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

项目总结/摘要 非酒精性脂肪性肝炎（NASH）是美国一种新兴的肝病流行病，是治疗非酒精性脂肪性肝炎的基础。 肝细胞癌发病率上升。NASH相关纤维化，不考虑其他组织学特征 例如炎症，是患者长期结果的主要预测因素。因此，有越来越多的- 许多临床药物试验减缓或逆转NASH患者的纤维化进展。然而，在这方面， 目前还没有药物被批准广泛使用。肝纤维化的直接纤维化介质是- 星形细胞（HSC），其被激活/分化成肌成纤维细胞，肌成纤维细胞存款过量的前胶原， 细胞外基质（ECM）蛋白在异常伤口愈合级联中的作用。由活化的纤维素产生的刚性基质 HSC导致肝细胞中主要功能的丧失。HSC向肌成纤维细胞的分化和 它们与NASH中肝细胞的相互作用是众多微环境之间复杂串扰的结果， 精神信号。因此，有效治疗NASH相关纤维化将需要理解和相互作用。 中断这种扭曲肝脏结构并导致肝脏失代偿的复杂串扰。 药物代谢和疾病途径的物种差异需要补充动物 与人类相关的体外试验数据。尽管在培养技术的发展方面取得了重要进展， 为了在培养物中稳定原代人肝细胞（PHH）的表型数周，需要 开发一个平台，使PHH-HSC相互作用的调查在生理和疾病 设置.我们已经开发了一种细胞微阵列，允许同时调节大小/组成， 模式化的ECM蛋白结构域，底物硬度和可溶性因子浓度，同时也使 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蛋白组成、底物硬度和可溶性因子共同起作用 调节PHH和HSC的表型及其在NASH样微环境中的相互作用。我们 这种方法将使假设驱动的研究纳入细胞外信号的受控扰动。在 目的1，我们将研究ECM成分和基质硬度对长期表型重建的影响。 PHH在正常和NASH诱导条件下的自发反应。在目标2中，我们将调查合作社 微环境调节原代人HSC的活化状态。在目标3中，我们将开发一个共同的 培养方法，以确定PHH和HSC之间的相互作用的作用，并建立一个 评估NASH相关治疗的平台。我们的研究将揭示表型 人类HSC和PHH的改变，包括生物化学和生物力学信号之间的相互联系， nals。这些努力将有助于开发旨在逆转纤维化的药物。

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.