Interaction between blood flow and ALK1 signaling in AVM development

项目总结 遗传性出血性毛细血管扩张症(HHT)是一种常染色体显性遗传性血管疾病， 1/5000是由ENG、ALK1或Smad4单倍体不足引起的。这些基因编码重要的蛋白质。 在内皮骨形态发生蛋白(BMP)信号转导中，这是预防血管内皮细胞癌发展所必需的 动脉和静脉之间脆弱的直接连接，或动静脉畸形(AVM)。在HHT中 在患者的一生中，动静脉畸形在皮肤、鼻粘膜、胃肠道(GI)和肝脏中发展，并可导致 到鼻出血、出血、贫血和高排血量心力衰竭。先天性肺和脑损害可能导致 脑部脓肿或中风。目前可用于HHT患者的药物可阻止血管生成或增强 凝血。这些疗法并不理想：它们减少了部分患者的鼻出血和消化道出血，但不是所有患者 对脑和肺中可能危及生命的先天性病变无效。此外， 这些药物可能会延迟伤口愈合，增加严重出血和血栓事件的风险。 因此，我们的研究计划的目标是了解HHT的发病机制，以支持开发 针对这种疾病的有针对性的药物治疗。使用斑马鱼alk1突变体作为HHT2模型，我们发现 AVM发展的两步机制。在步骤1中，流依赖的Alk1信令的丢失增强 血管内皮细胞向管腔内的流动方向迁移。这种异常的迁徙扭曲了 血管内皮细胞向更远端动脉节段分布并扩大管径。在步骤2中，通常 扩大的动脉节段下游的一过性动-静脉连接保留在依赖于血流的 方式，导致高流量的动静脉动静脉畸形。在这项工作中，我们将探索这两者背后的机制 独立的基于流的信号通路，其中第一条随着Alk1丢失而被废除，第二条 它是完好无损的，没有Alk1丢失。在目标1中，我们将结合发育生物学和生物力学方法。 确定流依赖的Alk1信号是否通过控制血管内皮细胞迁移来调控动脉内皮细胞迁移 活斑马鱼胚胎中平面细胞的极性或内皮张力的产生。在目标2中，我们将使用 斑马鱼胚胎和种植人内皮细胞的新型微流控平台 血流的两个组成部分-心脏来源的循环ALK1配体，BMP10，和 切应力-内流和Alk1依赖的动脉内皮细胞逆行迁移。在目标3中，我们将测试 动静脉动静脉畸形代表对血流动力改变的适应性反应的假设，并解决了 以流量为基础的动静脉动静脉畸形发展的信号机制。这些研究将为以下问题提供新的线索 两条不同的血流依赖通路对HHT相关的AVM的发展非常重要。机械论 从这项工作中收集的信息可以用来开发靶向疗法，1)阻止 通过修复血流依赖的ALK1信号和使内皮细胞迁移正常化而产生新的动静脉曲张，或者2)缓慢 通过防止现有动静脉动静脉畸形的血流依赖性扩大来进行表型进展。

PROJECT SUMMARY Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant vascular disorder with a prevalence of 1 in 5000 that is caused by ENG, ALK1, or SMAD4 haploinsufficiency. These genes encode proteins important in endothelial bone morphogenetic protein (BMP) signaling, which is required to prevent development of fragile, direct connections between arteries and veins, or arteriovenous malformations (AVMs). In HHT patients, AVMs develop throughout life in skin, nasal mucosa, gastrointestinal (GI) tract, and liver and can lead to epistaxis, hemorrhage, anemia, and high-output heart failure. Congenital lesions in lung and brain may lead to brain abscess or stroke. Currently available medications for HHT patients block angiogenesis or enhance clotting. These therapeutics are not ideal: they decrease epistaxis and GI bleeds in some but not all patients and are ineffective against potentially life-threatening congenital lesions in the brain and lung. Furthermore, these agents may delay wound healing and enhance risk of severe hemorrhage and thrombotic events. Therefore, the goal of our research program is to understand HHT disease mechanism to support development of targeted medical therapies for this disease. Using a zebrafish alk1 mutant as an HHT2 model, we uncovered a two-step mechanism of AVM development. In Step 1, loss of flow-dependent Alk1 signaling enhances endothelial cell migration in the direction of flow within lumenized arteries. This aberrant migration skews endothelial cell distribution toward and enlarges caliber of more distal arterial segments. In Step 2, normally transient artery-vein connections downstream of enlarged arterial segments are retained in a flow-dependent manner, resulting in high-flow AVMs. In this work, we will explore the mechanisms that underlie these two independent flow-based signaling pathways, the first of which is abrogated with Alk1 loss, and the second of which is intact with Alk1 loss. In Aim 1, we will combine developmental biology and biomechanics approaches to determine whether flow-dependent Alk1 signaling governs arterial endothelial cell migration via control of planar cell polarity or generation of endothelial tension in live zebrafish embryos. In Aim 2, we will use zebrafish embryos and a novel microfluidic platform seeded with human endothelial cells to dissect the roles of two components of blood flow—the heart-derived circulating ALK1 ligand, BMP10, and the mechanical force of shear stress—in flow- and Alk1-dependent retrograde arterial endothelial cell migration. In Aim 3, we will test the hypothesis that AVMs represent an adaptive response to altered hemodynamic force and address the signaling mechanisms that underlie flow-dependent AVM development. These studies will shed new light on two distinct flow-dependent pathways important for HHT-associated AVM development. Mechanistic information gleaned from this work can be used to develop targeted therapeutics that 1) stop development of new AVMs by repairing flow-dependent ALK1 signaling and normalizing endothelial cell migration, or 2) slow phenotype progression by preventing flow-dependent enlargement of existing AVMs.