Cellular Mechanisms of Lymphatic Muscle Contractility

描述(申请人提供)：美国目前有超过1000万人患有某种形式的淋巴水肿，其中包括从乳房切除术或重建手术中恢复的很高比例的患者。结合在慢性水肿性组织中促进淋巴管生成的方法，加强受影响区域的淋巴泵功能和引流的策略是必要的。淋巴管表现出与血管显著不同的收缩表型，后者的收缩以时相和紧张性成分为特征；血管以紧张性行为为主。大鼠肠系膜淋巴管作为一种典型的收集淋巴管模型，允许在体内和体外进行研究。令人惊讶的是，这些血管表达的收缩蛋白亚型通常只在横纹肌中存在：肌钙蛋白C(cTn-C)、1-横纹原肌球蛋白(1-TMstr)和肌球蛋白重链的快速2B亚型(SM-B)。它们在淋巴管中的功能作用尚不清楚。这种独特的表达模式和我们最近的研究发现，淋巴肌的缩短速度远远高于动脉或静脉平滑肌，这表明淋巴肌是血管平滑肌和心肌的混合体。我们建议验证SM-B、MHC、cTn-C和1-TMstr的表达能够使收集的淋巴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性和紧张性成分。这些方法将与短期血管培养和腺病毒转染法相结合，允许蛋白质过表达或siRNA介导的蛋白质敲除在长达14天的时间内单独或联合改变SM-B、TN-C和1-TM的表达。结合用于评估体外收缩的时相和紧张性成分的功能测试，靶标的消息/蛋白表达将通过RT/PCR、Western blotting和免疫荧光显微镜进行监测。我们预测，这三种蛋白的表达赋予淋巴肌肉独特的高收缩/松弛速率，内在的起搏器活动转化为有效的淋巴泵送所需的。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，从而在不对血管产生侧枝效应的情况下增强淋巴泵功能。 项目简介：毛细淋巴管与血管平行运行，捕获从毛细血管中过滤出来的多余液体。淋巴管逆着压力梯度向上输送液体，因此需要壁上肌肉细胞强大的、类似心脏的泵送活动。淋巴泵系统功能障碍与水肿、疼痛、行动不便和感染风险增加有关，这些情况在美国影响着1000多万人。这些研究将调查哪些蛋白质允许这些血管收缩，以便开发特定的治疗剂来纠正淋巴泵功能障碍和水肿组织的引流。

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.