Systemic Inflammation in Microphysiological Models of Muscle and Vascular Disease

摘要 动脉粥样硬化的发生和进展受全身炎症和个体的影响 患有自身免疫性疾病，例如类风湿性关节炎，患上这种疾病的风险会增加 心血管疾病。同样，类风湿性关节炎的慢性和全身性炎症也会诱发肌肉 消瘦和功能丧失。减少炎症的疗法可有效治疗类风湿性关节炎和 有可能降低心血管疾病的严重程度。克服动物的局限性 我们建立了复制一些关键疾病表型的模型，但没有复制潜在的机制 用于健康人体骨骼和心肌的功能性人体微生理系统（hMPS） 使用原代细胞和 iPS 衍生细胞构建内皮化组织工程血管 (eTBEV) 并进行评估 对药物和促炎细胞因子的反应。这些模型复制了结构和关键功能 天然组织并保持其结构和功能至少 4 周。这些体外组织 系统准确地模拟对药物的反应。我们在这个项目中的目标是开发临床相关的 hMPS 疾病模型用于检查类风湿性关节炎 (RA) 肌肉功能障碍和动脉粥样硬化的风险 以及运动在减轻疾病相关炎症中的作用。为了实现这一目标，我们将扩大我们的 开发和验证早期动脉粥样硬化模型的初步结果，该模型使用流动条件促进 内皮功能障碍、巨噬细胞积聚、泡沫细胞形成和血管活性改变。我们会 通过添加巨噬细胞和细胞因子在骨骼肌和心肌中再现 RA 表型 存在于 RA 中，并证明肌肉的模拟运动条件会产生肌因子，从而减少 该 RA 模型中的炎症。然后，我们将开发用于 eTEBV、骨骼的集成灌注系统 和心肌，并表明 RA 模型可以增加 eTEBV 中巨噬细胞的积累， 心脏束，并评估对运动和治疗动脉粥样硬化和炎症的药物的反应。 我们将利用CRISPR基因编辑技术产生前蛋白转化酶枯草杆菌蛋白酶/kexin的突变 9 型 (PCSK9) 和影响 IL-6 脱落的基因，以评估它们对内皮功能障碍和 eTEBV 中泡沫细胞的形成以及骨骼肌和心肌束中的炎症。我们将简介 RA 模型中的细胞因子和代谢物，并证明疾病进展和 在常见的抗炎治疗干预措施的存在下，生物标志物会减少 动脉粥样硬化，并评估运动的效果。同样，在 RA 肌肉模型中，我们将检查是否 基因变异导致细胞因子谱发生改变，影响肌肉功能和运动反应； 这些可能指向新的疾病相关生物标志物和治疗靶点。该项目的成果 将为动脉粥样硬化和自身免疫性疾病的体外建模提供一个总体框架 基因变异在疾病严重程度和药物开发中的作用。

ABSTRACT The initiation and progression of atherosclerosis is influenced by systemic inflammation and individuals suffering from autoimmune diseases, such as rheumatoid arthritis, have increased risk of developing cardiovascular diseases. Likewise, chronic and systemic inflammation in rheumatoid arthritis induces muscle wasting and loss of function. Therapies that reduce inflammation effectively treat rheumatoid arthritis and have the potential to reduce the severity of cardiovascular disease. To overcome limitations of animal models replicating some key disease phenotypes, but not the underlying mechanisms, we established functional human microphysiological systems (hMPS) for healthy human skeletal and cardiac muscle and endothelialized tissue-engineered blood vessels (eTBEVs) using primary and iPS-derived cells and assessed the response to drugs and pro-inflammatory cytokines. These models replicate the structure and key functions of the native tissue and maintain their structure and function for at least 4 weeks. These in vitro tissue systems accurately model the response to drugs. Our goal in this project is to develop clinically relevant hMPS disease models to examine rheumatoid arthritis (RA) risk for muscle dysfunction and atherosclerosis and the role of exercise in attenuating disease-associated inflammation. To meet this goal, we will expand our preliminary results to develop and validate an early atherosclerosis model that uses flow conditions promoting endothelial dysfunction, macrophage accumulation, foam cell formation, and altered vasoactivity. We will reproduce the RA phenotype in skeletal and cardiac muscle through addition of macrophages and cytokines present in RA, and demonstrate that simulated exercise conditions on muscle produce myokines that reduce inflammation in this RA model. Then, we will develop an integrated perfusion system for eTEBVs, skeletal and cardiac muscle and show that the RA model can increase macrophage accumulation in eTEBVs and cardiac bundles, and assess the response to exercise and drugs to treat atherosclerosis and inflammation. We will use CRISPR gene editing technology to generate mutations to proprotein convertase subtilisin/kexin type 9 (PCSK9) and genes that affect IL-6 shedding to assess their impact on endothelial dysfunction and foam cell formation in eTEBVs, and inflammation in skeletal and cardiac muscle bundles. We will profile cytokines and metabolites in the models with and without RA, and demonstrate that disease progression and biomarkers are reduced in the presence of common anti-inflammatory therapeutic interventions for atherosclerosis, and assess the effect of exercise. Likewise, in the RA muscle model, we will examine whether gene variants produce alterations in cytokine profiles impacting muscle function and response to exercise; these may point toward new disease-associated biomarkers and therapeutic targets. Results of this project will provide a general framework for in vitro modeling of atherosclerosis and autoimmune diseases and the role of gene variants in disease severity and drug development.