Therapeutic roles of hepatocyte exosomes in the liver

摘要 广泛的长期目标是改善肝脏疾病的治疗方法。肝纤维化是一种 发病率和死亡率的主要原因，影响世界各地数百万人，通常是一个特点， 慢性肝病，如过度饮酒引起的肝病。在美国，酗酒 肝病（ALD）是GI相关死亡的主要原因，约占75，000例肝病死亡的一半 每年都与饮酒有关。一个主要的限制是缺乏批准的治疗方法来治疗 肝纤维化或ALD。然而，一个新的线索已经出现在我们的研究外泌体， 肝细胞我们的总体目标是建立外泌体用于治疗肝病的治疗用途。我们 初步数据显示，肝细胞外泌体：（i）减弱调节纤维形成的基因的表达 或激活培养的原代肝星状细胞（HSC）;（ii）抑制纤维化途径和逆转 体内肝纤维化;（iii）减弱暴露于乙醇或CCl 4培养肝细胞中的损伤，（vi）含有 相对高水平的miR-532- 5 p或miR-214，这可能部分有助于它们在以下疾病中的治疗作用： 特别是当它们各自在肝脏中的组织水平被单独抑制时 在慢性损伤期间;和（V）通过细胞表面整联蛋白αvβ3和α5β1与靶肝细胞或HSC结合。我们 中心假设是miR-532- 5 p-/miR-214或特异性整联蛋白配体分别促成了 肝细胞外来体的治疗作用或细胞结合。检验这一假设的具体目的是： 目的1 -鉴定miR-532- 5 p作为肝细胞外泌体的治疗组分，其调节 通过使用纯化的外来体或细胞共培养测定，使用SMAD 3 3 3 '- UTR报告基因，CTGF启动子报告基因，SMAD 3下游靶标的表达，siRNA介导的SMAD 3 敲低和miR-532- 5 p过表达或拮抗作用，以显示SMAD 3在 目的2 -确定肝细胞外泌体和外泌体miR-214或miR-214的作用。 532在减轻乙醇诱导的肝损伤中的作用，通过证明外泌体在 体内乙醇饮食模型，对肝细胞或枯否细胞中乙醇/TNFα或LPS介导的通路的影响 通过证明miR-214对CTGF或ICAM-1的外泌体靶向或miR-214对SMAD 3的外泌体靶向， miR-532在体内和体外概括了外泌体的作用;目的3 -鉴定外泌体结合伴侣 通过确定肝细胞FN、VN或CTGF在接合整合素αvβ3或 α5β1在HSC或肝细胞上，从而介导靶细胞结合。理由是，目前的方法 治疗肝纤维化或ALD的方法是不够的，我们的方法是一种尖端和创新的解决方案， 利用外来体的天然疾病抑制特性。预期的结果将是一部小说 用于治疗纤维化或酒精诱导的细胞损伤和免疫功能改变的基于外泌体的疗法 肝脏积极的影响将是改善全球数百万慢性肝病患者的健康。

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.