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

项目摘要 中枢神经系统（CNS）内的血管发育通过极其活跃的 萌芽血管生成。在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未完全了解，血脑 屏障功能直到出生后才成熟。O-糖蛋白podoplanin（PDPN）与血小板 受体C型凝集素样受体2（CLEC-2）是发育中CNS血管完整性所必需的，但 导致Pdpn-/-和Clec-2-/-小鼠自发性CNS损伤的缺陷及其机制 PDPN-CLEC-2介导的血小板信号传导是未知的。初步观察表明，PDPN- CLEC-2导致有缺陷的维管芽侵入，伴随扩张的新生芽，这些芽是 - -观察还表明CLEC-2活化的血小板分泌产物能够 逆转VEGF诱导的VE-钙粘蛋白内化，Pdpn-/- CNS显著上调VEGF 信号通路基因表达。CLEC-2激活的血小板分泌分子鞘氨醇-1- 磷酸盐（S1 P）和血管生成素-1（Ang 1），但尚不清楚它们各自的信号传导机制 在发育过程中协同维持CNS血管完整性。这些初步结果 支持神经PDPN-CLEC-2介导的血小板活化和信号传导对于 维持CNS中发育中的脉管系统的结构稳定性。这将通过确定1） 在不存在PDPN-CLEC-2相互作用的情况下，血管缺陷导致血管老化的性质， 荧光血管报告小鼠和双光子共聚焦显微镜，以及确定缺陷， 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机制来进行测试， 发育过程中的血管完整性。初步观察支持S1 P和Ang 1 PDPN-CLEC-2介导的活化后从血小板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附蛋白连接处内化。这将由以下人员进行测试： 确定阈值浓度S1 P激动剂和Ang 1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，S1 P的下游效应物 和Ang 1信号传导，Rac 1和RhoA，将被抑制，以研究对血管生成的联合作用。 响应和结稳定性。如果拟议的研究支持这些假设，它将定义一个新的 组织特异性血小板信号传导在发育中的CNS中调节血管芽完整性的机制。

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.