Lipotoxicity and Liver Inflammation

项目摘要 本提案的总体目标是确定脂毒性期间肝细胞损伤与 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 其特征在于丰富的循环饱和游离脂肪酸（SFA），沿着肝细胞脂毒性， 单核细胞衍生的巨噬细胞介导的肝脏炎症。肝细胞脂毒性和肝损伤，部分， 由SFA及其细胞内代谢产物溶血磷脂酰胆碱（LPC）诱导。然而，细胞和 将肝细胞脂毒性与肝脏炎症联系起来的分子机制尚未完全了解。 新出现的数据表明，肝细胞脂毒性应激期间释放的细胞外囊泡（EV） 炎症在已发表的和初步的实验中，我们发现， 释放大量促炎性EV;这些EV富含粘附分子整合素 β1（ITGβ1），促进单核细胞与肝窦内皮细胞（LSECs）的粘附。我们也 表明ITGβ1配体，血管细胞粘附分子（VCAM）1在LSEC上的表达是 在脂毒性期间增加。基于这些新的观察，我们制定了中央 假设脂毒性肝细胞释放富含ITGβ1的EV，其募集并保留单核细胞， 肝脏促进炎症。我们将采用当前的生物化学和细胞生物学方法，包括 微流控技术，纳米级流式细胞术，和89锆同位素标记的电动汽车可视化与 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测试 三个综合假设。首先，我们将证明，肝细胞脂毒性诱导活性 ITGβ1的构象转换，增强其内吞运输和释放到EV中。第二，我们将定义 脂毒性过程中VCAM 1表达增加的机制。我们还将直接检验假设 脂毒性肝细胞来源的EV通过ITGβ1-VCAM 1结合介导单核细胞与LSEC的粘附 相互作用，在体外通过使用微流体技术。第三，使用NASH小鼠模型，我们将测试 假设整合素β1的药理学抑制或内皮VCAM 1的条件性缺失是 防止肝脏炎症。我们还将证明过继转移的脂毒性肝细胞- 衍生的EV通过其高亲和力整合素β 1货物归巢受体小鼠的LSEC。我们有 建立了必要的细胞和小鼠模型来研究脂毒性、整合素信号传导和EV生物学。这 该提案在技术上和概念上都是创新的，因为它试图整合分子机制， 潜在的肝细胞损伤，整合素活化和运输与肝脏炎症，并将肝脏 病理生理学与纳米医学这项研究有可能确定新的治疗策略， 即整联蛋白和VCAM 1抑制剂，以预防或逆转人NASH中的肝损伤和炎症。

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.