Conduction and coordination of electrical signals within the lymphatic vascular wall

描述（由申请方提供）：淋巴转运主要依赖于集合淋巴管壁中淋巴平滑肌（SM）细胞的内在收缩，以推动淋巴抵抗静水压力梯度。这些收缩必须在每个泵送单元（淋巴管）内协调，以有效地将淋巴液向下游推进到下一个节段。同步收缩依赖于淋巴SMC通过连接蛋白（Cx）-连接相邻细胞并允许电流转移的间隙连接蛋白的紧密电耦合。研究表明，受损或不协调的淋巴管收缩有助于先天性和后天性水肿，这强调了了解收缩如何协调的重要性。值得注意的是，具有Cx47突变的女性具有较高的乳腺癌相关的四肢水肿的发生率，并且具有Cx47或Cx43突变的患者发展原发性水肿，据说是由于集合淋巴管中的收缩协调缺陷。关于淋巴管壁细胞之间的Cx表达和电耦合知之甚少。更好地理解电信号如何在淋巴管壁内和沿着进行和协调，需要了解SM和内皮细胞（EC）层中表达的特定Cx亚型以及细胞之间电耦合的直接测量。我们最近开发了一种非常适合解决这些问题的制剂：小鼠的孤立腘淋巴管。我们可以控制压力/流量，测量SMC和EC的膜电位，并通过在定义的间隔处的两个记录微电极之间传递电流来定量评估细胞与细胞的耦合程度。我们的初步数据表明：1）淋巴管显示出EC特异性表达Cx 37和Cx43，但SM特异性表达Cx45; 2）存在稳健的EC-EC电耦合，良好的SM-SM耦合，但相对较差的SM-EC耦合。这与在小动脉中发现的形成鲜明对比。我们的中心假设是，淋巴管壁在瓣膜之间（但不是穿过瓣膜）表现出高度的SM-SM电耦合，以允许淋巴管内的动作电位（AP）的灶性产生、传导和同步;此外，我们假设SM层与EC层基本上是电解耦的，这是由于SM-ECCx表达差，并且被优化以传导去极化波而不是超极化波。该假设将通过3个特定目的进行检验：1）确定淋巴管壁中SM和EC细胞之间的电和Cx耦合程度; 2）确定淋巴管壁的哪些细胞启动电起搏; 3）确定控制淋巴起搏波方向和传导的因素。我们将利用GFP报告小鼠和特定Cx同种型的全局和组织特异性敲除。这些目标的实现将促进我们对淋巴收缩如何协调的理解，从而使潜在的药物拯救功能失调的淋巴泵在水肿。

DESCRIPTION (provided by applicant): Lymph transport relies critically on the intrinsic contractions of lymphatic smooth muscle (SM) cells in the walls of collecting lymphatic vessels to propel lymph against a hydrostatic pressure gradient. These contractions must be coordinated within each pumping unit, the lymphangion, to efficiently propel lymph downstream to the next segment. Synchronized contractions depend on tight electrical coupling of lymphatic SMCs through connexins (Cx) - gap junction proteins that link adjacent cells and allow the transfer of electrical current. The significance of understanding how contractions are coordinated is underscored by studies suggesting that impaired or uncoordinated lymphatic vessel contractions contribute to both congenital and acquired forms of lymphedema. Notably, women with Cx47 mutations have a higher incidence of breast cancer-related lymphedema of the extremities and patients with Cx47 or Cx43 mutations develop primary lymphedema, reputedly due to a contraction coordination deficit in collecting lymphatic vessels. Little is know about Cx expression and electrical coupling between the cells of the lymphatic wall. A better understanding of how electrical signals are conducted and coordinated within and along the lymphatic wall requires knowledge of the specific Cx isoforms expressed in the SM and endothelial cell (EC) layers as well as direct measures of electrical coupling between the cells. We have recently developed a preparation ideally suited to address these issues: the isolated popliteal lymphatic vessel of the mouse. We can control pressure/flow, measure membrane potential in both SMCs and ECs, and quantitatively assess the degree of cell-cell coupling by passing current between two recording microelectrodes at defined separations. Our preliminary data suggest 1) lymphatic vessels show EC-specific expression of Cx37 and Cx43 but SM-specific expression of Cx45; 2) there is robust EC-EC electrical coupling, good SM-SM coupling, but relatively poor SM-EC coupling. This is in stark contrast to what is found in arterioles. Our central hypothesis is that the lymphatic wall exhibits a high degree of SM-SM electrical coupling between, but not across, valves to allow focal generation, conduction and synchronization of action potentials (APs) within lymphangions; further, we hypothesize that the SM layer is essentially uncoupled electrically from the EC layer, due to poor SM-EC Cx expression, and is optimized to conduct a depolarizing wave rather than a hyperpolarization wave. This hypothesis will be tested by 3 specific aims: 1) Determine the extent of electrical and Cx coupling between SM and EC cells in the lymphatic wall; 2) Determine which cells of the lymphatic wall initiate electrical pacemaking; 3) Determine the factors controlling the direction and conduction of lymphatic pacemaker waves. We will take advantage of GFP reporter mice and global and tissue-specific knock-outs of specific Cx isoforms. Accomplishment of these aims will advance our understanding of how lymphatic contractions are coordinated, enabling potential pharmacological rescue of the dysfunctional lymphatic pump in lymphedema.