analysis and KEGG pathway analysis of the target genes of the identified exosomal mRNAs are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. A total of 3 GO terms (molecular function, cellular components, and biological processes) were found in the analysis. The results indicate that these differential genes are related to various enzyme activities, protein synthesis, etc., and are involved in various biological processes, such as retinal ganglion cell axon guidance, choline catabolic processes, mitotic sister chromatid segregation, spliceosomal tri-snRNP complex assembly, amyloid-beta metabolic processes, positive regulation of osteoblast differentiation, and cell responses to hepatocyte growth factor stimuli. KEGG pathway analysis revealed that metabolic pathways, cell adhesion molecules, epithelial signaling, and mitogen-activated protein kinase (MAPK) signaling pathways were significantly enriched in the exosomal mRNAs.\u003c/p\u003e\u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Candidate mRNAs screened by high-throughput sequencing and validated by qRT‒PCR Differentially expressed exosomal mRNAs\u003c/h2\u003e \u003cp\u003eAn analysis of the differentially expressed extracellular mRNAs was conducted, and a total of 1,077 mRNAs in exosomes were screened from plasma samples. The statistical results of the differentially expressed exosomal mRNAs are shown in a volcano plot (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Then, the differentially expressed exosomal mRNAs between the AAV group and HC group were screened according to the fold change (log2, multiple expression differences). The selection criteria were as follows: Q value ≤ 0.05 or |Log2FC|≥ 2; 34 exosomal mRNAs showed significantly upregulated expression levels; and 74 exosomal mRNAs had significantly downregulated expression levels (Supplementary Table S2 online). Moreover, we randomly selected six differentially expressed exosomal mRNAs (DEPDC1B, TPST1, LSM2, NSUN4, FBXO34, and AK4) and validated them by qRT–PCR (Table\u0026nbsp;2). The expression levels of DEPDC1B and TPST1 in the exosomes from MPO-AAV patients were significantly greater than those in the exosomes from normal controls, while those of NSUN4 and AK4 were significantly lower (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) in the patient group.\u003c/p\u003e \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"567\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"98.2363315696649%\" colspan=\"4\" valign=\"top\" style=\"width: 62.006%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTable 2.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eThe expression differences of 6 candidate\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eexosomal mRNAs\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;in the validation phase (\u003cem\u003en\u003c/em\u003e=22)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"28.395061728395063%\" valign=\"top\" style=\"width: 17.9227%;\"\u003e\n \u003cp\u003eExosomal mRNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.395061728395063%\" valign=\"top\" style=\"width: 17.9227%;\"\u003e\n \u003cp\u003eMPO-AAV\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.631393298059965%\" valign=\"top\" style=\"width: 16.8095%;\"\u003e\n \u003cp\u003eHC\u003cem\u003e\u003csup\u003e*\u003c/sup\u003e\u003c/em\u003e\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.57848324514991%\" valign=\"top\" style=\"width: 10.4642%;\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003csup\u003e#\u003c/sup\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"28.395061728395063%\" valign=\"top\" style=\"width: 17.9227%;\"\u003e\n \u003cp\u003eDEPDC1B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.395061728395063%\" style=\"width: 17.9227%;\"\u003e\n \u003cp\u003e1.73 (0.50,1.97)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.631393298059965%\" style=\"width: 16.8095%;\"\u003e\n \u003cp\u003e0.24 (0.03,0.32)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.57848324514991%\" style=\"width: 10.4642%;\"\u003e\n \u003cp\u003e0.002\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"28.395061728395063%\" valign=\"top\" style=\"width: 17.9227%;\"\u003e\n \u003cp\u003eTPST1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.395061728395063%\" valign=\"top\" style=\"width: 17.9227%;\"\u003e\n \u003cp\u003e1.78 (0.37,2.58)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.631393298059965%\" valign=\"top\" style=\"width: 16.8095%;\"\u003e\n \u003cp\u003e0.64 (0.20,1.47)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.57848324514991%\" style=\"width: 10.4642%;\"\u003e\n \u003cp\u003e0.008\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"28.395061728395063%\" valign=\"top\" style=\"width: 17.9227%;\"\u003e\n \u003cp\u003eLSM2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.395061728395063%\" valign=\"top\" style=\"width: 17.9227%;\"\u003e\n \u003cp\u003e1.48 (0.24,2.34)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.631393298059965%\" valign=\"top\" style=\"width: 16.8095%;\"\u003e\n \u003cp\u003e0.90 (0.65,1.24)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.57848324514991%\" style=\"width: 10.4642%;\"\u003e\n \u003cp\u003e\u0026nbsp;0.088\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"28.395061728395063%\" valign=\"top\" style=\"width: 17.9227%;\"\u003e\n \u003cp\u003eNSUN4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.395061728395063%\" valign=\"top\" style=\"width: 17.9227%;\"\u003e\n \u003cp\u003e1.95 (0.63,2.45)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.631393298059965%\" valign=\"top\" style=\"width: 16.8095%;\"\u003e\n \u003cp\u003e4.77 (1.55,9,53)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.57848324514991%\" style=\"width: 10.4642%;\"\u003e\n \u003cp\u003e\u0026nbsp;0.008\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"28.395061728395063%\" valign=\"top\" style=\"width: 17.9227%;\"\u003e\n \u003cp\u003eFBXO34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.395061728395063%\" valign=\"top\" style=\"width: 17.9227%;\"\u003e\n \u003cp\u003e2.78 (0.90,4.38)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.631393298059965%\" valign=\"top\" style=\"width: 16.8095%;\"\u003e\n \u003cp\u003e2.87 (0.20,5.26)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.57848324514991%\" style=\"width: 10.4642%;\"\u003e\n \u003cp\u003e\u0026nbsp;0.890\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"28.395061728395063%\" valign=\"top\" style=\"width: 17.9227%;\"\u003e\n \u003cp\u003eAK4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.395061728395063%\" valign=\"top\" style=\"width: 17.9227%;\"\u003e\n \u003cp\u003e19.91(5.80,19.14)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"26.631393298059965%\" valign=\"top\" style=\"width: 16.8095%;\"\u003e\n \u003cp\u003e56.56 (20.80,90.15)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.57848324514991%\" style=\"width: 10.4642%;\"\u003e\n \u003cp\u003e\u0026nbsp;0.013\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"98.2363315696649%\" colspan=\"4\" style=\"width: 62.006%;\"\u003e\n \u003cp\u003eAbbreviations: HC, healthy control; MPO-AAV,\u0026nbsp;antineutrophil cytoplasmic antibody against myeloperoxidase\u0026nbsp;associated vasculitis. *: data are presented as the median and 25\u003csup\u003eth\u003c/sup\u003e and 75\u003csup\u003eth\u003c/sup\u003e percentiles. #: the result of the Mann‒Whitney U test between the MPO-AAV and HC groups.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Relationships between the 6 selected mRNAs and MPO-AAV-related clinical indices\u003c/h2\u003e \u003cp\u003eThe results obtained from the univariate linear regression analysis of the 6 selected mRNAs and clinical or laboratory parameters in MPO-AAV patients are shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The results indicated that there were no statistically significant correlations between the expression of the 6 selected mRNAs and disease activity parameters (ESR, CRP level, and Birmingham vasculitis activity score version 3 (BVAS-V3)).\u003c/p\u003e\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"13\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eTable 3. Results of the correlation analysis between the 6 candidate mRNAs and the clinical indices\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.76595744680851%\" rowspan=\"2\"\u003e\n \u003cp\u003eClinical index\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.539007092198581%\" colspan=\"2\"\u003e\n \u003cp\u003eDEPDC1B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.539007092198581%\" colspan=\"2\"\u003e\n \u003cp\u003eTPST1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.539007092198581%\" colspan=\"2\"\u003e\n \u003cp\u003eLSM2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.539007092198581%\" colspan=\"2\"\u003e\n \u003cp\u003eNSUN4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.539007092198581%\" colspan=\"2\"\u003e\n \u003cp\u003eFBXO34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.539007092198581%\" colspan=\"2\"\u003e\n \u003cp\u003eAK4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"8.333333333333334%\"\u003e\n \u003cp\u003e\u003cem\u003er\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.333333333333334%\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.333333333333334%\"\u003e\n \u003cp\u003e\u003cem\u003er\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.333333333333334%\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.333333333333334%\"\u003e\n \u003cp\u003e\u003cem\u003er\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.333333333333334%\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.333333333333334%\"\u003e\n \u003cp\u003e\u003cem\u003er\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.333333333333334%\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.333333333333334%\"\u003e\n \u003cp\u003e\u003cem\u003er\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.333333333333334%\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.333333333333334%\"\u003e\n \u003cp\u003e\u003cem\u003er\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.333333333333334%\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.76595744680851%\"\u003e\n \u003cp\u003eDisease duration (months)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.2695035460992905%\"\u003e\n \u003cp\u003e-0.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.2695035460992905%\"\u003e\n \u003cp\u003e0.96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.2695035460992905%\"\u003e\n \u003cp\u003e-0.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.2695035460992905%\"\u003e\n \u003cp\u003e0.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.2695035460992905%\"\u003e\n \u003cp\u003e-0.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.2695035460992905%\"\u003e\n \u003cp\u003e0.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.2695035460992905%\"\u003e\n \u003cp\u003e-0.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.2695035460992905%\"\u003e\n \u003cp\u003e0.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.2695035460992905%\"\u003e\n \u003cp\u003e-0.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.2695035460992905%\"\u003e\n \u003cp\u003e0.43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.2695035460992905%\"\u003e\n \u003cp\u003e0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.2695035460992905%\"\u003e\n \u003cp\u003e0.52\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.76595744680851%\"\u003e\n \u003cp\u003eESR (mm/h)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.2695035460992905%\"\u003e\n \u003cp\u003e0.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.2695035460992905%\"\u003e\n \u003cp\u003e0.56\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.2695035460992905%\"\u003e\n \u003cp\u003e-0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.2695035460992905%\"\u003e\n \u003cp\u003e0.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.2695035460992905%\"\u003e\n \u003cp\u003e0.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.2695035460992905%\"\u003e\n \u003cp\u003e0.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.2695035460992905%\"\u003e\n \u003cp\u003e0.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.2695035460992905%\"\u003e\n \u003cp\u003e0.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.2695035460992905%\"\u003e\n \u003cp\u003e0.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.2695035460992905%\"\u003e\n \u003cp\u003e0.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.2695035460992905%\"\u003e\n \u003cp\u003e-0.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.2695035460992905%\"\u003e\n \u003cp\u003e0.34\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.76595744680851%\"\u003e\n \u003cp\u003eCRP level (mg/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.2695035460992905%\"\u003e\n \u003cp\u003e-0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.2695035460992905%\"\u003e\n \u003cp\u003e0.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.2695035460992905%\"\u003e\n \u003cp\u003e-0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.2695035460992905%\"\u003e\n \u003cp\u003e0.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.2695035460992905%\"\u003e\n \u003cp\u003e0.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.2695035460992905%\"\u003e\n \u003cp\u003e0.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.2695035460992905%\"\u003e\n \u003cp\u003e0.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.2695035460992905%\"\u003e\n \u003cp\u003e0.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.2695035460992905%\"\u003e\n \u003cp\u003e0.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.2695035460992905%\"\u003e\n \u003cp\u003e0.47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.2695035460992905%\"\u003e\n \u003cp\u003e-0.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.2695035460992905%\"\u003e\n \u003cp\u003e0.24\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.76595744680851%\"\u003e\n \u003cp\u003eBVAS-V3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.2695035460992905%\"\u003e\n \u003cp\u003e0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.2695035460992905%\"\u003e\n \u003cp\u003e0.59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.2695035460992905%\"\u003e\n \u003cp\u003e0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.2695035460992905%\"\u003e\n \u003cp\u003e0.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.2695035460992905%\"\u003e\n \u003cp\u003e0.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.2695035460992905%\"\u003e\n \u003cp\u003e0.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.2695035460992905%\"\u003e\n \u003cp\u003e0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.2695035460992905%\"\u003e\n \u003cp\u003e0.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.2695035460992905%\"\u003e\n \u003cp\u003e0.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.2695035460992905%\"\u003e\n \u003cp\u003e0.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.2695035460992905%\"\u003e\n \u003cp\u003e-0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.2695035460992905%\"\u003e\n \u003cp\u003e0.87\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"13\" valign=\"top\"\u003e\n \u003cp\u003eAbbreviations: BVAS-V3, Birmingham vasculitis activity score-version 3; CRP, C-reactive protein; ESR, erythrocyte sedimentation rate. *: The results of the Spearman rank correlation analysis.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn recent years, researchers have investigated miRNAs in AAV exosomes [\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e–\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], but studies on the mRNAs in AAV exosomes are lacking. The relationship between plasma-derived exosomal mRNA and inflammatory injury in vascular endothelial cells in patients with AAV is not clear. Exosomes enter receptor cells through various mechanisms, such as phagocytosis, fusion and signal transduction[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Vascular endothelial cells can produce and absorb extracellular vesicles. Therefore, exosomes may be captured by vascular endothelial cells throughout the body and cause cell damage, leading to the onset of AAV[\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e–\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Exosomes are widely distributed in the human body. They can carry proteins, lipids, and nucleic acids to exchange information between cells and regulate the activity of recipient cells. Studies have shown that exosomes can act as mediators of immune stimulation and regulation and participate in the regulation of a variety of immune processes, including antigen presentation, T-cell activation and polarization, and immunosuppression[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn the present study, we investigated the characteristics of mRNA expression profiles in the circulating exosomes of AAV patients. We isolated exosomes from peripheral blood for mRNA sequencing analysis and then identified candidate mRNAs by comparing the mRNA expression profiles between AAV patients and healthy controls.\u003c/p\u003e\u003cp\u003eThe RNA-sequencing data revealed that mRNAs may contribute to AAV disease progression. In this study, we identified three upregulated mRNAs (DEPDC1B, TPST1, and LSM2) and three downregulated mRNAs (NSUN4, FBXO34, and AK4) and validated the differential expression of these mRNAs by qRT‒PCR in 22 samples from AAV patients. The results indicated that the expression levels of DEPDC1B and TPST1 in the exosomes of AAV patients were significantly greater than those in the exosomes of normal controls, whereas those of NSUN4 and AK4 were significantly lower.\u003c/p\u003e\u003cp\u003eThe enrichment analysis results indicated that metabolic pathways, cell adhesion molecules, epithelial signaling, and MAPK signaling pathways are involved in AAV pathogenesis. Cell adhesion molecules and MAPK signaling pathways are considered to play crucial roles in autoimmune disease[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Studies have shown that plasma levels of extracellular vesicles increase when the vascular endothelium is activated or damaged[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Exosomes may damage endothelium-dependent vasodilation and reduce NO, leading to endothelial dysfunction[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In addition, exosomes can bind to the endothelium and activate it by increasing the production of reactive oxygen species (ROS)[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Neutosis is a special form of neutrophil death in which nuclear DNA is released from the broken nuclear membrane[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] and the broken plasma membrane forms NETs, which may lead to endothelial dysfunction through the activation of matrix metalloproteinases2[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], leading to vasculitis. Moreover, studies have confirmed a positive correlation between proinflammatory cytokines and adhesion molecules[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Therefore, we believe that endothelial injury and/or activation are essential features of AAV. Adhesion molecules in endothelial cells are upregulated during metabolism and inflammatory activation, which then mediate the adhesion and migration of white blood cells in blood vessels and promote inflammation and tissue damage. In general, cell adhesion molecules play a role in the pathogenesis of AAV.\u003c/p\u003e\u003cp\u003eMAPK signaling pathways are also considered to play a crucial role in autoimmune disease[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. In the vascular wall, macrophages produce proinflammatory cytokines (IL-1β and TNF-α). These proinflammatory cytokines aggravate vascular inflammation through phosphorylation of signaling pathways, such as the MAPK pathway. During inflammation, adhesion molecules, cytokines, and chemokines are regulated by the MAPK signaling pathway. Increasing evidence suggests that the inhibition of signaling cascade phosphorylation is effective in treating inflammatory diseases[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn AAV, ANCAs activate human neutrophils through the p38MAPK-mediated pathway, and this signaling cascade is responsible for the transfer of ANCA-specific antigens from cytoplasmic particles to the cell surface[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Many studies have confirmed that inflammatory damage to the vascular endothelium is caused by leukocyte infiltration in AAV[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. The patients we selected were all active AAV patients (with elevated BVAS-V3), and compared to those of healthy individuals, their ESR, CRP levels, and creatinine levels were significantly greater, indicating that the patients were experiencing a systemic inflammatory response. Considering the key role of inflammatory injury in endothelial cells in the pathogenesis of AAV, we speculate that the mRNAs from the exosomes of patients with active AAV may be absorbed by vascular endothelial cells and contribute to the induction of endothelial cell inflammation and neutrophil adhesion.\u003c/p\u003e\u003cp\u003eAn increase in the mRNAs DEPDC1B and TPST1 in AAV exosomes may promote the adhesion of neutrophils. DEPDC1B recognizes G protein-coupled receptors and regulates signaling pathways through effector and regulatory factors[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. DEPDC1B interacts with diverse signaling molecules, including splicing regulatory molecules and transmembrane proteins. Studies have shown that DEPDC1B participates in cell adhesion, cell proliferation, and cell cycle regulation[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Some studies have reported that the expression of the TPST1 gene may affect the tumor microenvironment and may even be related to the immunotherapy response in bladder cancer[\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Neutrophils express TPST1[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. TPST1 plays a role in the production of proinflammatory cytokines in LPS-induced macrophages, and the downregulation of TPST1 inhibits LPS-induced IL-6 production[\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. The expression of TPST1, which is related to the immune response, is upregulated in the bone marrow monocytes of patients with rheumatoid arthritis[\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Therefore, TPST1 may play an important role in the development of inflammation. Additionally, some studies have shown that the mRNA expression of NSUN4 and AK4 is related to tumors, but its role in autoimmune diseases needs further research[\u003cspan additionalcitationids=\"CR59\" citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e–\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe pathophysiological mechanisms of AAV are complex. According to the bioinformatics analysis of the differentially expressed mRNAs, we believe that the disease activity of AAV is related to an activation and imbalance of inflammation-related signaling pathways. Although these mRNAs can partially predict the onset of AAV, further studies are needed to understand the mechanisms underlying the effect of these target mRNAs on disease progression.\u003c/p\u003e\u003cp\u003eIn conclusion, this study revealed two mRNAs (DEPDC1B and TPST1) that were significantly upregulated in patients with AAV and two mRNAs (NSUN4 and AK4) that were significantly downregulated in patients with AAV. However, through correlation analysis, we found that these indicators were not related to disease activity, and these mRNA interactions may be involved in the development or progression of AAV. These data suggest that DEPDC1B, TPST1, NSUN4 and AK4 may be key regulators of AAV. Targeting these mRNAs may be useful for inhibiting the inflammatory response in AAV. However, the exact role of these mRNAs in the pathogenesis of AAV and their diagnostic and prognostic value for this disease are still unclear. This study detected the serum exosomal mRNAs of patients with AAV by high-throughput sequencing technology and further confirmed that exosomal mRNAs can be used for the early diagnosis and treatment of AAV. However, the sample size of this study was small, and thus should be further experimentally verified with an expanded sample size.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eANCA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eantineutrophil cytoplasmic antibody\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAAV\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eANCA-associated vasculitis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBVAS-V3\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBirmingham vasculitis activity score-version 3\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCRP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eC-reactive protein\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEGPA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eeosinophilic granuloma with polyangiitis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eESR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eerythrocyte sedimentation rate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGene Ontology\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehealthy control\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eKEGG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEncyclopedia of Genes and Genomes\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMAPK\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emitogen-activated protein kinase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003emiRNA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emicroRNA\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMPO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emyeloperoxidase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003emRNA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emessenger RNA\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMPA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emicroscopic polyangiitis,ncRNA,noncoding RNA\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNETs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eneutrophil extracellular traps\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePR3\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eneutral protease 3\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eqRT‒PCR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ereal-time quantitative polymerase chain reaction\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eROS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ereactive oxygen species\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003estandard deviation\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGPA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003egranuloma with polyangitis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTreg\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eT-regulated cell\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTh1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eT helper 1.\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\u003eSequence data that support the findings of this study have been deposited in the NCBI with the\u003c/p\u003e\n\u003cp\u003eprimary accession code PRJNA1114026.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclarations\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eof interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eparticipate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll subjects signed informed consent forms in accordance with the ethical principles of the Declaration of Helsinki. The research protocol was approved by the Ethics Committee of Anhui Medical University\u0026nbsp;(PJ2020-06-11).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding sources\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe current study received support from the Basic and Clinical Cooperative Research Promotion Program of Anhui Medical University (2021xkjT034).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank all the patients for their enthusiastic participation in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYF Chen contributed to the study design, experiment conduct, data analysis and drafting of the article. X Qian, Y Wang and SQ Ge contributed to sample collection, experiment conduct and data analysis. YF Chen and ZW Shuai reviewed and edited the article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKitching A. R., Anders H. J., Basu N., et al. ANCA-associated vasculitis. Nat Rev Dis Primers. 2020;6(1):71.\u003c/li\u003e\n\u003cli\u003eAlmaani S., Fussner L. A., Brodsky S., Meara A. S., Jayne D. ANCA-Associated Vasculitis: An Update. J Clin Med. 2021;10(7).\u003c/li\u003e\n\u003cli\u003eD Nakazawa, S Masuda, U Tomaru, A Ishizu, Id Orcid. - Pathogenesis and therapeutic interventions for ANCA-associated vasculitis. D - 101500080. (- 1759-4804 (Electronic)):- 91-101.\u003c/li\u003e\n\u003cli\u003eJennette J. C., Falk R. J., Bacon P. A., et al. 2012 revised International Chapel Hill Consensus Conference Nomenclature of Vasculitides. Arthritis Rheum. 2013;65(1):1-11.\u003c/li\u003e\n\u003cli\u003eSuppiah R., Robson J. C., Grayson P. 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(- 2001-1326 (Electronic)):- e953.\u003c/li\u003e\n\u003c/ol\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":"MPO-ANCA-associated vasculitis, exosomes, mRNA, pathogenesis","lastPublishedDoi":"10.21203/rs.3.rs-4622546/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4622546/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjective\u003c/h2\u003e \u003cp\u003eTo explore the expression patterns and potential roles of mRNAs in exosomes from patients with myeloperoxidase-specific anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (MPO-AAV).\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003ePlasma exosomes were isolated from MPO-AAV patients and healthy controls (HCs) to screen for differential mRNA expression via exosomal mRNA sequencing. The differentially expressed mRNAs in exosomes from the 2 groups were comparatively explored by bioinformatics analysis. The six most differentially expressed mRNAs were selected and validated in larger groups of MPO-AAV patients and HCs by real-time quantitative polymerase chain reaction (RT‒qPCR). The relationships between these selected mRNAs and patient characteristics were statistically analyzed.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eCompared with HCs, a total of 1,077 mRNAs in exosomes from MPO-AAV patients were found to be significantly upregulated, including DEPDC1B and TPST1, while NSUN4 and AK4 were significantly downregulated. Statistical analysis did not reveal any correlation between the six selected mRNAs and clinical indicators, including disease activity. GO enrichment analysis revealed that these differentially expressed genes participate in various enzyme activities, protein synthesis, etc. KEGG pathway analysis revealed that metabolic pathways, cell adhesion molecules, epithelial signaling, and mitogen-activated protein kinase (MAPK) signaling pathways were significantly enriched in the exosomal mRNAs.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThere were significant differences in the expression of exosomal mRNAs between MPO-AAV patients and HCs, which may be related to the occurrence and development of MPO-AAV. These findings provide clues for further investigations of MPO-AAV pathogenesis and the identification of new potential therapeutic targets.\u003c/p\u003e","manuscriptTitle":"Characteristic changes in the mRNA expression profile of plasma exosomes from patients with MPO-ANCA-associated vasculitis and its possible correlations with pathogenesis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-18 22:17:52","doi":"10.21203/rs.3.rs-4622546/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-07-07T17:34:50+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-07T15:49:48+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-04T11:45:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"175411325580990408092206582824368548946","date":"2024-06-26T10:03:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"131617207290248192185816468400272119271","date":"2024-06-26T07:34:55+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-06-25T18:32:27+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-24T06:50:04+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-06-24T06:48:57+00:00","index":"","fulltext":""},{"type":"submitted","content":"Clinical and Experimental Medicine","date":"2024-06-22T15:41:58+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":"d93e2400-794b-4a49-b77e-02155d8aab9d","owner":[],"postedDate":"July 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-09-23T16:09:56+00:00","versionOfRecord":{"articleIdentity":"rs-4622546","link":"https://doi.org/10.1007/s10238-024-01457-2","journal":{"identity":"clinical-and-experimental-medicine","isVorOnly":false,"title":"Clinical and Experimental Medicine"},"publishedOn":"2024-09-17 15:57:14","publishedOnDateReadable":"September 17th, 2024"},"versionCreatedAt":"2024-07-18 22:17:52","video":"","vorDoi":"10.1007/s10238-024-01457-2","vorDoiUrl":"https://doi.org/10.1007/s10238-024-01457-2","workflowStages":[]},"version":"v1","identity":"rs-4622546","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4622546","identity":"rs-4622546","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}