The lymphatic system plays important roles in body fluid circulation, macromolecular homeostasis, fat absorption, and immunity. A common lymphatic dysfunction, lymphedema is diversely expressed, and results in significant morbidity. Lymphedema can be associated with the long periods of enhanced lymph flow. Until now, very limited information about flow-mediated lymphatic responses and their impact has been available. Thus investigation of the mechanisms regulating lymph flow is extremely important to ongoing attempts to discover the pathogenesis and the effective treatment of lymphedema. The general objectives of proposal are to investigate the effects of lymph flow on lymphatic contractile function and to evaluate the mechanisms involved in these effects. The proposed studies will focus on: 1) Qualitatively and quantitatively evaluate the patterns and magnitudes of lymph flow and velocity in lymphatics under differing conditions in situ. We will measure lymphatic diameter, lymph pressures and velocities in rat mesenteric lymphatics under control conditions as well as during periods of enhanced lymph formation/flow by hypotonic volumeinfusion. Servo-null micropressure, optical velocimetry and video freeze-frame techniques will be used in these studies. 2) Investigate the quantitative and temporal patterns of flow-mediated alterations in lymphatic contractile function in isolated lymphatics. We will determine the pressure profiles needed in the isolated lymphatic to simulate the range of lymph pressures and velocities observed in situ and then evaluate the temporal pattern of flow-mediated effects on various aspects of lymphatic contractile pump function over the course of minutes to hours. Computerized servo controlled pumps and latex microspheres (about 5 mu m diameter) incorporated into perfusion system will be used to control the lymph pressure and velocities. 3) Investigate the cellular and molecular mechanisms that are responsible for the effects of flow on lymphatic contractile function. In isolated lymphatic experiments we will verify if the flow-mediated events are dependent upon an intact endothelium. Studies will then focus on 3 likely endothelial-dependent pathways (nitric oxide, prostanoid, and endothelial-dependent hyperpolarization factor) of the flow-mediated responses of lymphatics. Studies will be conducted using various blockers of these 3 pathways to determine if and to what degree these pathways might be involved in flow-mediated alteration of lymphatic contractile function. Endpoints of these experiments will include measurement of intracellular calcium of lymphatic endothelial and muscle cells in isolated lymphatics as well as lymph pressure, flow, velocity, shear, wall tension and lymphatic diameter.
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