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Mutations in many genes that regulate lymphatic valve development are associated with congenital lymphedema. Oscillatory shear stress (OSS) from lymph provides constant signals for the growth and maintenance of valve cells throughout life. The expression of valve-forming genes in lymphatic endothelial cells (LECs) is upregulated by OSS. The transcription factor FOXO1 represses lymphatic valve formation by inhibiting the expression of these genes, which makes FOXO1 a potential target for treating lymphedema. Here, we tested the ability of a FOXO1 inhibitor, AS1842856, to induce the formation of new lymphatic valves. Our quantitative RT-PCR and Western blot data showed that treatment of cultured human LECs with AS1842856 for 48 h significantly increased the expression levels of valve-forming genes. To investigate the function of AS1842856 in vivo, Foxc2+/− mice, the mouse model for lymphedema-distichiasis, were injected with AS1842856 for 2 weeks. The valve number in AS-treated Foxc2+/− mice was significantly higher than that of the vehicle-treated Foxc2+/− mice. Furthermore, since β-catenin upregulates the expression of Foxc2 and Prox1 during lymphatic valve formation, and AS1842856 treatment increased the level of active β-catenin in both cultured human LECs and in mouse mesenteric LECs in vivo, we used the mouse model with constitutive active β-catenin to rescue loss of lymphatic valves in Foxc2+/− mice. Foxc2+/− mice have 50% fewer lymphatic valves than control, and rescue experiments showed that the valve number was completely restored to the control level upon nuclear β-catenin activation. These findings indicate that pharmacological inhibition of FOXO1 can be explored as a viable strategy to resolve valve defects in congenital lymphedema.

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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.