Increased Lymphatic Vessels: A Risk Factor for Severe Renal Function Loss in Obstructive Nephropathy Patients.

Background: Obstructive nephropathy (ON), resulting from hindered urine flow, significantly contributes to both acute kidney injury (AKI) and chronic kidney disease (CKD). Research has consistently highlighted increased lymphatic vessels (LVs) density in diverse kidney diseases. However, the precise involvement of LVs in ON remains unclear. Methods: Patients diagnosed with ON were enrolled in this study from January 2020 to December 2023. LVs and histological pathology in renal biopsy tissues were detected through immunohistochemistry and Periodic Acid-Schiff staining. Patients were categorized into two cohorts based on their estimated glomerular filtration rate (eGFR) levels: one cohort included patients with eGFR < 90, while the other encompassed those with eGFR ≥ 90. Univariate and multivariable logistic regression analyses were conducted to determine the odds ratio (OR) and 95% confidence interval (CI) for the association between the two cohorts. Results: 239 patients were enrolled in the study. The density of LVs was elevated in ON, with even higher densities observed in patients with severe renal impairment. Additionally, several risk factors contributing to the deterioration of renal function in ON patients have been identified, including age, ureteral calculi (UC), alanine aminotransferase (ALT), and uric acid (UA). Furthermore, by leveraging LVs density, multiple robust models have been established to predict severe renal impairment in ON. Conclusions: Lymphatic vessels density is significantly elevated in ON, serving as an independent risk factor for the decline in renal function.

Influenza induces lung lymphangiogenesis independent of YAP/TAZ activity in lymphatic endothelial cells.

The lymphatic system consists of a vessel network lined by specialized lymphatic endothelial cells (LECs) that are responsible for tissue fluid homeostasis and immune cell trafficking. The mechanisms for organ-specific LEC responses to environmental cues are not well understood. We found robust lymphangiogenesis during influenza A virus infection in the adult mouse lung. We show that the number of LECs increases twofold at 7 days post-influenza infection (dpi) and threefold at 21 dpi, and that lymphangiogenesis is preceded by lymphatic dilation. We also show that the expanded lymphatic network enhances fluid drainage to mediastinal lymph nodes. Using EdU labeling, we found that a significantly higher number of pulmonary LECs are proliferating at 7 dpi compared to LECs in homeostatic conditions. Lineage tracing during influenza indicates that new pulmonary LECs are derived from preexisting LECs rather than non-LEC progenitors. Lastly, using a conditional LEC-specific YAP/TAZ knockout model, we established that lymphangiogenesis, fluid transport and the immune response to influenza are independent of YAP/TAZ activity in LECs. These findings were unexpected, as they indicate that YAP/TAZ signaling is not crucial for these processes.

The role of vascular and lymphatic networks in bone and joint homeostasis and pathology.

The vascular and lymphatic systems are integral to maintaining skeletal homeostasis and responding to pathological conditions in bone and joint tissues. This review explores the interplay between blood vessels and lymphatic vessels in bones and joints, focusing on their roles in homeostasis, regeneration, and disease progression. Type H blood vessels, characterized by high expression of CD31 and endomucin, are crucial for coupling angiogenesis with osteogenesis, thus supporting bone homeostasis and repair. These vessels facilitate nutrient delivery and waste removal, and their dysfunction can lead to conditions such as ischemia and arthritis. Recent discoveries have highlighted the presence and significance of lymphatic vessels within bone tissue, challenging the traditional view that bones are devoid of lymphatics. Lymphatic vessels contribute to interstitial fluid regulation, immune cell trafficking, and tissue repair through lymphangiocrine signaling. The pathological alterations in these networks are closely linked to inflammatory joint diseases, emphasizing the need for further research into their co-regulatory mechanisms. This comprehensive review summarizes the current understanding of the structural and functional aspects of vascular and lymphatic networks in bone and joint tissues, their roles in homeostasis, and the implications of their dysfunction in disease. By elucidating the dynamic interactions between these systems, we aim to enhance the understanding of their contributions to skeletal health and disease, potentially informing the development of targeted therapeutic strategies.

Mesenchymal Osr1+ cells regulate embryonic lymphatic vessel formation.

The lymphatic system is formed during embryonic development by the commitment of specialized lymphatic endothelial cells (LECs) and their subsequent assembly in primary lymphatic vessels. Although lymphatic cells are in continuous contact with mesenchymal cells during development and in adult tissues, the role of mesenchymal cells in lymphatic vasculature development remains poorly characterized. Here, we show that a subpopulation of mesenchymal cells expressing the transcription factor Osr1 are in close association with migrating LECs and established lymphatic vessels in mice. Lineage tracing experiments revealed that Osr1+ cells precede LEC arrival during lymphatic vasculature assembly in the back of the embryo. Using Osr1-deficient embryos and functional in vitro assays, we show that Osr1 acts in a non-cell-autonomous manner controlling proliferation and early migration of LECs to peripheral tissues. Thereby, mesenchymal Osr1+ cells control, in a bimodal manner, the production of extracellular matrix scaffold components and signal ligands crucial for lymphatic vessel formation.

Dynamic multispectral NIR/SWIR for in vivo lymphovascular architectural and functional quantification.

Significance: Although the lymphatic system is the second largest circulatory system in the body, there are limited techniques available for characterizing lymphatic vessel function. We report shortwave-infrared (SWIR) imaging for minimally invasive in vivo quantification of lymphatic circulation with superior contrast and resolution compared with near-infrared first window imaging. Aim: We aim to study the lymphatic structure and function in vivo via SWIR fluorescence imaging. Approach: We evaluated subsurface lymphatic circulation in healthy, adult immunocompromised salt-sensitive Sprague-Dawley rats using two fluorescence imaging modalities: near-infrared first window (NIR-I, 700 to 900 nm) and SWIR (900 to 1800 nm) imaging. We also compared two fluorescent imaging probes: indocyanine green (ICG) and silver sulfide quantum dots (QDs) as SWIR lymphatic contrast agents following intradermal footpad delivery in these rats. Results: SWIR imaging exhibits reduced scattering and autofluorescence background relative to NIR-I imaging. SWIR imaging with ICG provides 1.7 times better resolution and sensitivity than NIR-I, and SWIR imaging with QDs provides nearly two times better resolution and sensitivity with enhanced vessel distinguishability. SWIR images thus provide a more accurate estimation of in vivo vessel size than conventional NIR-I images. Conclusions: SWIR imaging of silver sulfide QDs into the intradermal footpad injection provides superior image resolution compared with conventional imaging techniques using NIR-I imaging with ICG dye.

Rictor, an mTORC2 Protein, Regulates Murine Lymphatic Valve Formation Through the AKT-FOXO1 Signaling.

BACKGROUND: Lymphatic valves are specialized structures in 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 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. Conventionally, in many cell types, AKT is phosphorylated at Ser473 by the mTORC2 (mammalian target of rapamycin complex 2). However, mTORC2 has not yet been implicated in lymphatic valve formation. METHODS: In vivo and in vitro techniques were used to investigate the role of Rictor, a critical component of mTORC2, in lymphatic endothelium. RESULTS: 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 dermal lymphatic endothelial cells not only 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 oscillatory shear stress. We further showed that the AKT target, FOXO1 (forkhead box protein O1), a repressor of lymphatic valve formation, had increased nuclear activity in Rictor knockout mesenteric lymphatic endothelial cells in vivo. Deletion of Foxo1 in Rictor knockout mice restored the number of valves to control levels in lymphatic vessels of the ear and mesentery. CONCLUSIONS: Our work identifies a novel role for RICTOR in the mechanotransduction signaling pathway, wherein it activates AKT and prevents the nuclear accumulation of the valve repressor, FOXO1, which ultimately enables the formation and maintenance of lymphatic valves.

Three-Dimensional Lymphatics-on-a-Chip Reveals Distinct, Size-Dependent Nanoparticle Transport Mechanisms in Lymphatic Drug Delivery.

Although nanoparticle-based lymphatic drug delivery systems promise better treatment of cancer, infectious disease, and immune disease, their clinical translations are limited by low delivery efficiencies and unclear transport mechanisms. Here, we employed a three-dimensional (3D) lymphatics-on-a-chip featuring an engineered lymphatic vessel (LV) capable of draining interstitial fluids including nanoparticles. We tested lymphatic drainage of different sizes (30, 50, and 70 nm) of PLGA-b-PEG nanoparticles (NPs) using the lymphatics-on-a-chip device. In this study, we discovered that smaller NPs (30 and 50 nm) transported faster than larger NPs (70 nm) through the interstitial space, as expected, but the smaller NPs were captured by lymphatic endothelial cells (LECs) and accumulated within their cytosol, delaying NP transport into the lymphatic lumen, which was not observed in larger NPs. To examine the mechanisms of size-dependent NP transports, we employed four inhibitors, dynasore, nystatin, amiloride, and adrenomedullin, to selectively block dynamin-, caveolin-, macropinocytosis-mediated endocytosis-, and cell junction-mediated paracellular transport. Inhibiting dynamin using dynasore enhanced the transport of smaller NPs (30 and 50 nm) into the lymphatic lumen, minimizing cytosolic accumulation, but showed no effect on larger NP transport. Interestingly, the inhibition of caveolin by nystatin decreased the lymphatic transport of larger NPs without affecting the smaller NP transport, indicating distinct endocytosis mechanisms used by different sizes of NPs. Macropinocytosis inhibition by amiloride did not change the drainage of all sizes of NPs; however, paracellular transport inhibition by adrenomedullin blocked the lymphatic transport of NPs of all sizes. We further revealed that smaller NPs were captured in the Rab7-positive late-stage lymphatic endosomes to delay their lymphatic drainage, which was reversed by dynamin inhibition, suggesting that Rab7 is a potential target to enhance the lymphatic delivery of smaller NPs. Together, our 3D lymphatics-on-a-chip model unveils size-dependent NP transport mechanisms in lymphatic drug delivery.

The emerging importance of lymphangiogenesis in aging and aging-associated diseases.

Lymphatic aging represented by cellular and functional changes, is involved in increased geriatric disorders, but the intersection between aging and lymphatic modulation is less clear. Lymphatic vessels play an essential role in maintaining tissue fluid homeostasis, regulating immune function, and promoting macromolecular transport. Lymphangiogenesis and lymphatic remodeling following cellular senescence and organ deterioration are crosslinked with the progression of some lymphatic-associated diseases, e.g., atherosclerosis, inflammation, lymphoedema, and cancer. Age-related detrimental tissue changes may occur in lymphatic vessels with diverse etiologies, and gradually shift towards chronic low-grade inflammation, so-called inflammaging, and lead to decreased immune response. The investigation of the relationship between advanced age and organ deterioration is becoming an area of rapidly increasing significance in lymphatic biology and medicine. Here we highlight the emerging importance of lymphangiogenesis and lymphatic remodeling in the regulation of aging-related pathological processes, which will help to find new avenues for effective intervention to promote healthy aging.

The Lymphatic Highway: How Lymphatics Drive Lung Health and Disease.

The pulmonary lymphatic system has emerged as a critical regulator of lung homeostasis and a key contributor to the pathogenesis of respiratory diseases. As the primary conduit responsible for maintaining fluid balance and facilitating immune cell trafficking, the integrity of lymphatic vessels is essential for preserving normal pulmonary structure and function. Lymphatic abnormalities manifest across a broad spectrum of pulmonary disorders, underscoring their significance in respiratory health and disease. This review provides an overview of pulmonary lymphatic biology and delves into the involvement of lymphatics in four major lung diseases: chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), asthma, and lung transplant rejection. We examine how lymphatic abnormalities manifest in each of these conditions and investigate the mechanisms through which lymphatic remodeling and dysfunction contribute to disease progression. Furthermore, we explore the therapeutic potential of targeting the lymphatic system to ameliorate these debilitating respiratory conditions. Despite the current knowledge, several crucial questions remain unanswered, such as the spatial and temporal dynamics of lymphatic changes, the molecular crosstalk between lymphatics and the lung microenvironment, and the distinction between protective versus detrimental lymphatic phenotypes. Unraveling these mysteries holds the promise of identifying novel molecular regulators, characterizing lymphatic endothelial phenotypes, and uncovering bioactive mediators. By harnessing this knowledge, we can pave the way for the development of innovative disease-modifying therapies targeting the lymphatic highway in lung disorders.

Meningeal lymphatic vessel dysfunction driven by CGRP signaling causes migraine-like pain in mice.

Migraines are a type of headache that occur with other neurological symptoms, but the pathophysiology remains unclear. In this issue of the JCI, Nelson-Maney and authors used constitutive and inducible knockouts of the CGRP receptor components, elegantly demonstrating an essential function of CGRP in modulating meningeal lymphatic vessels (MLVs) in migraine. CGRP was shown to induce rearrangement of membrane-bound gap junction proteins in MLVs, resulting in a reduced CSF flux into cervical lymph nodes. The authors also provided evidence of a primary role for CGRP in modulating neuro-immune function. Finally, by showing that blocking CGRP signaling in MLVs attenuated pain behavior associated with acute migraine in rodents, the authors provided a target for pharmacological blockade of CGRP in relation to primary headache disorders.

Lymphangiogenesis in the liver of biliary atresia.

BACKGROUND: Lymphatic vessels (LVs) play a crucial role in immune reactions by serving as the principal conduits for immune cells. However, to date, no study has analyzed the morphological changes in the LVs of patients with biliary atresia (BA). In this study, we aimed to determine the morphological changes in the LVs irrigating the liver in patients with BA, elucidate their correlations with the morphology of the portal vein (PV) branches, and discuss their etiopathogenetic significance. METHODS: Morphometric analyses of liver biopsy specimens from patients treated between 1986 and 2016 were performed. The parameters measured were as follows: the whole liver area of the specimen, fibrotic area, number of LVs, LVs without patent lumen (designated as Ly0) and PV branches, and diameters of the LVs with patent lumen and the PVs. RESULTS: The numbers of LVs, Ly0, and PV branches per unit area of the whole liver specimen were significantly higher in patients with BA than in control participants with liver disease and those with normal livers. However, no correlation was observed between the fibrotic area and the average diameter of LVs or PVs, and between the fibrotic area and the number of LVs or PV branches. Furthermore, no correlation was observed between the total number of LVs and the number of PV branches. CONCLUSIONS: The present study showed a significant increase in the number of total LVs and Ly0, characterized by a high Ly0 to total LVs ratio, suggesting that lymphangiogenesis occurs in the liver of patients with BA.

Preventing periprosthetic osteolysis in aging populations through lymphatic activation and stem cell-associated secretory phenotype inhibition.

With increases in life expectancy, the number of patients requiring joint replacement therapy and experiencing periprosthetic osteolysis, the most common complication leading to implant failure, is growing or underestimated. In this study, we found that osteolysis progression and osteoclast differentiation in the surface of the skull bone of adult mice were accompanied by significant expansion of lymphatic vessels within bones. Using recombinant VEGF-C protein to activate VEGFR3 and promote proliferation of lymphatic vessels in bone, we counteracted excessive differentiation of osteoclasts and osteolysis caused by titanium alloy particles or inflammatory cytokines LPS/TNF-α. However, this effect was not observed in aged mice because adipogenically differentiated mesenchymal stem cells (MSCs) inhibited the response of lymphatic endothelial cells to agonist proteins. The addition of the JAK inhibitor ruxolitinib restored the response of lymphatic vessels to external stimuli in aged mice to protect against osteolysis progression. These findings suggest that inhibiting SASP secretion by adipogenically differentiated MSCs while activating lymphatic vessels in bone offers a new method to prevent periprosthetic osteolysis during joint replacement follow-up.

Clearance of erythrocytes from the subarachnoid space through cribriform plate lymphatics in female mice.

BACKGROUND: Atraumatic subarachnoid haemorrhage (SAH) is associated with high morbidity and mortality. Proposed mechanisms for red blood cell (RBC) clearance from the subarachnoid space (SAS) are erythrolysis, erythrophagocytosis or through efflux along cerebrospinal fluid (CSF) drainage routes. We aimed to elucidate the mechanisms of RBC clearance from the SAS to identify targetable efflux pathways. METHODS: Autologous fluorescently-labelled RBCs along with PEGylated 40 kDa near-infrared tracer (P40D800) were infused via the cisterna magna (i.c.m.) in female reporter mice for lymphatics or for resident phagocytes. Drainage pathways for RBCs to extracranial lymphatics were evaluated by in vivo and in situ near-infrared imaging and by immunofluorescent staining on decalcified cranial tissue or dural whole-mounts. FINDINGS: RBCs drained to the deep cervical lymph nodes 15 min post i.c.m. infusion, showing similar dynamics as P40D800 tracer. Postmortem in situ imaging and histology showed perineural accumulations of RBCs around the optic and olfactory nerves. Numerous RBCs cleared through the lymphatics of the cribriform plate, whilst histology showed no relevant fast RBC clearance through dorsal dural lymphatics or by tissue-resident macrophage-mediated phagocytosis. INTERPRETATION: This study provides evidence for rapid RBC drainage through the cribriform plate lymphatic vessels, whilst neither fast RBC clearance through dorsal dural lymphatics nor through spinal CSF efflux or phagocytosis was observed. Similar dynamics of P40D800 and RBCs imply open pathways for clearance that do not impose a barrier for RBCs. This finding suggests further evaluation of the cribriform plate lymphatic function and potential pharmacological targeting in models of SAH. FUNDING: Swiss National Science Foundation (310030_189226), SwissHeart (FF191155).

The interplay between lymphatic vessels and macrophages in inflammation response.

Both lymphatic vessels and macrophages are key factors influencing the inflammatory response. During the inflammatory response, lymphatic vessels undergo dilation and growth, playing a beneficial role in alleviating inflammation by facilitating the drainage of exudate, inflammatory mediators, and leukocytes. Consequently, the promotion of lymphangiogenesis has emerged as a novel therapeutic approach to treating inflammation. Macrophages play a crucial role in promoting lymphangiogenesis by secreting several pro-lymphatic growth factors, including vascular endothelial growth factor (VEGF)-C, and undergoing transdifferentiation into lymphatic endothelial cell progenitors (LECP), which integrate into newly formed lymphatic vessels. Macrophages exhibit heterogeneity and perform diverse functions based on their phenotypes. The regulation of macrophage polarization is crucial in inflammatory responses. Notably, macrophages promote lymphangiogenesis, while lymphatic vessels, in turn, serve as a conduit for macrophages to drain out inflamed tissue and also affect macrophage polarization. Thus, there is an interactive relationship between them. In this review, we discuss current work on the effects of macrophages on lymphangiogenesis as well as lymphatic vessel recruitment of macrophages and regulation of macrophage polarization. Furthermore, we explore the roles of lymphatic vessels and macrophages in various inflammation-related diseases, emphasizing potential therapeutic targets within the context of lymphatic-macrophage interactions.

Platelet extracellular vesicles preserve lymphatic endothelial cell integrity and enhance lymphatic vessel function.

Lymphatic vessels are essential for preventing the accumulation of harmful components within peripheral tissues, including the artery wall. Various endogenous mechanisms maintain adequate lymphatic function throughout life, with platelets being essential for preserving lymphatic vessel integrity. However, since lymph lacks platelets, their impact on the lymphatic system has long been viewed as restricted to areas where lymphatics intersect with blood vessels. Nevertheless, platelets can also exert long range effects through the release of extracellular vesicles (EVs) upon activation. We observed that platelet EVs (PEVs) are present in lymph, a compartment to which they could transfer regulatory effects of platelets. Here, we report that PEVs in lymph exhibit a distinct signature enabling them to interact with lymphatic endothelial cells (LECs). In vitro experiments show that the internalization of PEVs by LECs maintains their functional integrity. Treatment with PEVs improves lymphatic contraction capacity in atherosclerosis-prone mice. We suggest that boosting lymphatic pumping with exogenous PEVs offers a novel therapeutic approach for chronic inflammatory diseases characterized by defective lymphatics.

ILC2-derived LIF licences progress from tissue to systemic immunity.

Migration and homing of immune cells are critical for immune surveillance. Trafficking is mediated by combinations of adhesion and chemokine receptors that guide immune cells, in response to chemokine signals, to specific locations within tissues and the lymphatic system to support tissue-localized immune reactions and systemic immunity1,2. Here we show that disruption of leukaemia inhibitory factor (LIF) production from group 2 innate lymphoid cells (ILC2s) prevents immune cells leaving the lungs to migrate to the lymph nodes (LNs). In the absence of LIF, viral infection leads to plasmacytoid dendritic cells (pDCs) becoming retained in the lungs where they improve tissue-localized, antiviral immunity, whereas chronic pulmonary allergen challenge leads to marked immune cell accumulation and the formation of tertiary lymphoid structures in the lung. In both cases immune cells fail to migrate to the lymphatics, leading to highly compromised LN reactions. Mechanistically, ILC2-derived LIF induces the production of the chemokine CCL21 from lymphatic endothelial cells lining the pulmonary lymphatic vessels, thus licensing the homing of CCR7+ immune cells (including dendritic cells) to LNs. Consequently, ILC2-derived LIF dictates the egress of immune cells from the lungs to regulate tissue-localized versus systemic immunity and the balance between allergen and viral responsiveness in the lungs.

The embryonic zebrafish brain is exclusively colonized by pu.1-dependent and lymphatic-independent population of microglia.

Microglia, the crucial immune cells inhabiting the central nervous system (CNS), perform a range of vital functions, encompassing immune defense and neuronal regulation. Microglia subsets with diverse functions and distinct developmental regulations have been identified recently. It is generally accepted that all microglia originate from hematopoiesis and depend on the myeloid transcription factor PU.1. However, a recent study reported the existence of mrc1+ microglia in zebrafish embryos, which are seemingly independent of Pu.1 and reliant on lymphatic vessels, sparking great interest in the possibility of lymphatic-originated microglia. To address this, we took advantage of a pu.1 knock-in zebrafish allele for a detailed investigation. Our results conclusively showed that almost all zebrafish embryonic microglia (~95% on average) express pu.1. Further, lineage tracing and mutant analysis revealed that these microglia neither emerged from nor depended on lymphatic vessels. In essence, our study refutes the presence of pu.1-independent but lymphatic-dependent microglia.