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.

Rictor induces AKT signaling to regulate lymphatic valve formation.

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.

Endothelial Nitric Oxide Synthase Regulates Lymphatic Valve Specification By Controlling ß - 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 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. Coimmunoprecipitation experiments reveal that eNOS forms a complex with ß-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 ß-catenin to regulate its nuclear translocation and thereby transcriptional activity.

Protocol for in vivo and in vitro study of lymphatic valve formation driven by shear stress signaling pathway.

Here we detail a protocol for the isolation and processing of lymphatic enriched tissue of mouse models for the purpose of immunostaining and quantification of lymphatic valves, vessel length, and vessel diameter. Furthermore, we describe an optimized protocol for exposing treated human dermal lymphatic endothelial cells to flow for the purpose of studying lymph shear stress responses via gene expression and protein detection methods. This approach is useful to study lymphatic valve formation driven by oscillatory shear stress. For complete details on the use and execution of this protocol, please refer to Scallan et al. (2021).1.

Pharmacological inhibition of FOXO1 promotes lymphatic valve growth in a congenital lymphedema mouse model.

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 ß-catenin upregulates the expression of Foxc2 and Prox1 during lymphatic valve formation, and AS1842856 treatment increased the level of active ß-catenin in both cultured human LECs and in mouse mesenteric LECs in vivo, we used the mouse model with constitutive active ß-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 ß-catenin activation. These findings indicate that pharmacological inhibition of FOXO1 can be explored as a viable strategy to resolve valve defects in congenital lymphedema.

Self-assembled miRNA-switch nanoparticles target denuded regions and prevent restenosis.

Cardiovascular disease is the leading cause of death and disability worldwide. Effective delivery of cell-selective therapies that target atherosclerotic plaques and neointimal growth while sparing the endothelium remains the Achilles heel of percutaneous interventions. The current study utilizes synthetic microRNA switch therapy that self-assembles to form a compacted, nuclease-resistant nanoparticle <200 nM in size when mixed with cationic amphipathic cell-penetrating peptide (p5RHH). These nanoparticles possess intrinsic endosomolytic activity that requires endosomal acidification. When administered in a femoral artery wire injury mouse model in vivo, the mRNA-p5RHH nanoparticles deliver their payload specifically to the regions of endothelial denudation and not to the lungs, liver, kidney, or spleen. Moreover, repeated administration of nanoparticles containing a microRNA switch, consisting of synthetically modified mRNA encoding for the cyclin-dependent kinase inhibitor p27Kip1 that contains one complementary target sequence of the endothelial cell-specific miR-126 at its 5' UTR, drastically reduced neointima formation after wire injury and allowed for vessel reendothelialization. This cell-selective nanotherapy is a valuable tool that has the potential to advance the fight against neointimal hyperplasia and atherosclerosis.