scholarly journals Meningococcal Internalization into Human Endothelial and Epithelial Cells Is Triggered by the Influx of Extracellular l-Glutamate via GltT l-Glutamate ABC Transporter inNeisseria meningitidis

2010 ◽  
Vol 79 (1) ◽  
pp. 380-392 ◽  
Author(s):  
Hideyuki Takahashi ◽  
Kwang Sik Kim ◽  
Haruo Watanabe
Keyword(s):  
Endothelial Cells ◽  
Abc Transporter ◽  
Glutamate Uptake ◽  
Umbilical Vein ◽  
Glutamate Transporter ◽  
Amino Acid Sequences ◽  
Host Cells ◽  
Epithelial Cell Line ◽  
Neighboring Gene ◽  
Experimental Conditions

ABSTRACTMeningococcal internalization into human cells is likely to be a consequence of meningococcal adhesion to human epithelial and endothelial cells. Here, we identified three transposon mutants ofNeisseria meningitidisthat were primarily defective in the internalization of human brain microvascular endothelial cells (HBMEC), with insertions occurring in thegltT(a sodium-independentl-glutamate transporter) gene or its neighboring gene,NMB1964(unknown function). NMB1964 was tentatively namedgltMin this study because of the presence of a mammalian cell entry (MCE)-related domain in the deduced amino acid sequences. The null ΔgltT-ΔgltM N. meningitidismutant was also defective in the internalization into human umbilical vein endothelial cells and the human lung carcinoma epithelial cell line A549, and the defect was suppressed by transcomplementation of the mutants withgltT+-gltM+genes. The intracellular survival of the ΔgltT-ΔgltMmutant in HBMEC was not largely different from that of the wild-type strain under our experimental conditions. Introduction of a1-bp deletion and amber or ochre mutations ingltT-gltMgenes resulted in the loss of efficient internalization into HBMEC. The defect in meningococcal internalization into HBMEC andl-glutamate uptake in the ΔgltT-ΔgltMmutant were suppressed only in strains expressing both GltT and GltM proteins. The efficiency of meningococcal invasion to HBMEC decreased underl-glutamate-depleted conditions. Furthermore, ezrin, a key membrane-cytoskeleton linker, accumulated beneath colonies of thegltT+-gltM+N. meningitidisstrain but not of the ΔgltT-ΔgltMmutant. These findings suggest thatl-glutamate influx via the GltT-GltMl-glutamate ABC transporter serves as a cue forN. meningitidisinternalization into host cells.

scholarly journals Multiple Functions of Glutamate Uptake via Meningococcal GltT-GltM l-Glutamate ABC Transporter in Neisseria meningitidis Internalization into Human Brain Microvascular Endothelial Cells

2015 ◽  
Vol 83 (9) ◽  
pp. 3555-3567 ◽  
Author(s):  
Hideyuki Takahashi ◽  
Tatsuo Yanagisawa ◽  
Kwang Sik Kim ◽  
Shigeyuki Yokoyama ◽  
Makoto Ohnishi
Keyword(s):  
Endothelial Cells ◽  
Human Brain ◽  
Neisseria Meningitidis ◽  
Abc Transporter ◽  
Glutamate Uptake ◽  
Underlying Mechanism ◽  
Host Cells ◽  
Wild Type ◽  
Content Type ◽  
Fetal Bovine

We previously reported thatNeisseria meningitidisinternalization into human brain microvasocular endothelial cells (HBMEC) was triggered by the influx of extracellularl-glutamate via the GltT-GltMl-glutamate ABC transporter, but the underlying mechanism remained unclear. We found that the ΔgltTΔgltMinvasion defect in assay medium (AM) was alleviated in AM without 10% fetal bovine serum (FBS) [AM(−S)]. The alleviation disappeared again in AM(−S) supplemented with 500 μM glutamate. Glutamate uptake by the ΔgltTΔgltMmutant was less efficient than that by the wild-type strain, but only upon HBMEC infection. We also observed that both GltT-GltM-dependent invasion and accumulation of ezrin, a key membrane-cytoskeleton linker, were more pronounced whenN. meningitidisformed larger colonies on HBMEC under physiological glutamate conditions. These results suggested that GltT-GltM-dependent meningococcal internalization into HBMEC might be induced by the reduced environmental glutamate concentration upon infection. Furthermore, we found that the amount of glutathione within the ΔgltTΔgltMmutant was much lower than that within the wild-typeN. meningitidisstrain only upon HBMEC infection and was correlated with intracellular survival. Considering that thel-glutamate obtained via GltT-GltM is utilized as a nutrient in host cells,l-glutamate uptake via GltT-GltM plays multiple roles inN. meningitidisinternalization into HBMEC.

Desmopressin (DDAVP) Enhances Platelet Adhesion to the Extracellular Matrix of Cultured Human Endothelial Cells through Increased Expression of Tissue Factor

1997 ◽  
Vol 77 (05) ◽  
pp. 0975-0980 ◽  
Author(s):  
Angel Gálvez ◽  
Goretti Gómez-Ortiz ◽  
Maribel Díaz-Ricart ◽  
Ginés Escolar ◽  
Rogelio González-Sarmiento ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Tissue Factor ◽  
Platelet Adhesion ◽  
Umbilical Vein ◽  
Procoagulant Activity ◽  
Experimental Conditions ◽  
Chromogenic Assay

SummaryThe effect of desmopressin (DDAVP) on thrombogenicity, expression of tissue factor and procoagulant activity (PCA) of extracellular matrix (ECM) generated by human umbilical vein endothelial cells cultures (HUVEC), was studied under different experimental conditions. HUVEC were incubated with DDAVP (1, 5 and 30 ng/ml) and then detached from their ECM. The reactivity towards platelets of this ECM was tested in a perfusion system. Coverslips covered with DD A VP-treated ECMs were inserted in a parallel-plate chamber and exposed to normal blood anticoagulated with low molecular weight heparin (Fragmin®, 20 U/ml). Perfusions were run for 5 min at a shear rate of 800 s1. Deposition of platelets on ECMs was significantly increased with respect to control ECMs when DDAVP was used at 5 and 30 ng/ml (p <0.05 and p <0.01 respectively). The increase in platelet deposition was prevented by incubation of ECMs with an antibody against human tissue factor prior to perfusion. Immunofluorescence studies positively detected tissue factor antigen on DDAVP derived ECMs. A chromogenic assay performed under standardized conditions revealed a statistically significant increase in the procoagulant activity of the ECMs produced by ECs incubated with 30 ng/ml DDAVP (p <0.01 vs. control samples). Northern blot analysis revealed increased levels of tissue factor mRNA in extracts from ECs exposed to DDAVP. Our data indicate that DDAVP in vitro enhances platelet adhesion to the ECMs through increased expression of tissue factor. A similar increase in the expression of tissue factor might contribute to the in vivo hemostatic effect of DDAVP.

RGDS peptide induces caspase 8 and caspase 9 activation in human endothelial cells

Blood ◽  
2004 ◽  
Vol 103 (11) ◽  
pp. 4180-4187 ◽  
Author(s):  
Maria Simona Aguzzi ◽  
Claudia Giampietri ◽  
Francesco De Marchis ◽  
Fabrizio Padula ◽  
Roberto Gaeta ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Umbilical Vein ◽  
Caspase 8 ◽  
Caspase 9 ◽  
Experimental Conditions ◽  
Biologic Effects ◽  
Independent Effects ◽  
Hoechst Staining ◽  
Umbilical Vein Endothelial Cells

Abstract Peptides containing the Arg-Gly-Asp (RGD) motif inhibit cell adhesion and exhibit a variety of other biologic effects including anticoagulant and antimetastatic activities. The aim of the present study was to examine the anchorage-independent effects of an RGD-containing peptide, Arg-Gly-Asp-Ser (RGDS), on human umbilical vein endothelial cells (HUVECs). Assays were performed on HUVECs seeded onto collagen IV; under these experimental conditions RGDS did not exert antiadhesive effects but significantly reduced FGF-2-dependent chemotaxis after 4 hours of treatment and reduced proliferation after 24 hours of treatment. Experiments carried out with caspase-specific inhibitors indicated that the observed antichemotactic effects required caspase 8 and caspase 9 activation. RGDS activated both caspase 8 and caspase 9 after 4 hours of treatment and caspase 3 after 24 hours of treatment, and markedly enhanced HUVEC apoptosis by transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL)/Hoechst staining and fluorescence-activated cell sorting (FACS) analysis. Finally, confocal microscopy showed that RGDS localizes in the cytoplasm of live HUVECs within 4 hours and in vitro experiments showed that RGDS directly interacts with recombinant caspases 8 and 9 in a specific way. In summary, these results indicate that RGDS directly binds and activates caspases 8 and 9, inhibits chemotaxis, and induces apoptosis of HUVECs with a mechanism independent from its antiadhesive effect.

Modulation Of PGI2-Like Activity Of Human Endothelial Cells By An Extracellular Collagen Matrix

1981 ◽  
Author(s):  
K M Spiegel ◽  
S F Mohammad ◽  
H Y K Chuang ◽  
R G Mason
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Umbilical Vein ◽  
Collagen Matrix ◽  
Significant Loss ◽  
Experimental Conditions ◽  
Cell Lysates ◽  
Collagen Coating ◽  
Umbilical Vein Endothelial Cells

Human umbilical vein endothelial cells were grown on several artificial matrices including collagen and on unmodified plastic dishes. Freeze thaw preparations of confluent monolayer endothelial cell cultures were assayed for PGI2-like activity by testing for inhibition of platelet a88regation. The cells grown on a collagen matrix expressed slightly less PGI2-like activity initially compared to the cells grown on plastic. When the PGI2like activity of the cell lysates was examined over a period of three hours after the initial preparation, endothelial cells grown on a collagen matrix exhibited a more rapid loss of activity. Preliminary quantitative evaluations suggest that lysates derived from cells grown on unmodified plastic retained 75% PGI2 like activity at 15 min, at which point collagen grown cells exhibited essentially no PGI2-like activity. Furthermore, as shown in the figure, under identical experimental conditions, cell lysates obtained from endothelial cells grown on unmodified plastic continued to express approximately 50% of the initial PGI2-like activity after one hour. Since addition of purified PGI2 or cell lysates in vitro to the collagen coating used as tissue culture substrate failed to cause any significant loss of platelet aggregation inhibitory activity beyond the normal rate of decay of PGI2 it appears likely that the reduction of PGI2-like activity may be associated with changes in the substrate collagen, possibly effected by the endothelial cell layer. Alternatively, the reduction of activity may be related to differences in the endothelial cells caused by growth on the collagen substrate.

scholarly journals Candida albicans Ecm33p Is Important for Normal Cell Wall Architecture and Interactions with Host Cells

2006 ◽  
Vol 5 (1) ◽  
pp. 140-147 ◽  
Author(s):  
Raquel Martinez-Lopez ◽  
Hyunsook Park ◽  
Carter L. Myers ◽  
Concha Gil ◽  
Scott G. Filler
Keyword(s):  
Endothelial Cells ◽  
Cell Wall ◽  
Candida Albicans ◽  
Epithelial Cells ◽  
Normal Cell ◽  
Cell Wall Integrity ◽  
Host Cells ◽  
Epithelial Cell Line ◽  
Oral Epithelial ◽  
Cell Wall Architecture

ABSTRACT Candida albicans ECM33 encodes a glycosylphosphatidylinositol-linked cell wall protein that is important for cell wall integrity. It is also critical for normal virulence in the mouse model of hematogenously disseminated candidiasis. To identify potential mechanisms through which Ecm33p contributes to virulence, we investigated the interactions of C. albicans ecm33Δ mutants with endothelial cells and the FaDu oral epithelial cell line in vitro. The growth rate of blastospores of strains containing either one or no intact copies of ECM33 was 50% slower than that of strains containing two intact copies of ECM33. However, all strains germinated at the same rate, forming similar-length hyphae on endothelial cells and oral epithelial cells. Strains containing either one or no intact copies of ECM33 had modestly reduced adherence to both types of host cells, and a markedly reduced capacity to invade and damage these cells. Saccharomyces cerevisiae expressing C. albicans ECM33 did not adhere to or invade epithelial cells, suggesting that Ecm33p by itself does not act as an adhesin or invasin. Examination of ecm33Δ mutants by transmission electron microscopy revealed that the cell wall of these strains had an abnormally electron-dense outer mannoprotein layer, which may represent a compensatory response to reduced cell wall integrity. The hyphae of these mutants also had aberrant surface localization of the adhesin Als1p. Collectively, these results suggest that Ecm33p is required for normal cell wall architecture as well as normal function and expression of cell surface proteins in C. albicans.

scholarly journals A linear amino acid sequence involved in the interaction of t-PA with its endothelial cell receptor

Blood ◽  
1989 ◽  
Vol 74 (6) ◽  
pp. 2034-2037
Author(s):  
DP Beebe ◽  
LA Miles ◽  
EF Plow
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Umbilical Vein ◽  
Cell Receptor ◽  
Amino Acid Sequences ◽  
Inhibitory Peptide ◽  
Tissue Plasminogen ◽  
Umbilical Vein Endothelial Cells

Endothelial cell receptors for tissue plasminogen activator (t-PA) have been demonstrated recently, and we have sought to identify a region of the t-PA molecule involved in its interaction with these receptors on human umbilical vein endothelial cells. Of three monoclonal antibodies against various regions of t-PA, one directed against the finger region inhibited 125I-t-PA binding to the cells. Synthetic peptides corresponding in amino acid sequences to segments from within the finger region were constructed, and one of these inhibited t-PA binding. This peptide corresponded to residues 7 through 17 of t-PA. The inhibition by this peptide was specific as other peptides from the finger region were inactive. The inhibitory peptide also did not affect the binding of another fibrinolytic ligand, urokinase, to the cells. Although a role for other regions of t-PA in binding to endothelial cells cannot be excluded, the results implicate a short span of linear amino acid sequence within the finger region in the interaction of t-PA with endothelial cells.

scholarly journals Exoproteome from Leptospira interrogans and Host Cells during Infection: Leptospiral Virulence Factors and Cellular Proteins Involved in Stress and Inflammation

2020 ◽  
Author(s):  
Weilin Hu ◽  
Muhammad Imran ◽  
Kai-Xuan Li ◽  
David M. Ojcius ◽  
Ai-Hua Sun ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Virulence Factors ◽  
Inflammatory Cytokines ◽  
Vascular Endothelial Cells ◽  
Umbilical Vein ◽  
Host Cells ◽  
Leptospira Interrogans ◽  
Vascular Endothelial ◽  
Macrophage Infection ◽  
Blood Coagulation Factors

Abstract Background: Leptospirosis, caused mainly by Leptospira interrogans, is a global zoonotic infectious disease. Macrophages and vascular endothelial cells are the main host cells for L. interrogans during infection, but the proteins released from the pathogen and the two host cells during infection remain mostly unknown.Results: Cellular supernatant proteins (CSPs) from human THP-1 macrophages or umbilical vein endothelial cells (HUVECs) infected with L. interrogans strain Lai were extracted by TCA/FASP methods. The exoproteins in the CSPs were identified by LC-MS/MS. Viability of the leptospires and host cells during infection was confirmed by confocal microscopy and MTT. The results showed that higher co-culture temperature (from 28°C to 37°C) and different biochemical environments cause a large change in the exoproteome of the spirochete. L. interrogans increased levels of leptospiral exoproteins related to stress, signal transduction and virulence factors, while the lipoprotein antigens LipL41, LipL21 and/or Loa22 were not detected. During infection of macrophages and endothelial cells, there was a large increase in host-cell exoproteins involved in stress response, complement pathways (C4/5/7/8), inflammatory cytokines (IL-6, TNF-α, MIF, MCP-1 and GM-CSF), extracellular matrix proteins (FN, LN and COLs), and blood coagulation factors. One-third of the leptospires and infected THP-1 macrophages died during macrophage infection, but nearly all the leptospires and endothelial cells remained viable during endothelial cell infection.Conclusions: Infection causes stress reponses for both leptospires and human macrophages and vascular endothelial cells and release of virulence factors, alteration of surface leptospiral lipoprotein antigens and secretion of complement components and inflammatory cytokines from host cells.

scholarly journals Proteasome-Independent Activation of Nuclear Factor κB in Cytoplasmic Extracts from Human Endothelial Cells byRickettsia rickettsii

1998 ◽  
Vol 66 (5) ◽  
pp. 1827-1833 ◽  
Author(s):  
Sanjeev K. Sahni ◽  
Daniel J. Van Antwerp ◽  
Marina E. Eremeeva ◽  
David J. Silverman ◽  
Victor J. Marder ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Umbilical Vein ◽  
Spotted Fever ◽  
Nuclear Factor Κb ◽  
Host Cells ◽  
Nuclear Factor ◽  
Rocky Mountain Spotted Fever ◽  
Free System

ABSTRACT Interaction of many infectious agents with eukaryotic host cells is known to cause activation of the ubiquitous transcription factor nuclear factor κB (NF-κB) (U. Siebenlist, G. Franzoso, and K. Brown, Annu. Rev. Cell Biol. 10:405–455, 1994). Recently, we reported a biphasic pattern of NF-κB activation in cultured human umbilical vein endothelial cells consequent to infection withRickettsia rickettsii, an obligate intracellular gram-negative bacterium and the etiologic agent of Rocky Mountain spotted fever (L. A. Sporn, S. K. Sahni, N. B. Lerner, V. J. Marder, D. J. Silverman, L. C. Turpin, and A. L. Schwab, Infect. Immun. 65:2786–2791, 1997). In the present study, we describe activation of NF-κB in a cell-free system, accomplished by addition of partially purified R. rickettsii to endothelial cell cytoplasmic extracts. This activation was rapid, reaching maximal levels at 60 min, and was dependent on the number ofR. rickettsii organisms added. Antibody supershift assays using monospecific antisera against NF-κB subunits (p50 and p65) confirmed the authenticity of the gel-shifted complexes and identified both p50-p50 homodimers and p50-p65 heterodimers as constituents of the activated NF-κB pool. Activation occurred independently of the presence of endothelial cell membranes and was not inhibited by removal of the endothelial cell proteasome. Lack of involvement of the proteasome was further confirmed in assays using the peptide-aldehyde proteasome inhibitor MG 132. Activation was not ATP dependent since no change in activation resulted from addition of an excess of the unhydrolyzable ATP analog ATPγS, supplementation with exogenous ATP, or hydrolysis of endogenous ATP with ATPase. Furthermore, Western blot analysis before and after in vitro activation failed to demonstrate phosphorylation of serine 32 or degradation of the cytoplasmic pool of IκBα. This lack of IκBα involvement was supported by the finding that R. rickettsii can induce NF-κB activation in cytoplasmic extracts prepared from T24 bladder carcinoma cells and human embryo fibroblasts stably transfected with a superrepressor phosphorylation mutant of IκBα, rendering NF-κB inactivatable by many known signals. Thus, evidence is provided for a potentially novel NF-κB activation pathway wherein R. rickettsii may interact with and activate host cell transcriptional machinery independently of the involvement of the proteasome or known signal transduction pathways.

An In Vitro Toxicity Assay for Human Endothelial Cells

1988 ◽  
Vol 16 (1) ◽  
pp. 38-41
Author(s):  
Rosella Sbarbati ◽  
Maria Luisa Schinetti ◽  
Maria Scarlattini
Keyword(s):  
Oxidative Stress ◽  
Endothelial Cells ◽  
Umbilical Vein ◽  
Vascular Diseases ◽  
In Vitro Toxicity ◽  
Human Endothelial Cells ◽  
Experimental Conditions ◽  
Umbilical Vein Endothelial Cells

Cultured human endothelial cells can replace living animals in studying the toxic role of noxious agents in the pathogenesis of vascular diseases and in the elucidation of the mechanism of action of protective drugs. Preliminary data are presented which examine the effects that oxidative stress produces on human endothelial cells in vitro. Human umbilical vein endothelial cells were subjected to an anoxia-re-oxygenation treatment and tested for the production of Super Oxide Dismutase (SOD)-inhibitable superoxide radicals. The results show that under our experimental conditions endothelial cells produce oxygen-free radicals and that the generation reaches a maximum after an anoxic challenge of 20 minutes. We conclude that the in vitro system presented in this paper could be a suitable tool for further studies on the effects of oxidative stress on the vascular endothelium, which mimics the in vivo conditions of re-perfusion after heart ischemia.

scholarly journals Effect of Interleukin-10 and Laminar Shear Stress on Endothelial Nitric Oxide Synthase and Nitric Oxide in African American Human Umbilical Vein Endothelial Cells

2015 ◽  
Vol 25 (4) ◽  
pp. 413 ◽  
Author(s):  
Dianne M. Babbitt ◽  
Ji-Seok Kim ◽  
Steven J. Forrester ◽  
Michael D. Brown ◽  
Joon-Young Park
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
African American ◽  
Umbilical Vein ◽  
Interleukin 10 ◽  
Laminar Shear Stress ◽  
Experimental Conditions ◽  
Umbilical Vein Endothelial Cells ◽  
Oxide Synthase ◽  
Endothelial Nitric Oxide

<p><strong>Background: </strong>African Americans have a pre­disposition to heightened systemic inflamma­tion and a high prevalence of hypertension.</p><p><strong>Objective: </strong>The purpose of this study was to evaluate the influence of interleukin-10 (IL- 10) and laminar shear stress (LSS) on African American endothelial cells by measuring to­tal endothelial nitric oxide synthase (eNOS) protein expression and its phosphorylated form (p-eNOS) at Serine 1177, and nitric oxide (NO) levels, in response to IL-10 incubation and high physiological levels of LSS, used as an <em>in vitro </em>mimetic for aerobic exercise training (AEXT).</p><p><strong>Design: </strong>Human umbilical vein endothelial cells (HUVEC) from an African American donor were cultured. The experimental conditions included <em>Static</em>, <em>Static with IL-10 Incubation, LSS at 20 dynes/cm</em><em>2</em><em>, and LSS at 20 dynes/cm</em><em>2 </em><em>with IL-10 Incubation</em>. West­ern blotting was used to measure eNOS and p-eNOS protein expression in the cells. A modified Griess assay was used to measure NO metabolites in the cell culture media.</p><p><strong>Results: </strong>There were significant increases in p-eNOS, eNOS, and NO in the <em>LSS at 20 dynes/cm</em><em>2 </em>and <em>LSS at 20 dynes/cm</em><em>2 </em><em>with IL-10 Incubation </em>experimental conditions when compared to the <em>Static </em>experimental condition. There were no other statistically significant differences demonstrating that IL- 10 did not have an additive effect on eNOS activity in our study.</p><p><strong>Conclusion: </strong>The significant increases in p-eNOS, eNOS, and NO as a result of LSS in African American HUVECs suggest that AEXT may be a viable, nonpharmacologic method to improve vascular inflamma­tion status and vasodilation, and thereby contribute to hypertension reduction in the African American population. <em>Ethn Dis. </em>2015;25(4):413-418; doi:10.18865/ ed.25.4.413</p>

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