Novel regulators, components and function of endothelial diaphragms

内皮隔膜的新型调节器、组件和功能

• 批准号: 10339380
• 负责人: RADU VIRGIL STAN
• 金额: $ 41万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-02-15 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10339380
• 关键词: Address; Adult; Animal Model; Antibodies; Antigens; B-Lymphocytes; Basement membrane; Biogenesis; Biological Assay; Biology; Blood; Blood Vessels; Blood capillaries; Caveolae; Cell Culture System; Cell Culture Techniques; Cell Line; Cell membrane; Cell model; Cellular Structures; Clinic; Clinical; Collaborations; Data; Defect; Diabetes Mellitus; Diarrhea; Discontinuous Capillary; Disease; Endocrine Glands; Endothelial Cells; Endothelium; Exocrine Glands; Extracellular Matrix; Extracellular Matrix Proteins; FGF5 gene; FGFR1 gene; Fenestrated Capillary; Fibrinogen; Gene Expression; Genetic Engineering; Genetically Engineered Mouse; Growth Factor; Homeostasis; Human; Immune System Diseases; Immune response; In Situ; Inflammation; Integrin Binding; Integrins; Investigation; KDR gene; Kidney Diseases; Kidney Failure; Kidney Glomerulus; Knowledge; Laboratories; Laminin; Lead; Leukocytes; Life; Literature; Liver; Liver diseases; MAP Kinase Gene; MAPK3 gene; MAPK8 gene; Maintenance; Malignant Neoplasms; Medical; Microvascular Permeability; Modeling; Molecular; Morphogenesis; Mus; Myocardial Infarction; Nutrient; Organ; Organelles; Outcome; Oxygen; Pathogenesis; Pathway interactions; Perinatal mortality demographics; Permeability; Phenotype; Physiological; Play; Positioning Attribute; Pre-Eclampsia; Process; Property; Protein-Losing Enteropathies; Proteins; Proto-Oncogene Protein c-kit; Reagent; Receptor Signaling; Regulation; Regulatory Pathway; Research; Respiratory Diaphragm; Retina; Retinal Diseases; Role; Signal Pathway; Signal Transduction; Structure of choroid plexus; Subcellular structure; Syndrome; Testing; Therapeutic; Tissues; Toxic Hepatitis; Validation; Vascular Endothelial Cell; Vascular Endothelial Growth Factors; Vascular Endothelium; Vascular Permeabilities; Vesicle; Viral; Visceral; Work; base; cancer immunotherapy; cancer therapy; gain of function; human disease; innovation; insight; interest; loss of function; loss of function mutation; lymph nodes; novel; novel therapeutic intervention; p38 Mitogen Activated Protein Kinase; receptor; reconstitution; tool; tumor growth; vascular bed; wasting

Endothelial diaphragms are protein barriers associated with vascular endothelial subcellular structures, such as fenestrae, transendothelial channels (TEC), caveolae and vesiculo-vacuolar organelles implicated in vascular permeability and immune response. Fenestrae are circular transcellular pores in vascular endothelial cells playing critical roles in the maintenance of basal permeability. Recent data in humans and mice demonstrate that fenestrae and their diaphragms play critical roles in the maintenance of normal endothelial barrier function, blood homeostasis and ultimately survival. Our group demonstrated that Plasmalemma Vesicle Associated Protein (PLVAP or PV1) is critical for diaphragm assembly, and that absence of PV1 in mice and humans causes abnormal fenestrae and perinatal lethality due to multiple vascular defects. PV1 is also critical in the immune response, and therapeutic strategies based on PV1 biology are being developed for myocardial infarction, immune diseases and cancer. While there is emerging interest in diaphragms and fenestrae in the clinic, there is a significant knowledge gap in our understanding of the components and regulatory pathways governing fenestrae and diaphragm morphogenesis, addressed in this proposal. Critically for this proposal, PV1 is the only known marker for diaphragms, and we will exploit this property in our investigation of regulation diaphragm morphogenesis. Based on our preliminary data and literature, our central hypothesis is that multiple signaling pathways synergize in vascular bed specific manner for diaphragm and fenestrae formation. Specifically, crosstalk between growth factors and laminin binding integrin receptors are critical for PV1 expression and fenestrae and diaphragm morphogenesis. This hypothesis is based on our key new observations including: 1) differential segmental and spatial control of fenestrae in different vascular beds; 2) Gene expression screens for factors involved in PV1 regulation, identified SCF, FGF-5, VEGF-A and specific laminins signaling via their respective c-KIT, FGFR1, VEGFR2, and ITGB1 receptors as necessary for PV1 expression in ERK1/2 dependent manner; and 3) the combination of laminin with SCF, FGF-5 and VEGF- A results in robust PV1 expression and de novo fenestrae formation We will test our hypothesis using a combination of functionally validated endothelial cell culture systems and genetically engineered animal models using loss of function, gain of function and reconstitution approaches. Our Aims will define the role and downstream signaling pathways of newly identified growth factors and laminins and their integrin receptors regulating PV1 expression and fenestrae morphogenesis. We are uniquely positioned both in terms of key and necessary expertise to complete this work. These investigations will define regulatory factors and pathways controlling the assembly for an intricate and physiologically relevant cellular structure, providing valuable insight that is currently lacking.

内皮隔膜是与血管内皮亚细胞结构相关的蛋白质屏障，例如 如窗孔、跨内皮通道 (TEC)、小窝和囊泡-液泡细胞器涉及 血管通透性和免疫反应。窗孔是血管内皮细胞中的圆形跨细胞孔 细胞在维持基础通透性方面发挥着关键作用。人类和小鼠的最新数据 证明窗孔及其隔膜在维持正常内皮细胞中发挥着关键作用 屏障功能、血液稳态和最终的生存。我们的小组证明了 Plasmalemma 囊泡相关蛋白（PLVAP 或 PV1）对于隔膜组装至关重要，并且 PV1 的缺失 小鼠和人类由于多种血管缺陷而导致窗孔异常和围产期死亡。 PV1 是 对于免疫反应也至关重要，基于 PV1 生物学的治疗策略正在开发中 心肌梗塞、免疫疾病和癌症。虽然人们对隔膜和 在临床上，我们对窗孔的成分和结构的理解存在很大的知识差距。 本提案讨论了控制窗孔和隔膜形态发生的调控途径。批判地 对于本提案，PV1 是唯一已知的隔膜标记，我们将在我们的项目中利用这一特性 调节隔膜形态发生的研究。根据我们的初步数据和文献，我们的中央 假设是多种信号通路以血管床特定方式协同作用，用于膈肌和 窗孔的形成。具体而言，生长因子和层粘连蛋白结合整联蛋白受体之间的串扰是 对于 PV1 表达以及窗孔和膈肌形态发生至关重要。这个假设是基于我们的关键 新的观察结果包括：1）不同血管床中窗孔的差异节段和空间控制； 2) 基因表达筛选参与PV1调节的因子，鉴定出SCF、FGF-5、VEGF-A和 通过各自的 c-KIT、FGFR1、VEGFR2 和 ITGB1 受体进行特定层粘连蛋白信号传导，这是必需的 PV1以ERK1/2依赖性方式表达； 3)层粘连蛋白与SCF、FGF-5和VEGF的组合- A 导致 PV1 表达强劲并从头形成窗孔 我们将使用 功能验证的内皮细胞培养系统和基因工程动物的结合 使用功能丧失、功能获得和重构方法的模型。我们的目标将定义角色和 新发现的生长因子和层粘连蛋白及其整合素受体的下游信号通路 调节 PV1 表达和窗孔形态发生。我们在关键和关键方面都处于独特的地位 完成这项工作所需的专业知识。这些调查将确定监管因素和途径 控制复杂且生理相关的细胞结构的组装，提供有价值的 目前缺乏的洞察力。