A novel microfluidic platform to study exosome biology in PAH.

内皮是覆盖整个循环系统内层的细胞单层。内皮细胞 功能障碍是肺动脉高压(PAH)的一个特征，是一种与 异常高的肺压力和慢性右心衰竭。由于可用静态电池的限制 文化和动物模型，我们对启动和坚持不懈的机制的理解 对PAH中内皮功能障碍的研究仍不完整。鉴于血管内皮细胞功能障碍是 PAH，了解适应不良内皮反应背后的机制可能有助于加速 发现治疗PAH的新疗法。目前，人们认为内皮细胞衍生的外切体有助于 PAH通过携带在损伤背景下触发适应不良的内皮反应的信号来实现。外切体是 细胞来源的小(~30-150 nm)细胞外小泡，携带蛋白质、代谢物和核酸 在各种生理和病理过程中。虽然已知外切体携带分子和 与血管生成、炎症和血管反应性相关的遗传因素，综合评估 目前的低产量外切体分离阻碍了健康和功能障碍的PMVECs的外切体运输 技巧。这些技术不能从肺组织中进行实时动态外显子分离 微血管内皮细胞(PMVECs)暴露于PAH相关应激源。为了解决这一未得到满足的需求， 我们设计了多功能外切体分类器(MFES)，它可以对整个外切体种群进行解剖 根据大小和表面标记划分为亚种群。MFES是第一个集成了以下功能的芯片实验室平台： 1)用于在大范围内实时表征PMVEC功能响应的片上舰船模块 生理和病理参数，2)用于基于大小的高产外切体分离的模块，3)a 基于表面标记的磁珠外切体分选，以及4)外切体的多组学表型 PMVEC。在这里，我们提出了一种技术，它可以广泛地研究两个主要定义 外体亚型的特征，即大小和表面标记，分别独立地和在 按顺序组合。我们将对健康和暴露的PAH PMVECs的外切体货物的变化进行表征 MFE中与剪应力相关的条件。我们将根据大小和大小分离外染色体的亚群 表面标记，并为其货物确定其特征(目标1)。然后，我们将确定外切体是否 应激来源的PMVECs可诱导微流控培养的健康PMVECs发生病变 培养芯片(目标2)。这项技术创新使研究内皮外切体生物学成为可能 表示与PAH关联的流动动力学。此外，尖端组学技术的使用， 生物信息学分析与机器学习算法相结合来分析纯化的外切体是可望的 产生一个全面的外系体货物轮廓数据集，并为研究 外切体在PAH病理生物学和新型治疗药物试验中的生物学作用。

The endothelium is the cellular monolayer that covers the inner lining of the entire circulatory system. Endothelial dysfunction is a feature of pulmonary arterial hypertension (PAH), a life-threatening disease associated with abnormally high pulmonary pressures and chronic right heart failure. Due to the limitations of available static cell culture and animal models, our understanding of the mechanisms that orchestrate the initiation and perseverance of endothelial dysfunction in PAH remains incomplete. Given that endothelial dysfunction is a common finding in PAH, an understanding of the mechanism behind maladaptive endothelial responses could help accelerate the discovery of novel therapies for PAH. Presently, it is believed that endothelial derived exosomes contribute to PAH by carrying signals that trigger maladaptive endothelial responses in the setting of injury. Exosomes are cell-derived small (~30-150 nm) extracellular vesicles that carry proteins, metabolites and nucleic acids involved in a variety of physiological and pathological processes. While it is known that exosomes carry molecular and genetic factors associated with angiogenesis, inflammation and vasoreactivity, a comprehensive assessment of exosome cargo of healthy and dysfunctional PMVECs has been hindered by current low-yield exosome isolation techniques. These techniques cannot perform real-time dynamic exosome isolation from pulmonary microvascular endothelial cells (PMVECs) exposed to PAH-associated stressors. To address this unmet need, we have designed the MFES (Multifunctional Exosome Sorter) that can dissect the whole exosome population into subpopulations based on size and surface markers. MFES is the first lab-on-a-chip platform that integrates: 1) a vessel-on-a-chip module for real-time characterization of PMVEC functional responses across a wide range of physiological and pathological parameters, 2) a module for high-yield exosome size-based isolation, 3) a surface marker based exosome sorting using magnetic beads, and 4) multi-omics phenotyping of exosomes of PMVECs. Here, we are proposing a technology that can enable broadly to investigate the two main defining characteristics of exosomal subtypes, i.e., size and surface markers, both separately independently, and in combination sequentially. We will characterize changes in exosome cargo in healthy and PAH PMVECs exposed to shear stress-related conditions in the MFES. We will isolate subpopulations of exosomes based on size and surface markers and characterize them for their cargo (Aim 1). Then, we will determine whether exosomes derived from stressed PMVECs can induce pathological changes in healthy PMVECs cultured in a microfluidic culture chip (Aim 2). This technological innovation enables to study endothelial exosome biology in a setting that represents the flow dynamics associated with PAH. Further, the use of cutting-edge -omics technologies, bioinformatic analysis integrated with machine learning algorithms to analyze the purified exosomes is expected to yield a comprehensive dataset of exosome cargo profiles and open exciting opportunities for investigating the biological role of exosomes in PAH pathobiology and the testing of novel therapeutic agents.