Podoplanin-mediated platelet activation and vascular integrity in the developing brain

 描述（由申请方提供）：在发育过程中，由血管内皮生长因子（VEGF）信号传导介导的血管生成在中枢神经系统（CNS）中非常活跃。新形成的血管通常不成熟，对出血敏感，因此需要特殊的机制来维持其内皮屏障的稳定性。然而，已知的血管完整性机制，如血脑屏障功能，在出生前尚未完全发育。CNS发育过程中血管稳定性的其他机制仍然难以捉摸。我们最近发现了一种新的血小板活化机制，其中O-糖蛋白podoplanin（PDPN）激活血小板C型凝集素样2（CLEC-2）受体。我们的初步实验表明，PDPN在早期发育的CNS中特异性表达于血管周围的神经细胞中。缺乏PDPN或CLEC-2（Pdpn-/-或Clec-2-/-）的小鼠主要在早期胚胎发育期间发育CNS特异性血管。此外，神经细胞中PDPN条件性缺失的小鼠也表现出自发性脑出血。这些初步结果支持了一个新的假设，即神经细胞PDPN介导的血小板活化是至关重要的新生血管的稳定性在早期发展的CNS。为了检验这一假设，我们将确定1）PDPN-CLEC-2介导的血小板活化是否保护早期发育的CNS中新形成的血管的完整性。我们发现，PDPN表达的神经细胞密切相关的血管在早期发育的中枢神经系统和血小板存在于血管外。这些数据支持了一个新的假设，即血管周围神经细胞上的PDPN和外渗血小板上的CLEC-2之间的相互作用对于早期CNS发育过程中新形成血管的血管完整性至关重要。我们将使用最先进的双光子共聚焦成像显微镜和血管渗透性测定来测试这一点;我们将确定2）从PDPN-CLEC-2活化的血小板释放的鞘氨醇1-磷酸（S1 P）是否平衡VEGF作用以维持发育中的CNS中的血管稳定性。我们假设PDPN-CLEC-2介导的活化后血小板释放S1 P通过平衡VEGF的功能对血管内皮屏障稳定性至关重要，并将使用缺乏S1 P功能的小鼠和药理学方法来解决这个问题。如果所提出的研究支持我们的假设，它将定义一个新的机制，组织特异性血小板活化调节血管完整性的发展中的中枢神经系统。这种新机制的鉴定可能为脑出血疾病提供新的见解，例如影响约35%的早产儿的脑出血基质-脑室内出血（GMH-IVH）。此外，Pdpn-/-或Clec-2-/-小鼠可用作测试促进血管完整性的新疗法的有价值的模型，所述新疗法靶向发育中的脑中的出血。

 DESCRIPTION (provided by applicant): During development, angiogenesis mediated by vascular endothelial growth factor (VEGF) signaling is extremely active in the central nervous system (CNS). The newly formed vessel is often immature and sensitive to bleeding and thus requires special mechanisms to maintain its endothelial barrier stability. However, known mechanisms for vascular integrity, such as blood-brain-barrier function, are not fully developed before birth. Additional mechanisms for vascular stability during CNS development remain elusive. We have recently identified a novel mechanism of platelet activation in which the O-glycoprotein podoplanin (PDPN) activates platelet C-type lectin-like 2 (CLEC-2) receptors. Our preliminary experiments show that PDPN is expressed specifically in neural cells surrounding vessels in the early developing CNS. Mice lacking PDPN or CLEC-2 (Pdpn-/- or Clec-2-/-) develop CNS-specific hemorrhages primarily during early embryonic development. Furthermore, mice with conditional deletion of PDPN in neural cells also exhibit spontaneous brain bleeding. These preliminary results support a novel hypothesis that neural cell PDPN-mediated platelet activation is essential for the stability of nascent vessels in the early developing CNS. To test this hypothesis, we will determine 1) whether PDPN-CLEC-2-mediated platelet activation protects integrity of newly formed vessels in the early developing CNS. We found that PDPN is expressed on neural cells closely associated with vessels in the early developing CNS and that platelets are present outside vessels. These data support a novel hypothesis that interactions between PDPN on perivascular neural cells and CLEC-2 on extravasated platelets are essential for vascular integrity of newly formed vessel during early CNS development. We will test this using the state-of-the-art two-photon confocal imaging microscopy and vascular permeability assays; we will determine 2) whether sphingoshine 1-phosphate (S1P) released from PDPN-CLEC-2-activated platelets balances VEGF action to maintain vascular stability in the developing CNS. We hypothesize that release of S1P from platelets after PDPN-CLEC-2-mediated activation is essential for vascular endothelial barrier stability by balancing the functin of VEGF, and will use mice lacking S1P functions and pharmacological approaches to address this question. If the proposed studies support our hypotheses, it will define a novel mechanism of tissue-specific platelet activation in regulation of vascular integrity in the developing CNS. Identification of such a new mechanism may provide new insights into brain bleeding disease, such as germinal matrix- intraventricular hemorrhage (GMH-IVH), which affects ~35% of premature human infants. In addition, Pdpn-/- or Clec-2-/- mice may be used as a valuable model for testing novel therapies promoting vascular integrity that target hemorrhage in the developing brain.