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，在这一过程中，小侧支小动脉重塑为较大的导管动脉，从而重新引导血液并改善流向缺血组织的流量。人类和小鼠的遗传背景强烈调节预先存在的络脉的能力及其闭塞后的生长，从而产生广泛的结果。这些侧支血管中血流动力学剪切应力的增加会促进动脉生成，但介导这种生长的分子途径和机械反应尚不清楚。 Notch 受体和配体功能丧失研究表明，Notch 信号通路对于现有血管的动脉生成是必需的。我们研究的目的是阐明动脉闭塞后内皮Notch信号传导的调节机制，并确定Notch信号传导在闭塞性损伤后增强脑侧支动脉生成中的作用。我们观察到，通过基因消融内皮细胞 (EC) 中的 Notch 信号，小鼠动脉闭塞后脑动脉生成减少。相反，我们观察到在 EC 中表达组成型活性 Notch4 (Notch4*) 的小鼠动脉闭塞后，脑侧支循环显着增大。我们的初步结果表明，脑侧支循环的动脉生成特别发生在向损伤区域输送增加流量的血管段中。我们假设增加的血流动力学剪切应力会激活内皮Notch信号传导，并且Notch是剪切诱导的动脉生成的必要且有效的增强剂。在目标 1 中，我们开发了方法，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，动态研究同一动物随时间的脑侧支扩大和血流。我们开发了新的分析方法来量化血流动力学和 WSS，具有高精度并改进了当前的近似值。在目标 2 中，我们将确定 EC 中的 Notch 信号传导是否由剪切应力激活。我们将使用典型 Notch 信号传导和免疫荧光的体内报告基因来确定 MCA 连接后 Notch 在内皮细胞中何时被激活。我们将确定 Notch 激活是否仅限于 WSS 增加的血管，我们观察到 WSS 与动脉生成相关。在目标 3 中，我们将确定 EC 中的 Notch 信号传导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 EC 中的 Notch 信号传导对于 MCA 连接后的动脉生成是否至关重要。我们还将确定 EC 中的 Notch4* 是否足以增强 MCA 连接后的动脉生成，以及是否还需要提高 WSS。该项目完成后，我们将加深对 Notch 介导的动脉生成机制的理解。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开发。 公共卫生相关性：闭塞性动脉疾病，包括中风和心脏病发作，是美国人死亡和发病的主要原因。人们对开发分子疗法来刺激血管生长并使血流返回濒临死亡的组织非常感兴趣。我们的研究可能阐明控制动脉生长的重要生物机制，有助于开发闭塞性动脉疾病的分子治疗方法。

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. PUBLIC HEALTH RELEVANCE: Occlusive arterial disease, including stroke and heart attack, is the leading cause of mortality and morbidity in Americans. There is significant interest in developing molecular therapy to stimulate blood vessel growth and return blood flow to dying tissue. Our research may elucidate an important biological mechanism that controls artery growth, helping in the development of molecular treatments for occlusive arterial disease.