Cilia as a biomarker of CNS vascular health

项目摘要 脑内皮细胞（EC）的作用，特别是纤毛的作用，微管流量传感器细胞器 在内皮细胞的顶面和面向管腔的表达在血液中的研究很少， 脑屏障（BBB）功能。内皮细胞和纤毛是与红细胞（RBC）接触的第一线， 血我们和其他人的研究表明，内皮细胞上的纤毛对血流介导的脑血管稳定性至关重要。 因此，流量如何通过纤毛将信号传递给EC，进而传递给神经血管（NVU）单元的细胞尚不清楚。 没有这些知识，我们产生模拟体内条件的BBB模型的能力将受到阻碍。的 EC纤毛及其在BBB功能中的作用的问题是临床相关的，因为镰状细胞病患者 (SCD)易患显性和隐性脑梗死，由镰状红细胞粘附于 内皮细胞镰状红细胞与内皮细胞的粘附可能有助于纤毛的物理去除 （decilation）从EC表面，最近在哺乳动物纤毛脱落的机制。在这个多PI 建议，血管生物学，镰状细胞生物学，人类诱导多能干细胞研究小组 （iPSC）衍生的BBB和NVU模型、纤毛生物学和啮齿动物损伤模型将研究总体的 一种假说认为，脑血流紊乱会触发纤毛的脱连和释放到血液中， 导致脱落的EC和包含NVU的周围细胞中的异常信号传导 导致对NVU和BBB的影响（图1）。本提案的目的是研究红细胞-EC-纤毛 相互作用及其对BBB模型开发的重要性。在R61阶段，我们将测试来自 镰状细胞病（小鼠模型和人类患者）将直接或间接触发脑中纤毛脱落 体外和体内的EC。我们将鉴定纤毛脱落片段中的蛋白质，并评估 EC纤毛对体外和体内BBB表型的影响，重点是BBB完整性。在成功建立 RBC-EC-纤毛相互作用对R61期BBB功能至关重要的里程碑， R33期的研究将为深入探讨红细胞-内皮细胞纤毛的作用机制和作用机制奠定基础 在SCD和创伤性脑损伤（TBI）啮齿动物模型中的相互作用。这个项目的意义在于 当脑EC被包括在流动介导的BBB模型中时，EC纤毛状态是重要的决定因素 体外发育创新之处在于，到目前为止，EC纤毛在BBB方案中基本上被忽视， 实现这一建议的目标将改变这一领域的现状。这一提议还将带来 我们距离使用RBC触发的EC纤毛脱落作为监测脑血管损伤又近了一步 血管健康的生物标志物，NIH任务相关的研究课题。

Project Summary The role of brain endothelial cells (ECs) and in particular the role of cilium, a microtubule flow sensor organelle expressed on the apical surface of ECs and facing the lumen has been poorly studied in the context of blood- brain-barrier (BBB) function. ECs and in turn cilia are the first line of contact with red blood cells (RBCs) in the blood. Studies from us and others suggest that cilia on ECs are critical for flow-mediated brain vessel stability. Thus, how flow relays signals to ECs via cilia and in turn to cells of the neurovascular (NVU) unit is not known. Without this knowledge, our ability to generate BBB models that mimic in vivo conditions will be hampered. The question of EC-cilia and its role in BBB function is clinically relevant given that patients with sickle cell disease (SCD) are predisposed to both overt and silent cerebral infarct, caused by sickle RBCs adhesion to the endothelium. The adhesion of sickle RBCs to endothelium may facilitate the physical removal of cilia (deciliation) from ECs surface, a mechanism recently identified in mammalian cilia shedding. In this multi-PI proposal, investigative team in vascular biology, sickle cell biology, human induced pluripotent stem cell (iPSC)-derived BBB and NVU models, cilia biology and rodent injury models will investigate the overarching hypothesis that disturbed cerebral blood flow triggers deciliation and release of cilia into the blood, thus resulting in aberrant signaling in the deciliated ECs and in the surrounding cells that comprise the NVU resulting in impact on NVU and BBB (Fig. 1). The objective of this proposal is to study RBCs-EC-cilia interaction and its importance to BBB model development. In the R61 phase, we will test whether RBCs from sickle cell disease (mouse models & human patients) will directly or indirectly trigger cilia shedding in brain ECs in vitro and in vivo. We will identify proteins in the cilia shed fragments, and also assess the importance of EC-cilia on BBB phenotypes in vitro and in vivo with emphasis on BBB integrity. Upon successfully establishing the milestone that RBC-EC-cilia interaction is critical for BBB function in the R61 phase, a go-decision in the R33 phase will initiate deeper probe into the underlying mechanisms and the role of the RBC-EC cilia interaction in SCD and traumatic brain injury (TBI) rodent models in vivo. The significance of this project is that EC-cilia status is an important determinant when brain ECs are included in flow-mediated BBB model development in vitro. The innovation is that until now, EC-cilia has been largely ignored in BBB protocols and accomplishing the objectives of this proposal will move the status quo in this field. This proposal will also bring us one step closer to monitoring cerebral vessel impairment using RBC-triggered EC-cilia shedding as a biomarker of vascular health, a NIH mission-related topic of research.