Notch Enhances Shear-mediated Arteriogenesis in Cerebral Vessels

描述（由申请人提供）：闭塞性动脉疾病仍然是美国人死亡和发病的主要原因，并对社会构成巨大的经济负担。动脉闭塞导致动脉生成，这是一个小的侧支小动脉重塑成更大的管道动脉的过程，该管道动脉改变血液的路线并改善流向缺血组织的血流。在人类和小鼠中，预先存在的侧枝的能力及其在闭塞后的生长受到遗传背景的强烈调节，从而导致广泛的结果。这些侧支血管中血流动力学切应力的增加促进了动脉生成，但介导这种生长的分子途径和机械反应还不清楚。Notch受体和配体功能丧失研究表明，Notch信号传导途径对于预先存在的血管的动脉生成是必需的。我们的研究目的是阐明动脉闭塞后内皮Notch信号调节的机制，并建立Notch信号在闭塞性损伤后增强脑侧支动脉形成中的作用。我们通过基因消融内皮细胞（EC）中的Notch信号，观察到动脉闭塞后小鼠脑动脉生成减少。相反，我们观察到动脉闭塞后，在EC中表达组成型活性Notch4（Notch4*）的小鼠中，脑侧支血管显著扩大。我们的初步研究结果表明，脑侧支动脉的发生，特别是在血管节段，提供更多的流量到受伤的地区。我们假设血流动力学剪切应力的增加激活了内皮Notch信号，并且Notch是剪切诱导的动脉生成的必要和有效的增强剂。在目标1中，我们开发了通过结合大脑中动脉（MCA）结扎手术模型和活体双光子激发荧光显微镜来动态研究相同动物随时间推移的脑侧支扩大和血流的方法。我们产生了新的分析方法来量化血流动力学和WSS，具有高精度和优于当前近似的改进。在目标2中，我们将确定内皮细胞中的Notch信号转导是否被剪切应力激活。我们将使用经典Notch信号传导和免疫荧光的体内报告物来确定MCA结扎后Notch何时在内皮中被激活。我们将确定Notch激活是否仅限于WSS增加的血管，我们已经观察到WSS增加与动脉生成相关。在目标3中，我们将确定内皮细胞中的Notch信号是否通过剪切响应程序控制脑动脉生成。具体来说，我们将确定内皮细胞中的Notch信号传导是否对MCA结扎后的动脉生成至关重要。我们还将确定EC中的Notch4* 是否足以增强MCA结扎后的动脉生成，以及是否还需要升高的WSS。完成这个项目后，我们将进一步了解Notch介导的动脉生成机制。从这项研究中获得的知识将有助于脑动脉闭塞相关疾病的治疗发展。

DESCRIPTION (provided by applicant): Occlusive arterial disease remains the leading cause of mortality and morbidity in Americans and constitutes a tremendous financial burden to society. Arterial occlusion results in arteriogenesis, a process by which small collateral arterioles remodel into larger conduit arteries that reroute blood and improve flow to the ischemic tissue. The capacity of pre-existing collaterals and their growth following occlusion is strongly modulated by genetic background in both humans and mice, resulting in a wide range of outcomes. Increased hemodynamic shear stress in these collateral vessels promotes arteriogenesis but the molecular pathways and mechano-responses mediating this growth are not well understood. Notch receptor and ligand loss-of-function studies demonstrate that the Notch signaling pathway is necessary for arteriogenesis of pre- existing vessels. The goal of our study is to elucidate the mechanism by which endothelial Notch signaling is regulated after arterial occlusion and to establish a role for Notch signaling in enhancing arteriogenesis of cerebral collaterals after occlusive injury. We observe decreased cerebral arteriogenesis after arterial occlusion in mice with genetic ablation of Notch signaling in endothelial cells (ECs). Conversely, we observe impressive enlargement of cerebral collaterals following arterial occlusion in mice with expression of constitutively active Notch4 (Notch4*) in ECs. Our preliminary results suggest that arteriogenesis of cerebral collaterals occurs specifically in vessel segments that deliver increased flow to the region of injury. We hypothesize that increased hemodynamic shear stress activates endothelial Notch signaling, and that Notch is a necessary and potent enhancer of shear-induced arteriogenesis. In aim 1, we develop methods to dynamically study cerebral collateral enlargement and blood flow in the same animals over time by combining a surgical model for middle cerebral artery (MCA) ligation and intravital two-photon excited fluorescence microscopy. We generate new analytical methods to quantify hemodynamics and WSS with high accuracy and improvement over current approximations. In aim 2, we will determine if Notch signaling in ECs is activated by shear stress. We will use an in vivo reporter of canonical Notch signaling and immunofluorescence to determine when Notch is activated in endothelium after MCA ligation. We will determine whether Notch activation is limited to vessels with increased WSS, which we have observed is correlated with arteriogenesis. In aim 3, we will determine whether Notch signaling in ECs controls cerebral arteriogenesis through a shear-responsive program. Specifically, we will determine whether Notch signaling in ECs is critical for arteriogenesis after MCA ligation. We will also determine whether Notch4* in ECs is sufficient to enhance arteriogenesis after MCA ligation and if elevated WSS is also required. Upon completion of this project, we will have advanced the mechanistic understanding of Notch-mediated arteriogenesis. Knowledge gained from this study will help therapeutic development for disease associated with cerebral arterial occlusion.