Cellular Mechanisms of Lymphatic Muscle Contractility

淋巴肌收缩力的细胞机制

基本信息

批准号：
7806457
负责人：
Michael John Davis
金额：
$ 33.34万
依托单位：
UNIVERSITY OF MISSOURI-COLUMBIA
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-05-01 至 2013-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7806457
关键词：
Abbreviations Address Adenoviruses Affect Agonist Behavior Blood Vessels Blood capillaries Caliber Cardiac Chronic Contractile Proteins Contracts Coupled Down-Regulation Drainage procedure Edema Equilibrium Exhibits Extracellular Fluid Figs - dietary Financial compensation Fluid Balance Frequencies Functional disorder Heart Hybrids Immunoblotting Immunofluorescence Microscopy Impairment In Vitro Infection Isometric Exercise Isotonic Exercise Laboratories Lead Length Light Liquid substance Lymphangiogenesis Lymphatic Lymphatic Capillaries Lymphatic System Lymphatic vessel Lymphedema MYLK gene Mastectomy Measures Mediating Mesentery Methods Modeling Molecular Profiling Molecular Target Monitor Muscle Muscle Cells Muscle Contraction Muscle function Myocardium Myosin Heavy Chains Myosin Light Chain Kinase Output Pacemakers Pain Patients Phenotype Physiological Physiology Play Preparation Protein Isoforms Protein Overexpression Proteins Protocols documentation Pump Rattus Reconstructive Surgical Procedures Regulation Relaxation Resistance Risk Role Running Small Interfering RNA Smooth Muscle Smooth Muscle Myosins Speed Striated Muscles Substance P System Testing Therapeutic Therapeutic Agents Therapeutic Intervention Thick Filament Thin Filament Time Tissues Transfection Transgenic Mice Translating Tropomyosin Troponin C Vascular Smooth Muscle Venous Western Blotting Work base citrate carrier genetic regulatory protein improved in vivo knock-down lymph flow lymphatic pump mouse model non-muscle myosin heavy chain-B overexpression pressure protein expression vector

项目摘要

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.

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，其中很大一部分患者从乳房切除术或重建手术中恢复过来。与促进慢性水肿组织中淋巴管生成的方法相结合，增强淋巴泵功能和受影响区域引流的策略是必要的。淋巴管表现出与血管显着不同的收缩表型，其中收缩的特征是阶段性和强直性成分；血管主要表现出紧张行为。大鼠肠系膜淋巴管作为典型的集合淋巴管模型，可进行体内和体外研究。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同种型：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 同种型 (SM-B)。它们在淋巴管中的功能作用尚不清楚。这种独特的表达谱以及我们最近发现淋巴肌比动脉或静脉平滑肌具有更高的缩短速度的发现表明，淋巴肌的功能是血管平滑肌和心肌之间的混合体。我们建议检验以下假设：SM-B MHC、cTn-C 和 1- TMstr 的表达使集合淋巴管能够经历正常淋巴泵功能所需的快速、阶段性收缩和舒张。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阶段性和紧张性成分。这些方法将与短期容器培养和腺病毒转染方法相结合，允许蛋白质过表达或 siRNA 介导的蛋白质敲低在一段时间内单独或组合改变 SM-B、Tn-C 和 1-TM 的表达长达 14 天。结合功能测试来评估体外收缩力的阶段性和强直性成分，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的表达赋予淋巴肌独特的高收缩/舒张率，这是将内在起搏器活动转化为有效的淋巴泵所需的。这项工作的完成将增进我们对淋巴收缩的理解，并制定治疗策略，从而在不对血管产生附带影响的情况下增强淋巴泵功能。项目叙述：毛细淋巴管与血管平行，捕获从毛细血管滤出的多余液体。淋巴管逆着压力梯度将液体向上输送，因此需要其壁上的肌肉细胞具有强大的、类似心脏的泵送活动。淋巴泵系统功能障碍与水肿、疼痛、行动不便以及感染风险增加有关，影响了超过 1000 万美国人口。这些研究将调查哪些蛋白质使这些血管收缩，以便开发出特定的治疗药物来纠正淋巴泵功能障碍和水肿组织的引流。