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Lymphatic valves are specialized structures of the collecting lymphatic vessels and are crucial for preventing retrograde lymph flow. Mutations in valve-forming genes have been clinically implicated in the pathology of congenital lymphedema. Lymphatic valves form when oscillatory shear stress (OSS) from lymph flow signals through the PI3K/AKT pathway to promote the transcription of valve-forming genes that trigger the growth and maintenance of lymphatic valves throughout life. Conventionally, in other tissue types, AKT activation requires dual kinase activity and the mammalian target of rapamycin complex 2 (mTORC2) commands this process by phosphorylating AKT at Ser473. Here we showed that embryonic and postnatal lymphatic deletion of Rictor , a critical component of mTORC2, led to a significant decrease in lymphatic valves and prevented the maturation of collecting lymphatic vessels. RICTOR knockdown in human lymphatic endothelial cells (hdLECs) not only significantly reduced the level of activated AKT and the expression of valve-forming genes under no-flow conditions, but also abolished the upregulation of AKT activity and valve-forming genes in response to flow. We further showed that the AKT target, FOXO1, a repressor of lymphatic valve formation, had increased nuclear activity in Rictor knockout mesenteric LECs, in vivo . Deletion of Foxo1 in Rictor knockout mice restored the number of valves to control levels in both mesenteric and ear lymphatics. Our work revealed a novel role of RICTOR signaling in the mechanotransduction signaling pathway, wherein it activates AKT and prevents the nuclear accumulation of the valve repressor, FOXO1, which ultimately allows the formation and maintenance of a normal lymphatic valve.

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.