The Role of Tie1 in Gut and Mesenteric Lymphatic Function

项目摘要 淋巴系统对于组织液稳态、脂质吸收和免疫监视至关重要， 不再被视为病理过程中的“被动旁观者”。消化淋巴系统，包括肠道和肠系膜淋巴管，在肥胖、代谢性疾病、血脂异常、水肿和炎症等多种病理过程中起着重要作用。3.4产前 出生后淋巴管的发育一直是一个研究热点，但对淋巴管的成熟、重塑以及结构和功能的维持等影响因素的认识还很有限。 我们的实验室最近表明，受体酪氨酸激酶TIE 1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特异性缺失Tie 1导致异常的LV形成。我们提出了新的初步数据，删除Tie 1 在出生后早期，导致营养不良，体重增加不佳，LV成熟异常和腹水。相反，我们发现成年动物中Tie 1缺失导致严重的LV功能障碍， 当用高脂肪饮食（HFD）攻击时，饮食诱导的肥胖（DIO）。我们假设TIE 1细胞自主和非细胞自主信号传导在协调出生后早期LV中起着独特的作用， 乳细胞成熟和功能，TIE 1信号传导对于维持LV和乳细胞是必需 成熟动物的结构和功能。因此，我们建议：（1）界定 TIE 1信号在出生后早期泌乳和LV成熟以及体内营养吸收中的作用。Tie 1层/层 小鼠和可诱导的LEC Cre系Prox 1CreERT将用于产生LEC特异性的，暂时缺失的 Tie 1（Tie 1 lecKO）在产后发育中的作用。定量和功能分析将用于表征 TIE 1在肠乳管和肠系膜LV中的衰减导致的独特表型。2)确定 TIE 1在维持成人淋巴功能中的作用，并表征其在介导 DIO。将Tie 1 lecKO和对照（Tie 1fl/fl）成年小鼠置于60%HFD或正常食物中6周， 他莫昔芬的暴露将测量LV结构和功能以及体重和 组成，循环激素胰岛素、瘦素和脂联素的水平，对葡萄糖和胰岛素的敏感性， 以及脂肪细胞形态和炎症状态。3)定义TIE 1介导的信号传导机制 所需的LV和乳重塑和维护，在体内。利用CRISPR/Cas9，我们开发了在Tie 1的关键细胞内结构域中具有错义突变的小鼠。独特的特定于域的影响， LV和泌乳结构和功能将在出生后早期和成年动物中表征， 动态重组和维护流程。scRNA-seq和 sc-UniFrac分析将 用于定义小区自主和非自主LEC子类型所需的TIE 1相关信令 转录签名调节出生后GI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.