Regulation of Lymphatic Endothelial Cell Junction and Drainage

项目总结 淋巴管(LV)的分化、发育和形态发生是维持液体的中心 动态平衡，调节宿主免疫，并运输饮食脂肪和神经元废物。所有这些功能 由淋巴引流控制，淋巴引流是间质液体通过最初的淋巴系统进入淋巴系统的一种运输。 LV和收集LV。初始LV呈现可渗透的纽扣状连接形态，并准备好 相反，收集LV具有拉链状连接结构，其渗透性较差， 这样收集的LV就能将“淋巴”输送到淋巴结而不会渗漏。淋巴引流受损 导致许多人类疾病，如淋巴水肿、免疫功能障碍、纤维化、肥胖、癌症、 和阿尔茨海默氏症。临床研究显示，虽然对左心室功能失调的原因知之甚少 炎症是淋巴功能障碍的主要原因之一。虽然功能失调 收集LV已经被广泛研究，炎症如何影响LV最初的发育和 形态发生尚不清楚，因为在我们目前的实验模型中，包括动物模型，我们经常 不能解偶联淋巴管内皮细胞中的多因子炎症因子。由于二维单元 研究人员称，培养未能概括淋巴管的三维组织结构 开发了LV的3D体外模型，展示了淋巴管的萌发、淋巴管网络的形成和 LV与其他细胞的相互作用。然而，这些以前的模型并没有创建3D淋巴结构 具有专门的LEC接头开发，能够通过纽扣状控制流体排放 关节和生理性炎症反应。在这项提案中，我们将使用一种体外生物工程3D 淋巴管系统，表现为LV的纽扣状连接形态发生和液体排出 通过研究ROCK1/2和整合素α5信号通路，了解细胞内皮细胞连接和引流的调控。 在目标1中，我们将研究岩石在LEC连接和排水中的作用。接下来，我们将仔细研究 晶体上皮细胞中岩石介导的结合部拉链机制。在目标2中，我们将研究整合素α5介导的 LEC连接和淋巴引流的调节。然后我们将确定信号转导通过 ROCKS和整合素α5及靶向ROCKS和整合素α5在淋巴系统中的疗效评价 体内功能障碍和炎症模型。总而言之，我们将使用3D淋巴管的生物工程模型 血管和液体运输提供对正常和炎症性淋巴引流的了解 条件。

PROJECT SUMMARY Lymphatic vessel (LV) differentiation, development, and morphogenesis are central in maintaining fluid homeostasis, regulating host immunity, and transporting dietary fat and neuronal waste. All these functions are governed by lymphatic drainage, a transport of interstitial fluid into the lymphatic system through the initial LVs and collecting LVs. The initial LVs show permeable button-like junction morphology and are ready to uptake interstitial fluid; by contrast, the collecting LVs are less permeable with zipper-like junction structure, so that the collecting LVs transport ‘lymph’ to lymph nodes without leaking. Impaired lymphatic drainage contributes to many human diseases, such as lymphedema, immune dysfunction, fibrosis, obesity, cancer, and Alzheimer’s disease. While little is known about why LVs become dysfunctional, clinical studies reveal that inflammation is one of the leading contributors to the lymphatic dysfunction. Although dysfunctional collecting LVs has been extensively studied, how inflammation impacts initial LV development and morphogenesis is unclear, because in our current experimental models, including animal models, we often cannot decouple multifactorial inflammatory factors in the lymphatic endothelium. Since two-dimensional cell culture has failed to recapitulate three-dimensional (3D) tissue architecture of lymphatics, researchers have developed 3D in vitro models of LVs, demonstrating lymphatic sprouting, lymphatic network formation, and LV interactions with other cells. However, these previous models have not created 3D lymphatic structure with specialized LEC junction development enabling controlled fluid drainage through the button-like junctions and physiological inflammatory response. In this proposal, we will use a bioengineered in vitro 3D lymphatic vascular system, exhibiting button-like junction morphogenesis of the LVs and fluid drainage to understand the regulation of LEC junction and drainage by focusing on ROCK1/2 and integrin α5 signaling. In Aim 1, we will examine the roles of ROCKs in LEC junction and drainage. Next, we will scrutinize the mechanisms of ROCKs-mediated junction zippering in LECs. In Aim 2, we will study integrin α5 mediated regulation of LEC junction and lymphatic drainage. We will then determine signal transduction through ROCKs and integrin α5 and evaluate therapeutic efficacy of targeting ROCKs and integrin α5 in lymphatic dysfunction and inflammation models in vivo. In summary, we will use a bioengineered model of 3D lymphatic vessels and fluid transport to provide an understanding of lymphatic drainage in normal and inflammatory conditions.