The Role of Tie1 in Gut and Mesenteric Lymphatic Function

项目总结 淋巴系统对组织液平衡、脂质吸收和免疫监视至关重要，是 不再被认为是病理过程中的“被动旁观者”。消化系统，包括肠道和肠系膜淋巴管，除了淋巴水肿和炎症外，还在肥胖、代谢性疾病、血脂异常等病理过程中发挥关键作用。3，4，同时产前 淋巴管发育一直是一个深入研究的领域，但对淋巴结构和功能的后天调节、淋巴结构和功能的重塑以及维持的因素的认识有限。 我们的实验室最近表明，受体酪氨酸激酶TIE1是淋巴重塑所必需的 和子宫内正常的淋巴功能，出生前发育中的淋巴管中特异的Tie1缺失会导致异常的LV形成。我们提供了新的初步数据，即Tie1的缺失 在出生后早期导致营养不良，体重增加不佳，左室成熟异常，并有腹水。相反，我们发现在成年动物中，Tie1缺失会导致严重的左心功能障碍和严重的 饮食诱导性肥胖(DIO)当挑战高脂肪饮食(HFD)时。我们推测，TIE1细胞自主性和非细胞自主性信号在协调出生后早期LV和 乳汁成熟和功能，TIE1信号对维持LV和乳汁是必不可少的 成熟动物的结构和功能。因此，我们建议：1)界定 TIE1信号在出生后早期乳汁和LV成熟以及体内营养吸收中的作用。第一层/第二层 小鼠和一个可诱导的LEC CRE系Prox1CreERT将被用于产生LEC特异的、暂时缺失的 Tie1(Tie1lecKO)基因在出生后淋巴管中的表达。将使用定量和功能分析来表征 肠道乳汁和肠系膜LV中TIE1衰减导致的独特表型。2)确定 TIE1在成人淋巴功能维持中的作用及其介导性研究 我的天。将Tie1lecKO和对照组(Tie1fl/fl)成年小鼠置于60%高脂饲料或正常饲料中，连续6周 暴露在三苯氧胺中。将测量左心室的结构和功能，以及体重和 组成，循环激素胰岛素、瘦素和脂联素水平，对葡萄糖和胰岛素的敏感性， 以及脂肪细胞的形态和炎症状态。3)定义TIE1介导的信号转导机制 体内左心室和乳管重塑和维持所需。利用CRISPR/Cas9，我们培育了Tie1关键胞内区错义突变的小鼠。独特的特定于领域的效果 LV和Lacteal的结构和功能在出生后早期和成年动物中将在 分别是动态重组和维护流程。ScRNA-seq和 SC-UniFrac分析将是 用于定义小区自主和非自主LEC亚型所需的TIE1依赖信令 转录信号调节出生后胃肠道淋巴系统的成熟和维持。

PROJECT SUMMARY The lymphatic system is critical for tissue fluid homeostasis, lipid absorption, and immune surveillance and is no longer considered a “passive bystander” in pathological processes. The digestive lymphatic system, includ-ing intestinal and mesenteric lymphatics, plays a critical role in several pathological processes including obe-sity, metabolic disease, and dyslipidemia in addition to lymphedema and inflammation. 3,4 While prenatal lym-phatic development has been an area of intensive investigation, insights into factors involved in post-natal reg-ulation of lymphatic maturation, remodeling, and maintenance of lymphatic structure and function is limited. Our laboratory has recently shown that the receptor tyrosine kinase TIE1 is required for lymphatic remodeling and normal lymphatic function in utero, and that deletion of Tie1 specifically from the developing lymphatic vas-culature prior to birth results in abnormal LV formation. We present new preliminary data that deletion of Tie1 in the early post-natal period results in malnutrition with poor weight gain, abnormal LV maturation, and asci-tes. In contrast, we show that Tie1 deletion in the adult animal results in severe LV dysfunction and profound diet-induced obesity (DIO) when challenged with a high fat diet (HFD). We hypothesize that TIE1 cell auton-omous and non-cell autonomous signaling plays a unique role in orchestrating early post-natal LV and lacteal maturation and function, and that TIE1 signaling is essential for maintenance of LV and lacteal structure and function in the mature animal. Therefore, we propose to: 1) Delineate the specific role of TIE1 signaling in early post-natal lacteal and LV maturation, and nutrient absorption, in vivo. Tie1fl/fl mice and an inducible LEC Cre line, Prox1CreERT, will be used to produce LEC-specific, temporal deletion of Tie1 (Tie1lecKO) in post-natal lymphatics. Quantitative and functional analyses will be used to characterize the unique phenotypes that result from TIE1 attenuation in intestinal lacteals and mesenteric LVs. 2) Determine the role of TIE1 in maintenance of lymphatic function in the adult and characterize its role in mediating DIO. Tie1lecKO and control (Tie1fl/fl) adult mice will be place on a 60% HFD or normal chow for 6 weeks following exposure to Tamoxifen. LV structure and function will be measured as well as alterations in body weight and composition, levels of circulating hormones insulin, leptin and adiponectin, sensitivity to glucose and insulin, and adipocyte morphology and inflammatory status. 3) Define the mechanisms of TIE1-mediated signaling required for LV and lacteal remodeling and maintenance, in vivo. Utilizing CRISPR/Cas9, we have devel-oped mice with missense mutations in critical intracellular domains of Tie1. Unique domain-specific effects on LV and lacteal structure and function will be characterized in the early post-natal and adult animals during the dynamic reorganization and maintenance processes, respectively. scRNA-seq and sc-UniFrac analysis will be employed to define TIE1-dependent signaling required for cell autonomous and non-autonomous LEC subtype transcriptional signatures regulating maturation and maintenance of the GI lymphatic system after birth.