Molecular pathogenesis of brain arteriovenous malformation

描述（由申请人提供）：脑动静脉（AV）畸形（bavm）可导致危及生命的中风，治疗选择有限。该项目的目标是阐明BAVM发病机制的细胞和分子机制，以确定新的候选治疗靶点来改善这种疾病。房颤的特征是异常的房颤分流使中间毛细血管移位。我们提出了一种跨学科的方法，融合了尖端的小鼠遗传学和成像技术，来验证我们的假设，即Notch突变可以重编程AV身份并改变AV差异一氧化氮（NO）信号，从而改变内皮功能障碍，从而引发bavm。Notch受体和配体在动脉中表达，而在静脉中不表达。Notch信号通过增强动脉和抑制静脉分子标记来促进动脉分化，以牺牲静脉分化为代价。我们已经报道了在小鼠中内皮表达组成活性Notch4突变（Notch4*）引发bavm。Notch4*重新编程静脉以获得动脉和失去静脉分子特性，纠正Notch4*导致已建立的bavm正常化。我们已经建立了一个定制的双光子显微镜，最适合活体小鼠的脑血管结构和血流动力学成像。因此，我们可以通过颅窗获得5D数据（3D加上随时间的血流速度），以揭示小鼠BAVM形成的过程。基于我们强大的背景和初步数据，我们提出：目的1 -确定内皮Notch4*对静脉内皮功能障碍和BAVM形成的影响。我们将验证我们的假设，即Notch4*上调静脉中的NO水平，改变静脉内皮对血流的反应，从而允许AVM形成。我们将研究Notch4*对静脉NO信号、内皮对血流的反应以及血流介导的BAVM形成的影响；目的2 -确定内皮Notch缺失对动脉重编程、动脉内皮功能障碍和BAVM形成的影响。我们将验证我们的假设，即内皮Notch基因功能的丧失使动脉重编程，失去动脉而获得静脉分子特性，减少动脉NO信号，导致动脉功能障碍，从而导致avm。我们将分析Notch缺失小鼠的BAVM病理、动脉NO信号和内皮对血流刺激的反应；目的3 -确定Notch与HHT在小鼠背主动脉和主静脉发育中的相互作用。我们将验证我们的假设，即Notch在遗传性出血性毛细血管扩张（HHT）下游发挥作用，介导HHT在AV规范中的作用。我们将使用双光子成像和3D渲染来比较Notch和HHT突变表型，并进行遗传修复。本研究结果将从概念上推进我们对AVM发病机制的细胞和分子机制的理解，揭示Notch在调节动脉和静脉独特生理中的新功能，并揭示Notch与HHT通路之间的相互作用。这项工作的成功将激发avm， Notch信号和血管病理生理学领域的新研究领域。

DESCRIPTION (provided by applicant): Brain arteriovenous (AV) malformations (BAVMs) can cause life-threatening strokes and have limited treatment options. The goal for this project is to elucidate the cellular and molecular mechanisms underlying BAVM pathogenesis to identify novel candidates as therapeutic targets to ameliorate this disease. AVMs are characterized by abnormal AV shunts that displace intervening capillaries. We propose a cross-disciplinary approach, fusing cutting-edge mouse genetics and imaging technologies, to test our hypothesis that Notch mutations can reprogram AV identity and alter AV differential nitric oxide (NO) signaling, and thus endothelial dysfunction, to elicit BAVMs. Notch receptors and ligands are expressed in arteries but not veins. Notch signaling promotes arterial at the expense of venous differentiation by enhancing arterial and suppressing venous molecular markers. We have reported that endothelial expression of a constitutively active Notch4 mutation (Notch4*) elicits BAVMs in mice. Notch4* reprograms veins to gain arterial and lose venous molecular identity, and correcting the causal Notch4* leads to normalization of established BAVMs. We have built a custom two-photon microscope, optimal for structural and hemodynamic imaging of cerebral vasculature in live mice. We can thus obtain 5D data (3D plus blood velocity over time) through a cranial window to reveal the process of BAVM formation in mice. Built on our strong background and preliminary data, we propose: Aim 1 - Determine the effect of endothelial Notch4* on venous endothelial dysfunction and BAVM formation. We will test our hypothesis that Notch4* upregulates NO levels in the veins, alters venous endothelial response to blood flow, and thus permits AVM formation. We will examine the effect of Notch4* on venous NO signaling, endothelial response to blood flow, and flow mediated BAVM formation; Aim 2 - Determine the effect of endothelial Notch deficiency on arterial reprogramming, arterial endothelial dysfunction, and BAVM formation. We will test our hypothesis that loss of endothelial Notch gene function reprograms arteries to lose arterial and gain venous molecular identity and reduces arterial NO signaling, leading to arterial dysfunction and thus AVMs. We will analyze mice with endothelial deletion of Notch for BAVM pathology, arterial NO signaling, and endothelial response to blood flow stimuli; Aim 3 - Ascertain the interaction between Notch and HHT in mouse dorsal aorta and cardinal vein development. We will test our hypothesis that Notch functions downstream of Hereditary Hemorrhagic Telangectasia (HHT) to mediate HHT function in AV specification. We will compare the Notch and HHT mutant phenotypes using two-photon imaging and 3D rendering and perform genetic rescue. The findings from this study will conceptually advance our understanding of the cellular and molecular mechanisms of AVM pathogenesis, reveal novel functions for Notch in regulating the unique physiology of arteries and veins, and uncover interactions between the Notch and HHT pathways. The success of this work will inspire new areas of investigation in the fields of AVMs, Notch signaling, and vascular pathophysiology.