Molecular mechanisms underlying flow sensing in lymphatic endothelial cells

 描述（由申请人提供）：我们的目的是确定液体流动引导淋巴瓣形成和生长的分子机制。瓣膜确保淋巴系统中的单向流体流动，并且对生理功能至关重要。关于淋巴瓣是如何形成的，我们知之甚少。这种知识差距，加上淋巴系统的再生能力有限，是开发有效治疗水肿的主要障碍，水肿是一种无法治愈的衰弱疾病。因此，了解淋巴瓣膜发生的分子基础将对治疗水肿和其他淋巴系统疾病产生变革性影响。依据：淋巴阀优先在血管连接处附近形成。这些区域具有复杂的再循环流动，其不存在于淋巴脉管系统的直部分中。我们假设，这些流动模式可能提供了一个关键的线索，触发瓣膜形成，特别是在这些位置。为了验证这一假设，我们开发了一种独特的体外试验，将淋巴管内皮细胞（LEC）暴露于模拟瓣膜形成部位的壁切应力（WSS）的空间梯度。值得注意的是，暴露于这种流动模式的LEC重演了体内瓣膜形成初始阶段发生的迁移、形态发生和信号传导事件。此外，我们发现，这些反应依赖于鞘氨醇-1-磷酸受体1（S1 PR 1）的激活，GPCR被血液内皮细胞中的流体流动激活，并且已知在淋巴管生成中发挥重要作用。这些和其他初步数据强烈表明WSS的空间模式在塑造淋巴瓣发育方面发挥着核心作用。策略：我们已经创建并表征了体外培养系统，该系统将LEC暴露于瓣膜形成部位的流动环境的关键属性。这些装置提供了对LEC所经历的流动刺激的定量控制，允许延时、多日成像，并提供高实验通量，这些能力在体内环境中难以实现。这种属性的组合在揭示LEC感知和响应流体流动的分子机制方面是独特的。我们的研究目标是：目标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梯度和振荡流在塑造淋巴管瓣膜发育中的作用（Aim 1a），并发现已知瓣膜发生所需的信号通路如何与LEC流量传感耦合（Aim 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，并使用这种强大的技术在体外重建瓣膜发生的关键属性。目标二。确定S1 PR 1介导LEC中流量传感的分子机制。我们将阐明流动激活的S1 PR 1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学方法来确定流动激活S1 PR 1的分子机制（目标2b和2c）。

 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).