Kidney Injury Causes Accumulation of Renal Sodium That Modulates Renal Lymphatic Dynamics.

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 23Na/1H-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.

Clinical variability in multifocal lymphangioendotheliomatosis with thrombocytopenia: a review of the literature.

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.

Feed­back loops integrating RELA, SOX18 and FAK mediate the break­down of the lymph­endothelial barrier that is triggered by 12(S)­HETE.

Metastatic cancer cells cross endothelial barriers and travel through the blood or lymphatic fluid to pre­metastatic niches, leading to their colonisation. 'S' stereoisomer 12S­hydroxy­5Z,8Z,10E,14Z­eicosatetraenoic 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)­HETE­treated lymph endothelial cells (LECs) with regard to their expression and mutual interaction with v­rel avian reticuloendotheliosis viral oncogene homolog A (RELA), intercellular adhesion molecule 1, SRY­box 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 transcription­quantitative 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 Bay11­7082, extended the mono­culture experiments to co­culture settings using HCT116 colon cancer cell spheroids that were placed on top of LEC monolayers to measure their retraction using the validated 'circular chemorepellent­induced 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 FAK­inhibitor defactinib and the NF­κB inhibitor Bay11­7082 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 pro­metastatic signalling cascade. Therefore, targeting the endothelial­specific activity of FAK through the pathway demonstrated herein may provide a potential therapeutic method to combat cancer dissemination via vascular routes.

Regulatory T Cells Condition Lymphatic Endothelia for Enhanced Transendothelial Migration.

Regulatory T cells (Tregs) express high levels of cell surface lymphotoxin alpha beta (LTα1ß2) to activate the LT beta receptor (LTßR) 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 LTα1ß2-LTßR 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.

Sonic hedgehog selectively promotes lymphangiogenesis after kidney injury through noncanonical pathway.

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.

Ectopic high endothelial venules in pancreatic ductal adenocarcinoma: A unique site for targeted delivery.

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.).

Small GTPase Rap1A/B Is Required for Lymphatic Development and Adrenomedullin-Induced Stabilization of Lymphatic Endothelial Junctions.

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 Gs-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.

Endothelial lineage-specific interaction of Mycobacterium tuberculosis with the blood and lymphatic systems.

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.

Burn Injury-Associated MHCII+ Immune Cell Accumulation Around Lymphatic Vessels of the Mesentery and Increased Lymphatic Endothelial Permeability Are Blocked by Doxycycline Treatment.

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.

Lymphotoxins Promote the Progression of Human Lymphatic Malformation by Enhancing Lymphatic Endothelial Cell Proliferation.

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 ß 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.

Intercellular Adhesion Molecule-1 and Vascular Cell Adhesion Molecule Are Induced by Ionizing Radiation on Lymphatic Endothelium.

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 TGFß in this process anti-TGFß 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- TGFß 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 TGFß. 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.

Efficacy of AdipoDren® in Reducing Interleukin-1-Induced Lymphatic Endothelial Hyperpermeability.

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-1ß) and the associated molecular mechanisms. AdipoDren® demonstrated a protective effect on dermal lymphatic endothelial cell permeability increased by IL-1ß. 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.

The histone deacetylase inhibitor trichostatin a decreases lymphangiogenesis by inducing apoptosis and cell cycle arrest via p21-dependent pathways.

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.

Blunted flow-mediated responses and diminished nitric oxide synthase expression in lymphatic thoracic ducts of a rat model of metabolic syndrome.

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.

The cooperative role of S1P3 with LYVE-1 in LMW-HA-induced lymphangiogenesis.

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.

Measurement of shear stress-mediated intracellular calcium dynamics in human dermal lymphatic endothelial cells.

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.

Lobatin B inhibits NPM/ALK and NF-κB attenuating anaplastic-large-cell-lymphomagenesis and lymphendothelial tumour intravasation.

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-Rß, 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.

The effect of podoplanin inhibition on lymphangiogenesis under pathological conditions.

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.

Involvement of histamine in endothelium-dependent relaxation of mesenteric lymphatic vessels.

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.

Involvement of H1 and H2 receptors and soluble guanylate cyclase in histamine-induced relaxation of rat mesenteric collecting lymphatics.

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.