Lymphatic pacemaking and pumping in lymphedema: function, dysfunction, and rescue

淋巴水肿中的淋巴起搏和泵送：功能、功能障碍和救援

• 批准号: 9887669
• 负责人: Michael John Davis
• 金额: $ 64.78万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9887669
• 关键词: Action Potentials; Address; Affect; Calcium; Caliber; Cations; Chronic; Clinical Research; Coupled; Data; Diastolic blood pressure; Disease; Endothelium; Event; Failure; Frequencies; Functional disorder; Funding; G-Protein-Coupled Receptors; Genetic; Goals; Gravitation; Heart; Hydrostatic Pressure; Impairment; Intercellular Fluid; Ion Channel; Knockout Mice; Limb structure; Lymph; Lymphatic; Lymphedema; Measures; Mediating; Membrane; Methods; Modeling; Mus; Muscle; Pacemakers; Peripheral; Play; Potassium Channel; Production; Property; Protocols documentation; Pump; Regulation; Rhodopsin; Role; Shapes; Site; Smooth Muscle; Smooth Muscle Myocytes; System; TRP channel; Testing; Tissues; Transgenic Mice; active control; lymphatic pump; lymphatic vessel; mouse model; optogenetics; patch clamp; pressure; prevent; response; therapeutic target; tool

Lymph transport occurs against a hydrostatic pressure gradient and thus relies critically on the intrinsic contractile function of lymphatic muscle, the “active lymphatic pump”. Failure of this pump is associated with many types of lymphedema. Little is known about why lymphatic vessels become dysfunctional, but clinical studies reveal an elevated lymphatic diastolic pressure, enlarged diameter, impaired or absent contractions, and incompetent valves. These findings point to both pacemaker and valve dysfunction as underlying causes. This R01 renewal continues to address the ionic mechanisms of pacemaking in lymphatic vessels, with the ultimate goal of developing methods to treat pacemaker and contractile dysfunction in lymphedema. In the previous funding period genetic methods were used to assess the roles of multiple ion channels in mouse lymphatic smooth muscle and a critical role for Ano1 (TMEM16A) was found. SM-specific deletion resulted in a 3-4-fold reduction in pacemaking frequency and blunting or abolition of the increase in frequency in response to pressure elevation. This proposal continues to use tissue-specific and global mouse KO models to answer questions about ion channels that act in concert with Ano1 to initiate pacemaking in lymphatic smooth muscle cells (LMCs). It addresses not only how Ano1 is activated but also pressure-sensing mechanisms through mechanosensitive ion channels or G-protein-coupled receptors (GPCRs). Optogenetic tools will be used extensively for measuring intracellular Ca2+ events, uncaging Ca2+ or IP3 and triggering depolarization with channel rhodopsin. The central hypothesis is that a membrane oscillator generates a repetitive cycle of depolarization/repolarization to trigger lymphatic action potentials (APs) and this cycle is modulated by mechanosensitive ionic conductances and by Gαq/11-mediated IP3 production / Ca2+ release. This hypothesis will be tested with 2 experimental aims and a numerical modeling aim to aid in the interpretation and integration of the underlying mechanisms: 1) Elucidate the Ca2+ sensitive ionic mechanisms that facilitate initiation of the lymphatic AP. Is another Ca2+-activated ion channel acting in combination with Ano1 to provide depolarization prior to AP firing? Is the slope of diastolic depolarization near the AP threshold determined by Ca2+ release events? Is a Ca2+-independent membrane oscillator involving HCN and Kv7 channels acting in combination with Ano1 as part of the pacemaking mechanism? 2) Determine the pressure- sensitive ionic mechanisms that regulate lymphatic pacemaking. Do mechanosensitive GPCRs coupled to Gαq/11 drive IP3 production to regulate Ano1? Do mechanosensitive ion channels modulate depolarization or repolarization? In parallel, 3) Numerical models will be used to predict and verify ionic mechanisms that govern pacemaking, including the shape of the LMC action potential, the effect of pressure on diastolic depolarization, factors determining pacemaker initiation sites, and properties of rectifying myoendothelial junctions that might prevent current shunting to the endothelial layer but allow endothelial modulation of LMCs.

淋巴运输是针对静水压力梯度发生的，因此准确依赖于内在的 淋巴肌肉的收缩功能，“活性淋巴泵”。该泵的故障与 多种类型的淋巴管道。关于为什么淋巴管功能失调，但临床知之甚少 研究表明，淋巴舒张压升高，直径肿大，收缩受损或缺乏，以及 无能的阀。这些发现将起搏器和阀功能障碍视为根本原因。这 R01更新继续解决淋巴管中起搏器的离子机制，最终 开发治疗淋巴水肿中的起搏器和收缩功能障碍的方法的目标。 在先前的资金期间，遗传方法用于评估多个离子通道在 发现小鼠淋巴平滑肌和ANO1（TMEM16A）的关键作用。 SM特定的删除 导致起搏频率的3-4倍降低，钝化或废除频率的增加 对压力升高的响应。该建议继续使用组织特异性和全局小鼠KO模型 回答有关与ANO1共同起作用的离子频道的问题，以启动淋巴光滑的起搏 肌肉细胞（LMC）。它不仅解决了ANO1的激活方式，还解决了通过压力激素机制 机械敏感的离子通道或G蛋白偶联受体（GPCR）。将使用光遗传学工具 广泛用于测量细胞内Ca2+事件，分解Ca2+或IP3，并触发沉积 通道视紫红质。中心假设是膜振荡器会产生一个重复的循环 去极化/重滤以触发淋巴作用电位（AP），并且该周期由 机械敏感的离子电导以及通过GαQ / 11介导的IP3生产 / Ca2+释放。 该假设将通过2个实验目的和数值建模旨在进行测试，以帮助 基本机制的解释和整合：1）阐明Ca2+敏感的离子机制 这有助于淋巴AP的主动性。是另一个Ca2+活化的离子通道与ANO1结合起作用 在AP射击之前提供去极化？是AP阈值附近的舒张期去极化的斜率 由CA2+发布事件确定？是涉及HCN和KV7的CA2+独立的膜振荡器 与ANO1结合起作用的通道作为起搏机制的一部分？ 2）确定压力 - 调节淋巴调节的敏感离子机制。做机理敏感的GPCR，耦合到GαQ/11 驱动IP3生产以调节ANO1？进行机械敏感的离子通道调节去极化或 复极？同时，3）数值模型将用于预测和验证控制的离子机制 起搏器，包括LMC动作电位的形状，压力对舒张期沉积的影响， 确定起搏器倡议站点的因素，以及纠正可能 防止电流分流到内皮层，但允许LMC的内皮调节。