Cellular Mechanisms of Lymphatic Muscle Contractility

描述（由申请人提供）：目前美国有超过1000万人患有某种形式的淋巴水肿，其中包括高百分比的乳房切除术或重建手术恢复期患者。结合促进慢性水肿组织淋巴管生成的方法，加强淋巴泵功能和受影响区域的引流是必要的。淋巴管表现出明显不同于血管的收缩表型，血管的收缩具有相位和张力两种成分；血管表现出主要的滋补行为。大鼠肠系膜淋巴管作为一种典型的收集淋巴管模型，可以在体内和体外进行研究。令人惊讶的是，这些血管表达通常只在横纹肌中发现的收缩蛋白异构体：肌钙蛋白C (cTn-C)， 1-横纹肌原肌球蛋白（1-TMstr）和肌球蛋白重链（SM-B）的快速2B异构体。它们在淋巴管中的功能作用尚不清楚。这种独特的表达谱和我们最近发现的淋巴肌比动脉或静脉平滑肌具有更高的缩短速度，表明淋巴肌的功能是血管平滑肌和心肌的混合体。我们提出验证SM-B MHC、cTn-C和1- TMstr的表达能够使淋巴血管经历正常淋巴泵功能所需的快速、阶段性收缩和松弛的假设。我们将使用我们实验室独有的等压、等长和等渗淋巴制剂的组合，使我们能够全面评估淋巴泵的相位和张力成分。这些方法将与短期血管培养和腺病毒转染方法相结合，这些方法允许蛋白过表达或sirna介导的蛋白敲低单独或联合改变SM-B， Tn-C和1-TM的表达，持续时间长达14天。结合功能测试来评估体外收缩性的相位和强直成分，将通过RT/PCR， Western blotting和免疫荧光显微镜监测目标的信息/蛋白质表达。我们预测，这三种蛋白的表达赋予淋巴肌肉独特的高收缩/舒张率，这是内在起搏器活动转化为有效淋巴泵送所必需的。这项工作的完成将促进我们对淋巴收缩的理解，并导致治疗策略，即淋巴泵功能可以在没有血管附带作用的情况下得到增强。

DESCRIPTION (provided by applicant): Over ten million people in the US currently suffer from some form of lymphedema, including a high percentage of patients recovering from mastectomy or reconstructive surgery. In conjunction with methods to promote lymphangiogenesis in chronically edematous tissue, strategies to enhance lymphatic pump function and drainage of the affected regions are necessary. Lymphatics display a dramatically different contractile phenotype than blood vessels, in which contractions are characterized by both phasic and tonic components; blood vessels exhibit predominantly tonic behavior. Rat mesenteric lymphatics serve as a prototypical collecting lymphatic vessel model, allowing both in vivo and in vitro studies. Surprisingly, those vessels express contractile protein isoforms typically found only in striated muscle: troponin C (cTn-C), 1-striated tropomyosin (1-TMstr), and the fast, 2B isoform of myosin heavy chain (SM-B). Their functional roles in lymphatics are unknown. This unique expression profile and our recent findings that lymphatic muscle has a much higher shortening velocity than arterial or venous smooth muscle, suggest that lymphatic muscle functions as a hybrid between vascular smooth muscle and cardiac muscle. We propose to test the hypothesis that expression of SM-B MHC, cTn-C and 1- TMstr enable collecting lymphatic vessels to undergo the rapid, phasic contractions and relaxations required for normal lymphatic pump function. We will use a combination of isobaric, isometric and isotonic lymphatic preparations unique to our laboratory that enable us to comprehensively assess both phasic and tonic components of the lymphatic pump. These methods will be combined with short-term vessel culture and adenoviral transfection methods that allow protein overexpression or siRNA-mediated protein knockdown to change the expression of SM-B, Tn-C and 1-TM, alone and in combination, over a period of up to 14 days. In conjunction with functional tests to assess phasic and tonic components of contractility in vitro, message/protein expression of the targets will be monitored by RT/PCR, Western blotting and immunofluorescence microscopy. We predict that the expression of these three proteins imparts the uniquely high rate of lymphatic muscle contraction/relaxation required for intrinsic pacemaker activity to be translated into efficient lymphatic pumping. Completion of this work will advance our understanding of lymphatic contraction and lead to therapeutic strategies whereby lymphatic pump function can be enhanced in the absence of collateral effects on blood vessels. Project Narrative: Lymphatic capillaries run in parallel to blood vessels and capture excess fluid filtered out of blood capillaries. Lymphatic vessels move fluid uphill against a pressure gradient and therefore require robust, heart-like, pumping activity of the muscle cells in their walls. Dysfunction of the lymphatic pump system is associated with edema, pain, lack of mobility, and increased risk of infection conditions that affect more than 10 million people in the USA. These studies will investigate which proteins allow these vessels to contract so that specific therapeutic agents can be developed to correct lymphatic pump dysfunction and drainage of edematous tissues.