Primary Cilia-Dependent Mechanisms of Lymphangiogenesis

淋巴管生成的初级纤毛依赖性机制

• 批准号: 10593088
• 负责人: Darci M. Fink
• 金额: $ 38.92万
• 依托单位: SOUTH DAKOTA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-20 至 2027-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10593088
• 关键词: Actins; Affect; Biological Assay; Biology; Blood; Carrier Proteins; Cell Line; Cell Polarity; Cell physiology; Cells; Chemotaxis; Chronic; Cilia; Clustered Regularly Interspaced Short Palindromic Repeats; Complement; Cornea; Corneal Neovascularization; Data; Defect; Development; Diameter; Elements; Endothelial Cells; Erinaceidae; Event; Fluorescence; Future; Genes; Goals; Graft Rejection; Immune; In Vitro; Inflammation; Intercellular Junctions; KDR gene; Keratoplasty; Knock-out; Knockout Mice; Light; Liquid substance; Lymphangiogenesis; Lymphatic; Lymphatic Diseases; Lymphatic Endothelial Cells; Lymphedema; Lymphocyte; Microscopy; Modeling; Mus; Ontology; Organelles; Pathology; Pathway Analysis; Pathway interactions; Pattern; Phase; Platelet-Derived Growth Factor alpha Receptor; Proliferating; Proteins; Refractory; Regulation; Reporter; Research; Research Personnel; Signal Pathway; Signal Transduction; Sorting; Structure; Surgical sutures; Technology; Testing; Time; Tissues; Translational Research; VEGFC gene; Validation; Vascular Endothelial Growth Factor C; Work; candidate identification; cell type; density; frontier; in vitro Assay; in vitro testing; in vivo; innovation; insight; intravital imaging; lymph nodes; lymphatic vessel; migration; mouse model; new therapeutic target; next generation sequencing; notch protein; novel therapeutics; prevent; programs; response; shear stress; spatiotemporal; targeted treatment; therapeutic target; transcriptome sequencing; transcriptomics; tumor; wound healing

PROJECT SUMMARY Properly patterned lymphatic vessels are critical regulators of fluid and cellular traffic to support an effective inflammation and wound healing response. However, the mechanisms that govern lymphatic vessel patterning are not fully understood, and new therapeutics are needed to treat dysregulations of lymphangiogenesis, for example in neovascularized corneal transplant rejection, lymphedema, and tumor-associated lymphangiogenesis. The long-term goal of this research program is to develop lymphatic-targeted strategies to promote wound healing and prevent or reverse refractory inflammation. In the course of work examining lymphatic vessel pathology, the current investigators recently identified primary cilia on lymphatic endothelial cells in vitro and in vivo. In other cell types, this organelle is a hub of cell signaling and regulates cellular processes such as proliferation, cell polarity, and migration. Preliminary data in knockout mouse models of IFT20, an intraflagellar transport protein important for primary cilia function, show that loss of primary cilia exacerbates lymphangiogenesis. This suggests that previously unappreciated primary cilia-dependent mechanisms may complement and modulate canonical mechanisms of lymphangiogenesis regulation. The central hypothesis of this proposal is that unknown primary cilia signaling pathways negatively regulate excessive lymphangiogenesis by suppressing sprouting, proliferation, and migration to form vasculature appropriately tuned to local demand. This hypothesis will be tested with two specific aims: (1) Identify the cellular mechanisms by which loss of primary cilia dysregulates lymphangiogenesis; and (2) Identify lymphatic endothelial cell signaling pathways that are disrupted by loss of primary cilia. In aim 1, intravital imaging in mice with IFT20 knockout and tdTomato reporter fluorescence along with in vitro lymphangiogenesis assays will be used to identify the timing and extent of dysregulation of specific lymphatic endothelial cell activities that comprise a functional lymphangiogenesis response, such as sprouting, cell-cell junction organization, and chemotaxis. In aim 2, two next generation sequencing technologies, RNA-Seq and spatial transcriptomics, will be used in combination with pathway analysis and gene ontology strategies and rational hypothesis-driven prioritization of hits to identify candidate primary cilia-dependent regulators of inflammation-associated corneal lymphangiogenesis. Candidates will be validated in vitro in CRISPR knockout lymphatic endothelial cell lines. The combination of intravital imaging, transcriptomics, gene editing, and a panel of in vitro lymphangiogenesis assays is an innovative strategy to comprehensively interrogate the cellular mechanistic basis of primary cilia regulation of lymphangiogenesis. The proposed project is significant because it will provide the first mechanistic insight into primary cilia function in lymphatic endothelial cells with the potential to provide novel therapeutic targets to control lymphangiogenesis as well as a framework for study of primary cilia regulation of other lymphatic remodeling events. This work may open a new frontier for the field of lymphatic vessel biology.

项目总结 正确设计的淋巴管是液体和细胞交通的关键调节器，以支持有效的 炎症和伤口愈合反应。然而，支配淋巴管构型的机制 目前还不完全清楚，需要新的疗法来治疗淋巴管生成的失调， 新生血管角膜移植排斥反应、淋巴水肿和肿瘤相关病例 淋巴管生成。这项研究计划的长期目标是开发针对淋巴的策略 促进伤口愈合，预防或逆转顽固性炎症。在工作审查过程中 淋巴管病理学，目前的研究人员最近在淋巴管内皮细胞上发现了初级纤毛 细胞在体外和体内。在其他类型的细胞中，这个细胞器是细胞信号的枢纽，并调节细胞 细胞增殖、细胞极性和迁移等过程。基因敲除小鼠模型的初步数据 IFT20是一种鞭毛内转运蛋白，对初级纤毛功能很重要，它表明初级纤毛的丧失 会加剧淋巴管生成。这表明，以前未被认识到的依赖于初级纤毛的 这些机制可以补充和调节淋巴管生成调节的典型机制。这个 这一建议的中心假设是未知的初级纤毛信号通路负调控 通过抑制淋巴管的萌发、增殖和迁移而形成血管的过度淋巴管生成 适当调整以适应当地需求。这一假设将以两个具体的目标进行检验：(1)确定 初级纤毛缺失导致淋巴管生成失调的细胞机制；以及(2)识别淋巴管 内皮细胞信号通路因初级纤毛的丧失而中断。在目标1中，活体成像 带有IFT20基因敲除和tdTomato报告荧光的小鼠的淋巴管生成实验 将用于确定特定淋巴管内皮细胞活动失调的时间和程度 包括功能性淋巴管生成反应，例如发芽、细胞-细胞连接组织，以及 趋化性。在目标2中，两项下一代测序技术，RNA-Seq和空间转录组学，将 与通径分析和基因本体论策略以及理性假设驱动相结合 确定炎症相关角膜的候选初级纤毛依赖调节因子的HITS优先顺序 淋巴管生成。候选基因将在CRISPR基因敲除的淋巴管内皮细胞系中进行体外验证。 活体成像、转录、基因编辑和一组体外淋巴管生成的结合 化验是全面探讨初生纤毛细胞机制基础的创新策略 淋巴管生成的调控。拟议中的项目意义重大，因为它将提供第一个 对淋巴管内皮细胞初级纤毛功能的机械性洞察可能提供新的 控制淋巴管生成的治疗靶点以及初级纤毛调节的研究框架 其他淋巴管重塑事件。这项工作可能会为淋巴管生物学领域开辟一个新的前沿。