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.

项目摘要/摘要 非酒精性脂肪肝病（NAFLD）是一种全球流行病，发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并增加心血管死亡率。 不幸的是，尚无药理疗法可用来治疗NASH和纤维化，因此需要 识别新型目标和方法。 RECK（诱导与Kazal诱导半胱氨酸蛋白质富含半胱氨酸的蛋白 主题）是一种独特的膜锚定蛋白，已显示出抑制涉及的多个介体 炎症和纤维化。我们的小说初步数据表明，肝reck蛋白水平明显 临床患者的NASH和纤维化严重程度的增加以及我们的临床前小鼠模型的降低 西方饮食（WD）诱导的NASH和纤维化。由于Reck Gene缺失是胚胎致死的，因此我们产生了 Reck Floxed（Reckfl/Fl）和Cag-Catflox-Reck转基因小鼠。我们的概念证明的试点研究表明 虽然AAV8介导的CRE重组酶的特定于肝细胞特定的RECK敲低加剧了Nash和 短期WD喂养小鼠的纤维化，其在肝细胞中的过表达钝化肝炎，kupffer细胞（KC） 和肝星细胞（HSC）激活。此外，我们在原代小鼠肝细胞中的初步体外数据， KCS和HSC在其中，RECK沉默或过表达的KCS和HSC支持我们的体内研究。这些初步 研究表明，维持RECK表达是肝保护剂。因此，我们假设Cre-lox 在肝细胞中特别专门的介导的RECK删除通过增强来增强促炎信号传导 Adam（含蛋白和金属蛋白酶结构蛋白的蛋白质）的两极分裂蛋白（AREG）裂解10- 和17，导致表皮生长因子受体（EGFR）和HSC激活增加增加 导致长期WD诱导的NASH和纤维化的恶化（AIM 1）。相反，转基因 划线的过表达，特别是在肝细胞中，将具有保护性。我们还将确定是否营救Reck 通过已建立的WD诱导的肝细胞特异性小鼠的异位过表达表达 NASH和纤维化可以逆转（AIM 1）。由于KC是主要的居民肝巨噬细胞，并且 HSC被认为是负责肝纤维化的主要细胞类型，我们将确定 在库普弗细胞中的删除和转基因过表达以及HSC在细胞损伤和激活中， 细胞外基质沉积和纤维化（AIM 2）。在这两个目的中，分子机制 炎症和纤维化将在使用原发性肝细胞，KCS和HSC的共同文化研究中研究 从这些改变基因的小鼠模型中分离出来。因此，我们提出的遗传和介入方法将 机械师将RECK建立为NASH和纤维化发病机理中新型上游调节剂 具有未来治疗的潜力。