Anti-inflammatory Signaling of RNA-binding Protein, Tristetraprolin, During Myocardial Infarction

RNA 结合蛋白 Tristetraprolin 在心肌梗死期间的抗炎信号传导

• 批准号: 10644962
• 负责人: Jonathan Yap
• 金额: $ 10万
• 依托单位: UNIVERSITY OF HAWAII AT MANOA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10644962
• 关键词: Acute; Adoptive Transfer; Affect; Anti-Inflammatory Agents; Antiinflammatory Effect; Attenuated; Binding; Calgranulin B; Cardiac; Cardiac Myocytes; Cardiomyopathies; Cell Death; Cell Line; Cell physiology; Cells; Cessation of life; ChIP-seq; Coculture Techniques; Comprehension; Coupled; Data; Development Plans; Echocardiography; Excision; Exposure to; Extracellular Matrix Degradation; Flow Cytometry; Gene Combinations; Gene Expression; Genes; Genetic Transcription; Histology; Hypoxia; Immune; Impairment; In Vitro; Inflammation; Inflammatory; Inflammatory Response; Injury; Ischemia; Knock-out; Leukocytes; Ligation; Link; LoxP-flanked allele; Macrophage; Measurement; Measures; Mediating; Messenger RNA; Methods; Mitochondria; Modality; Modeling; Molecular; Monitor; Mus; Mutate; Mutation; Myelogenous; Myeloid Cells; Myocardial Infarction; Myocarditis; Oxidative Stress; Pathology; Pathway interactions; Pattern; Phase; Play; Polymers; Prevention; Process; Production; Prognosis; Property; Proteins; RNA-Binding Proteins; Reactive Inhibition; Reactive Oxygen Species; Regulation; Reperfusion Injury; Reperfusion Therapy; Reporting; Research; Resolution; Role; S100A8 gene; S100A9 gene; Sampling; Signal Transduction; Site; System; TIS11 protein; Techniques; Testing; Therapeutic; Time; Tissues; Transcript; Treatment Efficacy; Ventricular Remodeling; Viral Vector; adeno-associated viral vector; cardioprotection; career development; cytokine; early onset; efficacy evaluation; experimental study; extracellular; gain of function; heart function; heart preservation; improved; in vivo; mRNA Expression; mitochondrial dysfunction; mouse model; mutant; next generation sequencing; novel; osteopontin; overexpression; polymerization; prevent; recruit; repaired; response; sound; spatiotemporal; tool; transcriptome; transcriptome sequencing; transcriptomics; vector

PROJECT SUMMARY Myocardial infarction (MI) initiates a dynamic cellular response almost immediately after reperfusion therapy. Inflammation during the early stages of ischemia reperfusion injury (IRI) initiates a cascade of immune cell signaling networks which contribute to immune cell recruitment and extracellular matrix (ECM) degradation. The paradoxical phenomenon of IRI results in massive cell death and is a primary determinant of adverse ventricular remodeling. Using a novel transcriptome-wide analysis of spatiotemporal gene expression, we determined that the RNA-binding protein, Tristetraprolin (TTP) has regulator effects on the expression of several key pro- inflammatory cytokines and genes related to reactive oxygen species (ROS). However, the role of TTP in early- onset MI is unknown despite its well-established anti-inflammatory properties. The research described in this proposal seeks to elucidate the cardioprotective mechanisms of TTP in early-onset MI. S100A8 and S100A9 act as damage associated molecular patterns (DAMPs) that are highly expressed after 3 days of IRI. Aim 1 investigates the regulatory role of TTP on S100A8/S100A9 expression and functionality. TTP knockout (TTP- KO) and TTP overexpressing (TTP-OE) in myeloid cell lines will be used to determine the effects of TTP on S100A8/S100A9 expression. Mutations to 6 specific AA residues has been shown to abrogate TTP binding of mRNA molecules. TTP mutant cell lines (TTP-Δ1-6) will be generated to determine whether TTP mediates S100A8/S100A9 signaling effects through mRNA destabilization or upstream regulation. The function of TTP and S100A8/S100A9 are closely tied to Ca2+ availability. Therefore, Ca2+ concentration will tested as an environmental variable. TTP has also been shown to diminish SPP1 and Cybb (NOX2) expression, inhibiting ROS production, oxidative stress, and mitochondrial dysfunction following IRI. Therefore, Aim 2 will explore the effects of simulated (sIRI) on ROS production in TTP-KO and TTP-OE myeloid cells in vitro. Mitochondrial function and ATP production will also be measured in cardiomyocytes upon exposure to conditioned media of sIRI-stimulated, TTP-KO and TTP-OE myeloid cells. Lastly Aim 3 will investigate the role of myeloid-specific TTP expression in mitigating adverse ventricular remodeling in vivo. To test this, TTP floxed mice and mice with a 136 bp floxed site upon LysMCre excision generate myeloid-specific TTP-KO and TTP-OE mice respectively. Experimental mice will be exposed to IRI treatment by temporary LAD ligation. Echocardiography will be regularly performed to monitor heart function and ventricular remodeling. Lastly, we will investigate the therapeutic potential of time-dependent TTP delivery by introduction of TTP-expressing adeno-associated viral vector-9 (AAV9) or adoptive transfer with TTP-OE macrophages after 3 days of IRI. The same measurements and assessments will be made to determine the efficacy of each treatment. The rationale behind this research is a sound comprehension of the anti-inflammatory properties and therapeutic potential of TTP in early-onset MI. Specifically, the effects of TTP expression on inflammation, mitochondrial dysfunction, and cardiomyopathy.

项目总结 心肌梗死(MI)在再灌注治疗后几乎立即启动动态细胞反应。 缺血再灌注损伤(IRI)早期炎症启动免疫细胞级联反应 有助于免疫细胞募集和细胞外基质(ECM)降解的信号网络。这个 IRI的矛盾现象导致大量细胞死亡，是不良室壁的主要决定因素 改建。使用一种新的转录组范围的时空基因表达分析，我们确定 RNA结合蛋白Tristetraprolin(TTP)对几种关键蛋白的表达具有调节作用。 炎性细胞因子和与活性氧(ROS)相关的基因。然而，TTP在早期- 尽管已有良好的抗炎特性，但发病心肌梗死尚不清楚。这篇文章中描述的研究 建议旨在阐明TTP在早发性心肌梗死中的心脏保护机制。S100A8和S100A9动作 作为损伤相关的分子模式(DAMP)，在IRI后3天高表达。目标1 探讨TTP对S100A8/S100A9基因表达和功能的调节作用。TTP基因敲除(TTP- KO)和TTP过表达(TTP-OE)在髓系细胞中的表达将被用来确定TTP对 S100A8/S100A9表达。6个特定氨基酸残基的突变已被证明取消了TTP与 信使核糖核酸分子将产生Ttp突变细胞系(Ttp-Δ1-6)以确定Ttp是否介导 S100A8/S100A9信号通过mRNA失稳或上游调控发挥作用。TTP的功能 S100A8/S100A9与钙离子可利用性密切相关。因此，钙离子浓度将作为一种 环境变量。TTP还被证明可以降低SPP1和Cybb(NOX2)的表达，抑制 IRI后ROS的产生、氧化应激和线粒体功能障碍。因此，目标2将探索 模拟Siri对体外培养的TTP-KO和TTP-OE髓系细胞产生ROS的影响线粒体 当心肌细胞暴露在条件培养液中时，功能和ATP的产生也将被测量 Siri刺激的TTP-KO和TTP-OE髓系细胞。最后，目标3将研究髓系特异性TTP的作用 在减轻体内不良室壁重构中的表达。为了测试这一点，TTP用牙线将小鼠和小鼠 切除LysMCre基因的136个碱基的牙线位点分别产生了髓系特异性的TTP-KO和TTP-OE小鼠。 实验小鼠将通过临时LAD结扎暴露于IRI治疗。超声心动图将定期 监测心功能和心室重构。最后，我们将调查治疗 TTP表达的腺相关病毒载体-9对时间依赖性TTP传递的影响 (AAV9)或在IRI后3d与TTP-OE巨噬细胞过继转移。相同的尺寸和 将进行评估，以确定每种治疗的疗效。这项研究背后的理论基础是 对TTP在早发性心肌梗死中的抗炎特性和治疗潜力有良好的理解。 具体地说，TTP的表达对炎症、线粒体功能障碍和心肌病的影响。