第一部分：外泌体miR-199a-5p调控大动脉炎 血管重塑和炎症浸润机制探究

                                                                   摘要

目的：尽管大动脉炎（Takayasu's arteritis, TAK）的治疗近年取得了一定进展，但亚临床血管炎症及由此引发的动脉重塑仍是TAK治疗中的难题。血管平滑肌细胞（vascular smooth muscle cell, VSMC）的表型转换被认为是血管病变的初始步骤，并在动脉重塑中发挥重要作用。外泌体通过转运和交换蛋白及特定核酸分子，在细胞间信息交流中发挥重要作用。本研究通过构建血清外泌体和VSMC共培养体系，探究血清外泌体在TAK中对VSMC表型转换及血管重塑的调控作用。

方法：从TAK患者中分离血清外泌体与VSMC共培养，探究外泌体对VSMC的潜在作用。通过转染miR-199a-5p模拟物和抑制剂来调控VSMC中microRNA的水平。采用CCK8实验和EdU实验评估VSMC增殖能力的改变；利用划痕实验和Transwell迁移实验评估VSMC迁移能力的改变；通过流式细胞术检测VSMC的凋亡情况。此外，双荧光素酶报告基因实验、RNA免疫共沉淀实验和荧光原位杂交实验被用来验证miR-199a-5p的靶基因。在TAK患者中，进一步评估了血清外泌体miR-199a-5p、血清MMP2和TIMP2的水平，以及其与临床参数之间的相关性。

结果：VSMC与血清外泌体共培养后去分化为合成表型。通过功能获得和抑制实验发现，miR-199a-5p的过表达显著增加了VSMC收缩表型相关基因的表达，抑制了VSMC的增殖和迁移；抑制内源性miR-199a-5p则表现出相反的效应。此外，miR-199a-5p的过表达抑制了VSMC的凋亡。进一步分析发现MMP2是miR-199a-5p的靶基因。相关性分析显示血管炎损伤指数与外泌体miR-199a-5p水平及血清MMP2呈负相关，需进一步在更大样本中验证。

结论：我们的研究表明，miR-199a-5p/MMP2通路在调控VSMC分化，抑制VSMC迁移、增殖和凋亡中发挥重要作用。MMP2的增多可能促使炎症细胞在血管壁内膜的浸润。我们的研究为同时干预炎症反应和内膜增生提供了一种新的治疗途径。此外，血清外泌体miR-199a-5p和MMP2具有作为血管损伤标志物的潜在应用前景。

第二部分：单细胞测序揭示肉芽肿性多血管炎中性粒细胞异质性

                                                               摘要

目的：抗中性粒细胞胞浆抗体（anti-neutrophil cytoplasmic antibody, ANCA）相关血管炎（ANCA-associated vasculitis , AAV）是一类特征为小血管损伤的自身免疫性疾病，常累及多个器官系统。尽管已有研究表明，中性粒细胞的异常激活在AAV的发病机制中发挥关键作用，但其异质性尚未被充分揭示。本研究基于单细胞RNA测序技术系统解析AAV患者外周血中性粒细胞的异质性特征及功能状态，并结合流式细胞术和实时定量聚合酶链式反应等多维检测手段，重点评估特定中性粒细胞亚群作为预测治疗应答及动态监测疾病进程的生物标志物的应用价值。

方法：本研究首先从三名活动期肉芽肿性多血管炎患者与三名健康对照者中采集外周血样本，应用单细胞RNA测序技术，系统分析中性粒细胞的异质性及其功能特征。在独立的临床队列中，采用流式细胞术和实时定量聚合酶链式反应对特定中性粒细胞亚群的存在进行了验证，并进一步探讨其在临床中的潜在意义。

结果：本研究成功分离并分析了28,907个中性粒细胞。这些中性粒细胞被划分为七个不同的亚群，每个亚群均涉及特异的中性粒细胞功能。其中，CXCL8高表达和TXNIP高表达的中性粒细胞亚群在多种炎症相关通路中呈显著富集，但CXCL8高表达亚群同时表达多种免疫调节分子以缓解过度炎症反应，可能代表健康个体中主要的中性粒细胞亚群。此外，MMP8高表达的中性粒细胞显示出增强的抗微生物防御功能；而IFIT1高表达亚群则呈现出与病毒感染或自身免疫反应相关的免疫激活特征。拟时序轨迹分析结果显示，PLA2G7高表达的中性粒细胞为终末分化状态。进一步的研究发现，该亚群的频率与疾病加重或复发，以及肾脏受累显著相关，提示其作为AAV进展中具有潜在临床价值的生物标志物。除中性粒细胞外，本研究还描绘了外周T细胞与自然杀伤细胞的转录特征，并构建了免疫细胞之间的通讯网络。本研究不仅加深了对AAV发病机制的理解，也为未来靶向治疗策略的开发提供了新的干预靶点。

结论：本研究首次应用单细胞RNA测序技术系统表征中性粒细胞的异质性，并评估其作为AAV患者疾病进展预后指标的潜在价值。这些新发现不仅加深了我们对中性粒细胞在AAV中动态功能作用的理解，也为开发靶向特定疾病相关中性粒细胞亚群的精准治疗策略提供了理论依据和研究基础。

Exosome miR-199a-5p modulated vascular remodeling and inflammatory infiltration of Takayasu's arteritis

                                                          ABSTRACT

Objectives: Advances in treatment that swiftly alleviate systemic inflammation of Takayasu’s arteritis (TAK), while subclinical vascular inflammation and the ensuing arterial remodeling continue to present unresolved challenges in TAK. The phenotypic switching of vascular smooth muscle cells (VSMC) is regarded as the first step in vascular pathology and contributed to arterial remodeling. Exosomes facilitate the transfer and exchange of proteins and specific nucleic acids, thereby playing a significant role in cell chat. This study investigated the regulatory effect of serum exosomes on VSMC phenotype transformation and vascular remodeling in TAK by constructing a co-culture system of serum exosomes and VSMC.

Methods: Serum exosomes isolated from TAK patients were co-cultured with VSMC to identify the modulation role of exosomes. VSMC were transfected with miR-199a 5p mimic and inhibitor. The CCK8 assays and EdU assays were performed to measure proliferative ability. The migration of VSMC was evaluated by scratch assays and transwell migration assays. The flow cytometry was employed to identify apoptosis of VSMC. Dual-luciferase reporter assays, RNA immunoprecipitation assays and fluorescence in situ hybridization were utilized to validate the target gene of miR-199a 5p. The correlational analysis was conducted among microRNA from exosomes, serum MMP2, TIMP2 and clinical parameters in TAK patients.

Results: The coculture of VSMC with serum exosome mediated dedifferentiation of VSMC. Through gain- and loss-of-function approaches, miR-199a-5p over-expression significantly increased expression of VSMC marker genes and inhibited VSMC proliferation and migration, whilst the opposite effect was observed when endogenous miR-199a-5p was knocked down. The overexpression of miR-199a-5p suppressed VSMC apoptosis. Further, MMP2 served as functional target gene of miR-199a-5p. The correlation analyses revealed an inverse correlation between Cardiovascular and Peripheral Vasculitis Damage Index and exosome miR-199a-5p level or serum MMP2 levels, which require validation in a larger cohort.

Conclusions: Our study indicated that the miR-199a-5p/MMP2 pathway played a role in regulating VSMC differentiation and inhibiting the migration, proliferation and apoptosis of VSMC. The increased secretion of MMP2 may potentially prompt the intimal infiltration of inflammatory cells within the vascular wall, offering a novel therapeutic opportunity by tackling both inflammatory responses and the neointimal overgrowth associated with TAK arterial damage. Moreover, exosome miR-199a-5p and MMP2 derived from serum possessed potential as future biomarkers for vascular injury.  

Single-cell sequencing highlights the heterogeneity of neutrophils in granulomatosis with polyangiitis

                                                          ABSTRACT

Objectives: Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) manifests as a heterogeneous autoimmune pathology characterized by small vessel injury and multisystem organ involvement. While the aberrant activation of neutrophils is recognized as a pivotal element in the pathogenesis of AAV, the heterogeneity of neutrophils remain poorly understood. This study is based on single-cell RNA sequencing technology to systematically analyze the heterogeneity characteristics and functional status of peripheral blood neutrophils in AAV patients, and combines multidimensional detection methods such as flow cytometry and quantitative real-time polymerase chain reaction to evaluate the application value of specific neutrophil subpopulations as biomarkers for predicting treatment response and dynamically monitoring disease progression.

Methods: Peripheral blood samples were collected from three patients with active granulomatosis with polyangiitis and three healthy controls, followed by single-cell RNA sequencing (single cell RNA sequencing, scRNA-seq) to investigate the heterogeneity and functional characteristics of neutrophils. In independent validation cohorts, flow cytometry and quantitative real-time polymerase chain reaction were employed to confirm the presence of specific neutrophil subtypes and to explore their potential clinical significance.

Results: A total of 28,907 neutrophils were grouped into seven distinct clusters, each contributing to a unique set of neutrophil functions. Among these, both CXCL8-high and TXNIP-high neutrophil subsets demonstrated inflammatory pathway enrichment, while CXCL8-high neutrophils expressed immunoregulatory molecules to mitigate excessive inflammation, potentially representing the predominant neutrophil subpopulation in healthy individuals. MMP8-high neutrophils displayed heightened antimicrobial defense mechanisms, whereas IFIT1-high neutrophils exhibited an enhanced immune response linked to viral infections or autoimmune conditions. PLA2G7-high neutrophils were identified as terminally differentiated neutrophils through pseudotime analysis. Further investigation revealed that PLA2G7-high neutrophils demonstrated significant correlations with disease exacerbation or relapse, and renal involvement, positioning them as clinically relevant biomarkers in AAV progression. Additionally, we delineated the transcriptional features of peripheral T cells and natural killing cells, while mapping systemic immune cell communication networks.

Conclusions: We initially employed scRNA-seq to characterize the heterogeneity of neutrophils and evaluate their potential as prognostic indicators of disease progression in AAV patients. These novel findings enhance our understanding of the dynamic functional roles of neutrophils in AAV and may facilitate the development of therapeutic strategies aimed at selectively targeting disease-specific neutrophil subsets.