A Bioengineered Model for Deciphering Lymphatic Dysfunction in Inflammation

项目摘要 淋巴管（LV）在维持体液平衡、调节宿主免疫和免疫功能方面起着重要作用。 运输食物脂肪和神经元废物。所有这些功能都是由淋巴引流， 间质液和免疫细胞通过初始LV和收集LV转运到淋巴系统中。 起始和集合淋巴管在维持淋巴引流方面具有不同的结构和作用。初始 LV具有可渗透的细胞-细胞连接，并且准备好摄取间质液和细胞;相比之下， 收集LV的渗透性较低，使得收集LV可以将“淋巴”运输到淋巴结，而不需要 漏水受损的淋巴引流导致LV相关的人类疾病，如水肿， 免疫功能障碍、纤维化、肥胖、癌症、阿尔茨海默病等。 变得功能失调，临床研究表明，炎症是其中一个主要贡献者， 淋巴功能障碍炎症如何影响初始和收集LV的机制， 功能障碍是不清楚的，因为在我们目前的实验模型，包括动物模型，我们经常 不能分离初始和收集LV中的多因子炎症因子。由于二维 研究人员说，细胞培养未能再现组织的三维结构， 已经开发了LV的3D体外模型，展示了淋巴发芽，淋巴网络形成， LV渗透性和LV与周围细胞的相互作用。然而，这些以前的模型没有 创建了具有LEC连接的3D解剖学，可实现受控引流、瓣膜形成或生理 炎症反应。此外，他们没有考虑初始和收集LV的不同作用。在这 建议，我们将建立一个生物工程体外三维淋巴管系统，包括初始和 收集LV，并确定炎症条件如何使初始LV和收集LV功能障碍。 在目标1中，我们将建立收集的LV芯片，考虑（i）紧密的细胞-细胞连接，（ii）管腔瓣膜， 和（iii）壁细胞覆盖率（目标1.1）。接下来，我们将联合收割机与初始LV合并， 证明在正常条件下液体和免疫细胞通过这些LV转运（目标1.2）。在目标2中， 我们将通过使用与下列因素相关的炎性细胞因子来确定炎性条件的影响： 初始和集合LV以及总体淋巴引流的淋巴功能障碍（目标2.1）。接下来我们就 通过筛选淋巴血管通路，确定逆转淋巴功能障碍的新靶点 机械转导（目的2.2）。然后，我们将在体内皮肤中验证机制和目标 炎症模型（目的2.3）。综上所述，我们将开发一种新的三维淋巴管生物工程模型， 血管系统和液体/免疫细胞通过初始和收集LV运输，并提供独特的 体外平台，以解决LV功能障碍如何在炎症中发生，并确定新的治疗方法 目标是扭转功能障碍。

PROJECT SUMMARY Lymphatic vessels (LVs) 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 and immune cells into the lymphatic system through initial LVs and collecting LVs. The initial and collecting LVs have different structures and roles in maintaining lymphatic drainage. The initial LVs have permeable cell-cell junctions and are ready to uptake interstitial fluid and cells; by contrast, the collecting LVs are less permeable, so that the collecting LVs can transport ‘lymph’ to lymph nodes without leaking. Impaired lymphatic drainage contributes to LV-related human diseases, such as lymphedema, immune dysfunction, fibrosis, obesity, cancer, Alzheimer’s disease, etc. While little is known about why LVs become dysfunctional, clinical studies reveal that inflammation is one of the leading contributors to the lymphatic dysfunction. Mechanisms of how inflammation affects both initial and collecting LVs making them dysfunctional are unclear, because in our current experimental models, including animal models, we often cannot decouple multifactorial inflammatory factors in the initial and collecting LVs. 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, LV permeability, and LV interactions with surrounding cells. However, these previous models have not created 3D lymphatics with LEC junctions enabling controlled drainage, valve formation, or physiological inflammatory response. Also, they have not considered the distinct roles of initial and collecting LVs. In this proposal, we will establish a bioengineered in vitro 3D lymphatic vascular system, including both initial and collecting LVs, and determine how inflammatory conditions make the initial and collecting LVs dysfunctional. In Aim 1, we will establish collecting LV-on-chip, considering (i) tightened cell-cell junctions, (ii) luminal valves, and (iii) mural cell coverage (Aim 1.1). Next, we will combine the collecting LVs with the initial LVs and demonstrate fluid and immune cell transport through these LVs in the normal condition (Aim 1.2). In Aim 2, we will determine the effects of inflammatory conditions by using inflammatory cytokines that are relevant to lymphatic dysfunction on the initial and collecting LVs and overall lymphatic drainage (Aim 2.1). Next, we will identify new targets to reverse the lymphatic dysfunction by screening pathways of lymphatic vascular mechanotransduction (Aim 2.2). We will then validate the mechanisms and targets in an in vivo skin inflammation model (Aim 2.3). In summary, we will develop a new bioengineered model of 3D lymphatic vascular system and fluid/immune cell transport through the initial and collecting LVs and provide a unique in vitro platform to address how the LV dysfunction occurs in inflammation, and identify new therapeutic targets to reverse the dysfunction.