Molecular mechanisms underlying flow sensing in lymphatic endothelial cells

 描述(申请人提供)：我们的目标是确定流体流动引导淋巴瓣形成和生长的分子机制。阀门确保淋巴系统中的单向液体流动，对生理功能是必不可少的。对于淋巴管瓣膜是如何形成的，人们知之甚少。这一知识差距，加上淋巴系统有限的再生能力，是开发有效治疗淋巴水肿的中心障碍，淋巴水肿是一种无法治愈的衰弱疾病。因此，了解淋巴瓣膜形成的分子基础将对淋巴水肿和其他淋巴系统疾病的治疗产生革命性的影响。原理：淋巴管瓣膜优先形成于血管交界处附近。这些区域的特点是淋巴管系统的直线部分不存在复杂的再循环血流。我们推测，这些流动模式可能提供了一个关键线索，特别是在这些位置触发了瓣膜的形成。为了验证这一假设，我们开发了一种独特的体外实验，将淋巴管内皮细胞(LECs)暴露在壁切应力(WSS)的空间梯度中，模拟瓣膜形成部位的空间梯度。值得注意的是，暴露在这种流动模式下的LEC重述了体内瓣膜形成初期发生的迁移、形态发生和信号传递事件。此外，我们发现这些反应依赖于鞘氨醇-1-磷酸受体1(S1PR1)的激活，S1PR1是一种由血液内皮细胞中的液体流动激活的GPCR，已知在淋巴管生成中发挥重要作用。这些和其他初步数据有力地表明，WSS的空间模式在塑造淋巴瓣发育过程中发挥着核心作用。策略：我们已经建立了体外培养系统，使LEC暴露在瓣膜形成部位发现的流动环境的关键属性中。这些设备提供对晶状体上皮细胞所经历的血流刺激的定量控制，允许延时、多天成像，并提供高实验吞吐量，这些能力是在活体环境中难以实现的。这种属性的组合在揭示晶状体上皮细胞感知和响应流体流动的分子机制方面是独一无二的。我们的研究目标是：目的1.阐明液体流动在引导淋巴瓣形成中的作用。我们将确定WSS梯度和振荡流在形成淋巴瓣发育中的作用(目标1a)，并发现已知的瓣膜形成所需的信号通路如何耦合到LEC流量传感(目标1b)。此外，我们将创建3D细胞培养系统，完全重现瓣膜生长部位的流动环境，并使用这项强大的技术在体外重建瓣膜形成的关键属性。目的2.确定S1PR1介导晶状体上皮细胞流感的分子机制。我们将阐明流激活的S1PR1信号在瓣膜形成中的作用(目标2a)，并使用细胞生物学和生物物理方法来确定流激活S1PR1(目标2b和2c)的分子机制。

 DESCRIPTION (provided by applicant): Our OBJECTIVE is to determine the molecular mechanisms by which fluid flow guides the formation and growth of lymphatic valves. Valves ensure one-way fluid flow in the lymphatic system, and are essential to physiological function. Relatively little is known about how lymphatic valves form. This knowledge gap, coupled with the limited regenerative ability of the lymphatic system, represents a central roadblock in the development of effective treatments for lymphedema, a debilitating condition for which there is no cure. Understanding the molecular basis of lymphatic valvulogenesis would thus have a transformative impact on the treatment of lymphedema and other diseases of the lymphatic system. RATIONALE: Lymphatic valves form preferentially near vessel junctions. These regions feature complex, recirculating flow that is not present in straight portions of the lymphatic vasculature. We hypothesized that these flow patterns might provide a critical cue that triggers valve formation specifically in these locations. To test this hypothesis, we developed a unique in vitro assay that exposes lymphatic endothelial cells (LECs) to spatial gradients in wall shear stress (WSS) that mimic those found at the sites of valve formation. Remarkably, LECs exposed to this flow pattern recapitulate the migratory, morphogenetic, and signaling events that occur during the initial stages of valve formation in vivo. Further, we find that these responses depend on activation of sphingosine-1-phosphate receptor 1 (S1PR1) a GPCR that is activated by fluid flow in blood endothelial cells, and that is known to play an important role in lymphangiogenesis. These and other preliminary data strongly suggest that spatial patterns in WSS play a central role in sculpting lymphatic valve development. STRATEGY: We have created and characterized in vitro culture systems that expose LECs to key attributes of the flow environment found at sites of valve formation. These devices provide quantitative control of the flow stimuli experienced by the LECs, allow time-lapse, multi-day imaging, and provide high experimental throughput, capabilities that are difficult to attain in an in vivo setting. This combination of attributes is nique in its ability to uncover the molecular mechanisms by which LECs sense and respond to fluid flow. The GOALS of our research are: Aim 1. Elucidate the role of fluid flow in guiding lymphatic valve formation. We will determine the role of WSS gradients and oscillating flow in shaping lymphatic valve development (Aim 1a), and discover how signaling pathways known to be required for valvulogenesis are coupled to LEC flow sensing (Aim 1b). Further, we will create 3D cell culture systems that fully recapitulate the flow environment found at sites of valve growth, and use this powerful technology to recreate key attributes of valvulogenesis in vitro. Aim 2. Determine the molecular mechanism by which S1PR1 mediates flow sensing in LECs. We will elucidate the role of flow-activated S1PR1 signaling in valve formation (Aim 2a), and use cell biological and biophysical approaches to determine the molecular mechanism by which flow activates S1PR1 (Aims 2b and 2c).