新型血小板受体CLEC-2维持脑血管完整性的机制研究

During early embryonic development, angiogenesis in the central nervous system (CNS) is extremely active, and the rapidly growing nascent vasculature requires special mechanisms to prevent leakage. The CNS develops and maintains a blood-brain barrier (BBB) to prevent leakage. However, BBB does not fully develop during embryonic development. Thus, additional mechanisms must be employed to prevent bleeding prior to the formation of BBB. Platelet activation serves as an important systemic mechanism for preventing hemorrhage after vessel injury. 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 in the developing CNS. Mice lacking PDPN or CLEC-2 (Pdpn-/-, or Clec-2-/-) develop CNS-specific hemorrhages primarily during embryonic development. The cerebral bleeding phenotype is reminiscent of that associated with germinal matrix-intraventricular hemorrhage (GHM-IVH), which occurs in about 35% of human premature infants with unknown mechanism. Based on our novel preliminary data, we hypothesize that PDPN-CLEC-2-mediated platelet activation is essential for the integrity of vasculature specifically in the developing CNS when BBB is incomplete. We propose to: 1) determine whether and how platelet CLEC-2 maintains vascular integrity of the developing CNS; 2) investigate whether PDPN in neuronal cells is required for integrity of nascent vessels in developing CNS; and 3) determine the role of sphingoshine-1-phosphate (S1P) released by platelets after PDPN-CLEC-2-dependent activation in vascular integrity. The proposed studies will investigate PDPN-CLEC-2-mediated platelet activation as a novel mechanism that protects the vulnerable CNS from bleeding during development. Identification of a novel role of PDPN and CLEC-2 in protecting newborns from brain bleeding may provide new insights into pathogenesis of diseases such as GMH-IVH potential therapies that target hemorrhage and thrombosis specifically in the brain.

血脑屏障在维持脑血管完整性及其功能方面发挥重要作用，但血脑屏障在胚胎发育期形成不完整，因此胚胎期脑血管完整性如何保持机制尚不清楚。我们前期研究显示一种新的血小板活化受体CLEC-2在维持胚胎发育期脑血管完整性发挥重要作用。CLEC-2及其配体PDPN基因敲除的小鼠出现生发层基质出血，这一表型与早产儿脑生发基质出血极为相似，提示PDPN和CLEC-2相互作用是保护胚胎发育期脑血管完整性的重要机制。本项目将运用动物模型进一步研究：（1）血小板CLEC-2在维持胚胎发育期大脑血管完整性的功能和机制；（2）脑组织中何种细胞表达PDPN并研究其在维持脑血管完整性方面作用机制；（3）是否CLEC-2信号通路介导血小板释放S1P而维持脑血管完整性。本研究将提出血小板维持胚胎期脑血管完整性的全新机制，并为胚胎期脑血管完整性的维持及早产儿脑出血的发生机理和治疗新靶点的研究打下基础，具有巨大的基础和临床意义。

脑室内出血(IVH)是早产、低体重儿最常见的并发症之一，发生率可达35%。因此，深入研究调控胚胎发育期脑血管完整性的分子机制将为积极预防和有效治疗早产儿IVH提供科学依据。血小板数量及/或功能异常是导致新生儿脑出血的主要诱因，但在胚胎发育过程中血小板如何参与、维护脑血管完整性尚缺乏机理研究，其中的一个重要原因就是缺乏有效的动物模型。本课题组利用基因敲除技术成功构建了新型血小板活化受体C-型凝聚素样受体2(简称CLEC-2)及其配体平足蛋白基因缺陷小鼠，并发现两种基因缺陷小鼠在胚胎发育期均出现严重的脑出血表型，部分存活至成年的小鼠发生脑积水，其临床表现及病理特征与早产新生儿的IVH极为相似，不但表明平足蛋白—CLEC-2信号轴介导的血小板活化是维持胚胎发育期脑血管完整性的重要机制，也为深入研究早产儿IVH的分子病理机制及干预策略提供了有效、可靠的动物模型。为深入研究平足蛋白—CLEC-2信号轴调控脑血管完整性的分子机理，课题组建立了条件性基因敲除小鼠模型并证实神经祖细胞表达的平足蛋白是一种新的血小板激活剂，可通过CLEC-2信号活化从新生、不成熟的血管渗漏的血小板，进而在胚胎脑组织中诱导血小板释放反应并保护内皮屏障。利用体外细胞模型，课题组发现CLEC-2信号活化血小板可释放血管保护生物活性介质，如1-磷酸鞘氨醇(S1P)和血管生成素-1，并可通过抑制VE-钙粘蛋白内吞从而保护内皮完整性；更为重要的是，通过母体给予选择性S1P受体1(S1PR1)激动剂部分保护CLEC-2缺陷小鼠胚胎的脑出血表型，提示S1P信号途径在胚胎发育阶段参与维护脑血管屏障。本研究有助于阐明IVH发生、发展的分子机制，为我们在后续工作中开展IVH及其它出血性疾病的防治奠定坚实的基础。