A novel microfluidic platform to study exosome biology in PAH.

内皮是覆盖整个循环系统的内层的细胞单层。内皮 功能障碍是肺动脉高压（PAH）的一个特征，PAH是一种与以下疾病相关的危及生命的疾病： 肺动脉压力异常高和慢性右心衰竭。由于现有静态单元的限制， 文化和动物模型，我们对协调启动和毅力的机制的理解， 肺动脉高压患者内皮功能障碍的研究尚不完整。鉴于内皮功能障碍是一种常见的发现， PAH，了解适应不良内皮反应背后的机制有助于加速 发现PAH的新疗法。目前，认为内皮来源的外泌体有助于 PAH通过携带在损伤情况下触发适应不良的内皮反应的信号。外来体是 细胞衍生的小（~30-150 nm）细胞外囊泡，携带相关蛋白质、代谢物和核酸 在各种生理和病理过程中。虽然已知外泌体携带分子和蛋白质， 与血管生成、炎症和血管反应性相关的遗传因素， 健康和功能障碍的PMVEC的外泌体货物已经被目前的低产量外泌体分离所阻碍 技术.这些技术不能从肺组织中进行实时动态外泌体分离。 微血管内皮细胞（PMVEC）暴露于PAH相关应激源。为了解决这一未满足的需求， 我们已经设计了MFES（多功能外泌体分选仪），可以解剖整个外泌体群体， 根据大小和表面标记分为亚群。MFES是第一个芯片实验室平台，它集成了： 1)一个芯片上的血管模块，用于在广泛的范围内实时表征PMVEC功能反应 生理和病理参数的模块，2）用于高产量的基于外泌体大小的分离的模块，3） 使用磁珠的基于表面标记的外泌体分选，和4） PMVEC。在这里，我们提出了一种技术，可以广泛地调查两个主要的定义 外泌体亚型的特征，即，尺寸和表面标记，两者都是独立的， 组合顺序。我们将描述健康和暴露于PAH的PMVEC中外泌体货物的变化 与MFES中的剪切应力相关的条件。我们将根据大小分离外泌体的亚群， 表面标记并确定其货物的特征（目标1）。然后，我们将确定外泌体是否 来源于应激的PMVEC可以诱导在微流体环境中培养的健康PMVEC的病理变化。 培养芯片（Aim 2）。这项技术创新使得能够在一种环境中研究内皮外泌体生物学， 表示与PAH相关的流动动力学。此外，使用尖端的组学技术， 生物信息学分析与机器学习算法相结合来分析纯化的外泌体是预期的 产生一个全面的外泌体货物概况数据集，并为研究 外泌体在PAH病理生物学中的生物学作用和新型治疗剂的测试。

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.