Cellular Mechanisms of Lymphatic Muscle Contractility

描述（由申请人提供）：美国目前有超过一千万人患有某种形式的水肿，包括高比例的乳房切除术或重建手术后恢复的患者。结合促进慢性水肿组织中淋巴管生成的方法，增强淋巴泵功能和受影响区域引流的策略是必要的。与血管相比，淋巴管显示出显著不同的收缩表型，其中收缩的特征在于相位和强直组分;血管主要表现出强直行为。大鼠肠系膜淋巴管作为一个原型收集淋巴管模型，允许在体内和体外研究。令人惊讶的是，这些血管表达通常仅在横纹肌中发现的收缩蛋白亚型：肌钙蛋白C（cTn-C），1-横纹肌球蛋白（1-TMstr）和肌球蛋白重链的快速2B亚型（SM-B）。它们在生物学中的功能作用尚不清楚。这种独特的表达谱和我们最近的发现，淋巴肌具有比动脉或静脉平滑肌高得多的缩短速度，表明淋巴肌作为血管平滑肌和心肌之间的杂交体起作用。我们建议测试的假设，SM-B MHC，cTn-C和1- TMstr的表达，使收集淋巴管进行快速，阶段性收缩和舒张所需的正常淋巴泵功能。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组分。这些方法将与短期血管培养和腺病毒转染方法相结合，这些方法允许蛋白质过表达或siRNA介导的蛋白质敲低，以在长达14天的时间内改变SM-B、Tn-C和1-TM单独和组合的表达。结合功能测试以评估体外收缩性的阶段性和紧张性组分，将通过RT/PCR、蛋白质印迹和免疫荧光显微镜监测靶标的信息/蛋白质表达。我们预测，这三种蛋白质的表达赋予了独特的高速率的淋巴肌肉收缩/舒张所需的内在起搏器活动被翻译成有效的淋巴泵。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，从而可以在没有对血管的附带影响的情况下增强淋巴泵功能。 项目叙述：淋巴毛细血管与血管平行，并捕获从毛细血管过滤出来的多余液体。淋巴管将液体逆着压力梯度向上移动，因此需要其壁中的肌肉细胞的强有力的、心脏样的泵送活动。淋巴泵系统功能障碍与水肿、疼痛、缺乏流动性和感染风险增加有关，影响美国超过1000万人。这些研究将调查哪些蛋白质允许这些血管收缩，以便可以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.