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.

淋巴液转运是逆着流体静压梯度发生的，因此关键依赖于内源性的 淋巴肌的收缩功能，“主动淋巴泵”。该泵的故障与 多种类型的水肿。关于淋巴管功能失调的原因知之甚少，但临床 研究表明淋巴舒张压升高，直径增大，收缩受损或缺失， 瓣膜功能不全这些发现指出起搏器和瓣膜功能障碍是根本原因。这 R 01更新继续解决淋巴管中起搏的离子机制， 目的是开发治疗水肿中起搏器和收缩功能障碍的方法。 在上一个资助期，遗传学方法被用于评估多种离子通道在 小鼠淋巴管平滑肌和Ano 1（TMEM 16 A）的关键作用。SM特异性缺失 导致起搏频率降低3-4倍， 对压力升高的反应。该提案继续使用组织特异性和全球小鼠KO模型， 回答有关与Ano 1协同作用以启动淋巴平滑起搏的离子通道的问题 肌细胞（LMCs）。它不仅解决了Ano 1是如何激活的，而且还解决了压力传感机制， 机械敏感性离子通道或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Ca 2+事件，释放Ca 2+或IP 3并触发去极化， 通道视紫红质核心假设是膜振荡器产生一个重复的周期， 去极化/复极化以触发淋巴细胞动作电位（AP），并且该周期由以下调节： 机械敏感性离子电导和Gαq/11介导的IP 3产生/Ca 2+释放。 该假设将通过2个实验目标和一个数值建模目标进行测试，以帮助 解释和整合潜在的机制：1）阐明钙离子敏感的离子机制 促进淋巴AP的启动。是另一种与Ano 1结合作用的Ca 2+激活离子通道 在AP发射前提供去极化舒张期去极化的斜率是否接近AP阈值 由Ca 2+释放事件决定？是一个涉及HCN和Kv 7的钙离子非依赖性膜振荡器 通道与Ano 1结合作为起搏机制的一部分？2)确定压力- 调节淋巴起搏的敏感离子机制。与Gαq/11偶联的机械敏感性GPCR 推动IP 3生产，以调节Ano 1？机械敏感离子通道调节去极化还是 复极化？同时，3）数值模型将用于预测和验证离子机制， 起搏，包括LMC动作电位的形状，压力对舒张期去极化的影响， 决定起搏点起始位点的因素，以及可能 阻止电流分流到内皮层，但允许内皮调节LMC。

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