Therapeutic roles of hepatocyte exosomes in the liver

摘要 长期目标是改进肝脏疾病的治疗方法。肝纤维化是一种 影响全世界数百万人的发病率和死亡率的主要原因，通常是 慢性肝病，如过度饮酒引起的肝病。在美国，酗酒者 肝病(ALD)是胃肠道相关死亡的主要原因，在75,000名肝病死亡中约有一半 每年都与饮酒有关。一个主要的限制是缺乏治疗这两种疾病的已获批准的疗法 肝纤维化或ALD。然而，从我们对外显体的研究中出现了新的线索，这些外显体是由 肝细胞。我们的总体目标是建立外切体用于治疗肝病的治疗用途。我们的 初步数据显示，肝细胞外切体：(I)减弱调节纤维化形成的基因的表达 或在原代培养的肝星状细胞(HSC)中激活；(Ii)抑制纤维化通路并逆转 体内肝纤维化；(Iii)减轻乙醇或CCl4对培养肝细胞的损伤，(Vi)含有 相对较高的miR-532-5p或miR-214水平，可能在一定程度上有助于它们在 纤维化或ALD，特别是当它们各自在肝脏中的组织水平被单独抑制时 在慢性损伤中；以及(V)通过细胞表面整合素αvβ3和α5β1与靶肝细胞或肝干细胞结合。 中心假设是miR-532-5P-/miR-214或特定的整合素配体分别对 肝细胞外切体的治疗作用或细胞结合。检验这一假设的具体目标是： 目的1-确定miR-532-5p是肝细胞外体的治疗成分，可调节 用纯化的外切体或细胞共培养试验，用Smad3 3‘- UTR记者，CTGF启动子记者，SMAD3下游靶点的表达，siRNA介导的SMAD3 和miR-532-5p的过度表达或拮抗显示Smad3在细胞中的直接功能调节 每个靶细胞；目标2-确定肝细胞外体和外体miR-214或miR-214的作用 532对小鼠酒精性肝损伤的治疗作用 乙醇饮食体内模型：乙醇/肿瘤坏死因子α或内毒素介导的肝细胞或枯否细胞途径 分别通过证明外体靶向CTGF或ICAM-1的miR-214或靶向SMAD 3的miR-214 MIR-532概述了胞外体在体内和体外的作用；并目标3-确定胞外体结合伙伴 通过确定肝细胞FN、VN或CTGF在参与整合素αvβ3或 α-5-β-1与肝星状细胞或肝细胞结合，从而介导靶细胞结合。其基本原理是目前的方法 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的创新解决方案， 利用外切体的天然抗病特性。预期的结果将是一个新奇的 以外切体为基础的治疗肝纤维化或酒精性细胞损伤及免疫功能改变 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.