Endothelial Nitric Oxide Synthase Regulates Lymphatic Valve Specification By Controlling β - catenin Signaling During Embryogenesis

OBJECTIVE: Lymphatic valves play a critical role in ensuring unidirectional lymph transport. Loss of lymphatic valves or dysfunctional valves are associated with several diseases including lymphedema, lymphatic malformations, obesity, and ileitis. Lymphatic valves first develop during embryogenesis in response to mechanotransduction signaling pathways triggered by oscillatory lymph flow. In blood vessels, eNOS (gene name: Nos3) is a well characterized shear stress signaling effector, but its role in lymphatic valve development remains unexplored. APPROACH AND RESULTS: We used global Nos3(−/−) mice and cultured hdLECs to investigate the role of eNOS in lymphatic valve development, which requires oscillatory shear stress signaling. Our data reveal a 45% reduction in lymphatic valve specification cell clusters and that loss of eNOS protein inhibited activation of β-catenin and its nuclear translocation. Genetic knockout or knockdown of eNOS led to downregulation of β-catenin target proteins in vivo and in vitro. However, pharmacological inhibition of NO production did not reproduce these effects. Coimmunoprecipitation experiments reveal that eNOS forms a complex with β-catenin and their association is enhanced by oscillatory shear stress. Finally, genetic ablation of the Foxo1 gene enhanced FOXC2 expression and rescued the loss of valve specification in the eNOS knockouts. CONCLUSION: In conclusion, we demonstrate a novel, nitric oxide-independent role for eNOS in regulating lymphatic valve specification and propose a mechanism by which eNOS forms a complex with β-catenin to regulate its nuclear translocation and thereby transcriptional activity.