Lipotoxicity and Liver Inflammation

项目总结 这项提案的总体目标是确定在脂肪毒性过程中肝细胞损伤与 非酒精性脂肪性肝炎(NASH)的肝脏炎症。NASH是最常见的儿科肝病 以循环中丰富的饱和游离脂肪酸(SFAs)为特征，并伴有肝细胞脂肪毒性和 单核细胞来源的巨噬细胞介导的肝脏炎症。肝细胞脂肪毒性和肝损伤在一定程度上是 由SFA及其胞内代谢物溶血磷脂酰胆碱(LPC)诱导。然而，蜂窝和 将肝细胞脂肪毒性与肝脏炎症联系起来的分子机制尚不完全清楚。 新数据显示肝细胞脂毒应激时释放的细胞外小泡(EV) 发炎。在已发表的和初步的实验中，我们发现，脂毒性肝细胞 释放大量促炎EV；这些EV富含黏附分子整合素 β-1(Itg-β-1)可促进单核细胞与肝窦内皮细胞的黏附。我们也 血管细胞黏附分子(β)1在LSECs上的表达 在脂肪毒性过程中增加。基于这些新奇的观察，我们制定了中心 假设脂毒性肝细胞释放富含ITGβ1的EV，从而在体内招募和保留单核细胞 肝促炎症。我们将采用目前的生化和细胞生物学方法，包括 微流控技术，纳米级流式细胞术，以及用可视化技术可视化的89Zr同位素标记的EVS 用正电子发射断层扫描(PET)来验证这一假设。我们独立的具体目标将考验 三个完整的假设。首先，我们将证明肝细胞脂毒性诱导活性 Itgβ1的构象转换，增强其在EVS内的转运和释放。第二，我们将定义 脂肪毒性时VCAM1表达增加的机制。我们还将直接检验这一假设 脂毒性肝细胞来源的EVS通过ITGβ1-VCAM1结合介导单核细胞与LSECs的黏附 利用微流控技术，在体外进行相互作用。第三，使用Nash的鼠标模型，我们将测试 假设整合素β1的药理抑制或有条件的内皮VCAM1缺失是 防止肝脏发炎。我们还将证明过继转移的脂毒性肝细胞- 通过其高亲和力整合素β1Cargo获得EV，并将其定位于受体小鼠的LSEC。我们有 建立了研究脂肪毒性、整合素信号和肠道病毒生物学所需的细胞和小鼠模型。这 该提议在技术和概念上都是创新的，因为它寻求整合分子机制 潜在的肝细胞损伤，整合素的激活和转运与肝脏炎症，并联系肝脏 纳米医学的病理生理学。这项研究有可能发现新的治疗策略， 即整合素和VCAM1抑制剂，用于预防或逆转人类NASH的肝损伤和炎症。

PROJECT SUMMARY Overall Objectives of this proposal are to define mechanisms linking hepatocyte injury during lipotoxicity with hepatic inflammation in nonalcoholic steatohepatitis (NASH). NASH is the most common pediatric liver disease characterized by abundant circulating saturated free fatty acids (SFAs), along with hepatocyte lipotoxicity and monocyte-derived macrophage mediated liver inflammation. Hepatocyte lipotoxicity and liver injury are, in part, induced by SFAs and their intracellular metabolite lysophosphatidyl choline (LPC). However, cellular and molecular mechanisms linking hepatocyte lipotoxicity to liver inflammation are not completely understood. Emerging data implicate extracellular vesicles (EVs) released during hepatocyte lipotoxic stress in liver inflammation. In published and preliminary experiments, we have discovered that, lipotoxic hepatocytes release a large number of proinflammatory EVs; these EVs are enriched with the adhesion molecule integrin β1 (ITGβ1) and promote monocytes adhesion to liver sinusoidal endothelial cells (LSECs) in vitro. We also demonstrated that the expression of ITGβ1 ligand, vascular cell adhesion molecule (VCAM) 1, on LSECs is increased during lipotoxicity. Based on these novel observations, we have formulated the CENTRAL HYPOTHESIS that lipotoxic hepatocytes release ITGβ1-enriched EVs that recruit and retain monocyte in the liver promoting inflammation. We will employ current biochemical and cell biological approaches that include microfluidic technology, Nanoscale flow cytometry, and 89Zirconium isotopically labelled EVs visualized with positron emission tomography (PET) scan to test this hypothesis. Our independent SPECIFIC AIMS will test three integrated hypotheses. First, we will demonstrate that hepatocyte lipotoxicity induces an active conformation switch of ITGβ1, enhancing its endocytic trafficking and release into EVs. Second, we will define the mechanism of increased VCAM1 expression during lipotoxicity. We will also directly test the hypothesis that lipotoxic hepatocyte-derived EVs mediate monocytes adhesion to LSECs, through ITGβ1-VCAM1 binding interaction, in vitro by using microfluidic technology. Third, using a mouse model of NASH, we will test the hypothesis that pharmacological inhibition of integrin β1 or conditional deletion of endothelial VCAM1 is protective against liver inflammation. We will also demonstrate that adoptively-transferred lipotoxic hepatocyte- derived EVs home to the LSECs of recipient mice through their high affinity integrin β1cargo. We have established the requisite cell and mouse models to study lipotoxicity, integrin signaling and EV biology. This proposal is technically and conceptually innovative, as it seeks to integrate the molecular mechanisms underlying hepatocyte injury, integrin activation and trafficking with liver inflammation, and links hepatic pathophysiology with nanomedicine. This research has the potential to identify new therapeutic strategies, namely integrin and VCAM1 inhibitors, to prevent or reverse liver injury and inflammation in human NASH.