The meningeal lymphatic network enables the drainage of cerebrospinal fluid (CSF) and facilitates the removal of central nervous system (CNS) waste. During aging and in Alzheimer’s disease, impaired meningeal lymphatic drainage promotes the buildup of toxic misfolded proteins in the CNS. Reversing this age-related dysfunction represents a promising strategy to augment CNS waste clearance; however, the mechanisms underlying this decline remain elusive. Here, we demonstrate that age-related alterations in meningeal immunity underlie this lymphatic impairment. Single-cell RNA sequencing of meningeal lymphatic endothelial cells from aged mice revealed their response to IFNγ, which was increased in the aged meninges due to T cell accumulation. Chronic elevation of meningeal IFNγ in young mice via AAV-mediated overexpression attenuated CSF drainage—comparable to the deficits observed in aged mice. Therapeutically, IFNγ neutralization alleviated age-related impairments in meningeal lymphatic function. These data suggest manipulation of meningeal immunity as a viable approach to normalize CSF drainage and alleviate the neurological deficits associated with impaired waste removal.

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.

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.

Lymphatic vascular permeability prevents lymph leakage that is associated with lymphedema, lymphatic malformations, obesity, and inflammation. However, the molecular control of lymphatic permeability remains poorly understood. Recent studies have suggested that adherens junctions and vesicle transport may be involved in regulating lymphatic vessel permeability. To determine the contribution of each transport pathway, we utilized an ex vivo permeability assay to directly measure the solute flux of various molecular weight solutes across a range of pressures in intact murine collecting lymphatic vessels. Pharmacological and biological tools were used to probe the relative contributions of vesicles and junction proteins in the lymphatic vasculature. We show that the permeability of collecting lymphatic vessels is inversely related to the solute molecular weight. Further, our data reveal that vesicles selectively transport BSA, as an inhibitor of vesicle formation significantly decreased the permeability to BSA (∼60% decrease, n = 8, P = 0.02), but not to 3 kDa dextran (n = 7, P = 0.41), α-lactalbumin (n = 5, P = 0.26) or 70 kDa dextran (n = 8, P = 0.13). In contrast, disruption of VE-cadherin binding with a function blocking antibody significantly increased lymphatic vessel permeability to both 3 kDa dextran (5.7-fold increase, n = 5, P < 0.0001) and BSA (5.8-fold increase, n = 5, P < 0.0001). Thus, in the lymphatic vasculature, adherens junctions did not exhibit selectivity for any of the solutes tested here, whereas vesicles specifically transport BSA. Overall, the findings suggest that disease states that disrupt VE-cadherin localization or expression will cause significant leakage of solutes and fluid from the lymphatic vasculature.

Somatic activating mutations in KRAS can cause complex lymphatic anomalies (CLAs). However, the specific processes that drive KRAS -mediated CLAs have yet to be fully elucidated. Here, we used single-cell RNA sequencing to construct an atlas of normal and Kras ^G12D -malformed lymphatic vessels. We identified 6 subtypes of lymphatic endothelial cells (LECs) in the lungs of adult wild-type mice (Ptx3, capillary, collecting, valve, mixed, and proliferating). To determine when the LEC subtypes were specified during development, we integrated our data with data from 4 stages of development. We found that proliferating and Ptx3 LECs were prevalent during early lymphatic development and that collecting and valve LECs emerged later in development. Additionally, we discovered that the proportion of Ptx3 LECs decreased as the lymphatic network matured but remained high in Kras ^G12D mice. We also observed that the proportion of collecting and valve LECs was lower in Kras ^G12D mice than in wild-type mice. Last, we found that immature lymphatic vessels in young mice were more sensitive to the pathologic effects of Kras ^G12D than mature lymphatic vessels in older mice. Together, our results expand the current model for the development of the lymphatic system and suggest that KRAS mutations impair the maturation of lymphatic vessels.