Therapeutic roles of hepatocyte exosomes in the liver

肝细胞外泌体在肝脏中的治疗作用

基本信息

批准号：
10362721
负责人：
DAVID R BRIGSTOCK
金额：
$ 42.05万
依托单位：
RESEARCH INST NATIONWIDE CHILDREN'S HOSP
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-03-01 至 2025-02-28
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10362721
关键词：
Adrenal Cortex Hormones Affect Alcohol consumption Alcoholic Hepatitis Alcoholic Liver Diseases Alcoholic beverage heavy drinker Alcohols Alternative Therapies Animals Apoptosis Applications Grants Attenuated Behavior Binding Biological Assay California Cause of Death Cell Survival Cell surface Cells Cessation of life Chronic Cicatrix Cirrhosis Clinic Coculture Techniques Data Deposition Diet Disease Ethanol FDA approved Fatty Liver Fibrillar Collagen Fibrosis Gene Expression Goals Health Heavy Drinking Hepatic Stellate Cell Hepatocyte Hypoxia In Vitro Incidence Individual Inflammatory Injury Integrin alpha5beta1 Integrin alphaVbeta3 Integrins Intercellular adhesion molecule 1 Knowledge Kupffer Cells Lead Ligands Liver Liver Cirrhosis Liver Fibrosis Liver diseases MADH3 gene Mediating Mediator of activation protein Medical Messenger RNA Methods Modeling Molecular Morbidity - disease rate National Institute on Alcohol Abuse and Alcoholism Ohio Outcome Pathology Pathway interactions Patients Persons Phenotype Primary carcinoma of the liver cells Production Progressive Disease Property Proteins Regulation Reporter Risk Factors Role Small Interfering RNA TNF gene Testing Therapeutic Therapeutic Effect Therapeutic Uses Tissues United States Universities Untranslated Regions Vesicle alcohol exposure antagonist base cell injury chronic alcohol ingestion chronic liver disease chronic liver injury connective tissue growth factor cytokine exosome extracellular fibrogenesis healing immune function improved in vivo in vivo Model innovation knock-down mortality nanovesicle novel programs promoter receptor side effect therapeutic candidate

项目摘要

ABSTRACT The broad long term objective is to improve methods of disease treatment in the liver. Hepatic fibrosis is a major cause of morbidity and mortality that affects millions of people world-wide and is usually a feature of chronic liver disease such as that caused by excessive alcohol consumption. In the United States, alcoholic liver disease (ALD) is a leading cause of GI-related deaths, with about half of the 75,000 liver disease deaths each year being related to alcohol use. A major limitation is the lack of approved therapeutics for treating either liver fibrosis or ALD. However, a new lead has emerged from our studies of exosomes that are produced by hepatocytes. Our overall objective is to establish therapeutic uses of exosomes for treating liver disease. Our Preliminary Data show that hepatocyte exosomes: (i) attenuate expression of genes that regulate fibrogenesis or activation in cultured primary hepatic stellate cells (HSC); (ii) suppress fibrogenic pathways and reverse hepatic fibrosis in vivo; (iii) attenuate damage in cultured hepatocytes exposed to ethanol or CCl4, (vi) contain relatively high levels of miR-532-5p or miR-214 that likely contribute, in part, to their therapeutic actions in fibrosis or ALD respectively especially as their respective tissue levels in the liver are individually suppressed during chronic injury; and (v) bind to target hepatocytes or HSC via cell-surface integrin αvβ3 and α5β1. Our central hypothesis is that miR-532-5p-/miR-214 or specific integrin ligands contribute to, respectively, the therapeutic actions or cellular binding of hepatocyte exosomes. The Specific Aims to test this hypothesis are: Aim 1 - Identify miR-532-5p as a therapeutic component of hepatocyte exosomes that modulates SMAD3 in hepatocytes or HSC by using purified exosomes or cell co-culture assays, employing SMAD3 3’- UTR reporters, CTGF promoter reporters, expression of SMAD3 downstream targets, siRNA-mediated SMAD3 knockdown, and miR-532-5p over-expression or antagonism to show direct functional regulation of SMAD3 in each target cell; Aim 2 - Determine the role of hepatocyte exosomes and of exosomal miR-214 or miR- 532 in attenuating ethanol-induced liver injury by demonstrating the therapeutic effect of exosomes in ethanol diet models in vivo, on ethanol/TNFα- or LPS-mediated pathways in hepatocytes or Kupffer cells respectively, and by demonstrating that exosomal targeting of CTGF or ICAM-1 by miR-214 or of SMAD 3 by miR-532 recapitulates exosomal action in vivo and in vitro; and Aim 3 - Identify exosomal binding partners of cellular integrins by establishing the role of hepatocyte FN, VN, or CTGF in engaging integrin αvβ3 or α5β1 on HSC or hepatocytes and thereby mediating target cell binding. The rationale is that current methods of treating liver fibrosis or ALD are inadequate and our approach is a cutting-edge and innovative solution that harnesses natural disease-suppressing properties of exosomes. The expected outcome will be a novel exosome-based therapy for treating fibrosis or alcohol-induced cell damage and altered immune function in the liver. The positive impact will be to improve the health of millions of people globally with chronic liver disease.

抽象的广义的长期目标是改善肝脏中疾病治疗方法。肝纤维化是发病率和死亡率的主要原因会影响全球数百万的人，通常是慢性肝病，例如由于饮酒过量引起的疾病。在美国，酒鬼肝病（ALD）是与胃肠道相关死亡的主要原因，其中75,000个肝病死亡中约有一半每年都与饮酒有关。一个主要限制是缺乏治疗的批准疗法肝纤维化或ALD。但是，我们对外泌体的研究产生了新的潜在客户肝细胞。我们的总体目标是建立外泌体治疗肝病的治疗用途。我们的初步数据表明肝细胞外泌体：（i）减弱调节纤维发生的基因的表达或在培养的原发性肝星状细胞（HSC）中激活；（ii）抑制纤维化途径和反向体内肝纤维化；（iii）减弱暴露于乙醇或CCL4的培养的肝细胞中的损害，（VI）包含相关高水平的miR-532-5p或miR-214可能部分促进其治疗作用纤维化或ALD分别尤其是由于它们各自的组织水平分别抑制在慢性受伤期间；（v）通过细胞表面整合蛋白αVβ3和α5β1与靶标肝细胞或HSC结合。我们的中心假设是miR-532-5p-/miR-214或特定整联蛋白配体分别有助于肝细胞外泌体的治疗作用或细胞结合。检验该假设的具体目的是：目标1-将miR-532-5p识别为调节肝细胞外泌体的治疗成分通过使用纯化的外泌体或单元培养分析，使用smad3 3'-- UTR记者，CTGF启动子记者，SMAD3下游目标的表达，siRNA介导的SMAD3 敲低和miR-532-5p过表达或对抗显示Smad3的直接功能调节每个目标细胞；目标2-确定肝细胞外泌体和外泌体miR-214或miR-的作用 532通过证明外泌体的治疗作用来衰减乙醇诱导的肝损伤乙醇饮食模型在体内，乙醇/TNFα-或LPS介导的肝细胞或库普夫细胞中的途径分别通过miR-214或SMAD 3对CTGF或ICAM-1的外泌体靶向。 miR-532在体内和体外概括了外泌体作用；目标3-识别外泌体约束伴侣通过建立肝细胞FN，VN或CTGF在使整联蛋白αVβ3或 HSC或肝细胞上的α5β1，从而介导靶细胞结合。理由是当前方法治疗肝纤维化或ALD的治疗不足，我们的方法是一种尖端和创新的解决方案利用外泌体的自然抑制特性。预期的结果将是一本小说基于外泌体的治疗治疗纤维化或酒精诱导的细胞损伤并改变了免疫功能的治疗肝。积极的影响是改善慢性肝病全球数百万人的健康。