Role of Neural Podoplanin-Activated Platelets in Vascular Development in the Brain

神经足足蛋白激活血小板在大脑血管发育中的作用

• 批准号: 9326735
• 负责人: Christopher Michael Hoover
• 金额: $ 3.23万
• 依托单位: UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-07 至 2021-08-06
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9326735
• 关键词: ANGPT1 gene; Affect; Agonist; Biological Assay; Birth; Blood - brain barrier anatomy; Blood Coagulation Disorders; Blood Platelets; Blood Vessels; Brain; C Type Lectin Receptors; C-Type Lectins; Cellular Morphology; Confocal Microscopy; Defect; Development; Embryo; Embryonic Development; Endothelial Cells; Gene Expression; Glycoproteins; Growth; Hemorrhage; Human; In Vitro; Individual; Lead; Measures; Mediating; Modeling; Morphology; Mus; Nature; Neuraxis; Neuroectoderm; Neurons; Pattern; Penetrance; Pericytes; Permeability; Phenotype; Phosphorylation; Platelet Activation; Premature Infant; Proteins; Regulation; Reporter; Role; Signal Pathway; Signal Transduction; Signaling Protein; Slice; Source; Structural defect; Surface; Testing; Tissues; Transmission Electron Microscopy; Vascular Endothelial Growth Factors; Vascular System; Very Low Birth Weight Infant; aged; angiogenesis; brain endothelial cell; cadherin 5; insight; intraventricular hemorrhage; lymph nodes; monolayer; neuroregulation; neurovascular; novel; podoplanin; prevent; receptor; relating to nervous system; response; solute; sphingosine 1-phosphate; therapeutic target; two-photon

PROJECT SUMMARY ABSTRACT Vascular development within the central nervous system (CNS) progresses through extremely active sprouting angiogenesis. Nascent vascular sprouts at the angiogenic front are sensitive to bleeding, but the mechanisms that maintain the developing neurovascular integrity are not fully understood, and the blood-brain barrier is not functionally mature until after birth. The O-glycoprotein podoplanin (PDPN) and its platelet receptor C-type lectin-like receptor 2 (CLEC-2) are required for vascular integrity in the developing CNS, but the defects that result in spontaneous CNS hemorrhages in Pdpn-/- and Clec-2-/- mice and the mechanism of PDPN-CLEC-2-mediated platelet signaling are unknown. Preliminary observations show that loss of PDPN- CLEC-2 results in defective vascular sprout ingression with dilated nascent sprouts that are a source of hemorrhaging. Observations also indicate that CLEC-2-activated platelet secretory products are able to reverse VEGF-induced VE-cadherin internalization and that Pdpn-/- CNS have significantly upregulated VEGF signaling pathway gene expression. CLEC-2-activated platelets secrete the molecules sphingosine-1- phosphate (S1P) and angiopoietin-1 (Ang1), but it is not known if their respective signaling mechanisms function in cooperation for maintaining CNS vascular integrity during development. These preliminary results support the hypothesis that neural PDPN-CLEC-2-mediated platelet activation and signaling is crucial for maintaining structural stability of the developing vasculature in the CNS. This will be tested by determining 1) the nature of the vascular defect leading to hemorrhaging in the absence of PDPN-CLEC-2 interaction using fluorescent vascular reporter mice and two-photon confocal microscopy, as well as determining defects in the endothelial morphology of nascent sprouts using transmission electron microscopy. In addition, the hypothesis will be tested by determining 2) the mechanism of PDPN-CLEC-2-mediated platelet signaling that regulates vascular integrity during development. Preliminary observations support the hypothesis that S1P and Ang1 released from platelets after PDPN-CLEC-2-mediated activation cooperate to counteract VEGF-signaling- induced VE-cadherin phosphorylation and internalization from the adherins junction. This will be tested by determining if threshold concentration S1P agonist and Ang1 signaling cooperates in an in vitro endothelial permeability assay. In addition, using an ex vivo embryonic brain slice model, the downstream effectors of S1P and Ang1 signaling, Rac1 and RhoA, will be inhibited in order to study the combined effects on angiogenic response and junction stability. If the proposed studies support these hypotheses, it will define a novel mechanism of tissue-specific platelet signaling in regulation of vascular sprout integrity in the developing CNS.

项目概要 摘要 中枢神经系统 (CNS) 内的血管发育通过极其活跃的过程进行 血管生成发芽。血管生成前沿的新生血管芽对出血敏感，但 维持发育中的神经血管完整性的机制尚不完全清楚，血脑 直到出生后，屏障的功能才成熟。 O-糖蛋白足足蛋白 (PDPN) 及其血小板 受体 C 型凝集素样受体 2 (CLEC-2) 是中枢神经系统发育中血管完整性所必需的，但是 导致 Pdpn-/- 和 Clec-2-/- 小鼠自发性 CNS 出血的缺陷及其机制 PDPN-CLEC-2 介导的血小板信号传导尚不清楚。初步观察表明，PDPN-的丢失 CLEC-2 导致有缺陷的维管芽侵入，新生芽扩张，这是 出血。观察结果还表明，CLEC-2 激活的血小板分泌产物能够 逆转 VEGF 诱导的 VE-钙粘蛋白内化，并且 Pdpn-/- CNS 显着上调 VEGF 信号通路基因表达。 CLEC-2 激活的血小板分泌鞘氨醇-1-分子 磷酸盐（S1P）和血管生成素-1（Ang1），但尚不清楚它们各自的信号机制是否 在发育过程中协同维持中枢神经系统血管完整性。这些初步结果 支持以下假设：神经 PDPN-CLEC-2 介导的血小板激活和信号传导对于 维持中枢神经系统中正在发育的脉管系统的结构稳定性。这将通过确定 1) 进行测试 在没有 PDPN-CLEC-2 相互作用的情况下导致出血的血管缺陷的性质，使用 荧光血管报告小鼠和双光子共聚焦显微镜，以及确定血管缺陷 使用透射电子显微镜观察新生芽的内皮形态。此外，假设 将通过确定 2) PDPN-CLEC-2 介导的血小板信号传导的机制进行测试 发育过程中血管的完整性。初步观察结果支持 S1P 和 Ang1 的假设 PDPN-CLEC-2 介导的激活后从血小板中释放的细胞协同抵消 VEGF 信号传导 诱导 VE-钙粘蛋白磷酸化和从粘着蛋白连接的内化。这将通过以下方式进行测试 确定阈值浓度 S1P 激动剂和 Ang1 信号传导是否在体外内皮细胞中协同作用 渗透性测定。此外，使用离体胚胎脑切片模型，S1P的下游效应器 Ang1 信号传导、Rac1 和 RhoA 将被抑制，以研究对血管生成的综合影响 响应和结稳定性。如果拟议的研究支持这些假设，它将定义一种新颖的 组织特异性血小板信号传导调节发育中中枢神经系统血管芽完整性的机制。