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

项目摘要/摘要 非酒精性脂肪性肝炎(NASH)是一种在美国新出现的肝病流行，是一种 肝细胞癌发病率上升。NASH相关性纤维化，与其他组织学特征无关 例如炎症，是患者长期结果的主要预测因素。相应地，增加了- 临床药物试验的数量，以减缓或逆转NASH患者的纤维化进程。然而， 目前还没有药物被批准广泛使用。肝纤维化的直接致纤维化介质为HE-1。 Patic星状细胞(HSCs)被激活/分化为肌成纤维细胞，后者分泌过量的exp-2。 异常创面愈合级联反应中的细胞基质(ECM)蛋白。由活化产生的刚性基质 HSCs导致肝细胞主要功能的丧失。肝干细胞向肌成纤维细胞分化的实验研究 在NASH中，它们与肝细胞的相互作用是众多微血管之间复杂串扰的结果。 环境信号。因此，有效地治疗NASH相关的纤维化需要理解和相互理解。 打破这种复杂的串扰，扭曲肝脏结构，导致肝脏失代偿。 不同物种在药物代谢和疾病途径上的差异需要动物的补充 数据与人体相关的体外分析。尽管养殖技术的发展取得了重大进展 为了使原代人肝细胞(PHH)在培养数周内的表型稳定，有必要 开发一个能够研究PHH-HSC在生理和疾病中相互作用的平台 设置。我们已经开发了一种细胞微阵列，它允许同时调节大小/成分 图案化的ECM蛋白结构域、底物硬度和可溶性因子浓度，同时还使 细胞表型和收缩能力的平行测量。在这里，我们将使这种细胞微阵列 测试我们的假设，即ECM蛋白组成、底物硬度和可溶性因子共同作用 调节PHHs和HSCs的表型及其在纳什样微环境中的相互作用。我们的 这种方法将使纳入受控细胞外信号扰动的假设驱动研究成为可能。在……里面 目的1，我们将研究细胞外基质成分和底物硬度对长期表型再分化的影响。 正常和NASH诱导条件下PHHs的应答。在目标2中，我们将调查合作社 原代人肝星状细胞激活状态的微环境调节。在目标3中，我们将开发一种联合- 文化方法，以确定PHH和HSC之间的相互作用的作用，并建立 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HSCs和PHHs的改变，包括生化和生物力学信号之间的相互联系。 纳尔。这些努力将有助于旨在逆转纤维化的药物的开发。

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.