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

 描述（由申请人提供）：淋巴转运逆着流体静压梯度发生，因此关键依赖于淋巴肌的内在收缩功能，即“淋巴泵”。这种泵系统的故障与许多类型的水肿有关。关于淋巴管功能障碍的原因知之甚少，但临床研究表明淋巴管舒张压升高，直径增大，收缩受损或缺失，以及似乎是瓣膜功能不全。总的来说，这些发现指出了淋巴起搏器、泵和瓣膜的潜在问题。在这个提议中，我们将测试在水肿发展过程中淋巴泵所经历的压力下导致收缩和瓣膜功能障碍的机制。 近年来，我们发展了在离体、加压的小鼠淋巴管中测量淋巴管平滑肌（LSM）膜电位的方法和研究LSM细胞中选择性离子电流的膜片钳方法。我们将把这些方法应用于转基因小鼠模型，以研究驱动自发和压力调节收缩的电起搏器的离子基础。我们的初步结果挑战了现有的关于LSM起搏机制的教条，并建议它是由电压门控Ca 2+通道和KCNQ K+通道之间的相互作用控制的。我们的新数据显示，相同的通道在人类LSM中运行。 我们已经开发了方法来测试从小鼠的孤立的单个淋巴管的阀门和泵功能时，血管受到不断增加的输出压力，模拟在一个依赖的肢体血管上的负载。我们的研究结果为研究压力升高时淋巴泵如何失效提供了新的见解。泵要么逐渐减弱，直到它不能弹出，或输出阀“锁定”打开灾难性的。重要的是，我们发现这两种类型的泵衰竭可以通过给予去甲肾上腺素（NE）激活起搏器来纠正，同时保留正常瓣膜门控的收缩强度和张力。虽然泵故障发生在一些正常的血管受到高压，失败的易感性增加的小鼠缺乏转录因子FOXC 2，控制淋巴阀的发展和维护。Foxc 2 +/-小鼠重现人类疾病双股水肿。 我们的中心假设是淋巴起搏、泵强度和瓣膜功能是密切相关的，任何一个的失效都会使淋巴泵失效并导致水肿。我们建议，泵功能障碍可以通过药物干预正常，Foxc 2 +/-和诱导型Foxc 2-/-小鼠获救。该假设将通过3个目的进行检验：1）确定淋巴动作电位和起搏的离子基础。2)确定压力调节淋巴起搏点的离子机制。3)确定NE调节淋巴起搏器的机制，并可以挽救因压力负荷升高或瓣膜缺陷而功能障碍的淋巴管。这种治疗淋巴泵衰竭的方法代表了一种潜在的新策略，用于治疗水肿的关键潜在原因。

 DESCRIPTION (provided by applicant): Lymph transport occurs against a hydrostatic pressure gradient and thus relies critically on the intrinsic contractile function of lymphatic muscle, the "lymphatic pump". Failure of this pump system 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 what appear to be incompetent valves. Collectively these findings point to underlying problems with the lymphatic pacemaker, pump and valves. In this proposal we will test the mechanisms leading to contraction and valve dysfunction at pressures experienced by the lymph pump during the development of lymphedema. Recently we developed methods to measure the membrane potential of lymphatic smooth muscle (LSM) in isolated, pressurized mouse lymphatics and patch clamp methods to study selected ion currents in LSM cells. We will apply those methods to transgenic mouse models to study the ionic basis of the electrical pacemaker that drives spontaneous and pressure-regulated contractions. Our preliminary results challenge the existing dogma about the pacemaking mechanism in LSM and suggest it is controlled by interactions between voltage- gated Ca2+ channels and KCNQ K+ channels. Our new data show the same channels operate in human LSM. We have developed methods to test valve and pump function in isolated single lymphangions from the mouse when the vessel is subjected to increasing output pressure, simulating the load on a vessel in a dependent extremity. Our results provide new insights into how the lymph pump fails when pressure is elevated. The pump either gradually weakens until it cannot eject, or the output valve "locks" open catastrophically. Importantly, we find that both types of pump failure can be corrected by the administration of norepinephrine (NE) to activate the pacemaker while preserving contractile strength and tone for normal valve gating. Although pump failure occurs in some normal vessels subjected to elevated pressure, susceptibility to failure is increased of mice deficient in the transcription factor FOXC2, which controls the development and maintenance of lymphatic valves. Foxc2+/- mice recapitulate the human disease lymphedema distichiasis. Our central hypothesis is that lymphatic pacemaking, pump strength, and valve function are closely interrelated such that failure of any one disables the lymph pump and leads to lymphedema. We propose that pump dysfunction can be rescued by pharmacological intervention in normal, Foxc2+/- and inducible Foxc2-/- mice. This hypothesis will be tested by 3 aims: 1) Determine the ionic basis of the lymphatic action potential and pacemaking. 2) Determine the ionic mechanisms by which pressure modulates the lymphatic pacemaker. 3) Determine the mechanisms by which NE modulates the lymphatic pacemaker and can rescue a lymphatic vessel that is made dysfunctional by an elevated pressure load or valve defect. This approach to treating a failed lymph pump represents a potential new strategy for treating a key underlying cause of lymphedema.