A Bioengineered Model for Deciphering Lymphatic Dysfunction in Inflammation

项目总结 淋巴管(LV)在维持体液平衡、调节宿主免疫以及 运输膳食脂肪和神经元废物。所有这些功能都是由淋巴引流控制的， 间质液体和免疫细胞通过初始LV和收集LV进入淋巴系统。 起始LV和收集LV在维持淋巴引流方面具有不同的结构和作用。首字母 LV有可渗透的细胞-细胞连接，并准备吸收间质液体和细胞；相比之下，LV 收集LV的通透性较差，因此收集LV可以将淋巴输送到淋巴结，而不需要 漏水了。淋巴引流受损会导致与LV相关的人类疾病，如淋巴水肿， 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导致 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中，包括动物模型，我们经常 不能使初始和收集LV中的多因素炎症因子解偶联。由于二维 研究人员称，细胞培养未能概括淋巴管的三维组织结构 建立了LV的3D体外模型，展示了淋巴管的萌发、淋巴管网络的形成 LV通透性，以及LV与周围细胞的相互作用。然而，这些以前的型号并没有 创建了带有LEC连接的3D淋巴管，可以控制引流、瓣膜形成或生理 炎症反应。此外，他们也没有考虑初始LV和收集LV的不同角色。在这 建议，我们将建立一个体外生物工程三维淋巴管系统，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LV-on-Chip，考虑(I)紧密的细胞-细胞连接，(Ii)管腔瓣膜， 和(3)壁细胞覆盖率(目标1.1)。接下来，我们将把收集的LV与初始的LV组合在一起，并 在正常情况下演示液体和免疫细胞通过这些LV的传输(目标1.2)。在目标2中， 我们将通过使用与以下相关的炎性细胞因子来确定炎症条件的影响 淋巴功能障碍的初始和收集LV和总体淋巴引流(目标2.1)。接下来，我们将 通过筛选淋巴管通路寻找逆转淋巴功能障碍的新靶点 机械转导(目标2.2)。然后我们将在活体皮肤上验证这些机制和靶点 炎症模型(AIM 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的LV运输并提供独特的 体外平台解决炎症中左心功能障碍的发生，并寻找新的治疗方法 目标是扭转这种功能障碍。

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.