Synergistic effects of ECM and heterotypic crosstalk on cellular responses in non-alcoholic fatty liver disease

摘要/项目摘要 非酒精性脂肪性肝炎(NASH)是一种迅速流行的肝病，易患上 受影响的个体发展为晚期纤维化和肝细胞癌。以细胞介体为目标的策略 对于肝纤维化，如活化的肝星状细胞(HSCs)，由于不完全的原因，仍然有限 肌成纤维细胞分化的机制及肝与肝之间的信号转导 实质细胞和非实质细胞(NPC)类型。微环境信号串扰的重要性， 包括细胞外基质(ECM)蛋白组成与细胞机械硬度之间的相互作用 表型变化，越来越受到重视。尽管动物模型提供了对纳什的洞察， 不同物种在药物代谢和疾病途径上存在显著差异。因此，有必要 与人类相关的体外方法，使研究肝细胞表型在 生理和纳什样的微环境，并可以促进高通量发现新的 治疗学。这个项目的目标是实现工程化的文化平台，以选择性地调节 微环境信号，并利用这些系统揭示关键的表型编程途径和 纳什类微环境背景下的相互作用机制。我们的方法将使 假说驱动的研究结合了细胞外信号的受控扰动。在目标1中，我们将 探讨肌成纤维细胞表型的微环境调控。利用定义的ECM成分 和机械僵硬的机制，我们将检查表观遗传基因调控的作用 组合微环境线索调控表观遗传变化的机制和检验假说 在正常情况下，对人肝星状细胞的肌成纤维细胞编程是至关重要的 和纳什一样的可溶触发器。在目标2中，我们将研究ECM的组成和硬度对 库普弗细胞(KC)和原代人肝细胞(PHH)的功能，包括细胞间的相互作用 纳氏类可溶刺激的协同效应。我们的方法将促进模块化控制和 ECM成分、衬底硬度和可溶信号的反卷积效应。在《目标3》中，我们将 开发和实现用于系统分析多细胞表型的多细胞3D肝脏显微组织 在类似疾病的ECM改变的背景下的监管。我们将研究异型蜂窝串扰 非实质细胞类型(HSC、KC和肝窦内皮细胞)和它们之间的机制 集体对人类原代肝细胞功能的影响。我们将进一步建立评估能力 多细胞肝平台内的NASH相关治疗药物。拟议的研究将提供一个) 对细胞表型背后的微环境线索和信号机制的基本见解 NASH的变化，这可能有助于开发新的药物疗法，以及b)新的工程肝脏 培养平台，可作为广泛的生理和疾病结果的支持工具。

ABSTRACT / PROJECT SUMMARY Non-alcoholic steatohepatitis (NASH) represents a rapidly growing epidemic of liver disease that can predispose affected individuals to advanced fibrosis and hepatocellular carcinoma. Strategies for targeting cellular mediators of liver fibrosis, such as activated hepatic stellate cells (HSCs), remain limited due to an incomplete understanding of the mechanisms governing myofibroblastic differentiation and the signaling between liver parenchymal and non-parenchymal cell (NPC) types. The importance of microenvironmental signal crosstalk, including interactions between extracellular matrix (ECM) protein composition and mechanical stiffness in cell phenotypic alterations, is increasingly appreciated. Although animal models have provided insights into NASH, significant differences across species in drug metabolism and disease pathways exist. Thus, there is a need for human-relevant in vitro approaches that enable the investigation of hepatocellular phenotypes within physiological and NASH-like microenvironments and could facilitate the high-throughput discovery of novel therapeutics. The goal of this project is to implement engineered culture platforms for selectively modulating microenvironmental signals and utilize these systems to reveal key phenotypic programming pathways and interaction mechanisms within the context of a NASH-like microenvironment. Our approach will enable hypothesis-driven studies incorporating controlled perturbations of extracellular signals. In Aim 1, we will investigate the microenvironmental regulation of myofibroblastic phenotype. Utilizing defined ECM compositions and mechanical stiffness regimes in engineered cultures, we will examine the role of epigenetic gene regulatory mechanisms and test the hypothesis that combinatorial microenvironmental cues regulate epigenetic changes that are critical for the myofibroblastic programming of human hepatic stellate cells within the context of normal and NASH-like soluble triggers. In Aim 2, we will examine the influence of ECM composition and stiffness on Kupffer cell (KC) and primary human hepatocyte (PHH) functions including reciprocal intercellular interactions and cooperative effects of NASH-like soluble stimuli. Our approach will facilitate modular control and deconvolution of the effects of ECM composition, substrate stiffness, and soluble signals. In Aim 3, we will develop and implement multicellular 3D liver microtissues for the systematic analysis of multicellular phenotype regulation in the context of disease-like ECM alterations. We will investigate heterotypic cellular crosstalk mechanisms between non-parenchymal cell types (HSC, KC, and liver sinusoidal endothelial cells), and their collective influence on primary human hepatocyte functions. We will further establish capabilities for assessing NASH-relevant therapeutics within the multicellular liver platform. The proposed studies will provide a) fundamental insights into the microenvironmental cues and signaling mechanisms underlying cellular phenotypic alterations in NASH, which could aid the development of novel drug therapies, and b) novel engineered liver culture platforms that can serve as enabling tools for a broad range of physiological and disease outcomes.