RECK regulation of NASH and fibrosis

RECK 对 NASH 和纤维化的调节

• 批准号: 10616763
• 负责人: Chandrasekar Bysani
• 金额: $ 55.19万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-15 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10616763
• 关键词: Amino Acid Motifs; Amphiregulin; Anti-Inflammatory Agents; Attenuated; Cardiac; Cardiovascular Diseases; Cardiovascular system; Cell Separation; Cells; Cholesterol; Clinical; Coculture Techniques; Cre-LoxP; Data; Deposition; Development; Disease; Disintegrins; Down-Regulation; Ectopic Expression; Embryo; Enterobacteria phage P1 Cre recombinase; Epidemic; Epidermal Growth Factor Receptor; Extracellular Matrix; Fatty acid glycerol esters; Fibroblasts; Fibrosis; Future; GPI Membrane Anchors; Gene Deletion; Genes; Genetic; Growth Factor; Healthcare; Hepatic; Hepatic Stellate Cell; Hepatocyte; In Vitro; Infiltration; Inflammation; Inflammatory; Intervention; Investigation; Kupffer Cells; Ligands; Liver; Liver Fibrosis; Liver diseases; LoxP-flanked allele; Macrophage; Mediating; Mediator; Membrane; Metalloproteases; Molecular; Molecular Analysis; Mouse Strains; Mus; Myofibroblast; Organ; Outcome; Pathogenesis; Pathway interactions; Patients; Phenotype; Pilot Projects; Play; Prevention strategy; Primary carcinoma of the liver cells; Process; Production; Proteins; Receptor Activation; Regulation; Reporting; Role; Severities; Signal Transduction; Sucrose; System; Testing; Therapeutic; Transgenic Mice; Transgenic Organisms; cell injury; cell type; chemokine; cysteine rich protein; cytokine; gain of function; hepatoprotective; improved; in vivo; innovation; knock-down; liver inflammation; loss of function; migration; mortality; mouse model; non-alcoholic fatty liver disease; nonalcoholic steatohepatitis; novel; overexpression; pharmacologic; pre-clinical; protein expression; receptor-mediated signaling; therapeutic target; therapeutically effective; western diet

PROJECT SUMMARY/ABSTRACT Nonalcoholic fatty liver disease (NAFLD) is a global epidemic, progresses to nonalcoholic steatohepatitis (NASH) and fibrosis, and results in the development of hepatocellular carcinoma and increased cardiovascular mortality. Unfortunately, no pharmacological therapies are available yet to treat NASH and fibrosis, necessitating the identification of novel targets and approaches. RECK (Reversion Inducing Cysteine Rich Protein with Kazal Motifs), a unique membrane-anchored protein, has been shown to inhibit multiple mediators involved in inflammation and fibrosis. Our novel preliminary data demonstrate that hepatic RECK protein levels are markedly reduced with increasing severity of NASH and fibrosis in clinical patients and in our pre-clinical mouse model of western diet (WD)-induced NASH and fibrosis. Since RECK gene deletion is embryonically lethal, we generated RECK floxed (RECKfl/fl) and CAG-CATflox-RECK transgenic mice. Our proof-of-concept pilot studies demonstrate that while hepatocyte-specific RECK knockdown by AAV8-mediated Cre recombinase exacerbates NASH and fibrosis in short-term WD-fed mice, its overexpression in hepatocytes blunts liver inflammation, Kupffer cells (KC) and hepatic stellate cell (HSC) activation. Moreover, our preliminary in vitro data in primary mouse hepatocytes, KCs and HSCs in which RECK is either silenced or overexpressed support our in vivo studies. These preliminary studies suggest that sustaining RECK expression is hepatoprotective. Therefore, we hypothesize that Cre-Lox mediated RECK deletion specifically in hepatocytes enhances pro-inflammatory signaling by enhancing amphiregulin (AREG) cleavage by ADAM (A Disintegrin And Metalloproteinase domain-containing protein) 10- and 17, leading to increased epidermal growth factor receptor (EGFR) and HSC activation collectively contributing to worsening of long-term WD-induced NASH and fibrosis (Aim 1). Conversely, transgenic overexpression of RECK, specifically in hepatocytes, will be protective. We will also determine if rescuing RECK expression by ectopic overexpression in hepatocyte-specific RECK deficient mice with established WD-induced NASH and fibrosis can be reversed (Aim 1). Since KCs are the predominant resident liver macrophages and HSCs are considered the principal cell type responsible for hepatic fibrosis, we will establish the importance of RECK deletion and transgenic overexpression in Kupffer cells and HSCs on cellular injury and activation, extracellular matrix deposition, and fibrosis (Aim 2). In both Aims, molecular mechanisms underlying inflammation and fibrosis will be investigated in co-culture studies using primary hepatocytes, KCs and HSCs isolated from these gene-altered mouse models. Thus, our proposed genetic and interventional approaches will mechanistically establish RECK as a novel upstream regulator in the pathogenesis of both NASH and fibrosis with potential as a future therapeutic.

项目总结/摘要 非酒精性脂肪性肝病（NAFLD）是一种全球性流行病，进展为非酒精性脂肪性肝炎（NASH） 和纤维化，并导致肝细胞癌的发展和心血管死亡率的增加。 不幸的是，还没有药理学疗法可用于治疗NASH和纤维化，这使得需要使用药物治疗NASH和纤维化。 确定新的目标和方法。RECK（逆转诱导富含半胱氨酸的蛋白质与Kazal 基序），一种独特的膜锚定蛋白，已被证明可以抑制多种介质参与 炎症和纤维化。我们的新的初步数据表明，肝脏RECK蛋白水平显着 在临床患者和我们的临床前小鼠模型中， 西方饮食（WD）诱导的NASH和纤维化。由于RECK基因缺失具有胚胎致死性，因此我们生成了 RECK floxed（RECKfl/fl）和CAG-CATflox-RECK转基因小鼠。我们的概念验证试点研究表明， 虽然通过AAV 8介导的Cre重组酶的肝细胞特异性RECK敲低加剧NASH， 在短期WD喂养的小鼠中的纤维化，其在肝细胞中的过表达减弱了肝脏炎症，枯否细胞（KC） 和肝星状细胞（HSC）活化。此外，我们在原代小鼠肝细胞中的初步体外数据， RECK沉默或过表达的KC和HSC支持我们的体内研究。这些初步 研究表明，持续的RECK表达具有保肝作用。因此，我们假设Cre-Lox 肝细胞中特异性介导的RECK缺失通过增强促炎信号传导， 双调蛋白（AREG）被ADAM（一种含有去整合素和金属蛋白酶结构域的蛋白质）10- 和17，导致表皮生长因子受体（EGFR）和HSC活化共同增加 导致长期WD诱导的NASH和纤维化恶化（目的1）。相反，转基因 RECK的过表达，特别是在肝细胞中，将是保护性的。我们还将确定是否要拯救雷克 肝细胞特异性RECK缺陷小鼠中异位过表达，并建立WD诱导 NASH和纤维化可以逆转（目的1）。由于KC是主要的常驻肝巨噬细胞， HSC被认为是导致肝纤维化的主要细胞类型，我们将建立HSC的重要性， 库普弗细胞和HSC中RECK缺失和转基因过表达对细胞损伤和活化的影响， 细胞外基质沉积和纤维化（目的2）。在这两个目标中， 将在使用原代肝细胞、KC和HSC的共培养研究中研究炎症和纤维化 从这些基因改变的小鼠模型中分离出来。因此，我们提出的遗传和干预方法将 在NASH和纤维化的发病机制中将RECK机制确立为一种新型上游调节因子 有潜力成为未来的治疗药物