Abstract 546: L-NAME Attenuates the Cardiotonic Steroids-induced Monolayer Permeability in Lymphatic Endothelial Cells

Hypertension ◽  
2013 ◽  
Vol 62 (suppl_1) ◽  
Author(s):  
Juan Castor ◽  
Darijana Horvat ◽  
Walter E Cromer ◽  
Thomas J Kuehl ◽  
David C Zawieja ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Protein Expression ◽  
Tight Junctions ◽  
Cell Monolayer ◽  
Hypertensive Disorder ◽  
Lymphatic Endothelial Cells ◽  
Vascular Leak Syndrome ◽  
Vascular Leak ◽  
Monolayer Permeability

Objective: Preeclampsia (preE) is a hypertensive disorder unique to pregnancy. Cardiotonic steroids (CTS) such as marinobufagenin (MBG), cinobufotalin (CINO), and ouabain (OUB) are Na + /K + ATPase inhibitors. MBG is elevated in a rat model and patients with preE. MBG causes a vascular leak syndrome in vivo and increases endothelial cell monolayer permeability. Edema is a common syndrome of preE. To assess whether CTS are involved in the leakage of lymphatic endothelial cells (LECs) lining during preE, we evaluated the effect of these CTS on monolayer permeability of LECs in culture. Methods: LECs were isolated from a rat mesenteric collecting lymphatic vessel. The cells were treated with DMSO (vehicle), MBG, CINO, or OUB (1, 10 or 100 nM). Some LECs were pretreated with L-NAME (N-Nitro-L-Arginine Methyl Ester) at a concentration of 1μM before treatment with 100 nM MBG or CINO. Monolayer permeability of CTS-induced LECs was measured by using a fluorescent dye that was quantified on a fluorescence plate reader. The expression of β-catenin and VE-cadherin in the CTS-treated LECs was measured by immunofluorescence. Western blot was performed to measure β-catenin, VE-cadherin, and LYVE-1 protein levels. Statistical comparisons were performed using analysis of variance with Dunnett's post hoc tests. Results: MBG (≥ 1 nM, p<0.05) and CINO (≥ 10 nM, p<0.05) significantly increased the monolayer permeability of LECs compared to DMSO while OUB had no effect. Pretreatment of LECs with 1μM L-NAME attenuated the monolayer permeability of LECs treated with either 100 nM of MBG (p<0.05) or 100 nM of CINO (p<0.05). The β-catenin protein expression in LECs was downregulated by both MBG (p<0.05) and CINO (p<0.05) treatment. However, CTS did not cause any disruption of the LECs tight junctions. CINO (p<0.05) downregulated the VE-cadherin and LYVE-1 protein expression, but MBG did not. Conclusions: We have demonstrated that bufadienolides, MBG and CINO, caused an increase in the monolayer permeability of LECs which was attenuated by L-NAME pretreatment. Moreover, the β-catenin protein expression was downregulated by MBG and CINO treatment with no significant effect on tight junctions. These data suggest that CTS may be involved in the vascular leak syndrome in the LEC lining in preE.

scholarly journals HIV-1 Matrix Protein p17 Promotes Lymphangiogenesis and Activates the Endothelin-1/Endothelin B Receptor Axis

2014 ◽  
Vol 34 (4) ◽  
pp. 846-856 ◽  
Author(s):  
Francesca Caccuri ◽  
Christine Rueckert ◽  
Cinzia Giagulli ◽  
Kai Schulze ◽  
Daniele Basta ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Lymph Node ◽  
Structure Formation ◽  
Highly Active Antiretroviral Therapy ◽  
Matrix Protein ◽  
Lymphatic Endothelial Cells ◽  
Endothelin 1 ◽  
Tumor Dissemination ◽  
Hiv 1

Objective— AIDS-related lymphomas are high grade and aggressively metastatic with poor prognosis. Lymphangiogenesis is essential in supporting proliferation and survival of lymphoma, as well as tumor dissemination. Data suggest that aberrant lymphangiogenesis relies on action of HIV-1 proteins rather than on a direct effect of the virus itself. HIV-1 matrix protein p17 was found to accumulate and persist in lymph nodes of patients even under highly active antiretroviral therapy. Because p17 was recently found to exert a potent proangiogenic activity by interacting with chemokine (C-X-C motif) receptors 1 and 2, we tested the prolymphangiogenic activity of the viral protein. Approach and Results— Human primary lymph node–derived lymphatic endothelial cells were used to perform capillary-like structure formation, wound healing, spheroids, and Western blot assays after stimulation with or without p17. Here, we show that p17 promotes lymphangiogenesis by binding to chemokine (C-X-C motif) receptor-1 and chemokine (C-X-C motif) receptor-2 expressed on lymph node–derived lymphatic endothelial cells and activating the Akt/extracellular signal–regulated kinase signaling pathway. In particular, it was found to induce capillary-like structure formation, sprout formation from spheroids, and increase lymph node–derived lymphatic endothelial cells motility. The p17 lymphangiogenic activity was, in part, sustained by activation of the endothelin-1/endothelin receptor B axis. A Matrigel plug assay showed that p17 was able to promote the outgrowth of lymphatic vessels in vivo, demonstrating that p17 directly regulates lymphatic vessel formation. Conclusions— Our results suggest that p17 may generate a prolymphangiogenic microenvironment and plays a role in predisposing the lymph node to lymphoma growth and metastasis. This finding offers new opportunities to identify treatment strategies in combating AIDS-related lymphomas.

scholarly journals Direct Current Stimulation Disrupts Endothelial Glycocalyx and Tight Junctions of the Blood-Brain Barrier in vitro

2021 ◽  
Vol 9 ◽  
Author(s):  
Yifan Xia ◽  
Yunfei Li ◽  
Wasem Khalid ◽  
Marom Bikson ◽  
Bingmei M. Fu
Keyword(s):  
Endothelial Cells ◽  
Tight Junction ◽  
Tight Junctions ◽  
Blood Brain Barrier ◽  
Direct Current ◽  
Endothelial Glycocalyx ◽  
Microvascular Endothelial Cells ◽  
Bbb Permeability

Transcranial direct current stimulation (tDCS) is a non-invasive physical therapy to treat many psychiatric disorders and to enhance memory and cognition in healthy individuals. Our recent studies showed that tDCS with the proper dosage and duration can transiently enhance the permeability (P) of the blood-brain barrier (BBB) in rat brain to various sized solutes. Based on the in vivo permeability data, a transport model for the paracellular pathway of the BBB also predicted that tDCS can transiently disrupt the endothelial glycocalyx (EG) and the tight junction between endothelial cells. To confirm these predictions and to investigate the structural mechanisms by which tDCS modulates P of the BBB, we directly quantified the EG and tight junctions of in vitro BBB models after DCS treatment. Human cerebral microvascular endothelial cells (hCMECs) and mouse brain microvascular endothelial cells (bEnd3) were cultured on the Transwell filter with 3 μm pores to generate in vitro BBBs. After confluence, 0.1–1 mA/cm2 DCS was applied for 5 and 10 min. TEER and P to dextran-70k of the in vitro BBB were measured, HS (heparan sulfate) and hyaluronic acid (HA) of EG was immuno-stained and quantified, as well as the tight junction ZO-1. We found disrupted EG and ZO-1 when P to dextran-70k was increased and TEER was decreased by the DCS. To further investigate the cellular signaling mechanism of DCS on the BBB permeability, we pretreated the in vitro BBB with a nitric oxide synthase (NOS) inhibitor, L-NMMA. L-NMMA diminished the effect of DCS on the BBB permeability by protecting the EG and reinforcing tight junctions. These in vitro results conform to the in vivo observations and confirm the model prediction that DCS can disrupt the EG and tight junction of the BBB. Nevertheless, the in vivo effects of DCS are transient which backup its safety in the clinical application. In conclusion, our current study directly elucidates the structural and signaling mechanisms by which DCS modulates the BBB permeability.

Appearance of surfactant-like particles in apical medium of Caco-2 cells may occur via tight junctions

1995 ◽  
Vol 268 (6) ◽  
pp. C1401-C1413 ◽  
Author(s):  
M. J. Engle ◽  
M. L. Grove ◽  
M. J. Becich ◽  
A. Mahmood ◽  
D. H. Alpers
Keyword(s):  
Tight Junctions ◽  
Secretory Pathway ◽  
Cell Monolayer ◽  
Cytochalasin D ◽  
Total Cell ◽  
Intestinal Lumen ◽  
Intestinal Alkaline Phosphatase ◽  
Particle Content ◽  
Quantitative Image ◽  
Monolayer Permeability

Intestinal alkaline phosphatase (AP) is secreted by Caco-2 cells bound to surfactant-like particles (SLP), which can be localized by electron microscopy to the basolateral space and the intestinal lumen, especially over tight junctions. To investigate the hypothesis that SLP are secreted basolaterally and enter the lumen through the tight junction, Caco-2 cells were incubated with agents known to increase permeability at tight junctions. Cytochalasin D and phorbol 12-myristate 13-acetate increased Caco-2 cell monolayer permeability and the appearance of particles in apical medium two- to threefold, as monitored by mannitol movement and AP activity, respectively. Blocking the apical secretory pathway by nocodazole or colchicine had no effect on either parameter. Estimation of SLP content demonstrated an increase in apical media particles similar to that determined by AP activity. Quantitative image analysis established that apical SLP content increased 4-10 times, whereas total cell particle content remained unchanged. These data indicate that SLP may be secreted initially into the basolateral space and then transported to the intestinal lumen through the tight junctions.

scholarly journals Attenuation of Glomerular Endothelial Cells from High Glucose-Induced Injury by Blockade of MAD2B

2015 ◽  
Vol 35 (1) ◽  
pp. 61-70 ◽  
Author(s):  
Yu-Mei Wang ◽  
Yu Hao ◽  
Xian-Fang Meng ◽  
Fang-Fang He ◽  
Shan Chen ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Endothelial Cells ◽  
High Glucose ◽  
Cell Monolayer ◽  
No Production ◽  
Protein Levels ◽  
Glomerular Endothelial Cells ◽  
Monolayer Permeability ◽  
Glucose Treatment ◽  
In Cells

Background/Aims: To assess the role of mitotic arrest-deficient 2-like protein 2 (MAD2B) in high glucose-induced injury in mouse glomerular endothelial cells (GEnCs). Methods: GEnCs were cultured in vitro, and MAD2B protein levels were measured by Western blot in cells stimulated with high glucose (30 mM) for various periods of time. MAD2B and scrambled shRNA were introduced into GEnCs by liposomal transfection. Cell proliferation, apoptosis, nitric oxide (NO) production, and monolayer permeability were then measured in cells grown in the following conditions: control, high glucose treatment, MAD2B shRNA transfection with high glucose treatment, and scrambled shRNA transfection with high glucose treatment. Results: High glucose increased the protein levels of MAD2B in GEnCs. Compared with control cells, apoptosis was increased by high glucose treatment, which was attenuated by transfection with MAD2B shRNA transfection. Cells treated with high glucose produced less NO than control cells, whereas MAD2B shRNA transfection increased NO production. Cell monolayer permeability was enhanced in high glucose treated cells, but MAD2B shRNA transfection reduced permeability. Conclusion: High glucose levels induced the expression of MAD2B in GEnCs, whereas suppressing its expression reduced high glucose-induced endothelial cell apoptosis and high permeability, and promoted cell proliferation and NO production.

Combined temozolomide and ionizing radiation induces galectin-1 and galectin-3 expression in a model of human glioma

2015 ◽  
Vol 2 (1) ◽  
Author(s):  
Lauren A. Bailey ◽  
Azemat Jamshidi-Parsian ◽  
Tulsi Patel ◽  
Nathan A. Koonce ◽  
Alan B. Diekman ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Ionizing Radiation ◽  
Protein Expression ◽  
Glioma Cell ◽  
Glioma Cells ◽  
Human Glioma ◽  
Glioma Recurrence ◽  
Galectin 3

AbstractBackground Despite aggressive treatment for glioblastoma multiforme (GBM), including surgical resection, radiotherapy and temozolomide (TMZ) chemotherapy, over 90% of patients experience tumor recurrence. Galectins are carbohydrate-binding proteins that are overexpressed in the stroma of GBM tumors, and are potent modulators of GBM cell migration and angiogenesis. The objective of this study was to analyze glioma and endothelial cell galectin expression in response to combined chemoradiation. Methodology The effects of TMZ, ionizing radiation, or combined chemoradiation on galectin protein secretion and expression were assessed in U87 orthotopically grown GBM tumors in mice, as well as in vitro in U87 human glioma cells and human umbilical vein endothelial cells (HUVECs). Results We found that combination chemoradiation increased galectin-1 and galectin-3 protein expression in U87 glioma cells. In response to radiation alone, U87 cells secreted significant levels of galectin-1 and galectin-3 into the microenvironment. HUVEC co-culture increased U87 galectin-1 and galectin-3 protein expression 14 - 20% following chemoradiation, and conferred a radioprotective benefit to U87 glioma cells. In vivo, radiation alone and combination chemoradiation significantly increased tumor galectin-1 expression in an orthotopic murine model of GBM. Conclusions Glioma cell galectin expression increased following combined chemoradiation, both in vitro and in vivo. The presence of endothelial cells further increased glioma cell galectin expression and survival, suggesting that crosstalk between tumor and endothelial cells in response to standard chemoradiation may be an important factor in mediating glioma recurrence, potentially via galectin upregulation.

scholarly journals Epigenetic upregulation by valproic acid of transferrin receptor 1, and not glucose transporter 1 or CD98hc, at the blood-brain barrier

2022 ◽  
Author(s):  
Steinunn Sara Helgudóttir ◽  
Kasper Bendix Johnsen ◽  
Lisa Juul Routhe ◽  
Charlotte L.M. Rasmussen ◽  
Azra Karamehmedovic ◽  
...  
Keyword(s):  
Valproic Acid ◽  
Endothelial Cells ◽  
Protein Expression ◽  
Transferrin Receptor ◽  
Glucose Transporter ◽  
Brain Capillaries ◽  
Glucose Transporter 1 ◽  
Transferrin Receptor 1

Abstract BackgroundThe objectives of the present study were to investigate whether the expression of transferrin receptor 1 (TfR1), glucose transporter 1 (Glut1), or Cluster of Differentiation 98 Heavy Chain (CD98hc) is epigenetically regulated in brain capillary endothelial cells (BCECs) denoting the blood-brain barrier (BBB).MethodsThe expression of these targets was investigated both in vitro and in vivo following treatment with the histone deacetylase inhibitor (HDACi) valproic acid (VPA). Mice were injected intraperitoneally with VPA followed by analysis of isolated brain capillaries, and the capillary depleted brain samples. Brain tissue, isolated brain capillaries, and cultured primary endothelial cells were analyzed by RT-qPCR, immunolabeling and ELISA for expression of TfR1, Glut1 and CD98hc. We also studied the vascular targeting in VPA-treated mice injected with monoclonal anti-transferrin receptor (Ri7) conjugated with 1.4 nm gold nanoparticles. ResultsValidating the effects of VPA on gene transcription in BCECs, transcriptomic analysis identified 24,371 expressed genes, of which 305 were differentially expressed with 192 upregulated and 113 downregulated genes. In vitro using BCECs co-cultured with glial cells, the mRNA expression of Tfrc was significantly higher after VPA treatment for 6 h with its expression returning to baseline after 24 h. Conversely, the mRNA expression of Glut1 and Cd98hc was unaffected by VPA treatment. In vivo, the TfR1 protein expression in brain capillaries increased significantly after treatment with both 100 mg/kg and 400 mg/kg VPA. Conversely, VPA treatment did not increase GLUT1 or CD98hc. Using ICP-MS-based quantification, the brain uptake of nanogold conjugated anti-TfR1 antibodies was non-significant in spite of increased expression of TfR1. ConclusionsWe report that VPA treatment upregulates TfR1 at the BBB both in vivo and in vitro in isolated primary endothelial cells. In contrast, VPA treatment does not influence the expression of GLUT1 and CD98hc. The increase in the overall TfR1 protein expression however does not increase transport of TfR-targeted monoclonal antibody and indicates that targeted delivery using the transferrin receptor should aim for increased mobilization of already available transferrin receptor molecules to improve trafficking through the BBB.

scholarly journals Phomaketide A Inhibits Lymphangiogenesis in Human Lymphatic Endothelial Cells

Marine Drugs ◽  
2019 ◽  
Vol 17 (4) ◽  
pp. 215 ◽  
Author(s):  
Huai-Ching Tai ◽  
Tzong-Huei Lee ◽  
Chih-Hsin Tang ◽  
Lei-Po Chen ◽  
Wei-Cheng Chen ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Protease Inhibitors ◽  
Cancer Metastasis ◽  
New Drugs ◽  
Tumor Xenograft ◽  
Specific Marker ◽  
Lymphatic Endothelial Cells ◽  
Matrix Metalloproteinase 1

Lymphangiogenesis is an important biological process associated with cancer metastasis. The development of new drugs that block lymphangiogenesis represents a promising therapeutic strategy. Marine fungus-derived compound phomaketide A, isolated from the fermented broth of Phoma sp. NTOU4195, has been reported to exhibit anti-angiogenic and anti-inflammatory effects. However, its anti-lymphangiogenic activity has not been clarified to date. In this study, we showed that phomaketide A inhibited cell growth, migration, and tube formation of lymphatic endothelial cells (LECs) without an evidence of cytotoxicity. Mechanistic investigations revealed that phomaketide A reduced LECs-induced lymphangiogenesis via vascular endothelial growth factor receptor-3 (VEGFR-3), protein kinase Cδ (PKCδ), and endothelial nitric oxide synthase (eNOS) signalings. Furthermore, human proteome array analysis indicated that phomaketide A significantly enhanced the protein levels of various protease inhibitors, including cystatin A, serpin B6, tissue factor pathway inhibitor (TFPI), and tissue inhibitor matrix metalloproteinase 1 (TIMP-1). Importantly, phomaketide A impeded tumor growth and lymphangiogenesis by decreasing the expression of LYVE-1, a specific marker for lymphatic vessels, in tumor xenograft animal model. These results suggest that phomaketide A may impair lymphangiogenesis by suppressing VEGFR-3, PKCδ, and eNOS signaling cascades, while simultaneously activating protease inhibitors in human LECs. We document for the first time that phomaketide A inhibits lymphangiogenesis both in vitro and in vivo, which suggests that this natural product could potentially treat cancer metastasis.

scholarly journals Epoxyeicosatrienoic acids are part of the VEGF-activated signaling cascade leading to angiogenesis

2008 ◽  
Vol 295 (5) ◽  
pp. C1292-C1301 ◽  
Author(s):  
Anke C. Webler ◽  
U. Ruth Michaelis ◽  
Rüdiger Popp ◽  
Eduardo Barbosa-Sicard ◽  
Andiappan Murugan ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Growth Factor ◽  
Protein Expression ◽  
Second Messengers ◽  
Tube Formation ◽  
Dominant Negative ◽  
Endothelial Cell Proliferation ◽  
Angiogenic Response

Cytochrome P-450 (CYP) epoxygenases metabolize arachidonic acid to epoxyeicosatrienoic acid (EET) regioisomers, which activate several signaling pathways to promote endothelial cell proliferation, migration, and angiogenesis. Since vascular endothelial growth factor (VEGF) plays a key role in angiogenesis, we assessed a possible role of EETs in the VEGF-activated signal transduction cascade. Stimulation with VEGF increased CYP2C promoter activity in endothelial cells and enhanced CYP2C8 mRNA and protein expression resulting in increased intracellular EET levels. VEGF-induced endothelial cell tube formation was inhibited by the EET antagonist 14,15-epoxyeicosa-5( Z)-enoicacid (14,15-EEZE), which did not affect the VEGF-induced phosphorylation of its receptor or basic fibroblast growth factor (bFGF)-stimulated tube formation. Moreover, VEGF-stimulated endothelial cell sprouting in a modified spheroid assay was reduced by CYP2C antisense oligonucleotides. Mechanistically, VEGF stimulated the phosphorylation of the AMP-activated protein kinase (AMPK), which has also been linked to CYP induction, and the overexpression of a constitutively active AMPK mutant increased CYP2C expression. On the other hand, a dominant-negative AMPK mutant prevented the VEGF-induced increase in CYP2C RNA and protein expression in human endothelial cells. In vivo (Matrigel plug assay) in mice, endothelial cells were recruited into VEGF-impregnated plugs; an effect that was sensitive to 14,15-EEZE and the inclusion of small interfering RNA directed against the AMPK. The EET antagonist did not affect responses observed in plugs containing bFGF. Taken together, our data indicate that CYP2C-derived EETs participate as second messengers in the angiogenic response initiated by VEGF and that preventing the increase in CYP expression curtails the angiogenic response to VEGF.

scholarly journals Endothelin and nitric oxide synthase in lymphatic endothelial cells: Immunolocalization in vivo and in vitro

1997 ◽  
Vol 248 (4) ◽  
pp. 490-497 ◽  
Author(s):  
Carla Marchetti ◽  
Andrea Casasco ◽  
Amalia Di Nucci ◽  
Marcella Reguzzoni ◽  
Simone Rosso ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Nitric Oxide Synthase ◽  
Lymphatic Endothelial Cells ◽  
Oxide Synthase
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