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: 10533796
• 负责人: Michael John Davis
• 金额: $ 62.93万
• 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 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10533796
• 关键词: Action Potentials; Address; Affect; Calcium; Cations; Chronic; Clinical Research; Coupled; Data; Diameter; Diastolic blood pressure; Disease; Endothelium; Event; Failure; Frequencies; Functional disorder; Funding; G-Protein-Coupled Receptors; Genetic; Goals; Heart; Hydrostatic Pressure; Impairment; Intercellular Fluid; Intrinsic drive; Ion Channel; Knockout Mice; Limb structure; Lymph; Lymphatic; Lymphatic System; Lymphatic function; Lymphedema; Measures; Mediating; Membrane; Methods; Modeling; Mus; Muscle; Obesity; Pacemakers; Peripheral; Persons; 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 dysfunction; 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.

淋巴运输发生在流体静压梯度的情况下，因此严重依赖于固有的 淋巴肌肉的收缩功能，“主动淋巴泵”。这个泵的故障与 多种类型的淋巴水肿。淋巴管功能障碍的原因目前知之甚少，但临床上是这样。 研究显示淋巴舒张压升高，直径增大，收缩受损或消失，以及 不合格的瓣膜。这些发现指出起搏器和瓣膜功能障碍都是潜在的原因。这 R01更新继续解决淋巴管起搏的离子机制，最终 目的开发治疗淋巴水肿性起搏器和收缩功能障碍的方法。 在以前的资助期间，使用遗传方法来评估多个离子通道在 小鼠淋巴管平滑肌和Ano1(TMEM16A)的关键作用被发现。SM特定删除 导致起搏频率减少3-4倍，并使起搏频率增加变钝或取消 对压力升高的反应。该提案继续使用特定于组织和全局的小鼠KO模型来 回答有关与ANO1协同作用启动淋巴顺畅起搏的离子通道的问题 肌细胞(LMC)。它不仅阐述了Ano1是如何被激活的，而且还阐述了通过 机械敏感离子通道或G蛋白偶联受体(GPCRs)。将使用光遗传工具 广泛用于测量细胞内钙事件，阻断钙或IP3，并触发去极化 通道视紫红质。中心假设是，膜振荡器产生一个重复的周期 去极化/复极化触发淋巴动作电位(AP)，这一循环受 机械敏感离子电导和G-αQ/11介导的IP3产生/钙释放。 这一假设将通过两个实验目标和一个数值模拟目标进行验证，以帮助 对潜在机制的解释和整合：1)阐明钙离子敏感的离子机制 促进淋巴AP的启动。是另一种钙激活的离子通道与ANO1结合作用 在AP发射前提供去极化？是接近AP阈值的舒张期除极斜率 由钙离子释放事件决定？是一种不依赖于钙离子的膜振荡器，涉及HCN和KV7 与作为起搏机制一部分的ANO1共同作用的通道？2)决定压力- 调节淋巴起搏的敏感离子机制。机械敏感型GPCR是否与GαQ/11偶联 推动IP3生产调控Ano1？机械敏感离子通道是否调制去极化或 复极化？同时，数值模型将被用来预测和验证支配离子机制。 起搏，包括LMC动作电位的形状，压力对舒张期去极化的影响， 决定起搏器起始位置的因素，以及可能存在的整流肌-内皮连接的特性 防止电流分流到内皮层，但允许内皮细胞调节LMCs。

项目成果

Emerging trends in the pathophysiology of lymphatic contractile function.

• DOI: 10.1016/j.semcdb.2015.01.005
• 发表时间: 2015-02
• 期刊: SEMINARS IN CELL & DEVELOPMENTAL BIOLOGY
• 影响因子: 7.3
• 作者: Chakraborty, Sanjukta;Davis, Michael J.;Muthuchamy, Mariappan
• 通讯作者: Muthuchamy, Mariappan

Electric field stimulation unmasks a subtle role for T-type calcium channels in regulating lymphatic contraction.

电场刺激揭示了 T 型钙通道在调节淋巴收缩中的微妙作用。

• DOI: 10.21203/rs.3.rs-2938440/v1
• 发表时间: 2023
• 期刊: Research square
• 作者: Davis,MichaelJ;Castorena-Gonzalez,JorgeA;Zawieja,ScottD
• 通讯作者: Zawieja,ScottD

Lymphatic muscle cells are the innate pacemaker cells regulating mouse lymphatic collecting vessel contractions.

淋巴肌细胞是调节小鼠淋巴集合管收缩的先天起搏细胞。

• DOI: 10.1101/2023.08.24.554619
• 发表时间: 2023
• 期刊: bioRxiv : the preprint server for biology
• 作者: Zawieja,SD;Pea,GA;Broyhill,SE;Bromert,KH;Norton,CE;Kim,HJ;Li,M;Castorena-Gonzalez,JA;Drumm,BT;Davis,MJ
• 通讯作者: Davis,MJ

Complementary Wnt Sources Regulate Lymphatic Vascular Development via PROX1-Dependent Wnt/β-Catenin Signaling.

• DOI: 10.1016/j.celrep.2018.09.049
• 发表时间: 2018-10-16
• 期刊: Cell reports
• 影响因子: 8.8
• 作者: Cha B;Geng X;Mahamud MR;Zhang JY;Chen L;Kim W;Jho EH;Kim Y;Choi D;Dixon JB;Chen H;Hong YK;Olson L;Kim TH;Merrill BJ;Davis MJ;Srinivasan RS
• 通讯作者: Srinivasan RS

Effects of Elevated Downstream Pressure and the Role of Smooth Muscle Cell Coupling through Connexin45 on Lymphatic Pacemaking.

• DOI: 10.3390/biom10101424
• 发表时间: 2020-10-08
• 期刊: Biomolecules
• 影响因子: 5.5
• 作者: Castorena-Gonzalez JA;Li M;Davis MJ
• 通讯作者: Davis MJ