eNOS Regulates Lymphatic Valve Specification by Controlling ß-Catenin Signaling During Embryogenesis in Mice.

BACKGROUND: 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 (endothelial NO synthase; gene name: Nos3) is a well-characterized shear stress signaling effector, but its role in lymphatic valve development remains unexplored. METHODS: We used global Nos3-/- mice and cultured human dermal lymphatic endothelial cells to investigate the role of eNOS in lymphatic valve development, which requires oscillatory shear stress signaling. RESULTS: Our data reveal a 45% reduction in lymphatic valve specification cell clusters and that loss of eNOS protein inhibited activation of ß-catenin and its nuclear translocation. Genetic knockout or knockdown of eNOS led to downregulation of ß-catenin target proteins in vivo and in vitro. However, pharmacological inhibition of NO production did not reproduce these effects. Co-immunoprecipitation and proximity ligation assays reveal that eNOS directly binds to ß-catenin and their binding is enhanced by oscillatory shear stress. Finally, genetic ablation of the Foxo1 gene enhanced FOXC2 expression and partially rescued the loss of valve specification in the eNOS knockouts. CONCLUSIONS: In conclusion, we demonstrate a novel, NO-independent role for eNOS in regulating lymphatic valve specification and propose a mechanism by which eNOS directly binds ß-catenin to regulate its nuclear translocation and thereby transcriptional activity.

VEGFR3 is required for button junction formation in lymphatic vessels.

Lymphatic capillaries develop discontinuous cell-cell junctions that permit the absorption of large macromolecules, chylomicrons, and fluid from the interstitium. While excessive vascular endothelial growth factor 2 (VEGFR2) signaling can remodel and seal these junctions, whether and how VEGFR3 can alter lymphatic junctions remains incompletely understood. Here, we use lymphatic-specific Flt4 knockout mice to investigate VEGFR3 signaling in lymphatic junctions. We show that loss of Flt4 prevents specialized button junction formation in multiple tissues and impairs interstitial absorption. Knockdown of FLT4 in human lymphatic endothelial cells results in impaired NOTCH1 expression and activation, and overexpression of the NOTCH1 intracellular domain in Flt4 knockout vessels rescues the formation of button junctions and absorption of interstitial molecules. Together, our data reveal a requirement for VEGFR3 and NOTCH1 signaling in the development of button junctions during postnatal development and may hold clinical relevance to lymphatic diseases with impaired VEGFR3 signaling.