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.

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.

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.

Lymphatic Valves and Lymph Flow in Cancer-Related Lymphedema.

Lymphedema is a complex disease caused by the accumulation of fluid in the tissues resulting from a dysfunctional or damaged lymphatic vasculature. In developed countries, lymphedema most commonly occurs as a result of cancer treatment. Initially, impaired lymph flow causes edema, but over time this results in inflammation, fibrotic and fatty tissue deposition, limited mobility, and bacterial infections that can lead to sepsis. While chronically impaired lymph flow is generally believed to be the instigating factor, little is known about what pathophysiological changes occur in the lymphatic vessels to inhibit lymph flow. Lymphatic vessels not only regulate lymph flow through a variety of physiologic mechanisms, but also respond to lymph flow itself. One of the fascinating ways that lymphatic vessels respond to flow is by growing bicuspid valves that close to prevent the backward movement of lymph. However, lymphatic valves have not been investigated in cancer-related lymphedema patients, even though the mutations that cause congenital lymphedema regulate genes involved in valve development. Here, we review current knowledge of the regulation of lymphatic function and development by lymph flow, including newly identified genetic regulators of lymphatic valves, and provide evidence for lymphatic valve involvement in cancer-related lymphedema.

Impact of hyperoxia on cardiac pathophysiology.

Mechanical ventilation with high oxygen therapy (hyperoxia) is widely implemented in critical care and ICU settings. Although supplemental oxygen is beneficial to treat hypoxia, its use is also associated with poor outcomes and high mortality in patients. Lung injury due to hyperoxia exposure has been well-documented in patients, including in adults and neonates. Thus, lung injury due to hyperoxia has been extensively researched in both preclinical and clinical studies. However, hyperoxia has also been shown to be associated with hemodynamic changes in patients in ICU, including reductions in heart rate, stroke volume, and cardiac output. In addition, certain experimental studies report that hyperoxia exposure in neonates results in cardiac dysfunction in later adult life. Despite this, until recently, the impact of hyperoxia within the heart has not been well studied, or reported, specifically in adult experimental models. To close this significant gap, our lab has sought to clarify hyperoxia-induced cardiac pathophysiology in adult murine models. This review discusses the current findings regarding the cardiovascular impact of hyperoxia exposure.