Septins in intestinal fibrosis

肠道纤维化中的脓毒症

• 批准号: 10656661
• 负责人: Andrei Ivanovich Ivanov
• 金额: $ 63.01万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-01 至 2027-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10656661
• 关键词: Acceleration; Actomyosin; Affect; Animal Model; Anti-Inflammatory Agents; Atlases; Attenuated; Automobile Driving; Binding; Biology; Cell Differentiation process; Cell physiology; Cells; Collagen; Complex; Complication; Crohn' s disease; Cytoskeletal Proteins; Cytoskeleton; Data; Deposition; Development; Disease; Disease Progression; Effector Cell; Endocytosis; Excision; Exposure to; Extracellular Matrix; Extracellular Matrix Proteins; Fibroblasts; Fibronectins; Fibrosis; Filament; Flagellin; Fostering; GTP-Binding Proteins; Gene Expression; Generations; Genetic; Genetic Induction; Goals; Growth Factor; Health Benefit; Human; Immune; In Vitro; Incidence; Inflammation; Intestinal Fibrosis; Intestinal Obstruction; Intestines; Investigation; Link; LoxP-flanked allele; Maps; Mediating; Membrane; Microfilaments; Molecular; Motor; Mus; Myofibroblast; Myosin ATPase; Operative Surgical Procedures; Organ; Organelles; Patients; Phenotype; Polymers; Prevention; Production; Property; Protein Isoforms; Proteins; Receptor Signaling; Regulation; Resolution; Role; Signal Transduction; Small Interfering RNA; Structure; Testing; Thick; Tissue Sample; Tissues; Transforming Growth Factors; Translations; United States National Institutes of Health; Up-Regulation; Vesicle; antifibrotic treatment; cell motility; fibrogenesis; gain of function; genetic approach; gut inflammation; improved; in vivo; loss of function; migration; mouse model; novel; novel strategies; novel therapeutic intervention; novel therapeutics; overexpression; pharmacologic; posttranscriptional; prevent; scaffold; self assembly; single-cell RNA sequencing; therapeutically effective; trafficking; translatome

ABSTRACT Over their disease course more than half of Crohn’s disease (CD) patients develop fibrosis-induced intestinal obstruction and ultimately require surgery. No specific anti-fibrotic therapies are available. Despite advances of anti-inflammatory therapies the incidence of strictures remains high, suggesting that inflammation- independent mechanisms are crucial in the progression of the disease. The main effector cell mediating fibrosis is the myofibroblast that is activated by multiple pro-fibrotic growth factors, such as transforming growth factor (TGF)-1. Such activation results in accelerated secretion of extracellular matrix (ECM) and remodeling of the actomyosin cytoskeleton. Septins are understudied cytoskeletal proteins that regulate secretory and actomyosin- dependent cellular functions. No data on the roles and regulation of the septin cytoskeleton in intestinal fibrosis exists. Our preliminary data shows a high gene expression of septins 2, 6, 7, 8, 9, 10, 11 in human intestinal tissues with septin 7 as the most predominant isoform, which is upregulated in CD. Pharmacologic or genetic disruption of the septin cytoskeleton inhibited TGF-β1-dependent increase in ECM production (Collagen I & fibronectin) and migration in immortalized and primary human myo/fibroblasts. Preliminary evidence suggests this is post-transcriptionally regulated. Septin modulation improved experimental murine fibrosis. We hence propose to investigate the hypothesis that remodeling of the septin cytoskeleton is a driver of intestinal fibrosis and targeting the septin cytoskeleton is a novel approach to therapy of fibrostenosing Crohn’s disease. This hypothesis will be tested by three specific aims: AIM1. Characterization of alterations in septin expression and distribution in tissue samples of IBD patients. This includes development of a high-resolution map of septin expression profiles in human intestinal tissues and primary human intestinal myofibroblasts, including generation of the first full thickness single cell RNA sequencing gut atlas for stricturing CD and controls. AIM2. Investigation of the roles and mechanisms of septin dependent regulation of pro-fibrotic myofibroblast activation. We will test if septin disruption or overexpression modulates TGF-β1-signaling, intracellular vesicular trafficking or the translatome and post-transcriptionally regulated networks using a loss-of-function and gain-of- function approach. AIM3. Functional exploration if targeting septins ameliorates intestinal fibrosis in vivo. We will modulate septins in vivo using a pharmacologic and genetic approach and induce experimental fibrosis in two different animal models. We will temporally control the deletion of the central septin 7 prior to (prevention) and after induction (reversal) of experimental intestinal fibrosis specifically in Col I positive cells. If successful, this proposal will challenge the paradigm of purely immune-driven ECM deposition driving stricture formation and provide proof-of-concept for a novel mechanism to prevent or treat stricture associated intestinal obstruction in CD patients.

摘要 在他们的病程中，超过一半的克罗恩病(CD)患者发展为纤维化诱导的肠道 梗阻，最终需要手术。目前还没有特效的抗纤维化治疗方法。尽管取得了进步 在抗炎治疗中，狭窄的发生率仍然很高，这表明炎症- 独立的机制在疾病的发展过程中至关重要。介导纤维化的主要效应细胞 是由多种促纤维化生长因子激活的肌成纤维细胞，如转化生长因子 (转化生长因子)-1。这种激活导致细胞外基质的加速分泌和血管重塑。 肌动蛋白细胞骨架。间隔蛋白是一种未被研究的细胞骨架蛋白，它调节分泌和肌动球蛋白。 依赖细胞功能。目前尚无有关Septin细胞骨架在肠道中的作用和调控的数据 纤维化是存在的。我们的初步数据显示，在人类中，Septins 2，6，7，8，9，10，11有高水平的基因表达 肠组织中Septin 7是最主要的异构体，在CD中表达上调。药理或 SEPTIN细胞骨架的基因破坏抑制了转化生长因子-β1依赖的细胞外基质(胶原)产量的增加 纤维连接蛋白)和在永生化和原代人肌/成纤维细胞中的迁移。初步证据表明 这是转录后调控的。Septin调节改善实验性小鼠纤维化。因此，我们 建议研究间隔蛋白细胞骨架重塑是肠道驱动因素的假说。 纤维化和靶向Septin细胞骨架是治疗纤维狭窄克罗恩病的新途径 疾病。这一假设将通过三个具体目标进行检验：AIM1。Septin中变化的特征 IBD患者组织标本的表达和分布。这包括开发高分辨率的 人肠组织和原代人肠肌成纤维细胞Septin表达谱图， 包括制作第一个用于限制CD和对照的全厚度单细胞RNA测序肠道图谱。 AIM2.Septin依赖的促纤维化肌成纤维细胞调控作用及机制的研究 激活。我们将测试Septin是否破坏或过度表达调节转化生长因子-β1信号，细胞内囊泡 使用功能丧失和获得-的贩运或翻译基因组和转录后调控网络 函数法。AIM3.体内靶向Septins改善肠纤维化的功能探讨。我们会 用药理学和遗传学方法在体内调节间隔蛋白并诱导两例实验性纤维化 不同的动物模型。我们将在预防和预防之前暂时控制中央间隔蛋白7的缺失 诱导(逆转)实验性肠纤维化后，特异性地定位于I型胶原阳性细胞。如果成功，这将是 提案将挑战纯免疫驱动的ECM沉积驱动狭窄的范式 形成并为预防或治疗相关狭窄的新机制提供概念验证 CD患者的肠梗阻。