Cellular Mechanisms of Lymphatic Muscle Contractility

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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.