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Lymphatic vessels are highly responsive to changes in the interstitial environment. Previously, we showed renal lymphatics express the Na-K-2Cl cotransporter. Since interstitial sodium retention is a hallmark of proteinuric injury, we examined whether renal sodium affects NKCC1 expression and the dynamic pumping function of renal lymphatic vessels. Puromycin aminonucleoside (PAN)-injected rats served as a model of proteinuric kidney injury. Sodium 23 Na/ 1 H-MRI was used to measure renal sodium and water content in live animals. Renal lymph, which reflects the interstitial composition, was collected, and the sodium analyzed. The contractile dynamics of isolated renal lymphatic vessels were studied in a perfusion chamber. Cultured lymphatic endothelial cells (LECs) were used to assess direct sodium effects on NKCC1. MRI showed elevation in renal sodium and water in PAN. In addition, renal lymph contained higher sodium, although the plasma sodium showed no difference between PAN and controls. High sodium decreased contractility of renal collecting lymphatic vessels. In LECs, high sodium reduced phosphorylated NKCC1 and SPAK, an upstream activating kinase of NKCC1, and eNOS, a downstream effector of lymphatic contractility. The NKCC1 inhibitor furosemide showed a weaker effect on ejection fraction in isolated renal lymphatics of PAN vs controls. High sodium within the renal interstitium following proteinuric injury is associated with impaired renal lymphatic pumping that may, in part, involve the SPAK-NKCC1-eNOS pathway, which may contribute to sodium retention and reduce lymphatic responsiveness to furosemide. We propose that this lymphatic vessel dysfunction is a novel mechanism of impaired interstitial clearance and edema in proteinuric kidney disease.

Multifocal lymphangioendotheliomatosis with thrombocytopenia (MLT) is a recently recognized disorder characterized by vascular lesions marked by distinct endothelial proliferation. Lesions affect multiple tissues, and MLT can be associated with refractory thrombocytopenia resulting in life-threatening bleeding. Diagnosing MLT may be challenging given its rarity and phenotypic variability. There is no consensus on the optimal management or treatment duration. We report a 4-month-old male who presented with multiple vascular malformations involving the gastrointestinal tract, lung, bones, choroid plexus, and spleen, with minimal cutaneous involvement and no thrombocytopenia. Wedge resection of a pulmonary nodule was strongly positive for lymphatic vessel endothelial hyaluronan receptor 1 favoring MLT despite the lack of thrombocytopenia. The patient's clinical symptoms and vascular lesions improved on sirolimus therapy. We review the literature to highlight the clinical variability of MLT and discuss the diagnostic and therapeutic options for MLT.

Metastatic cancer cells cross endothelial barriers and travel through the blood or lymphatic fluid to pre‑metastatic niches, leading to their colonisation. 'S' stereoisomer 12S‑hydroxy‑5Z,8Z,10E,14Z‑eicosatetraenoic acid [12(S)‑HETE] is secreted by a variety of cancer cell types and has been indicated to open up these barriers. In the present study, another aspect of the endothelial unlocking mechanism was elucidated. This was achieved by investigating 12(S)‑HETE‑treated lymph endothelial cells (LECs) with regard to their expression and mutual interaction with v‑rel avian reticuloendotheliosis viral oncogene homolog A (RELA), intercellular adhesion molecule 1, SRY‑box transcription factor 18 (SOX18), prospero homeobox 1 (PROX1) and focal adhesion kinase (FAK). These key players of LEC retraction, which is a prerequisite for cancer cell transit into vasculature, were analysed using western blot analysis, reverse transcription‑quantitative PCR and transfection with small interfering (si)RNA. The silencing of a combination of these signalling and executing molecules using siRNA, or pharmacological inhibition with defactinib and Bay11‑7082, extended the mono‑culture experiments to co‑culture settings using HCT116 colon cancer cell spheroids that were placed on top of LEC monolayers to measure their retraction using the validated 'circular chemorepellent‑induced defect' assay. 12(S)‑HETE was indicated to induce the upregulation of the RELA/SOX18 feedback loop causing the subsequent phosphorylation of FAK, which fed back to RELA/SOX18. Therefore, 12(S)‑HETE was demonstrated to be associated with circuits involving RELA, SOX18 and FAK, which transduced signals causing the retraction of LECs. The FAK‑inhibitor defactinib and the NF‑κB inhibitor Bay11‑7082 attenuated LEC retraction additively, which was similar to the suppression of FAK and PROX1 (the target of SOX18) by the transfection of respective siRNAs. FAK is an effector molecule at the distal end of a pro‑metastatic signalling cascade. Therefore, targeting the endothelial‑specific activity of FAK through the pathway demonstrated herein may provide a potential therapeutic method to combat cancer dissemination via vascular routes.

Regulatory T cells (Tregs) express high levels of cell surface lymphotoxin alpha beta (LTα1β2) to activate the LT beta receptor (LTβR) on the lymphatic endothelial cells (LECs), modulating LEC adhesion molecules, intercellular junctions, and chemokines. We demonstrate a role for Tregs through this pathway to condition the permissiveness of lymphatic endothelia for transendothelial migration (TEM), thus gating leukocyte traffic. Human Tregs share the same property with murine Tregs. Activation of TLR2 on Tregs during inflammation specifically augments LTα1β2-LTβR signaling, which further enhances the permissiveness of LECs to facilitate TEM. The conditioning of endothelia may promote the resolution of inflammation by directing leukocytes out of tissues to lymphatic vessels and draining lymph nodes (dLNs). Thus, Tregs interact with lymphatic endothelia under homeostasis and inflammation and dictate endothelial permissiveness and gating mechanisms for subsequent leukocyte migration through endothelial barriers., Competing Interests: Declaration of Interests The authors declare no competing interests., (Published by Elsevier Inc.)

Kidney fibrosis is associated with an increased lymphangiogenesis, characterized by the formation and expansion of new lymphatic vessels. However, the trigger and underlying mechanism responsible for the growth of lymphatic vessels in diseased kidney remain poorly defined. Here, we report that tubule-derived sonic hedgehog (Shh) ligand is a novel lymphangiogenic factor that plays a crucial role in mediating lymphatic endothelial cell proliferation and expansion. Shh was induced in renal tubular epithelium in various models of fibrotic chronic kidney disease, and this was accompanied by an expansion of lymphatic vessels in adjacent areas. In vitro, Shh selectively promoted the proliferation of human dermal lymphatic endothelial cells (HDLECs) but not human umbilical vein endothelial cells, as assessed by cell counting, MTT assay, and bromodeoxyuridine incorporation. Shh also induced the expression of vascular endothelial growth factor receptor-3, cyclin D1, and proliferating cell nuclear antigen in HDLECs. Shh did not affect the expression of Gli1, the downstream target and readout of canonical hedgehog signaling, but activated ERK-1/2 in HDLECs. Inhibition of Smoothened with small-molecule inhibitor or blockade of ERK-1/2 activation abolished the lymphatic endothelial cell proliferation induced by Shh. In vivo, inhibition of Smoothened also repressed lymphangiogenesis and attenuated renal fibrosis. This study identifies Shh as a novel mitogen that selectively promotes lymphatic, but not vascular, endothelial cell proliferation and suggests that tubule-derived Shh plays an essential role in mediating lymphangiogenesis after kidney injury.

Background: Nanomedicine offers an excellent opportunity to tackle treatment-refractory malignancies by enhancing the delivery of therapeutics to the tumor site. High endothelial venules (HEVs) are found primarily in lymph nodes or formed de novo in peripheral tissues during inflammatory responses. They express peripheral node addressin (PNAd), which is recognized by the monoclonal antibody MECA79., Methods: Here, we demonstrated that HEVs form de novo in human pancreatic ductal adenocarcinoma (PDAC). We engineered MECA79 coated nanoparticles (MECA79-NPs) that recognize these ectopic HEVs in PDAC., Findings: The trafficking of MECA79-NPs following intravenous delivery to human PDAC implanted in a humanized mouse model was more robust than non-conjugated NPs. Treatment with MECA79-Taxol-NPs augmented the delivery of Paclitaxel (Taxol) to the tumor site and significantly reduced the tumor size. This effect was associated with a higher apoptosis rate of PDAC cells and reduced vascularization within the tumor., Interpretation: Targeting the HEVs of PDAC using MECA79-NPs could lay the ground for the localized delivery of a wide variety of drugs including chemotherapeutic agents. FUND: National Institutes of Health (NIH) grants: T32-EB016652 (B·B.), NIH Cancer Core Grant CA034196 (L.D.S.), National Institute of Allergy and Infectious Diseases grants R01-AI126596 and R01-HL141815 (R.A.)., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Objective- Maintenance of lymphatic permeability is essential for normal lymphatic function during adulthood, but the precise signaling pathways that control lymphatic junctions during development are not fully elucidated. The G s -coupled AM (adrenomedullin) signaling pathway is required for embryonic lymphangiogenesis and the maintenance of lymphatic junctions during adulthood. Thus, we sought to elucidate the downstream effectors mediating junctional stabilization in lymphatic endothelial cells. Approach and Results- We knocked-down both Rap1A and Rap1B isoforms in human neonatal dermal lymphatic cells (human lymphatic endothelial cells) and genetically deleted the mRap1 gene in lymphatic endothelial cells by producing 2 independent, conditional Rap1a/b knockout mouse lines. Rap1A/B knockdown caused disrupted junctional formation with hyperpermeability and impaired AM-induced lymphatic junctional tightening, as well as rescue of histamine-induced junctional disruption. Less than 60% of lymphatic- Rap1a/b knockout embryos survived to E13.5 exhibiting interstitial edema, blood-filled lymphatics, disrupted lymphovenous valves, and defective lymphangiogenesis. Consistently, inducible lymphatic- Rap1a/b deletion in adult animals prevented AM-rescue of histamine-induced lymphatic leakage and dilation. Conclusions- Rap1 (Ras-related protein) serves as the dominant effector downstream of AM to stabilize lymphatic junctions. Rap1 is required for maintaining lymphatic permeability and driving normal lymphatic development.

Mycobacterium tuberculosis (Mtb) has plagued humanity for tens of thousands of years, yet still remains a threat to human health. Its pathology is largely associated with pulmonary tuberculosis with symptoms including fever, hemoptysis, and chest pain. Mtb, however, also manifests in other extrapulmonary organs, such as the pleura, bones, gastrointestinal tract, central nervous system, and lymph nodes. Compared to the knowledge of pulmonary tuberculosis, extrapulmonary pathologies of Mtb are quite understudied. Lymph node tuberculosis is one of the most common extrapulmonary manifestations of tuberculosis, and presents significant challenges in its diagnosis, management, and treatment due to its elusive etiologies and pathologies. The objective of this review is to overview the current understanding of the tropism and pathogenesis of Mtb in endothelial cells of the extrapulmonary tissues, particularly, in lymph nodes. Lymphatic endothelial cells (LECs) are derived from blood vascular endothelial cells (BECs) during development, and these two types of endothelial cells demonstrate substantial molecular, cellular and genetic similarities. Therefore, systemic comparison of the differential and common responses of BECs vs. LECs to Mtb invasion could provide new insights into its pathogenesis, and may promote new investigations into this deadly disease., (Copyright © 2018. Published by Elsevier Ltd.)

It is theorized that toxic agents are transported from the hyperpermeable gut of burn victims through the lymph, to the systemic circulation, causing global injury. We believe that immune cells respond to leakage of "toxic lymph" following trauma causing the attraction of these cells to the perilymphatic space. To test this, we utilized a model of burn on rats to examine changes in a single immune cell population associated with mesenteric lymphatic dysfunction. We examined the ability of serum from these animals to increase permeability in lymphatic endothelial monolayers and disrupt cellular junctions. We also treated burn animals with doxycycline, an inhibitor of microvascular permeability, and observed the effects on immune cell populations, morphometry, and lymphatic endothelial permeability. Burn injury increased the number of MHCII + immune cells along the vessel (>50%). The size and shape of these cells also changed significantly following burn injury. Serum from burn animals increased lymphatic endothelial permeability (∼1.5-fold) and induced breaks in VE-cadherin staining. Doxycycline treatment blocked the accumulation of immune cells along the vessel, whereas serum from doxycycline-treated animals failed to increase lymphatic endothelial permeability. The size of cells along the vessel in doxycycline-treated burn animals was not affected, suggesting that the cells already present on the lymphatic vessels still respond to substances in the lymph. These findings suggest that factors produced during burn can induce lymphatic endothelial barrier disruption and lymph produced during traumatic injury can influence the attraction and morphology of immune cell populations along the vessel.

Formation of inflammation-related tertiary lymphoid organs promotes human lymphatic malformation (LM) development. However, the role of lymphotoxins (LTs) and LT-related inducible ligand, the crucial mediators for tertiary lymphoid organ formation, is undetermined in LMs. Herein, we show that LTs and LT-related inducible ligand promote LM development by enhancing lymphatic endothelial cell (LEC) proliferation via activating NF-κB pathways. The expression of LTs and their receptors was increased in LMs, especially the infected ones, when compared with normal skins. Nuclear translocation of p65, p52, and RelB in the LECs of LMs indicated the activation of classic and alternative NF-κB pathways. Pearson's correlation and cluster analysis suggested the close relationship between LEC proliferation and NF-κB activation. Moreover, in vitro data demonstrated LTs accelerated the proliferation of human dermal LECs (HdLECs) through activation of NF-κB. In addition, lipopolysaccharide (LPS) up-regulated LT receptor expression in HdLECs, leading to increased sensitivity to LTs. Suppression of LT receptors hampered LPS-enhanced HdLEC proliferation, indicating the crucial role of LT pathways in inflammatory lymphangiogenesis. Besides, evidence from the LM rat models demonstrated LTα and LPS enhanced LEC proliferation, therefore promoting LM development. Blocking LT pathways by neutralizing antibodies against LTα and lymphotoxin β receptor may decelerate the growth of the disease. In summary, our present study demonstrated activation of LT signaling pathways in LECs contributed to the progression of LMs., (Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Purpose/objectives: The goal of this study was to assess the effects of ionizing radiation on the expression of the integrin ligands ICAM-1 and VCAM that control leucocyte transit by lymphatic endothelial cells., Materials/methods: Confluent monolayers of primary human lymphatic endothelial cells (LEC) were irradiated with single dose of 2, 5, 10 or 20 Gy, with 6 MeV-x-rays using a Linear-Accelerator. ICAM-1 and VCAM expression was determined by flow cytometry. Human tissue specimens received a single dose of 20 Gy with 15 MeV-x-rays. MC38, B16-OVA or B16-VEGF-C tumors grown in C57BL/6 mice were irradiated with single dose of 20Gy using a Linear-Accelerator fitted with a 10mm Radiosurgery collimator. Clinical samples were obtained from patients previous and 4 weeks after complete standard radiotherapy. ICAM-1 and VCAM expression was detected in all tissue specimens by confocal microscopy. To understand the role of TGFβ in this process anti-TGFβ blocking mAb were injected i.p. 30min before radiotherapy. Cell adhesion to irradiated LEC was analyzed in adhesion experiments performed in the presence or in the absence of anti- TGFβ and /or anti-ICAM1 blocking mAb., Results: We demonstrate that lymphatic endothelial cells in tumor samples experience induction of surface ICAM-1 and VCAM when exposed to ionizing radiation in a dose- and time-dependent manner. These effects can be recapitulated in cultured LEC, and are in part mediated by TGFβ. These data are consistent with increases in ICAM-1 and VCAM expression on LYVE-1+ endothelial cells in freshly explanted human tumor tissue and in mouse transplanted tumors after radiotherapy. Finally, ICAM-1 and VCAM expression accounts for enhanced adherence of human T lymphocytes to irradiated LEC., Conclusion: Our results show induction of ICAM-1 and VCAM on LVs in irradiated lesions and offer a starting point for elucidating the biological and therapeutic implications of targeting leukocyte traffic in combination to immunotherapy., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: The formation of new lymphatic vessels provides an additional route for tumour cells to metastasize. Therefore, inhibiting lymphangiogenesis represents an interesting target in cancer therapy. First evidence suggests that histone deacetylase inhibitors (HDACi) may mediate part of their antitumor effects by interfering with lymphangiogenesis. However, the underlying mechanisms of HDACi induced anti-lymphangiogenic properties are not fully investigated so far and in part remain unknown., Methods: Human lymphatic endothelial cells (LEC) were cultured in vitro and treated with or without HDACi. Effects of HDACi on proliferation and cell cycle progress were analysed by BrdU assay and flow cytometry. Apoptosis was measured by quantifying mono- and oligonucleosomes in the cytoplasmic fraction of cell lysates. In vitro lymphangiogenesis was investigated using the Matrigel short term lymphangiogenesis assay. The effects of TSA on cell cycle regulatory proteins and apoptosis-related proteins were examined by western blotting, immunofluorescence staining and semi-quantitative RT-PCR. Protein- and mRNA half-life of p21 were analysed by western blotting and quantitative RT-PCR. The activity of the p21 promoter was determined using a dual luciferase assay and DNA-binding activity of Sp1/3 was investigated using EMSA. Furthermore, siRNA assays were performed to analyse the role of p21 and p53 on TSA-mediated anti-lymphangiogenic effects., Results: We found that HDACi inhibited cell proliferation and that the pan-HDACi TSA induced G0/G1 arrest in LEC. Cell cycle arrest was accompanied by up-regulation of p21, p27 and p53. Additionally, we observed that p21 protein accumulated in cellular nuclei after treatment with TSA. Moreover, we found that p21 mRNA was significantly up-regulated by TSA, while the protein and mRNA half-life remained largely unaffected. The promoter activity of p21 was enhanced by TSA indicating a transcriptional mechanism. Subsequent EMSA analyses showed increased constitutive Sp1/3-dependent DNA binding in response to HDACi. We demonstrated that p53 was not required for TSA induced p21 expression and growth inhibition of LECs. Interestingly, siRNA-mediated p21 depletion almost completely reversed the anti-proliferative effects of TSA in LEC. In addition, TSA induced apoptosis by cytochrome c release contributed to activating caspases-9, -7 and -3 and downregulating the anti-apoptotic proteins cIAP-1 and -2., Conclusions: In conclusion, we demonstrate that TSA - a pan-HDACi - has distinct anti-lymphangiogenic effects in primary human lymphatic endothelial cells by activating intrinsic apoptotic pathway and cell cycle arrest via p21-dependent pathways.

Shear-dependent inhibition of lymphatic thoracic duct (TD) contractility is principally mediated by nitric oxide (NO). Endothelial dysfunction and poor NO bioavailability are hallmarks of vasculature dysfunction in states of insulin resistance and metabolic syndrome (MetSyn). We tested the hypothesis that flow-dependent regulation of lymphatic contractility is impaired under conditions of MetSyn. We utilized a 7-wk high-fructose-fed male Sprague-Dawley rat model of MetSyn and determined the stretch- and flow-dependent contractile responses in an isobaric ex vivo TD preparation. TD diameters were tracked and contractile parameters were determined in response to different transmural pressures, imposed flow, exogenous NO stimulation by S-nitro-N-acetylpenicillamine (SNAP), and inhibition of NO synthase (NOS) by l-nitro-arginine methyl ester (l-NAME) and the reactive oxygen species (ROS) scavenging molecule 4-hydroxy-tempo (tempol). Expression of endothelial NO synthase (eNOS) in TD was determined using Western blot. Approximately 25% of the normal flow-mediated inhibition of contraction frequency was lost in TDs isolated from MetSyn rats despite a comparable SNAP response. Inhibition of NOS with l-NAME abolished the differences in the shear-dependent contraction frequency regulation between control and MetSyn TDs, whereas tempol did not restore the flow responses in MetSyn TDs. We found a significant reduction in eNOS expression in MetSyn TDs suggesting that diminished NO production is partially responsible for impaired flow response. Thus our data provide the first evidence that MetSyn conditions diminish eNOS expression in TD endothelium, thereby affecting the flow-mediated changes in TD lymphatic function., (Copyright © 2016 the American Physiological Society.)

Lymphatic leakage can be seen as a detrimental phenomenon associated with fluid retention and deposition as well as gain of weight. Moreover, lymphatic dysfunction is associated with an inflammatory environment and can be a substrate for other health conditions. A number of treatments can ameliorate lymphatic vasculature: natural substances have been used as treatment options particularly suitable for their consolidated effectiveness and safety profile. Here we report the protective effect of AdipoDren®, an association of a series of plant-derived natural complexes, on lymphatic endothelium permeability promoted by interleukin-1 beta (IL-1β) and the associated molecular mechanisms. AdipoDren® demonstrated a protective effect on dermal lymphatic endothelial cell permeability increased by IL-1β. Reduced permeability was due to the maintenance of tight junctions and cell-cell localisation of occludin and zonula occludens-1 (ZO-1). Moreover, AdipoDren® reduced the expression of the inflammatory key element cyclooxygenase-2 (COX-2), while not altering the levels of endothelial and inducible nitric oxide synthases (eNOS and iNOS). The upregulation of antioxidant enzymatic systems (catalase and superoxide dismutase-1, SOD-1) and the downregulation of pro-oxidant markers (p22 phox subunit of NADPH oxidase) were also evident. In conclusion, AdipoDren® would be useful to ameliorate conditions of altered lymphatic vasculature and to support the physiological functionality of the lymphatic endothelium., (© 2016 The Author(s) Published by S. Karger AG, Basel.)

Lymphangiogenesis, the formation of new lymph vessels, plays a significant role in the development and metastasis of various cancers. We and others have demonstrated that low molecular weight hyaluronan (LMW-HA) promotes lymphangiogenesis. However, the underlying mechanisms are poorly defined. In this study, using immunofluorescence and co-immunoprecipitation, we found that LMW-HA increased the colocalization of lymphatic vessel endothelial HA receptor (LYVE-1) and sphingosine 1-phosphate receptor (S1P3) at the cell surface. Silencing of either LYVE-1 or S1P3 decreased LMW-HA-mediated tube formation in lymphatic endothelial cells (LECs). Furthermore, silencing of either LYVE-1 or S1P3 significantly inhibited LMW-HA-induced tyrosine phosphorylation of Src kinase and extracellular signal-regulated kinase (ERK1/2). In summary, these results suggest that S1P3 and LYVE-1 may cooperate to play a role in LMW-HA-mediated lymphangiogenesis. This interaction may provide a useful target for the intervention of lymphangiogenesis-associated tumor progression., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

The shear stress applied to lymphatic endothelial cells (LEC) by lymph flow changes dramatically under normal conditions as well as in response to disease conditions and immune reactions. In general, LEC are known to regulate the contraction frequency and strength of lymphatic pumping in response to shear stress. Intracellular calcium concentration ([Ca(2+)]i) is an important factor that regulates lymphatic contraction characteristics. In this study, we measured changes in the [Ca(2+)]i under different shear stress levels and determined the source of this calcium signal. Briefly, human dermal LEC were cultured in custom-made microchannels for 3 days before loading with 2 µM fura-2 AM, a ratiometric calcium dye to measure [Ca(2+)]i. Step changes in shear stress resulted in a rapid increase in [Ca(2+)]i followed by a gradual return to the basal level and sometimes below the initial baseline (45.2 ± 2.2 nM). The [Ca(2+)]i reached a peak at 126.2 ± 5.6 nM for 10 dyn/cm(2) stimulus, whereas the peak was only 71.8 ± 5.4 nM for 1 dyn/cm(2) stimulus, indicating that the calcium signal depends on the magnitude of shear stress. Removal of the extracellular calcium from the buffer or pharmocological blockade of calcium release-activated calcium (CRAC) channels significantly reduced the peak [Ca(2+)]i, demonstrating a role of extracellular calcium entry. Inhibition of endoplasmic reticulum (ER) calcium pumps showed the importance of intracellular calcium stores in the initiation of this signal. In conclusion, we demonstrated that the shear-mediated calcium signal is dependent on the magnitude of the shear and involves ER store calcium release and extracellular calcium entry., (Copyright © 2015 the American Physiological Society.)

An apolar extract of the traditional medicinal plant Neurolaena lobata inhibited the expression of the NPM/ALK chimera, which is causal for the majority of anaplastic large cell lymphomas (ALCLs). Therefore, an active principle of the extract, the furanoheliangolide sesquiterpene lactone lobatin B, was isolated and tested regarding the inhibition of ALCL expansion and tumour cell intravasation through the lymphendothelium. ALCL cell lines, HL-60 cells and PBMCs were treated with plant compounds and the ALK inhibitor TAE-684 to measure mitochondrial activity, proliferation and cell cycle progression and to correlate the results with protein- and mRNA-expression of selected gene products. Several endpoints indicative for cell death were analysed after lobatin B treatment. Tumour cell intravasation through lymphendothelial monolayers was measured and potential causal mechanisms were investigated analysing NF-κB- and cytochrome P450 activity, and 12(S)-HETE production. Lobatin B inhibited the expression of NPM/ALK, JunB and PDGF-Rβ, and attenuated proliferation of ALCL cells by arresting them in late M phase. Mitochondrial activity remained largely unaffected upon lobatin B treatment. Nevertheless, caspase 3 became activated in ALCL cells. Also HL-60 cell proliferation was attenuated whereas PBMCs of healthy donors were not affected by lobatin B. Additionally, tumour cell intravasation, which partly depends on NF-κB, was significantly suppressed by lobatin B most likely due to its NF-κB-inhibitory property. Lobatin B, which was isolated from a plant used in ethnomedicine, targets malignant cells by at least two properties: I) inhibition of NPM/ALK, thereby providing high specificity in combating this most prevalent fusion protein occurring in ALCL; II) inhibition of NF-κB, thereby not affecting normal cells with low constitutive NF-κB activity. This property also inhibits tumour cell intravasation into the lymphatic system and may provide an option to manage this early step of metastatic progression., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Objectives: The knowledge of the basic principles of lymphatic function, still remains, to a large degree, rudimentary and will require significant research efforts. Recent studies of the physiology of the MLVs suggested the presence of an EDRF other than NO. In this study, we tested the hypothesis that lymphatic endothelium-derived histamine relaxes MLVs., Methods: We measured and analyzed parameters of lymphatic contractility in isolated and pressurized rat MLVs under control conditions and after pharmacological blockade of NO by L-NAME (100 μM) or/and histamine production by α-MHD (10 μM). Effectiveness of α-MHD was confirmed immunohistochemically. We also used immunohistochemical labeling and Western blot analysis of the histamine-producing enzyme, HDC. In addition, we blocked HDC protein expression in MLVs by transient transfection with vivo-morpholino oligos., Results: We found that only combined pharmacological blockade of NO and histamine production completely eliminates flow-dependent relaxation of lymphatic vessels, thus confirming a role for histamine as an EDRF in MLVs. We also confirmed the presence of HDC and histamine inside lymphatic endothelial cells., Conclusions: This study supports a role for histamine as an EDRF in MLVs., (© 2014 John Wiley & Sons Ltd.)

Objective: This study investigated the roles of the H1 and H2 histamine receptors, NO synthase, and sGC cyclase in histamine-induced modulation of rat mesenteric collecting lymphatic pumping., Methods: Isolated rat mesenteric collecting lymphatics were treated with 1- to 100-μM histamine. Histamine receptors were blocked with either the H1 antagonist mepyramine or the H2 antagonist cimetidine. The role of NO/sGC signaling was tested using the arginine analog L-NAME, the sGC inhibitor ODQ, and SNP as a positive control., Results: Histamine applied at 100 μM decreased tone and CF of isolated rat mesenteric collecting lymphatics. Pharmacologic blockade of either H1 or H2 histamine receptors significantly inhibited the response to histamine. Pretreatment with ODQ, but not L-NAME, completely inhibited the histamine-induced decrease in tone. ODQ pretreatment also significantly inhibited SNP-induced lymphatic relaxation., Conclusions: H1 and H2 histamine receptors are both involved in histamine-induced relaxation of rat mesenteric collecting lymphatics. NO synthesis does not appear to contribute to the histamine-induced response. However, sGC is critical for the histamine-induced decrease in tone and contributes to the drop in CF., (© 2014 John Wiley & Sons Ltd.)

Purpose: Podoplanin has been shown to be a reliable marker of lymphatic endothelium, but its role in the lymphatic system has not been well investigated. The purpose of this study is to investigate the role of podoplanin in lymphangiogenesis and macrophage functions under inflammatory conditions., Methods: Mouse corneal suture and ear section models were used to induce lymphangiogenesis and macrophage infiltration. Antilymphatic vessel endothelial hyaluronan-1 (Anti-LYVE-1) antibody was used to visualize lymphatic vessels. Thioglycollate-induced macrophages (mps) were collected and cultured with lipopolysaccharide (LPS), IFN-γ, and anti-mouse podoplanin antibody (PMab-1). Podoplanin, NF-κB, and mitogen-activated protein kinase (MAPK) pathway expression were detected by Western blot analysis. The TNF-α secretion was measured by ELISA., Results: Administration of PMab-1, reduced lymphangiogenesis in the corneal suture and ear wound healing models. Also, PMab-1 suppressed mps infiltration at the site of wound healing. Moreover, administration of PMab-1 led to a significant suppression of the rejection reaction in the corneal transplantation model. Our in vitro experiments showed that PMab-1 suppressed TNF-α secretion from mps under inflamed conditions, especially secretion caused by LPS stimulation. We confirmed the effect of PMab-1 on mps under inflamed conditions with a Western blot experiment, which clearly showed that the phosphorylation signal of the MAPK and NF-κB pathways was suppressed by PMab-1., Conclusions: Podoplanin neutralization resulted in inhibition of lymphatic growth associated with corneal and ear wound healing as well as mps inflammation. These data suggest that podoplanin is a novel therapeutic target for suppressing lymphangiogenesis and inflammation., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

Background: Cilostazol, an inhibitor of phosphodiesterase type III, is an antiplatelet agent and vasodilator. Some clinical reports have suggested that this drug can improve progressive and refractory lymphedema., Objective: In this study, we investigated whether cilostazol has the potential to proliferate lymphatic vessels and to improve lymphatic function using human lymphatic endothelial cells (LECs) and mouse lymphedema models., Methods: Human LECs were counted at several time points while they were cultured in the presence of cilostazol and/or protein kinase A inhibitor. After receiving a diet including 0.1% cilostazol or control diet, skin tissue and lymphatic function of k-cyclin transgenic (kCYC(+/-)) mice, which have pernicious lymphatic dysfunction, was analyzed. A different lymphedema model was generated in wild type mice by excising circumferential tail skin to remove the superficial lymphatics. After oral administration of cilostazol, tail lymphedema was examined in this mouse model., Results: Proliferation of LECs was promoted in a dose-dependent manner, which was partially inhibited by a protein kinase A inhibitor. Lymphatic vessel count increased in the cilostazol-treated kCYC(+/-) mice over that in the non-treated mice. Lymph flow improved in cilostazol-fed kCYC(+/-) mice as assessed by subcutaneous injection of Evans blue dye into the footpad. Oral administration of cilostazol also decreased lymphedema in a tail of wild type mice., Conclusion: Cilostazol promoted growth of human LECs and improved lymph flow and lymphedema in two different mouse lymphedema models. These results suggest that cilostazol would be a promising agent for the treatment of lymphedema., (Copyright © 2014 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.)

Purpose: Ranibizumab (Lucentis(®) ) is a Fab-Fragment of a recombinant, humanized, monoclonal VEGF (anti-vascular endothelial growth factor) antibody. This study analyzed the ability of topical Ranibizumab to inhibit lymphangiogenesis in addition to hemangiogenesis after acute corneal inflammation in vivo. In addition, the effect of Ranibizumab on the proliferation of human lymphatic endothelial cells (LECs) and blood endothelial cells (BECs) in vitro was studied., Methods: The inhibitory effect of Ranibizumab on LECs and BECs was studied in vitro using a proliferation enzyme-linked immunosorbent assay (ELISA) assay. To study the in vivo effects of Ranibizumab, the mouse model of suture induced inflammatory corneal neovascularization was used. Study mice received topical Ranibizumab as eye drops. After 1 week excised corneas were stained with LYVE-1 and CD31. Hemangiogenesis and lymphangiogenesis were analyzed morphometrically by using a semiautomatic method based on the image analyzing program Cell^F., Results: An antiproliferative effect of Ranibizumab was seen in vitro on both human BECs and LECs with a significance of p < 0.0001 and p < 0.0004, respectively. In vivo experiments showed that topical application of Ranibizumab significantly inhibits both hemangiogenesis (p = 0.0026) and lymphangiogenesis (p = 0.0026) in the cornea., Conclusion: Ranibizumab is a potent inhibitor of inflammatory corneal hemangiogenesis and lymphangiogenesis in vivo with a direct inhibitory effect on both endothelial cell types in vitro. This study for the first time demonstrates an inhibitory effect of Ranibizumab on lymphatic vessels which could have a wider range of clinical applications., (© 2012 The Authors. Acta Ophthalmologica © 2012 Acta Ophthalmologica Scandinavica Foundation.)

Purpose of Review: The review summarizes the current knowledge of the roles played by the vascular and lymphatic endothelium throughout the gut in the pathogenesis of inflammatory bowel disease (IBD) and gives an update on emerging strategies targeting both vasculatures., Recent Findings: Enormous efforts have been made to understand the mechanisms underlining the origin, development and maintenance of intestinal chronic inflammation. In particular, new studies focused their attention on the role played by the microvascular and lymphatic endothelium in the pathogenesis of IBD. During inflammation, whereas the microvasculature is responsible for the entry and distribution of immune cells in the mucosa, the lymphatic system controls leukocyte exit, bacterial clearance and edema absorption. The study of these events, which are aberrant during chronic inflammation, has resulted in the identification and validation of several targets for the treatment of experimental colitis, some of which have translated into effective treatments for patients with IBD., Summary: Although much attention has been paid to the microvascular endothelium and to antiangiogenic therapies, specific studies on the lymphatic vasculature and its functions in IBD are still at the initial stage, and other molecular mechanisms, genes, molecules and new pathways must definitely be explored.

Lymphatic vessels are thought to contribute to metastasis primarily by serving as a transportation system. It is widely believed that tumor cells enter lymph nodes passively by the flow of lymph. We demonstrate that lymph node lymphatic sinuses control tumor cell entry into the lymph node, which requires active tumor cell migration. In human and mouse tissues, CCL1 protein is detected in lymph node lymphatic sinuses but not in the peripheral lymphatics. CCR8, the receptor for CCL1, is strongly expressed by human malignant melanoma. Tumor cell migration to lymphatic endothelial cells (LECs) in vitro is inhibited by blocking CCR8 or CCL1, and recombinant CCL1 promotes migration of CCR8(+) tumor cells. The proinflammatory mediators TNF, IL-1β, and LPS increase CCL1 production by LECs and tumor cell migration to LECs. In a mouse model, blocking CCR8 with the soluble antagonist or knockdown with shRNA significantly decreased lymph node metastasis. Notably, inhibition of CCR8 led to the arrest of tumor cells in the collecting lymphatic vessels at the junction with the lymph node subcapsular sinus. These data identify a novel function for CCL1-CCR8 in metastasis and lymph node LECs as a critical checkpoint for the entry of metastases into the lymph nodes.

Background: Many cancers spread through lymphatic routes, and mechanistic insights of tumour intravasation into the lymphatic vasculature and targets for intervention are limited. The major emphasis of research focuses currently on the molecular biology of tumour cells, while still little is known regarding the contribution of lymphatics., Methods: Breast cancer cell spheroids attached to lymphendothelial cell (LEC) monolayers were used to investigate the process of intravasation by measuring the areas of 'circular chemorepellent-induced defects' (CCID), which can be considered as entry gates for bulky tumour intravasation. Aspects of tumour cell intravasation were furthermore studied by adhesion assay, and siRNA-mediated knockdown of intracellular adhesion molecule-1 (ICAM-1). Replacing cancer spheroids with the CCID-triggering compound 12(S)-hydroxyeicosatetraenoic acid (HETE) facilitated western blot analyses of Bay11-7082- and baicalein-treated LECs., Results: Binding of LECs to MCF-7 spheroids, which is a prerequisite for CCID formation, was mediated by ICAM-1 expression, and this depended on NF-κB and correlated with the expression of the prometastatic factor S100A4. Simultaneous inhibition of NF-κB with Bay11-7082 and of arachidonate lipoxygenase (ALOX)-15 with baicalein prevented CCID formation additively., Conclusion: Two mechanisms contribute to CCID formation: ALOX15 via the generation of 12(S)-HETE by MCF-7 cells, which induces directional migration of LECs, and ICAM-1 in LECs under control of NF-κB, which facilitates adhesion of MCF-7 cells to LECs.

Background: As metastasis is the prime cause of death from malignancies, there is vibrant interest to discover options for the management of the different mechanistic steps of tumour spreading. Some approved pharmaceuticals exhibit activities against diseases they have not been developed for. In order to discover such activities that might attenuate lymph node metastasis, we investigated 225 drugs, which are approved by the US Food and Drug Administration., Methods: A three-dimensional cell co-culture assay was utilised measuring tumour cell-induced disintegrations of the lymphendothelial wall through which tumour emboli can intravasate as a limiting step in lymph node metastasis of ductal breast cancer. The disintegrated areas in the lymphendothelial cell (LEC) monolayers were induced by 12(S)-HETE, which is secreted by MCF-7 tumour cell spheroids, and are called 'circular chemorepellent induced defects' (CCIDs). The putative mechanisms by which active drugs prevented the formation of entry gates were investigated by western blotting, NF-κB activity assay and by the determination of 12(S)-HETE synthesis., Results: Acetohexamide, nifedipin, isoxsuprine and proadifen dose dependently inhibited the formation of CCIDs in LEC monolayers and inhibited markers of epithelial-to-mesenchymal-transition and migration. The migration of LECs is a prerequisite of CCID formation, and these drugs either repressed paxillin levels or the activities of myosin light chain 2, or myosin-binding subunit of myosin phosphatase. Isoxsuprine inhibited all three migration markers, and isoxsuprine and acetohexamide suppressed the synthesis of 12(S)-HETE, whereas proadifen and nifedipin inhibited NF-κB activation. Both the signalling pathways independently cause CCID formation., Conclusion: The targeting of different mechanisms was most likely the reason for synergistic effects of different drug combinations on the inhibition of CCID formation. Furthermore, the treatment with drug combinations allowed also a several-fold reduction in drug concentrations. These results encourage further screening of approved drugs and their in vivo testing.

In treatment of various-aetiology chronic oedemas, of great importance is a possibility of exerting an influence on the resorptional, transportation and transmission functions of the lymphatic system, as well as on the microcirculatory blood channel, because they all maintain the balance of fluid exchange in tissues. There exist various causes of chronic oedema, which requires a differentiated approach to administration of bioflavonoids possessing different mechanisms of antioedemic action. With this in mind we carried out experimental histomorphometric studies on 24 rats, aimed at determining variously directed effects of agents belonging to the class of bioflavonoids - phlebodia 600 (diosmin) and troxerutin on regeneration ability of the lymphatic endothelium and permeability of blood capillaries. It was determined that phlebodia 600 exerts a direct influence upon the functional state of the lymphatic system, activating proliferation of the lymphatic endothelium by means of gemmation, which leads to the formation of new capillary lymphatic networks with the resulting increase in both the total absorption area of the lymphatic capillary networks and the volume of lymph reabsorption. Troxerutin acts predominantly on the endothelium of blood capillaries, decreasing permeability in the arterial segment of the capillary, thus lowering the total volume of fluid in the interstitial space and accordingly the load on the lymphatic system.

Objective: We studied the effects of S1P on the diameter and spontaneous contraction of murine iliac collecting lymph vessels., Methods: The isolated lymph vessel was cannulated with two glass micropipettes and then pressurized to 4 cmH(2) O at the intraluminal pressure. The changes in lymph vessel diameter were measured using a custom-made diameter-detection device. Immunohistochemical studies were also performed to confirm S1P receptors on the lymph vessels., Results: S1P (10(-7) M) had no significant effect on the frequency or amplitude of the lymph vessels' spontaneous contractions. In contrast, S1P (10(-8) -10(-6) M) produced a concentration-related reduction in lymph vessel diameter (tonic contraction). Pretreatment with 10(-4) M l-NAME or 10(-5) M aspirin had no significant effect on the S1P-induced tonic contraction of the lymph vessels. To evaluate the intracellular signal transduction pathway responsible for the S1P-induced tonic contractions and their Ca(2+) -dependence, we investigated the effects of JTE013, VPC23019, U-73122, xestospongin C, and nifedipine on the S1P-induced tonic contractions. All of these inhibitors except VPC23019 and nifedipine significantly reduced the S1P-induced tonic contractions. S1P (5x10(-7) M) also induced significant tonic contractions in the lymph vessels that had been superfused with high K(+) Krebs-bicarbonate solution or Ca(2+) -free high K(+) Krebs solution containing 1 mM EGTA. S1P2 receptors were immunohistochemically detected in the lymph vessels., Conclusion: These findings suggest that neither endogenous NO nor prostaglandins are involved in the S1P-induced tonic contraction of lymph vessels, which is mainly caused by Ca(2+) release from intracellular Ca(2+) stores through the activation of S1P2 and 1,4,5 IP(3) receptors., (© 2012 John Wiley & Sons Ltd.)

Lymph node metastasis of tumors is a crucial early step in the metastatic process. Tumor lymphangiogenesis plays an important role in promoting tumor metastasis to regional lymph nodes. Norcantharidin (NCTD) has been reported to possess potent anti-angiogenesis and antitumor properties in several cell lines and xenograft tumor models. However, its role in tumor-associated lymphangiogenesis and lymphatic metastasis remains unclear. Here, we investigated the effect of NCTD on proliferation, apoptosis, migration, invasion and the lymphatic tube formation, lymphangiogenesis, of human lymphatic endothelial cells (HLECs) in vitro by MTT, proliferation assay, Hoechst staining and flow cytometry, scraping line method, Matrigel invasion assay, inverted or fluorescence microscope and transmission electron microscope. Moreover, the underlying mechanisms, such as VEGF-C, VEGF-D, VEGFR-3 at protein and mRNA levels in lymphangiogenesis using 3-dimensional (3-D) culture of HLECs were measured by immunohistochemistry, western blotting and real-time polymerase chain reaction (RT-PCR). It was shown that NCTD inhibited proliferation, migration, invasion and lymphatic tube formation (forming-lymphatic and/or formed-lymphatic) of HLECs, induced HLEC apoptosis (all P<0.01) significantly, in a dose- and time-dependent manner (IC50 6.8 µg/ml); and downregulated the expression of VEGF-C, VEGF-D and VEGFR-3 at protein or/and mRNA levels (P<0.01) in HLEC lymphatic tube formation. Thus, we identified for the first time that NCTD inhibited HLEC lymphangiogenesis by simultaneously blocking VEGF-C and VEGF-D/VEGFR-3 in vitro. The present findings may be of importance to explore the therapeutical target or strategy of NCTD for tumor lymphangiogenesis and lymphatic metastasis.

Dendritic cell (DC) migration via lymphatic vessels to draining lymph nodes (dLNs) is crucial for the initiation of adaptive immunity. We imaged this process by intravital microscopy (IVM) in the ear skin of transgenic mice bearing red-fluorescent vasculature and yellow-fluorescent DCs. DCs within lymphatic capillaries were rarely transported by flow, but actively migrated within lymphatics and were significantly faster than in the interstitium. Pharmacologic blockade of the Rho-associated protein kinase (ROCK), which mediates nuclear contraction and de-adhesion from integrin ligands, significantly reduced DC migration from skin to dLNs in steady-state. IVM revealed that ROCK blockade strongly reduced the velocity of interstitial DC migration, but only marginally affected intralymphatic DC migration. By contrast, during tissue inflammation, ROCK blockade profoundly decreased both interstitial and intralymphatic DC migration. Inhibition of intralymphatic migration was paralleled by a strong up-regulation of ICAM-1 in lymphatic endothelium, suggesting that during inflammation ROCK mediates de-adhesion of DC-expressed integrins from lymphatic-expressed ICAM-1. Flow chamber assays confirmed an involvement of lymphatic-expressed ICAM-1 and DC-expressed ROCK in DC crawling on lymphatic endothelium. Overall, our findings further define the role of ROCK in DC migration to dLNs and reveal a differential requirement for ROCK in intralymphatic DC crawling during steady-state and inflammation.

Endothelial cells of initial lymphatics have discontinuous button-like junctions (buttons), unlike continuous zipper-like junctions (zippers) of collecting lymphatics and blood vessels. Buttons are thought to act as primary valves for fluid and cell entry into lymphatics. To learn when and how buttons form during development and whether they change in disease, we examined the appearance of buttons in mouse embryos and their plasticity in sustained inflammation. We found that endothelial cells of lymph sacs at embryonic day (E)12.5 and tracheal lymphatics at E16.5 were joined by zippers, not buttons. However, zippers in initial lymphatics decreased rapidly just before birth, as buttons appeared. The proportion of buttons increased from only 6% at E17.5 and 12% at E18.5 to 35% at birth, 50% at postnatal day (P)7, 90% at P28, and 100% at P70. In inflammation, zippers replaced buttons in airway lymphatics at 14 and 28 days after Mycoplasma pulmonis infection of the respiratory tract. The change in lymphatic junctions was reversed by dexamethasone but not by inhibition of vascular endothelial growth factor receptor-3 signaling by antibody mF4-31C1. Dexamethasone also promoted button formation during early postnatal development through a direct effect involving glucocorticoid receptor phosphorylation in lymphatic endothelial cells. These findings demonstrate the plasticity of intercellular junctions in lymphatics during development and inflammation and show that button formation can be promoted by glucocorticoid receptor signaling in lymphatic endothelial cells., (Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background: Loco-regional spread of disease causes high morbidity and is associated with the poor prognosis of malignant oral tumors. Better understanding of mechanisms underlying the establishment of lymph node metastasis is necessary for the development of more effective therapies for patients with oral cancer. The aims of this work were to evaluate a possible correlation between endothelial cell Bcl-2 and lymph node metastasis in patients with oral squamous cell carcinoma (OSCC), and to study signaling pathways that regulate Bcl-2 expression in lymphatic endothelial cells., Methods: Endothelial cells were selectively retrieved from paraffin-embedded tissue sections of primary human OSCC from patients with or without lymph node metastasis by laser capture microdissection. RT-PCR was used to evaluate Bcl-2 expression in tumor-associated endothelial cells and in tumor cells. In vitro, mechanistic studies were performed to examine the effect of vascular endothelial growth factor (VEGF)-C on the expression of Bcl-2 in primary human lymphatic endothelial cells., Results: We observed that Bcl-2 expression is upregulated in the endothelial cells of human oral tumors with lymph node metastasis as compared to endothelial cells from stage-matched tumors without metastasis. VEGF-C induced Bcl-2 expression in lymphatic endothelial cells via VEGFR-3 and PI3k/Akt signaling. Notably, OSCC cells express VEGF-C and induce Bcl-2 in lymphatic endothelial cells., Conclusions: Collectively, this work unveiled a mechanism for the induction of Bcl-2 in lymphatic endothelial cells and suggested that endothelial cell Bcl-2 contributes to lymph node metastasis in patients with oral squamous cell carcinoma., (© 2011 John Wiley & Sons A/S.)

Angiopoietin (Ang)-2, a ligand of the receptor tyrosine kinase Tie2, is known to be involved in the regulation of embryonic lymphangiogenesis. However, the role of Ang-2 in postnatal pathological lymphangiogenesis, such as inflammation, is largely unknown. We used a combination of imaging, molecular, and cellular approaches to investigate whether Ang-2 is involved in inflammatory lymphangiogenesis. We observed strong and continuous expression of Ang-2 on newly generated lymphatic vessels for 2 wk in sutured corneas of BALB/c mice. This expression was concurrent with an increased number of lymphatic vessels. TNF-α expression also increased, with peak TNF-α expression occurring before peak Ang-2 expression was reached. In vitro experiments showed that TNF-α stimulates Ang-2 and Tie2 and ICAM-1 expression on human lymphatic endothelial cells (LECs) and blood vascular endothelial cells (BECs). Ang-2 alone did not affect the biological behavior of LECs, whereas Ang-2 combined with TNF-α significantly promoted the proliferation of LECs but not BECs. In mouse models, blockade of Ang-2 with L1-10, an Ang-2-specific inhibitor, significantly inhibited lymphangiogenesis but promoted angiogenesis. These results clearly indicate that Ang-2 acts as a crucial regulator of inflammatory lymphangiogenesis by sensitizing the lymphatic vasculature to inflammatory stimuli, thereby directly promoting lymphangiogenesis. The involvement of Ang-2 in inflammatory lymphangiogenesis provides a strong rationale for the exploitation of anti-Ang-2 treatment in the prevention and treatment of tumor metastasis and transplant rejection.

Donor-derived mesenchymal stem cells (MSC) can induce long-term acceptance in a rat heart transplantation model when injected prior to transplantation in combination with mycophenolate mofetil (MMF). In contrast, MSC alone cause accelerated graft rejection. To better understand these conflicting data we studied the effects of MSC and MMF on lymphocyte populations in heart allografts and secondary lymphatic organs. Allogeneic MSC injected prior to transplantation are immunogenic in this model because activated CD4+ and CD8+ cells emerged earlier in secondary lymphatic organs of MSC- and MSC/MMF-treated animals, compared to animals not treated with MSC. Consequently T cells infiltrated the grafts of MSC-only treated animals promptly causing accelerated graft rejection. However, few T cells or antigen-presenting cells (APC) infiltrated the grafts of animals treated with MSC and MMF. Consistent with this finding, intercellular adhesion molecule 1 (ICAM-1) and E-selectin was down-regulated exclusively in MSC/MMF-treated grafts, indicating that MSC together with MMF interfere with endothelial activation. Additionally, the presence of interferon-gamma (IFN-γ) enhanced MSC capabilities to suppress T cell proliferation in vitro. Interestingly, MMF did not influence serum IFN-γ levels in vivo. Together, our data indicate that MSC pre-activate T cells, but co-treatment with MMF eliminates these T cells, decreases intragraft APC and T cell trafficking by inhibiting endothelial activation, and allows IFN-γ stimulation of suppressive MSC., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Objectives: To analyze whether topical application of corticosteroids inhibits inflammatory corneal lymphangiogenesis and to study the potential underlying antilymphangiogenic mechanisms., Methods: Inflammatory corneal neovascularization was induced by suture placement, and the corneas were then treated with topical fluorometholone, prednisolone acetate, or dexamethasone sodium phosphate. After 1 week, the corneas were stained with lymphatic vessel endothelial hyaluronan receptor 1 for detection of pathological corneal lymphangiogenesis. The effect of these corticosteroids on macrophage recruitment was assessed via fluorescence-activated cell sorting analysis. The effect of these corticosteroids on proinflammatory cytokine expression by peritoneal exudate cells was tested via real-time polymerase chain reaction. Furthermore, the effect of steroid treatment on the proliferation of lymphatic endothelial cells was assessed via enzyme-linked immunosorbent assay., Results: Treatment with corticosteroids resulted in a significant reduction of inflammatory corneal lymphangiogenesis. The antilymphangiogenic effect of fluorometholone was significantly weaker than that of prednisolone and dexamethasone. Corneal macrophage recruitment was also significantly inhibited by the application of topical steroids. Treatment of peritoneal exudate cells with corticosteroids led to a significant downregulation of the RNA expression levels of tumor necrosis factor and interleukin 1β. Additionally, proliferation of lymphatic endothelial cells was also inhibited., Conclusions: Corticosteroids are strong inhibitors of inflammatory corneal lymphangiogenesis, with significant differences between various corticosteroids in terms of their antilymphangiogenic potency. The main mechanism of the antilymphangiogenic effect seems to be through the suppression of macrophage infiltration, proinflammatory cytokine expression, and direct inhibition of proliferation of lymphatic endothelial cells., Clinical Relevance: Steroids block corneal lymphangiogenesis, the main risk factor for immune rejections after corneal transplantation. The different antilymphangiogenic potency of these drugs should be taken into account when using steroids in clinical practice (eg, after keratoplasty).

Lymphatic vessel growth requires extensive remodeling of the extracellular matrix, a process hypothesized to be related to the expression and function of the matrix metalloproteinases. We used a protein based screening strategy to demonstrate increased matrix matalloproteinase-10 expression in human lymphatic endothelial cells undergoing collagen I induced tubulogenesis. Knock-down experiments showed that matrix metalloproteinase-10 regulated lymphatic endothelial cell tubulogenesis. β1 integrin signaling via the ERK/MAPK pathway increased matrix metalloproteinase-10 mRNA and protein expression in human lymphatic endothelial cells. These findings demonstrate a novel mechanism by which β1 integrin regulates matrix metalloproteinase-10 expression during lymphatic vessel remodeling., (Copyright © 2011 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.)

To investigate if local lymphoid tissues are a target of FA, nasopharynx-associated lymphoid tissues (NALT) and upper-respiratory tract-draining lymph nodes were examined in a 28-day inhalation study with FA vapor in Fischer-344 rats and B6C3F1 mice. Paraffin-embedded tissues were sectioned and stained with H&E or stained immunohistochemically for cell proliferation (BrdU incorporation). Light microscopy revealed simple hyperplasia of NALT lymphoepithelium of rats exposed to 15 ppm and an increased proliferation rate of the epithelial cells. Principal component (discriminant) analysis of rat NALT and lymph nodes data did not reveal other effects or effects at lower exposure levels. Mice tissues were not affected. It was concluded that hyperplasia of the lymphoepithelium of NALT of rats exposed to 15 ppm was the only distinct effect of FA vapor on local lymphoid tissues (NALT and lymph nodes) of Fischer-344 rats and B3C3F1 mice., (Copyright © 2009 Elsevier GmbH. All rights reserved.)

There is growing evidence that vascular endothelial growth factor-A (VEGF-A), a ligand of the receptor tyrosine kinases VEGFR1 and VEGFR2, promotes lymphangiogenesis. However, the underlying mechanisms by which VEGF-A induces the growth of lymphatic vessels remain poorly defined. Here we report that VEGFR2, not VEGFR1, is the primary receptor regulating VEGF-A-induced lymphangiogenesis. We show that specific inhibition of VEGF-A/VEGFR2 signaling with the fully human monoclonal antibody r84 significantly inhibits lymphangiogenesis in MDA-MB-231 tumors. In vitro experiments with primary human dermal lymphatic endothelial cells (LECs) demonstrate that blocking VEGF-A activation of VEGFR2, not VEGFR1, significantly inhibits VEGF-A-induced proliferation and migration of LECs. We show that VEGF-A stimulation of LECs leads to the phosphorylation of VEGFR2 (Tyr 951, 1054, 1059, 1175, and 1214) which subsequently triggers PKC dependent phosphorylation of ERK1/2 and PI3-K dependent phosphorylation of Akt. Additionally, we demonstrate that inhibitors that suppress the phosphorylation of ERK1/2 and Akt significantly block VEGF-A- induced proliferation and migration of LECs. Together, these results shed light on the mechanisms regulating VEGF-A-induced proliferation and migration of LECs, reveal that VEGFR2 is the primary signaling VEGF-A receptor on lymphatic endothelium, and suggest that therapeutic agents targeting the VEGF-A/VEGFR2 axis could be useful in blocking the pathological formation of lymphatic vessels.

Background: Inflammatory cytokines dysregulate microvascular function, yet how cytokines affect lymphatic endothelial cells (LEC) are unclear., Methods and Results: We examined effects of TNF-α, IL-1 beta, and IFN-gamma on LEC proliferation, endothelial cell adhesion molecule (ECAM) expression, capillary formation, and barrier changes in murine (SV-LEC) and human LECs (HMEC-1a)., Results: All cytokines induced ICAM-1, VCAM-1, MAdCAM-1, and E-selectin in SV-LECs; TNF-α, IL-1 beta; and IFN-gamma induced ECAMs (but not MAdCAM-1) in HMEC-1a. IL-1 beta increased, while IFN-gamma and TNF-α reduced SV-LEC proliferation. While TNF-α induced, IFN-gamma decreased, and IL-1 beta did not show any effect on HMEC-1a proliferation. TNF-α, IL-1 beta, and IFN-gamma each reduced capillary formation in SV-LEC and in HMEC-1a. TNF-α and IL-1 beta reduced barrier in SV-LEC and HMEC-1a; IFN-gamma did not affect SV-LEC barrier, but enhanced HMEC-1a barrier. Inflammatory cytokines alter LEC growth, activation and barrier function in vitro and may disturb lymphatic clearance increasing tissue edema in vivo., Conclusion: Therapies that maintain or restore lymphatic function (including cytokines blockade), may represent important strategies for limiting inflammation.

Background: To characterize lymphangiogenesis in early-stage hamster tongue carcinoma development, morphological features and spatial relationships of lymphatic vessels., Methods: Lymphatic vessels were examined histochemically, using 5'-Nase-ALPase enzyme and combined light and electron microscopy to measure lymphatic vessel area (LVA) and lymphatic vessel density (LVD)., Results: In atypical hyperplastic tissues, LVA was found to be 1429.97 and LVD was found to be 39, in carcinoma in situ LVA was 2538.33 and LVD was 48, and in micro-invasive carcinoma LVA was 5733.74 and LVD was 59. Increased lymphangiogenesis was seen in pre-neoplastic states and in early-stage oral squamous cell carcinoma (OSCC). Small regular lymphatic vessels predominated in atypical hyperplasia, and large, irregular lymphatic vessels in early-stage OSCC. Lymphatic endothelial vessels were stretched and porous over large areas., Conclusions: Newly formed lymphatics and patulous intercellular junctions may be optimally suited for tumor cell metastasis through lymphatic channels in early- and middle-phase carcinogenesis. Lymphatic capillary LVA and LVD became enlarged, and positively correlated, with malignancy, but show no correlation with 7,12-dimethylbenz[a]anthracene-induced time.

Vitamin E, a micronutrient (comprising alpha-, beta-, gamma- and delta-tocopherols, alpha-, beta-, gamma- and delta-tocotrienols), has documented antioxidant and non-antioxidant effects, some of which inhibit inflammation and angiogenesis. We compared the abilities of alpha-, gamma- and delta-tocopherols to regulate human blood cytotoxicity (BEC) and lymphatic endothelial cytotoxicity (LEC), proliferation, invasiveness, permeability, capillary formation and suppression of TNF-alpha-induced VCAM-1 as in vitro models of inflammatory angiogenesis. alpha-, gamma- and delta-tocopherols were not toxic to either cell type up to 40 microM. In BEC, confluent cell density was decreased by all concentrations of delta- and gamma-tocopherol (10-40 microM) but not by alpha-tocopherol. LEC showed no change in cell density in response to tocopherols. delta-Tocopherol (40 microM), but not other isomers, decreased BEC invasiveness. In LEC, all doses of gamma-tocopherol, as well as the highest dose of alpha-tocopherol (40 microM), decreased cell invasiveness. delta-Tocopherol had no effect on LEC invasiveness at any molarity. delta-Tocopherol dose dependently increased cell permeability at 48 h in BEC and LEC; alpha- and gamma-tocopherols showed slight effects. Capillary tube formation was decreased by high dose (40 microM) concentrations of alpha-, gamma- and delta-tocopherol, but showed no effects with smaller doses (10-20 microM) in BEC. gamma-Tocopherol (10-20 microM) and alpha-tocopherol (10 microM), but not delta-tocopherol, increased LEC capillary tube formation. Lastly, in BEC, alpha-, gamma- and delta-tocopherol each dose-dependently reduced TNF-alpha-induced expression of VCAM-1. In LEC, there was no significant change to TNF-alpha-induced VCAM-1 expression with any concentration of alpha-, gamma- or delta-tocopherol. These data demonstrate that physiological levels (0-40 microM) of alpha-, gamma- and delta-tocopherols are nontoxic and dietary tocopherols, especially delta-tocopherol, can limit several BEC and LEC endothelial behaviors associated with angiogenesis. Tocopherols may therefore represent important nutrient-signals that limit cell behaviors related to inflammation/angiogenesis, which when deficient, may predispose individuals to risks associated with elevated angiogenesis such as inflammation and cancer; further differences seen from the tocopherols may be due to their blood or lymphatic cell origin., ((c) 2010 Elsevier Inc. All rights reserved.)

Purpose: Compounds regulating intracellular thiol redox status, such as N,N-diacetyl-L-cystine dimethylester (NM(2)), were shown to prolong corneal graft survival in a penetrating keratoplasty (PKP) model. However, the effect of NM(2) on hemangiogenesis and lymphangiogenesis has not been investigated. The effect of manipulating ambient thiol redox status on riskier (higher rejection rate) transplantation models, such as limbal graft survival and hemangiogenesis and lymphangiogenesis in a corneal suture model, were investigated., Methods: C57BL/10 mice that received BALB/c corneas were treated by subconjunctival injection of NM(2), and limbal graft survival was assessed. Sutured C57BL/6 received daily intraperitoneal injections of NM(2), glutathione diethylester (GSH-OEt), or PBS. Lymphatic endothelial cell (LEC) and peritoneal mps were treated with NM(2) or GSHOEt, and then VEGFR3, neuropilin-2, podoplanin, and LYVE-1 expression were analyzed. Supernatants were collected for analysis of TNF-alpha and VEGF-A levels by ELISA., Results: Significantly less cellular infiltration was detected in mice with corneal limbal transplant-treated NM(2)-treated mice. Hemangiogenesis and lymphangiogenesis were suppressed in the NM(2)-treated mice. NM(2) treatment of mps led to reduced levels of VEGFR3, neuropilin-2, podoplanin, and LYVE-1 expression compared with PBS- or GSHOEt- treated mps, lower levels of TNF-alpha, and increased secretion of VEGF. Moreover, NM(2)-treated LECs had reduced levels of LYVE-1 and Prox-1., Conclusions: Reduction of ambient redox status reduced inflammatory cell infiltrates. Consequently, reduced inflammatory response might have contributed to both the observed prolonged corneal limbal graft survival and the attenuated hemangiogenesis and lymphangiogenesis in cornea.

To clarify the roles of lymphatic endothelial cells (LEC) in the regulation of endothelial constitutive nitric oxide synthase (ecNOS) expression, we examined the effects of shear stress on ecNOS immunohistochemical staining and mRNA and protein expression in human LEC as well as on ATP release from these cells. Shear stress at 0.5 or 1.0 dyn/cm(2) increased ecNOS immunohistochemical staining and ecNOS mRNA and protein expression in cultured LEC. The same strength of shear stress produced a significant release of ATP from the LEC. Exogenous ATP ranging in concentration from 10(-9) to 10(-6) M produced a significant increase in ecNOS immunohistochemical expression in a dose-dependent manner. The increase in ecNOS expression mediated by 10(-6)M ATP was significantly reduced by 10(-5) M suramin. Suramin (10(-5) M) caused a significant reduction in the shear stress-mediated increases in ecNOS immunohistochemical staining and mRNA expression. The shear stress-mediated increases in ecNOS expression were significantly reduced by 3 mM tetraethylammonium, 10(-4) M apamin, 10(-9) M iberiotoxin, 10(-5) M 2-aminoethoxydephenyl borate, or 10(-5)M xestospongin C, but not 10(-5) M glybenclamide or 10(-5) M nifedipine. The shear stress-mediated increases in ecNOS expression were significantly potentiated by pinacidil or NS1619 in a dose-dependent manner. The immunohistochemical expression of small- (SK(Ca)) and big-conductance (BK(Ca)) Ca(2+)-activated K(+) channels was confirmed on the surfaces of human LEC. These findings suggest that shear stress produces a significant release of ATP from LEC, which activates the purinergic P2X/2Y receptor, thereby facilitating ecNOS mRNA and protein expression through inositol 1,4,5-trisphosphate-mediated release of intracellular Ca(2+) ions and the activation of Ca(2+)-activated K(+) channels in LEC.

Adrenomedullin (AM) and gastrin releasing peptide (GRP) are neuroendocrine peptides that have been previously implicated as regulators of angiogenesis and lymphangiogenesis. Using an immortalized human dermal microvascular lymphatic endothelial cell line stably transfected with red fluorescent protein (LEC/RFP), we demonstrate the ability of AM and GRP to augment tube formation complexity of this target cell in a dose-dependent manner. Maximum tube density was initiated at 1 nM for both peptides, and as concentrations exceeded 10 nM a decrease in tube formation was noted, hence following a classic rise/fall biological response curve. In addition, we show that appropriate small molecule mimetics to neutralizing monoclonal antibodies of AM or GRP, at 1 microM concentration, can function to either inhibit (antagonist) or enhance (super agonist) peptide-induced tube formation of LEC/RFP. Our small molecule reagents by themselves have no activity, but in the presence of their respective peptides can mediate a positive or negative response, hence the super agonist designation. These compounds represent new regulatory drugs of the lymphatic system with possible patient application in the clinical management of edema and metastatic disease.

IL-20 is an arteriogenic cytokine that remodels collateral networks in vivo, and plays a role in cellular organization. Here, we investigate its role in lymphangiogenesis using a lymphatic endothelial cell line, hTERT-HDLEC, which expresses the lymphatic markers LYVE-1 and podoplanin. Upon stimulation of hTERT-HDLEC with IL-20, we found an increase in the intracellular free calcium concentration, in Akt and eNOS phosphorylations as well as in perinuclear NO production. We found that eNOS phosphorylation and NO synthesis are highly dependent on the PI3K/Akt signalling pathway. We also found an IL-20 induced phosphorylation of Erk1/2 and mTOR, and using the MEK inhibitor PD98059 and mTOR complex inhibitor rapamycin we demonstrated the importance of these signalling pathways in IL-20-mediated proliferation. IL-20 triggered actin polymerization and morphological changes resulting in elongated cell structures, and in matrigels, IL-20 caused tube formations of hTERT-HDLEC in a PI3K- and mTOR dependent way. In a sprouting assay we found that IL-20 caused cell migration within 24 h at a rate comparable to VEGF-C, and this migration could be inhibited by wortmannin and rapamycin. These data show that IL-20 activates cell signalling resulting in lymphangiogenic processes including migration, proliferation and tube formation. Thus, IL-20 is a cytokine that has the potential of activating or modulating the formation of lymphatic vessels.

NFATc1 transcription factor is critical for lineage selection in T-cell differentiation, cardiac valve morphogenesis and osteoclastogenesis. We identified a role for calcineurin-NFAT signaling in lymphatic development and patterning. NFATc1 was colocalized with lymphatic markers Prox-1, VEGFR-3 and podoplanin on cardinal vein as lymphatic endothelial cells (LEC) are specified and as they segregate into lymph sacs and mature lymphatics. In NFATc1 null mice, Prox-1, VEGFR-3 and podoplanin positive endothelial cells sprouted from the cardinal vein at E11.5, but poorly coalesced into lymph sacs. NFAT activation requires the phosphatase calcineurin. Embryos treated in utero with the calcineurin inhibitor cyclosporine-A showed cytoplasmic NFATc1, diminished podoplanin and FGFR-3 expression by the lymphatics and irregular patterning of the LEC sprouts coming off the jugular lymph sac, which suggests a role for calcineurin-NFAT signaling in lymphatic patterning. In a murine model of injury-induced lymphangiogenesis, NFATc1 was expressed on the neolymphatics induced by lung-specific overexpression of VEGF-A. Mice lacking the calcineurin Abeta regulatory subunit, with diminished nuclear NFAT, failed to respond to VEGF-A with increased lymphangiogenesis. In vitro, endogenous and VEGF-A-induced VEGFR-3 and podoplanin expression by human microvascular endothelial cells was reduced by siRNA to NFATc1, to levels comparable to reductions seen with siRNA to Prox-1. In reporter assays, NFATc1 activated lymphatic specific gene promoters. These results demonstrate the role of calcineurin-NFAT pathway in lymphangiogenesis and suggest that NFATc1 is the principle NFAT involved.

G protein-coupled receptors (GPCRs) are widely expressed cell surface receptors that have been successfully exploited for the treatment of a variety of human diseases. Recent studies in genetically engineered mouse models have led to the identification of several GPCRs important for lymphatic vascular development and function. The adrenomedullin receptor, which consists of an oligomer between calcitonin receptor-like receptor and receptor activity modifying protein 2, is required for normal lymphatic vascular development and regulates lymphatic capillary permeability in mice. Numerous studies also suggest that lysophospholipid receptors are involved in the development of lymphatic vessels and lymphatic endothelial cell permeability. Given our current lack of pharmacological targets for the treatment of lymphatic vascular diseases like lymphedema, the continued identification and study of GPCRs in lymphatic endothelial cells may eventually lead to major breakthroughs and new pharmacological strategies for the treatment of lymphedema.

Objective: Although diverse functions of angiopoietin-2 (Ang2) have been revealed, little is known about upstream signaling molecules regulating Ang2 exocytosis. We therefore investigated the mechanism of Ang2 exocytosis in human blood and lymphatic endothelial cells (BECs and LECs) by stimulation with sphingosine-1-phosphate (S1P)., Methods and Results: By immunostaining and ELISA analyses using our newly developed human Ang2-specific antibodies, Ang2 exocytosis from human endothelial cells was examined. Both exogenous and endogenous S1P trigger rapid Ang2 exocytosis in time- and dose-dependent manners. Intriguingly, S1P-induced Ang2 exocytosis is higher in LECs than BECs. These effects of S1P are mainly mediated by the endothelial differentiation gene receptor 1, which subsequently activates its downstream phospholipase C and intracellular calcium mobilization to trigger Ang2 exocytosis. Consistently, S1P also dramatically stimulates Ang2 exocytosis from the ECs of ex vivo-incubated blood vessels., Conclusion: These results imply that the rapid secretion of Ang2 by exocytosis from endothelial cells is another possible mechanism underlying S1P-induced angiogenesis and inflammation.

Lymphatic metastasis of tumors is one of the most important prognostic factors and provides valuable information for decisions on appropriate surgical protocols. Recent studies have demonstrated that lymphangiogenesis of lymphatic vascular endothelial cells into tumors is a key event in lymphatic metastasis. Therefore, control of lymphangiogenesis is a promising strategy for treatment or prevention of tumor metastasis and lymphatic disorders. However, mechanisms of lymphangiogenesis or its specific inhibition are not well-understood. In this study we examined effects of various types of signaling inhibitors on tube formation in human lymphatic microvascular endothelial cells (LECs) and blood microvascular endothelial cells (BECs) in vitro. We found that tube formation of LECs was specifically inhibited by 2-methoxyestradiol (2ME). This observation is of potential benefit in understanding the molecular mechanism of lymphangiogenesis. Furthermore, 2ME could therefore offer specific protection against lymphatic metastasis and lymphangiogenesis-related diseases.

The lymphatic vascular system functions to maintain fluid homeostasis by removing fluid from the interstitial space and returning it to venous circulation. This process is dependent upon the maintenance and modulation of a semi-permeable barrier between lymphatic endothelial cells of the lymphatic capillaries. However, our understanding of the lymphatic endothelial barrier and the molecular mechanisms that govern its function remains limited. Adrenomedullin (AM) is a 52 amino acid secreted peptide which has a wide range of effects on cardiovascular physiology and is required for the normal development of the lymphatic vascular system. Here, we report that AM can also modulate lymphatic permeability in cultured dermal microlymphatic endothelial cells (HMVEC-dLy). AM stimulation caused a reorganization of the tight junction protein ZO-1 and the adherens protein VE-cadherin at the plasma membrane, effectively tightening the endothelial barrier. Stabilization of the lymphatic endothelial barrier by AM occurred independently of changes in junctional protein gene expression and AM(-/-) endothelial cells showed no differences in the gene expression of junctional proteins compared to wildtype endothelial cells. Nevertheless, local administration of AM in the mouse tail decreased the rate of lymph uptake from the interstitial space into the lymphatic capillaries. Together, these data reveal a previously unrecognized role for AM in controlling lymphatic endothelial permeability and lymphatic flow through reorganization of junctional proteins.