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 非酒精性脂肪性肝病(NAFLD)是一种全球性的流行病，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增加心血管死亡率。 不幸的是，目前还没有治疗NASH和纤维化的药物疗法，这使得 确定新的目标和方法。RECK(用Kazal逆转诱导富含半胱氨酸的蛋白 基序)，一种独特的膜锚定蛋白，已被证明抑制参与 炎症和纤维化。我们新的初步数据表明，肝脏RECK蛋白水平显著 在临床患者和我们的临床前小鼠模型中，随着NASH和纤维化严重程度的增加而降低 西方饮食(WD)诱导的NASH和纤维化。由于RECK基因缺失在胚胎上是致命的，我们产生了 RECKfl/f1和CAG-CATflx-RECK转基因小鼠。我们的概念验证试点研究表明 AAV8介导的Cre重组酶在肝细胞特异性RECK基因敲除的同时加剧NASH和 短期喂食WD的小鼠肝纤维化，其在肝细胞中的过度表达抑制肝脏炎症，Kupffer细胞(KC) 和肝星状细胞(HSC)激活。此外，我们在原代小鼠肝细胞中的初步体外数据， RECK被沉默或过度表达的KCS和HSCs支持我们的活体研究。这些初步的 研究表明，持续表达RECK对肝脏有保护作用。因此，我们假设Cre-Lox 肝细胞特异性介导的RECK缺失通过增强 ADAM(去整合素和金属蛋白酶结构域蛋白)10-裂解双调节蛋白(AREG) 和17，导致表皮生长因子受体(EGFR)和HSC共同激活 导致WD引起的长期NASH和纤维化恶化(目标1)。相反，转基因 RECK的过度表达，特别是在肝细胞中的过度表达，将具有保护作用。我们还将确定是否营救雷克 WD诱导的肝细胞特异性RECK缺陷小鼠异位过表达 NASH和纤维化是可以逆转的(目标1)。由于KCs是主要的驻留肝巨噬细胞， HSCs被认为是导致肝纤维化的主要细胞类型，我们将确定 RECK缺失和转基因在Kupffer细胞和HSC中的过表达对细胞损伤和激活的影响 细胞外基质沉积和纤维化(目标2)。在这两个目标中，潜在的分子机制 炎症和纤维化将在使用原代肝细胞、KCs和HSCs的共培养研究中进行研究。 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 从机制上证实RECK在NASH和纤维化的发病机制中是一种新的上游调节因子 有可能成为未来的治疗药物。