Conduction and coordination of electrical signals within the lymphatic vascular wall

描述（由申请人提供）：淋巴运输主要依赖于收集淋巴管壁上淋巴平滑肌（SM）细胞的内在收缩，以推动淋巴对抗静水压力梯度。这些收缩必须在每个泵送单元（淋巴管）内协调，以有效地推动淋巴下游到下一个节段。同步收缩依赖于淋巴SMCs通过连接蛋白（Cx）的紧密电偶联，Cx是连接相邻细胞并允许电流传递的间隙连接蛋白。研究表明，受损或不协调的淋巴管收缩会导致先天性和获得性淋巴水肿，这强调了理解收缩如何协调的重要性。值得注意的是，携带Cx47突变的女性患乳腺癌相关肢体淋巴水肿的几率更高，携带Cx47或Cx43突变的患者发生原发性淋巴水肿，据说是由于收集淋巴管的收缩协调缺陷。关于Cx的表达和淋巴壁细胞之间的电偶联知之甚少。为了更好地理解电信号是如何在淋巴壁内和沿淋巴壁传导和协调的，需要了解在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：小鼠的分离腘窝淋巴管。我们可以控制压力/流量，测量SMCs和ec中的膜电位，并通过在两个记录微电极之间以确定的分离传递电流来定量评估细胞-细胞耦合程度。我们的初步数据表明：1)淋巴管显示ec特异性表达Cx37和Cx43，而sm特异性表达Cx45；2) EC-EC电耦合较强，SM-SM耦合较好，SM-EC耦合相对较差。这与在小动脉中发现的情况形成鲜明对比。我们的中心假设是，淋巴壁表现出高度的SM-SM电耦合，而不是跨瓣膜，以允许淋巴细胞内动作电位（APs）的集中产生，传导和同步；此外，我们假设由于SM-EC Cx表达差，SM层本质上与EC层电不耦合，并且优化为传导去极化波而不是超极化波。这一假设将通过3个特定目的来验证：1)确定淋巴壁SM和EC细胞之间电和Cx耦合的程度；2)确定淋巴壁的哪些细胞启动电起搏；3)确定控制淋巴起搏器波方向和传导的因素。我们将利用GFP报告小鼠和特定Cx亚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，使淋巴水肿中功能失调的淋巴泵的潜在药理拯救成为可能。

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.