Clinical manifestations of severe COVID-19 include coagulopathies that are exacerbated by the formation of neutrophil extracellular traps (NETs). Here, we report that pulmonary lymphatic vessels, which traffic neutrophils and other immune cells to the lung-draining lymph node (LDLN), can also be blocked by fibrin clots in severe COVID-19. Immunostained tissue sections from COVID-19 decedents revealed widespread lymphatic clotting not only in the lung but also in the LDLN, where the extent of clotting correlated with the presence of abnormal, regressed, or missing germinal centers (GCs). It strongly correlated with the presence of intralymphatic NETs. In mice, tumor necrosis factor α induced intralymphatic fibrin clots; this could be inhibited by DNase I, which degrades NETs. In vitro, TNF-α induced lymphatic endothelial cell upregulation of ICAM-1 and CXCL8, among other neutrophil-recruiting factors, as well as thrombomodulin downregulation; in decedents, lymphatic clotting in LDLNs. In a separate cohort of hospitalized patients, serum levels of Myeloperoxidase-DNA (MPO-DNA, a NET marker) inversely correlated with antiviral antibody titers, but D-dimer levels, indicative of blood thrombosis, did not correlate with either. Patients with high MPO-DNA but low D-dimer levels generated poor antiviral antibody titers. This study introduces lymphatic coagulation in lungs and LDLNs as a clinical manifestation of severe COVID-19 and suggests the involvement of NETosis of lymphatic-trafficking neutrophils. It further suggests that lymphatic clotting may correlate with impaired formation or maintenance of GCs necessary for robust antiviral antibody responses, although further studies are needed to determine whether and how lymphatic coagulation affects adaptive immune responses.

The ability of lymph to clot indicates that, like blood vessels, lymphatics must have means to counteract this process. Here, we analyzed lymphatic hemostatic properties, tailoring them for potential therapeutic applications. Inflammatory stimuli induced tissue factor-dependent focal lymph clotting while blocking thrombomodulin leading to widespread but transient occlusion of collecting vessels. Decellularization of lymphatics resulted in tissue factor-independent lymphatic occlusion by widespread and persistent lymph clots. In occluded decellularized ‘ghost’ vessels, fibrin was eventually reperfused. During the regeneration, ghost lymphatics were filled with granuloma-like clusters of antigen-presenting cells and T cells. Despite that, immune response against allografts placed under non-drained skin did not develop as long lymphatics remained occluded, the effect that could be prolonged by delaying regeneration of the decellularized collectors. When the lymph clotting was blocked, decellularized lymphatics could still drain macromolecules and leukocytes, showing that lymphatic endothelium is not necessary for the classic lymphatic functions. The control of excessive clotting emerges as the essential function of lymphatics that could explain the seeming spandrel presence of lymphatic networks in organs such as the kidney or heart, contribute to microvascular thrombosis during infection, and can be exploited to induce immune ignorance of the subcutaneous endocrine grafts.

The ability of lymph to clot indicates that similar to blood vessels lymphatics must have means to counteract this process. We analyzed their hemostatic properties, tailoring them for the potential therapeutic applications. Inflammatory stimuli induced tissue factor-dependent focal lymph clotting while blocking thrombomodulin lead to widespread but also transient occlusion of collecting vessels. Decellularization of lymphatics resulted in tissue factor-independent lymphatic occlusion by widespread and persistent lymph clots. These decellularized basement membrane remnants of collectors were capable of macromolecules drainage and leukocytes transit only when lymph clotting was inhibited with heparin. In occluded ‘ghost’ vessels, fibrin was replaced with transient basement membrane-rich inclusions to be eventually reperfused. During regeneration, ghost vessels were filled with granuloma-like clusters of antigen-presenting and T cells. Despite that, immune response against allografts placed under non-drained skin was not developed as long lymphatics remained occluded, and graft survival was prolonged together with the delay of lymphatic regeneration with anti-lymphangiogenic therapy. Other potential applications of lymphatic hemostatic control functions include blocking pathogen or metastasis spread but also should be considered in the treatment of lymphedema.

Controlled infection with intestinal nematodes has therapeutic potential for preventing the symptoms of allergic and autoimmune diseases. Here, we engineered larvae of the filarial nematode Litomosoides sigmodontis as a vaccine strategy to induce adaptive immunity against a foreign, crosslinked protein, chicken egg ovalbumin (OVA), in the absence of an external adjuvant. The acylation of filarial proteins with fluorescent probes or biotin was not immediately detrimental to larval movement and survival, which died 3 to 5 days later. At least some of the labeled and skin-inoculated filariae migrated through lymphatic vessels to draining lymph nodes. The immunization potential of OVA-biotin-filariae was compared to that of an OVA-bound nanoparticulate carrier co-delivered with a CpG adjuvant in a typical vaccination scheme. Production of IFNγ and TNFα by restimulated CD4+ cells but not CD8+ confirmed the specific ability of filariae to stimulate CD4 + T cells. This alternative method of immunization exploits the intrinsic adjuvancy of the attenuated nematode carrier and has the potential to shift the vaccination immune response towards cellular immunity.

Lymphatic endothelial cells (LECs) chemoattract naïve T cells and promote their survival in the lymph nodes, and can cross-present antigens to naïve CD8 + T cells to drive their proliferation despite lacking key costimulatory molecules. However, the functional consequence of LEC priming of CD8 + T cells is unknown. Here, we show that while many proliferating LEC-educated T cells enter early apoptosis, the remainders comprise a long-lived memory subset, with transcriptional, metabolic, and phenotypic features of central memory and stem cell-like memory T cells. In vivo, these memory cells preferentially home to lymph nodes and display rapid proliferation and effector differentiation following memory recall, and can protect mice against a subsequent bacterial infection. These findings introduce a new immunomodulatory role for LECs in directly generating a memory-like subset of quiescent yet antigen-experienced CD8 + T cells that are long-lived and can rapidly differentiate into effector cells upon inflammatory antigenic challenge.

Prior to metastasis, tumor-draining lymph nodes and distant organs are altered by a number of tumor-derived factors that help to both suppress host immunity as well as form an environment that supports metastatic tumor cells, i.e., a pre-metastatic niche. Recent studies suggest that pre-metastatic niches are induced by vesicles, including exosomes, secreted by tumor cells and cells of the tumor microenvironment. Here, we investigated the contribution of lymphatic vessels, particularly the lymphatic endothelial cells (LECs), in the active regulation of tumor exosome production and trafficking. To track exosomes, we purified and fluorescently labeled exosomes from in vitro cultures of the mouse melanoma cell line B16 F10 and of the human melanoma cell line Me260LN. Upon injection into the mouse ear dermis, fluorescent exosomes were observed within minutes in draining lymphatics, but not in surrounding blood vessels. Using the B16 F10 melanoma model overexpressing the lymphangiogenic growth factor VEGF-C, we found that upon intratumoral injection of exosomes, LECs within the tumor microenvironment abundantly take up exosomes, and at higher levels than blood endothelial cells, delivering exosomes to draining lymph nodes and eventually to the blood. In transgenic mice lacking dermal lymphatics, exosomes injected intratumorally were absent in the lymph nodes and present in far lower amounts in the blood compared to those injected into wild type mice. Additionally, while the density of endogenous blood exosomes increased upon tumor growth in wild type mice, no significant change in blood exosome density was observed in transgenic mice. Using an in vitro model where LECs are seeded onto porous inserts, we found that LEC transport of tumor exosomes occurs preferentially from their basal to apical side, and that some of these exosomes were taken up and stored intracellularly. Together, these findings suggest that lymphatic vessels constitute a major route of tumor exosome distribution to the systemic circulation and that LECs actively regulate exosome transport by active uptake.Citation Format: Lea Maillat, Lambert Potin, Witold W. Kilarski, Melody A. Swartz. Lymphatic vessels regulate exosome trafficking from tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4520.

Filariases are diseases caused by arthropod-borne filaria nematodes. The related pathologies depend on the location of the infective larvae when their migration, the asymptomatic and least studied phase of the disease, comes to an end. To determine factors assisting in filariae dissemination, we image Litomosoides sigmodontis infective larvae during their escape from the skin. Burrowing through the dermis filariae exclusively enter pre-collecting lymphatics by mechanical disruption of their wall. Once inside collectors, their rapid and unidirectional movement towards the lymph node is supported by the morphology of lymphatic valves. In a microfluidic maze mimicking lymphatic vessels, filariae follow the direction of the flow, the first biomechanical factor capable of helminth guidance within the host. Finally, non-infective nematodes that rely on universal morpho-physiological cues alone also migrate through the dermis, and break in lymphatics, indicating that the ability to spread by the lymphatic route is an ancestral trait rather than acquired parasitic adaptation.

The common experimental use of B16-F10 melanoma cells focuses on exploring their metastatic potential following intravenous injection into mice. In this study, B16-F10 cells are used to develop a primary tumor model by implanting them directly into the ears of C57BL/6J mice. The model represents a reproducible and easily traceable tool for local tumor growth and for making additional in vivo observations, due to the localization of the tumors. This model is relatively simple and involves (i) surgical opening of the ear skin, (ii) removal of a square-piece of cartilage followed by (iii) the implantation of tumor cells with fibrin gel. The remodeling of the fibrin gel within the cartilage chamber, accompanying tumor proliferation, results in the formation of blood vessels, lymphatics and tissue matrix that can be readily distinguished from the pre-existing skin structures. Moreover, this method avoids the injection-enforced artificial spread of cells into the pre-existing lymphatic vessels. The tumors have a highly reproducible exponential growth pattern with a tumor doubling time of around 1.8 days, reaching an average volume of 85mm3 16 days after implantation. The melanomas are densely cellular with proliferative indices of between 60 and 80%. The induced angiogenesis and lymphangiogenesis resulted in the development of well-vascularized tumors. Different populations of immunologically active cells were also present in the tumor; the population of macrophages decreases with time while the population of T cells remained quasi constant. The B16-F10 tumors in the ear frequently metastasized to the cervical lymph nodes, reaching an incidence of 75% by day 16. This newly introduced B16-F10 melanoma model in the ear is a powerful tool that provides a new opportunity to study the local tumor growth and metastasis, the associated angiogenesis, lymphangiogenesis and tumor immune responses. It could potentially be used to test different treatment strategies.

Postdevelopmental lymphangiogenesis occurs in chronic inflammation and wound healing, and here we describe a window preparation in the mouse ear in which lymphangiogenesis can be observed and manipulated. This model has many advantages, including access for intravital immunostaining and imaging to assess morphological features and regeneration kinetics, as well as functional assays such as lymphatic clearance. We describe five procedures: (1) the creation of a collagen-fibrin-filled window in the mouse ear as a model for regenerative lymphangiogenesis, (2) intravital immunostaining for live analysis of morphology and structure, (3) lymphatic clearance assay for functional quantification, (4) whole-mount imaging with tissue clearing for confocal imaging, and (5) postmortem lymphangiography. These procedures allow for identification of morphological and functional abnormalities in both preexisting and newly formed lymphatic vessels.

Lymphangiogenesis occurs in inflammation and wound healing, yet its functional roles in these processes are not fully understood. Consequently, clinically relevant strategies for therapeutic lymphangiogenesis remain underdeveloped, particularly using growth factors. To achieve controlled, local capillary lymphangiogenesis with protein engineering and determine its effects on fluid clearance, leukocyte trafficking, and wound healing, we developed a fibrin-binding variant of vascular endothelial growth factor C (FB-VEGF-C) that is slowly released upon demand from infiltrating cells. Using a novel wound healing model, we show that implanted fibrin containing FB-VEGF-C, but not free VEGF-C, could stimulate local lymphangiogenesis in a dose-dependent manner. Importantly, the effects of FB-VEGF-C were restricted to lymphatic capillaries, with no apparent changes to blood vessels and downstream collecting vessels. Leukocyte intravasation and trafficking to lymph nodes were increased in hyperplastic lymphatics, while fluid clearance was maintained at physiological levels. In diabetic wounds, FB-VEGF-C-induced lymphangiogenesis increased extracellular matrix deposition and granulation tissue thickening, indicators of improved wound healing. Together, these results indicate that FB-VEGF-C is a promising strategy for inducing lymphangiogenesis locally, and that such lymphangiogenesis can promote wound healing by enhancing leukocyte trafficking without affecting downstream lymphatic collecting vessels.

Rationale: The transport of interstitial fluid and solutes into lymphatic vessels is important for maintaining interstitial homeostasis and delivering antigens and soluble factors to the lymph node for immune surveillance. Transendothelial transport across lymphatic endothelial cells (LECs) is commonly considered to occur paracellularly, or between cell–cell junctions, and driven by local pressure and concentration gradients. However, emerging evidence suggests that LECs also play active roles in regulating interstitial solute balance and can scavenge and store antigens, raising the possibility that vesicular or transcellular pathways may be important in lymphatic solute transport. Objective: The aim of this study was to determine the relative importance of transcellular (vesicular) versus paracellular transport pathways by LECs and how mechanical stress (ie, fluid flow conditioning) alters either pathway. Methods and Results: We demonstrate that transcellular transport mechanisms substantially contribute to lymphatic solute transport and that solute uptake occurs in both caveolae- and clathrin-coated vesicles. In vivo, intracelluar uptake of fluorescently labeled albumin after intradermal injection by LECs was similar to that of dermal dendritic cells. In vitro, we developed a method to differentially quantify intracellular solute uptake versus transendothelial transport by LECs. LECs preconditioned to 1 µm/s transmural flow demonstrated increased uptake and basal-to-apical solute transport, which could be substantially reversed by blocking dynamin-dependent vesicle formation. Conclusions: These findings reveal the importance of intracellular transport in steady-state lymph formation and suggest that LECs use transcellular mechanisms in parallel to the well-described paracellular route to modulate solute transport from the interstitium according to biomechanical cues.

Photodynamic therapy (PDT) has been shown to destroy tumour-associated lymphatic vessels. Therefore, we sought to investigate the functional outcomes of PDT-mediated damage to the lymphatic vessels. We observed that PDT with verteporfin, completely but transiently, blocks the functional lymphatic drainage in the orthotopic mammary tumour models. Sustained inhibition of lymphatic vessels regeneration induced by lenalidomide or the soluble form of vascular endothelial growth factor receptor 3 (sVEGFR3) that neutralises lymphangiogenic vascular endothelial growth factor C (VEGF-C), significantly impaired antitumour efficacy of PDT. Antilymphangiogenic compounds also significantly inhibited the ability of intratumourally inoculated dendritic cells (DCs) to translocate to local lymph nodes and diminished the number of tumour-infiltrating interferon-γ-secreting or tumour antigen-specific CD8+ T cells. Lenalidomide also abrogated antitumour effects of the combination immunotherapy with PDT and anti-programmed death-ligand 1 (PD-L1) antibodies. Altogether, these findings indicate that PDT-mediated damage to the lymphatic vessels negatively affects development of antitumour immunity, and that drugs that impair lymphatic vessel regeneration might not be suitable for the use in combination with PDT.

We present a protocol for the specific labeling and isolation of proteins from the membrane surface of endothelial cells and the surrounding extracellular matrix of organs, experimental wounds and tumors using chicken embryos. Proteins are deglycosylated on streptavidin resin and purified after gentle elution and trypsin digestion. Peptides are analyzed by spectroscopy and reverse proteomic fingerprinting. The major advantages of this protocol include reductions in both the background and overrepresentation of single proteins that would otherwise mask less well-represented proteins in the mass spectroscopy analysis. We also present methods to identify putative vascular and endothelial cell targets from isolated chicken membranes and extracellular proteins. The use of human genome and transcriptome data facilitates this analysis. Human orthologs of isolated chicken proteins are identified using best hit BLAST searches against the Human Reference Sequence Database. The expression of Human orthologs is then assessed for endothelial and non-endothelial cell enrichment using second generation RNA-seq sequenced libraries. Scanning of the published literature then provides a ranking score of those genes most likely involved in cancer or having a link to angiogenesis.

Lymphatic vasculature plays a crucial role in the immune response, enabling transport of dendritic cells (DCs) and antigens (Ags) into the lymph nodes. Unfortunately, the lymphatic system has also a negative role in the progression of cancer diseases, by facilitating the metastatic spread of many carcinomas to the draining lymph nodes. The lymphatics can promote antitumor immune response as well as tumor tolerance. Here, we review the role of lymphatic endothelial cells (LECs) in tumor progression and immunity and mechanism of action in the newest anti-lymphatic therapies, including photodynamic therapy (PDT).

Objective-Perivascular cells, including pericytes, macrophages, smooth muscle cells, and other specialized cell types, like podocytes, participate in various aspects of vascular function. However, aside from the well-established roles of smooth muscle cells and pericytes, the contributions of other vascular-associated cells are poorly understood. Our goal was to ascertain the function of perivascular macrophages in adult tissues under nonpathological conditions.Approach and Results-We combined confocal microscopy, in vivo cell depletion, and in vitro assays to investigate the contribution of perivascular macrophages to vascular function. We found that resident perivascular macrophages are associated with capillaries at a frequency similar to that of pericytes. Macrophage depletion using either clodronate liposomes or antibodies unexpectedly resulted in hyperpermeability. This effect could be rescued when M2-like macrophages, but not M1-like macrophages or dendritic cells, were reconstituted in vivo, suggesting subtype-specific roles for macrophages in the regulation of vascular permeability. Furthermore, we found that permeability-promoting agents elicit motility and eventual dissociation of macrophages from the vasculature. Finally, in vitro assays showed that M2-like macrophages attenuate the phosphorylation of VE-cadherin upon exposure to permeability-promoting agents.Conclusions-This study points to a direct contribution of macrophages to vessel barrier integrity and provides evidence that heterotypic cell interactions with the endothelium, in addition to those of pericytes, control vascular permeability.

Background: Photodynamic therapy (PDT) has been shown to induce ablation and functional occlusion of tumor-associated lymphatic vessels. However, direct effects of PDT on lymphatic endothelial cells (LECs) have not been studied so far. The aim of this study was to elucidate molecular mechanisms of cell death induced by PDT in human LECs.Methods: Verteporfin was used as a photosensitizer to investigate PDT-mediated damage of lymphatic vessels in mice using immunofluorescent staining and stereomicroscopy. In vitro dose-response studies were carried-out with crystal violet staining. Immunofluorescence, flow cytometry, immunoblotting and DNA electrophoresis were used to investigate the mechanisms of cell death in human LECs undergoing PDT.Results: PDT induced an increase in the number of propidium iodide positive lymphatic endothelial cells in the mouse dermis. In in vitro studies dose-dependent cytotoxic effects of PDT towards LECs were observed. Typical hallmarks of apoptotic cell death, including Annexin V binding, loss of mitochondrial membrane potential, caspase activation, cleavage of PARP as well as DNA fragmentation were observed in LECs when PDT was used at high irradiation conditions, causing >80% cell death. At lower light fluencies causing <50% cell death PDT induced autophagy rather than apoptosis, as revealed by conversion of LC3-I to the autophagosomal LC3-II and formation of LC3 puncta. Z-VAD-FMK, a caspase inhibitor, prevented cell death induced by high-dose PDT only, while 3-methyladenine, an autophagy suppressor, inhibited cell death induced by low-dose PDT.Conclusions: Both apoptosis and autophagy are involved in cell death induced by verteporfin-PDT in LECs. (C) 2016 Elsevier B.V. All rights reserved.

Nat. Cell Biol. 17, 1193–1204 (2015); published online 24 August 2015; corrected after print 28 August 2015 An error in the print version of this Article meant that Witold W. Kilarski's name was incorrect. This has been corrected in all online versions.

In natural situations, growth factors such as VEGF and PDGF are present bound to the ECM, yet therapeutic use of such growth factors has focused on application in soluble forms. To explore ECM immobilization of growth factors, we have explored complexation with recombinant variants of EMC proteins such as fibronectin and engineering growth factors fused to a domain that binds strongly to ECM proteins. To explore noncovalent immobilization upon matrices, we have engineered a fibrin‐binding domain of fibronectin, containing the 12th‐14th type III repeat (which was known to bind VEGF‐A). In studies of the 12th‐14th type III repeat, we determined that the growth factor binding activity of this domain was promiscuous. Incorporation of this domain into fibrin, through engineered transglutaminase activity, provides a powerful and generalizable method to retain such growth factors into surgical matrices. Examination of promiscuous growth factor binding to ECM proteins revealed broad binding to not only fibronectin but also fibrinogen, vitronectin, osteopontin, and tenascin C, among others. We examined these affinities to determine that placenta growth factor‐2 (PlGF‐2) binds the mentioned ECM molecules very strongly. We identified the binding domain and engineered it into other growth factors to confer nM affinity to them, demonstrating enhanced efficacy in the context of angiogenesis, skin repair in chronic wound models, and bone repair. In this way, using variant growth factors that have super‐affinity for ECM proteins, either a fibrin matrix or a tissue itself may be turned into a reservoir for growth factor sequestration and presentation.

Tumor-associated lymphatic vessels actively participate in tumor progression and dissemination. ADAM17, a sheddase for numerous growth factors, cytokines, receptors, and cell adhesion molecules, is believed to promote tumor development, facilitating both tumor cell proliferation and migration, as well as tumor angiogenesis. In this work we addressed the issue of whether ADAM17 may also promote tumor lymphangiogenesis. First, we found that ADAM17 is important for the migratory potential of immortalized human dermal lymphatic endothelial cells (LEC). When ADAM17 was stably silenced in LEC, their proliferation was not affected, but: (i) single-cell motility, (ii) cell migration through a 3D Matrigel/collagen type I matrix, and (iii) their ability to form sprouts in a 3D matrix were significantly diminished. The differences in the cell motility between ADAM17-proficient and ADAM17-silenced cells were eliminated by inhibitors of EGFR and HER2, indicating that ADAM17-mediated shedding of growth factors accounts for LEC migratory potential. Interestingly, ADAM17 depletion affected the integrin surface expression/functionality in LEC. ADAM17-silenced cells adhered to plastic, type I collagen, and fibronectin faster than their ADAM17-proficient counterparts. The difference in adhesion to fibronectin was abolished by a cyclic RGD peptide, emphasizing the involvement of integrins in the process. Using a soluble receptor array, we identified BIG-H3 among several candidate proteins involved in the phenotypic and behavioral changes of LEC upon ADAM17 silencing. In additional assays, we confirmed the increased expression of BIG-H3, as well as TGFβ2 in ADAM17-silenced LEC. The antilymphangiogenic effects of ADAM17 silencing in lymphatic endothelial cells suggest further relevance of ADAM17 as a potential target in cancer therapy.