Elucidating chemo-mechanical determinants of human hepatocyte and stellate cell responses in non-alcoholic fatty liver disease

阐明非酒精性脂肪肝患者肝细胞和星状细胞反应的化学机械决定因素

• 批准号: 10092152
• 负责人: Salman R Khetani
• 金额: $ 34.45万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-02-08 至 2023-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10092152
• 关键词: Adult; Animal Model; Architecture; Biochemical; Biomechanics; Cell Communication; Cells; Clinical; Clinical Trials; Coculture Techniques; Communities; Complex; Culture Techniques; Data; Deposition; Development; Disease; Disease Pathway; Drug Screening; Engineering; Epidemic; Evaluation; Extracellular Matrix Proteins; Fibrosis; Gene Expression; Genes; Glass; Goals; Growth Factor; Hepatic; Hepatic Stellate Cell; Hepatocyte; Histologic; Human; In Vitro; Incidence; Inflammation; Inflammatory; Interruption; Investigation; Liver; Liver Fibrosis; Liver diseases; Measurement; Mechanics; Mediating; Mediator of activation protein; Modulus; Myofibroblast; Normal Range; Outcome; Pathway interactions; Patients; Pattern; Pharmaceutical Preparations; Pharmacologic Substance; Phenotype; Physiological; Polystyrenes; Primary carcinoma of the liver cells; Protein-Lysine 6-Oxidase; Proteins; Proteoglycan; Regulation; Research Personnel; Role; Scientist; Signal Transduction; Supplementation; Surface; Tertiary Protein Structure; Testing; Therapeutic; Transforming Growth Factors; animal data; cell type; combinatorial; cytokine; drug development; drug efficacy; drug metabolism; efficacy evaluation; experimental study; extracellular; in vitro Assay; insight; man; mechanotransduction; non-alcoholic fatty liver disease; nonalcoholic steatohepatitis; novel; novel therapeutics; paracrine; response; rho GTP-Binding Proteins; small molecule; stellate cell; therapeutic candidate; tool; wound healing

PROJECT SUMMARY/ABSTRACT Non-alcoholic steatohepatitis (NASH) is an emerging epidemic of liver disease in the US and the basis for a rising incidence of hepatocellular carcinoma. NASH-associated fibrosis, regardless of other histologic features such as inflammation, is the major predictor of long-term outcomes in patients. Accordingly, there are increas- ing numbers of clinical drug trials to slow down or reverse fibrosis progression in patients with NASH. However, no drugs have been approved yet for widespread use. The direct fibrogenic mediators of liver fibrosis are he- patic stellate cells (HSCs), which become activated/differentiate into myofibroblasts that deposit excessive ex- tracellular matrix (ECM) proteins in an aberrant wound healing cascade. The stiff matrix produced by activated HSCs leads to the loss of major functions in hepatocytes. The differentiation of HSCs into myofibroblasts and their interactions with hepatocytes in NASH is the result of the complex crosstalk between numerous microen- vironmental signals. Thus, treating NASH-associated fibrosis effectively will require understanding and inter- rupting this complex crosstalk that distorts liver architecture and leads to liver decompensation. Differences across species in drug metabolism and disease pathways necessitate supplementation of animal data with human-relevant in vitro assays. Despite important progress in the development of culture techniques to stabilize the phenotype of primary human hepatocytes (PHHs) in culture for several weeks, there is a need to develop a platform that enables the investigation of PHH-HSC interactions within physiological and disease settings. We have developed a cellular microarray that allows simultaneous modulation of the size/composition of patterned ECM protein domains, substrate stiffness, and soluble factor concentrations, while also enabling parallel measurements of cellular phenotype and contractility. Here, we will adapt this cellular microarray to test our hypothesis that the ECM protein composition, substrate stiffness, and soluble factors act collectively to modulate the phenotypes of PHHs and HSCs and their interactions in an NASH-like microenvironment. Our approach will enable hypothesis-driven studies incorporating controlled perturbations of extracellular signals. In aim 1, we will examine the effects of ECM composition and substrate stiffness on long-term phenotypic re- sponses of PHHs under normal and NASH-inducing conditions. In aim 2, we will investigate the cooperative microenvironmental regulation of the activation states of primary human HSCs. In aim 3, we will develop a co- culture approach to determine the roles of reciprocal interactions between PHHs and HSCs and establish a platform for evaluating NASH-relevant therapeutics. Our studies will reveal mechanisms underlying phenotypic alterations of human HSCs and PHHs, including interconnections between biochemical and biomechanical sig- nals. These efforts will aid the development of drugs aimed at reversing fibrosis.

项目概要/摘要 非酒精性脂肪性肝炎 (NASH) 是美国一种新出现的肝病流行病，也是全球性肝病的基础 肝细胞癌的发病率上升。 NASH 相关纤维化，无论其他组织学特征如何 例如炎症，是患者长期结果的主要预测因素。因此，有增加—— 正在进行大量减缓或逆转 NASH 患者纤维化进展的临床药物试验。然而， 尚未批准广泛使用的药物。肝纤维化的直接纤维化介质是he- 肝星状细胞 (HSC)，被激活/分化为肌成纤维细胞，沉积过量的前体细胞 异常伤口愈合级联中的微量细胞基质（ECM）蛋白。活化后产生的刚性基体 HSC 导致肝细胞主要功能丧失。 HSC 分化为肌成纤维细胞和 NASH 中它们与肝细胞的相互作用是众多微环境之间复杂串扰的结果。 环境信号。因此，有效治疗 NASH 相关纤维化需要了解和相互了解 破坏这种复杂的串扰，扭曲肝脏结构并导致肝脏失代偿。 不同物种在药物代谢和疾病途径方面存在差异，因此需要补充动物饲料 与人类相关的体外测定的数据。尽管养殖技术的发展取得了重要进展 为了稳定培养中原代人肝细胞（PHH）的表型数周，需要 开发一个平台，能够研究 PHH-HSC 在生理和疾病中的相互作用 设置。我们开发了一种细胞微阵列，可以同时调节尺寸/成分 图案化的 ECM 蛋白结构域、基质刚度和可溶性因子浓度，同时还能够 细胞表型和收缩性的平行测量。在这里，我们将调整这个细胞微阵列 检验我们的假设，即 ECM 蛋白质组成、基质硬度和可溶性因子共同作用 调节 PHH 和 HSC 的表型及其在 NASH 样微环境中的相互作用。我们的 该方法将使假设驱动的研究纳入细胞外信号的受控扰动。在 目标 1，我们将检查 ECM 成分和基质刚度对长期表型重新形成的影响 PHH 在正常和 NASH 诱导条件下的反应。在目标 2 中，我们将调查合作社 原代人类 HSC 激活状态的微环境调节。在目标 3 中，我们将开发一个共同 文化方法来确定 PHH 和 HSC 之间相互作用的作用，并建立一个 评估 NASH 相关疗法的平台。我们的研究将揭示表型的潜在机制 人类 HSC 和 PHH 的改变，包括生化和生物力学信号之间的相互联系 纳尔斯。这些努力将有助于开发旨在逆转纤维化的药物。