PROJECT SUMMARY/ABSTRACT The lymphatic vascular system controls tissue fluid homeostasis and intestinal lipid uptake. Proper lymphatic function positively correlates with favorable outcomes for patients with cardiovascular and metabolic disorders, which accentuates the importance of this system in maintaining systemic homeostasis. Our long-term goal is to uncover molecular mechanisms and critical regulators that govern lymphatic function in health and disease, with the hope of offering new therapeutic targets to combat cardiovascular and metabolic diseases. In the previous funding period, we discovered that the Forkhead Box C2 (Foxc2) transcription factor antagonizes vascular endothelial growth factor receptor 3 (VEGFR3) signaling by inducing the expression of epsins; endocytic adaptor proteins critical for VEGFR3 degradation and vascular endothelial growth factor C (VEGF-C) signal attenuation in lymphatic endothelial cells (LECs). We also discovered that the Forkhead Box C2 transcription factor (Foxc2) was an important regulator of obesity and that restoration of lymphatic function was a potential strategy to treat metabolic diseases. As a result, in this renewal application, we sought to identify and study additional regulatory molecules of lymphatic function. We determined that the micro-ribonucleic acid miR-22 regulates lymphatic function in normal and diseased conditions. Despite its prominence in governing lymphatic pathophysiology, little is known about the role that miR-22 plays in regulating the function of this vascular system. Consequently, we generated novel, inducible lymphatic endothelial cell (LEC)-specific miR-22 loss-of-function mice and discovered that the deficiency of this molecule dramatically increased developmental lymphangiogenesis and increased the expression of the master regulator of lymphatic differentiation and fate determination Prox1 as well as fortifying VEGF-C/VEGFR3 signaling and increasing the expression of metabolic regulatory genes. Therefore, our central hypothesis is that lymphatic miR-22 represses Prox1, constrains VEGFR3 signaling, and stymies energy production by suppressing metabolic programming. Conversely, loss of lymphatic miR-22 elevates Prox1 expression, VEGFR-3 signaling, and metabolic bioenergetics; thereby, mending impaired lymphangiogenesis and lymphatic function in cardiovascular and metabolic disorders. To test our hypothesis and determine how miR-22 inhibition exerts a pro-lymphangiogenic stimulus to ameliorate cardiovascular and metabolic disease, we propose the following related, but independent, Specific Aims: 1) to determine the role of miR-22 in governing metabolic programming and VEGFR3 signaling, 2) to determine molecular mechanisms by which miR-22 governs lymphatic function in the adult, and 3) to determine the therapeutic potential of targeting miR-22 and epsins in lymphatic systems. Our findings will identify novel molecular mechanisms underlying metabolic regulation and signaling t...