PROJECT SUMMARY / ABSTRACT Patients with congenital heart disease frequently exhibit increased pulmonary blood flow (PBF) that leads to altered respiratory mechanics, respiratory distress and poor growth. Although it has been known for decades that these patients also have altered pulmonary lymphatics, and that lymphatic dysfunction can contribute to the disease course, investigations into the mechanisms that underlie these lymphatic aberrations are sparse. The ultimate goal of this proposal is to better understand the mechanisms that regulate lymphatic function and growth, in order to develop new treatments for these vulnerable patients. Using our established clinically relevant ovine model of a congenital heart defect with increased PBF (shunt), we have shown that the resulting increase in pulmonary lymph flow leads to impaired lymphatic endothelial signaling and disrupts the normal post-natal function and development of the pulmonary lymphatic system. Specifically, we have found that chronically increased PBF resulted in (1) impaired pulmonary lymphatic tone and flow associated with decreased bioavailable NO; and, (2) aberrations in pulmonary lymphatic development associated with HIF- and c-MYC-dependent accelerated growth and metabolic reprogramming. Importantly, an expanding body of literature indicates that lymphatic vessel capacitance and pumping primarily dictate lymphatic function, and supports the idea that endothelial NO signaling is an important modulator of lymphatic pump activity and flow. Additionally, metabolic alterations are associated with pulmonary vascular remodeling in the setting of pulmonary hypertension. Based on our findings, our overall hypothesis is that chronic exposure to increased pulmonary blood and lymph flow leads to lymphatic endothelial dysfunction characterized by disrupted NO signaling, resulting in decreased pulmonary lymphatic flow and abnormal postnatal pulmonary lymphatic growth and development associated with metabolic remodeling. By integrating whole animal, isolated vessel, cell culture, molecular and biochemical investigations, the proposal seeks to: 1) elucidate the mechanisms underlying alterations in pulmonary lymphatic tone and flow induced by chronically increased PBF; and 2) elucidate the mechanisms whereby HIF- and c-MYC interact to promote abnormal postnatal pulmonary lymphatic growth and development in response to chronically increased PBF. These experiments will establish an interdisciplinary research program aimed at incorporating novel insights with respect to lymphatic biology into the care of critically ill children born with congenital heart disease. Our clinical expertise, large animal model-driven research, cell culture expertise and molecular and metabolic research capabilities uniquely position us to translate our findings into the care of these patients. Importantly, our preliminary data suggest a possible therapeutic strategy to support lymphatic function in this setting.