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

 描述(申请人提供)：在发育过程中，由血管内皮生长因子(VEGF)信号介导的血管生成在中枢神经系统(CNS)非常活跃。新形成的血管往往不成熟，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，在出生前并未完全发育。中枢神经系统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激活机制，即O-糖蛋白泊多拉宁(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，在早期发育的中枢神经系统中，PDPN在血管周围的神经细胞中特异表达。缺乏PDPN或CLEC-2(Pdpn-/-或CLEC-2-/-)的小鼠主要在胚胎发育早期出现中枢神经系统特异性出血。此外，神经细胞中PDPN有条件删除的小鼠也会出现自发性脑出血。这些初步结果支持一种新的假说，即神经细胞PDPN介导的血小板激活对于早期发育的中枢神经系统新生血管的稳定性是必不可少的。为了验证这一假设，我们将确定1)PDPN-CLEC-2介导的血小板激活是否保护了早期发育的CNS中新形成的血管的完整性。我们发现，在中枢神经系统发育早期，PDPN表达于与血管密切相关的神经细胞上，而血小板则存在于血管外。这些数据支持一种新的假设，即在中枢神经系统发育的早期，血管周围神经细胞上的PDPN与外渗血小板上的CLEC-2之间的相互作用对于新形成的血管的完整性是必不可少的。我们将使用最先进的双光子共聚焦成像显微镜和血管通透性分析来测试这一点；我们将确定2)从PDPN-CLEC-2激活的血小板释放的S1P是否平衡了血管内皮生长因子的作用，以维持发育中的中枢神经系统的血管稳定。我们假设，在PDPN-CLEC-2介导的激活后，从血小板释放S1P对于平衡血管内皮生长因子的功能对于血管内皮屏障的稳定性是必不可少的，并将使用缺乏S1P功能的小鼠和药理学方法来解决这一问题。如果拟议的研究支持我们的假设，它将定义一种新的机制，即在发育中的中枢神经系统中，组织特异性血小板激活在调节血管完整性方面的作用。这种新机制的发现可能会为脑出血疾病提供新的见解，例如生发基质-脑室出血(GMH-IVH)，它影响到约35%的早产儿。此外，Pdpn-/-或CLEC-2/-小鼠可能被用作测试促进血管完整性的新疗法的有价值的模型，这些疗法针对发育中的大脑出血。

 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.