Gamma Interferon Confers Resistance to Infection with Staphylococcus aureus in Human Vascular Endothelial Cells by Cooperative Proinflammatory and Enhanced Intrinsic Antibacterial Activities

ABSTRACT Vascular endothelium is an exposed target in systemic endovascular Staphylococcus aureus infections. We reported earlier that the proinflammatory and procoagulant activities of primary human umbilical vein endothelial cells (ECs) after binding and ingestion of S. aureus organisms provide the cells effective means for leukocyte-mediated bacterial elimination. Expanding on this, we now show that these ECs exhibit a modest intrinsic capacity for eliminating intracellular S. aureus that was influenced by cytokines relevant to S. aureus infections. Using various EC infection assays, we showed that gamma interferon (IFN-γ), applied to cultures of ECs prior to or after infection with S. aureus, markedly reduced the level of infection, illustrated by lower percentages of S. aureus-infected ECs and less intracellular bacteria per infected cell. IFN-γ-activated ECs had unaltered abilities to bind S. aureus and processed ingested bacteria by a seemingly conventional phagocytic pathway. IFN-γ treatment rescued EC monolayers from severe injury by virulent clinical S. aureus strains or excessive bacterial numbers. Mechanistically, IFN-γ controls S. aureus infection via IFN-γ receptor, most likely through stimulation of intrinsic endothelial antibacterial mechanisms but independent of processes that deprive bacteria of intracellular l-tryptophan or iron. The antibacterial activity of IFN-γ-stimulated ECs coincided with sustained or slightly elevated endothelial proinflammatory responses that supported monocyte recruitment. In conclusion, we identify IFN-γ as a potent regulatory Th1 cytokine possessing exclusive abilities to augment intrinsic antistaphylocccal effector mechanisms in human ECs without ablating the S. aureus-induced proinflammatory EC responses and, as such, coordinating a protective efficacy of ECs against blood-borne S. aureus infection.

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In vitro ability of Staphylococcus aureus isolates from bacteraemic patients with and without metastatic complications to invade vascular endothelial cells

Journal of Medical Microbiology ◽

10.1099/jmm.0.46765-0 ◽

2007 ◽

Vol 56 (10) ◽

pp. 1290-1295 ◽

Cited By ~ 3

Author(s):

Wan Beom Park ◽

Sung Han Kim ◽

Cheol-in Kang ◽

Jae Hyun Cho ◽

Ji Whan Bang ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Endothelial Cells ◽

Vascular Endothelial Cells ◽

Umbilical Vein ◽

Control Group ◽

Vascular Endothelial ◽

Tertiary Referral Hospital ◽

Metastatic Infection ◽

Umbilical Vein Endothelial Cells

Invasion of vascular endothelial cells is thought to be a critical step in the development of metastatic infections in patients with Staphylococcus aureus bacteraemia. This study was designed to evaluate the association between the ability to invade endothelial cells and metastatic infection by S. aureus. Patients with metastatic infection were identified among those with community-acquired S. aureus bacteraemia in a tertiary referral hospital. Patients with simple bacteraemia caused by S. aureus over the same period served as the control group. The ability of each clinical isolate to invade endothelial cells was evaluated by counting the number of intracellular organisms 1 h after inoculation onto human umbilical vein endothelial cells in vitro. The cytotoxic activity of intracellular S. aureus was determined 24 h after internalization, and expressed as the percentage of cells killed. The clinical isolates varied in invasiveness and cytotoxicity. The median invasiveness, relative to S. aureus reference strain ATCC 29213, was 145 % in the cases (n=10) [interquartile range (IQR) 103–160] and 153 % (IQR 111–173) in the controls (n=11; P=0.44). The median cytotoxicity was 59.4 % (IQR 47–68) in the cases and 65.2 % (IQR 50–74) in the controls (P=0.44). Differences in the ability of S. aureus to invade and destroy vascular endothelial cells in vitro were not associated with the development of metastatic complications in patients with S. aureus bacteraemia. This implies that the invasiveness and toxicity of S. aureus for endothelial cells may not be major determinants of metastatic infection.

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Modulation of Arachidonic Acid Metabolism in Human Endothelial Cells by Glucocorticoids

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661566 ◽

1986 ◽

Vol 55 (03) ◽

pp. 369-374 ◽

Cited By ~ 15

Author(s):

Raffaele De Caterina ◽

Babette B Weksler

Keyword(s):

Endothelial Cells ◽

Vascular Endothelial Cells ◽

Umbilical Vein ◽

Arachidonic Acid Metabolism ◽

Protein Synthesis Inhibitor ◽

Synthesis Inhibitor ◽

Prolonged Incubation ◽

Dependent Inhibition ◽

Human Thrombin ◽

Umbilical Vein Endothelial Cells

SummaryTo learn whether glucocorticoids inhibit prostaglandin (PG) production in vascular endothelial cells, we investigated the effects of glucocorticoids on PG synthesis by cultured human umbilical vein endothelial cells (EC). Pretreatment of EC with dexamethasone (DX, 10-9 to 5 x 10-5 M) caused a dose-dependent inhibition of PGI2 production when PG synthesis from endogenous arachidonate was stimulated by human thrombin (0.25-2 U/ml) or ionophore A 23187 (1-5 μM). The inhibition was detectable at 10-7 M DX and maximal at 10-5 M (4.0 ± 0.7 vs. control: 7.7 ± 1.9 ng/ml, mean ± S.D., P <0.01). The production of PGE2 and the release of radiolabelled arachidonate (AA) from prelabelled cells were similarly inhibited. Prolonged incubation of EC with glucocorticoids was required to inhibit PG production or arachidonate release: ranging from 8% inhibition at 5 h to 44% at 38 h. In contrast, prostaglandin formation from exogenous AA was not altered by DX treatment. When thrombin or ionophore-stimulated EC were restimulated with exogenous AA (25 μM), DX-treated cells released more PGI2 than control cells (5.7 ± 0.5 vs. 4.1 ± 0.6 ng/ml, P <0.01). Both the decrease in PGI2 production after thrombin/ionophore and the increase after re-stimulation with AA were blunted in the presence of the protein synthesis inhibitor cycloheximide (0.1-0.2 μg/ml). Thus, incubation of EC with glucocorticoids inhibits PG production at the step of phospholipase activation. The time requirement for these steroid effects and their blunting by cycloheximide are consistent with the induction of regulatory proteins, possibly lipocortins, in endothelial cells.

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Citrus bergamiaRisso Elevates Intracellular Ca2+in Human Vascular Endothelial Cells due to Release of Ca2+from Primary Intracellular Stores

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2013/759615 ◽

2013 ◽

Vol 2013 ◽

pp. 1-7 ◽

Cited By ~ 1

Author(s):

Purum Kang ◽

Seung Ho Han ◽

Hea Kyung Moon ◽

Jeong-Min Lee ◽

Hyo-Keun Kim ◽

...

Keyword(s):

Endothelial Cells ◽

Channel Blocker ◽

Vascular Endothelial Cells ◽

Umbilical Vein ◽

Dependent Manner ◽

Vascular Endothelial ◽

Concentration Dependent Manner ◽

Carbonyl Cyanide ◽

Intracellular Ca ◽

Umbilical Vein Endothelial Cells

The purpose of the present study is to examine the effects of essential oil ofCitrus bergamiaRisso (bergamot, BEO) on intracellular Ca2+in human umbilical vein endothelial cells. Fura-2 fluorescence was used to examine changes in intracellular Ca2+concentration[Ca2+]i. In the presence of extracellular Ca2+, BEO increased[Ca2+]i, which was partially inhibited by a nonselective Ca2+channel blocker La3+. In Ca2+-free extracellular solutions, BEO increased[Ca2+]iin a concentration-dependent manner, suggesting that BEO mobilizes intracellular Ca2+. BEO-induced[Ca2+]iincrease was partially inhibited by a Ca2+-induced Ca2+release inhibitor dantrolene, a phospholipase C inhibitor U73122, and an inositol 1,4,5-triphosphate (IP3)-gated Ca2+channel blocker, 2-aminoethoxydiphenyl borane (2-APB). BEO also increased[Ca2+]iin the presence of carbonyl cyanide m-chlorophenylhydrazone, an inhibitor of mitochondrial Ca2+uptake. In addition, store-operated Ca2+entry (SOC) was potentiated by BEO. These results suggest that BEO mobilizes Ca2+from primary intracellular stores via Ca2+-induced and IP3-mediated Ca2+release and affect promotion of Ca2+influx, likely via an SOC mechanism.

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Hypoxia-induced expression of complement receptor type 1 (CR1, CD35) in human vascular endothelial cells

AJP Cell Physiology ◽

10.1152/ajpcell.1999.276.2.c450 ◽

1999 ◽

Vol 276 (2) ◽

pp. C450-C458 ◽

Cited By ~ 39

Author(s):

Charles D. Collard ◽

Cuneyt Bukusoglu ◽

Azin Agah ◽

Sean P. Colgan ◽

Wende R. Reenstra ◽

...

Keyword(s):

Endothelial Cells ◽

Protein Expression ◽

Vascular Endothelial Cells ◽

Umbilical Vein ◽

Receptor Type ◽

Complement Receptor ◽

Complement Receptor Type ◽

Umbilical Vein Endothelial Cells ◽

Complement Receptor Type 1

Reoxygenation of hypoxic human umbilical vein endothelial cells (HUVECs) increases protein expression of the complement regulators CD46 and CD55. As the receptor for C3b is known to be present on injured bovine endothelial cells, we investigated whether hypoxia or inflammatory mediators induce complement receptor type 1 (CR1; CD35) expression on HUVECs. CR1 protein expression increased 3.7 ± 0.6-fold as measured by ELISA on HUVECs following hypoxia (48 h, 1% O2). Colocalization of CD35 and von Willebrand factor by confocal microscopy confirmed that CD35 was predominantly intracellular. Lipopolysaccharide or tumor necrosis factor-α also significantly increased HUVEC CR1 protein expression. Western blot analysis of neutrophil or hypoxic HUVEC lysates revealed a 221-kDa CR1 band under nonreducing conditions. RT-PCR of hypoxic HUVEC mRNA revealed a single band that, after sequencing, was identified as CD35. In situ hybridization of hypoxic HUVECs, but not normoxic HUVECs or fibroblasts, demonstrated increased CD35 mRNA. Hypoxic HUVECs bound immune complexes and acted as a cofactor for factor I-mediated cleavage of C3b. Thus hypoxia induces functional HUVEC CR1 expression.

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Small- and intermediate-conductance Ca2+-activated K+ channels directly control agonist-evoked nitric oxide synthesis in human vascular endothelial cells

AJP Cell Physiology ◽

10.1152/ajpcell.00036.2007 ◽

2007 ◽

Vol 293 (1) ◽

pp. C458-C467 ◽

Cited By ~ 84

Author(s):

Jian-Zhong Sheng ◽

Andrew P. Braun

Keyword(s):

Nitric Oxide ◽

Endothelial Cells ◽

Vascular Endothelial Cells ◽

Umbilical Vein ◽

Early Event ◽

No Production ◽

Primary Role ◽

Membrane Hyperpolarization ◽

Intracellular Ca ◽

Umbilical Vein Endothelial Cells

The contribution of small-conductance (SKCa) and intermediate-conductance Ca2+-activated K+ (IKCa) channels to the generation of nitric oxide (NO) by Ca2+-mobilizing stimuli was investigated in human umbilical vein endothelial cells (HUVECs) by combining single-cell microfluorimetry with perforated patch-clamp recordings to monitor agonist-evoked NO synthesis, cytosolic Ca2+ transients, and membrane hyperpolarization in real time. ATP or histamine evoked reproducible elevations in NO synthesis and cytosolic Ca2+, as judged by 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM) and fluo-3 fluorescence, respectively, that were tightly associated with membrane hyperpolarizations. Whereas evoked NO synthesis was unaffected by either tetraethylammonium (10 mmol/l) or BaCl2 (50 μmol/l) + ouabain (100 μmol/l), depleting intracellular Ca2+ stores by thapsigargin or removing external Ca2+ inhibited NO production, as did exposure to high (80 mmol/l) external KCl. Importantly, apamin and charybdotoxin (ChTx)/ triarylmethane (TRAM)-34, selective blockers SKCa and IKCa channels, respectively, abolished both stimulated NO synthesis and membrane hyperpolarization and decreased evoked Ca2+ transients. Apamin and TRAM-34 also inhibited an agonist-induced outwardly rectifying current characteristic of SKCa and IKCa channels. Under voltage-clamp control, we further observed that the magnitude of agonist-induced NO production varied directly with the degree of membrane hyperpolarization. Mechanistically, our data indicate that SKCa and IKCa channel-mediated hyperpolarization represents a critical early event in agonist-evoked NO production by regulating the influx of Ca2+ responsible for endothelial NO synthase activation. Moreover, it appears that the primary role of agonist-induced release of intracellular Ca2+ stores is to trigger the opening of both KCa channels along with Ca2+ entry channels at the plasma membrane. Finally, the observed inhibition of stimulated NO synthesis by apamin and ChTx/TRAM-34 demonstrates that SKCa and IKCa channels are essential for NO-mediated vasorelaxation.

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CRIF1 Deficiency Increased Homocysteine Production by Disrupting Dihydrofolate Reductase Expression in Vascular Endothelial Cells

Antioxidants ◽

10.3390/antiox10111645 ◽

2021 ◽

Vol 10 (11) ◽

pp. 1645

Author(s):

Ikjun Lee ◽

Shuyu Piao ◽

Seonhee Kim ◽

Harsha Nagar ◽

Su-Jeong Choi ◽

...

Keyword(s):

Endothelial Cells ◽

Dihydrofolate Reductase ◽

Vascular Endothelial Cells ◽

Umbilical Vein ◽

Plasma Concentrations ◽

Vascular Endothelial ◽

Cell Dysfunction ◽

Key Enzyme ◽

Umbilical Vein Endothelial Cells ◽

Folate Cycle

Elevated plasma homocysteine levels can induce vascular endothelial dysfunction; however, the mechanisms regulating homocysteine metabolism in impaired endothelial cells are currently unclear. In this study, we deleted the essential mitoribosomal gene CR6 interacting factor 1 (CRIF1) in human umbilical vein endothelial cells (HUVECs) and mice to induce endothelial cell dysfunction; then, we monitored homocysteine accumulation. We found that CRIF1 downregulation caused significant increases in intracellular and plasma concentrations of homocysteine, which were associated with decreased levels of folate cycle intermediates such as 5-methyltetrahydrofolate (MTHF) and tetrahydrofolate (THF). Moreover, dihydrofolate reductase (DHFR), a key enzyme in folate-mediated metabolism, exhibited impaired activity and decreased protein expression in CRIF1 knockdown endothelial cells. Supplementation with folic acid did not restore DHFR expression levels or MTHF and homocysteine concentrations in endothelial cells with a CRIF1 deletion or DHFR knockdown. However, the overexpression of DHFR in CRIF1 knockdown endothelial cells resulted in decreased accumulation of homocysteine. Taken together, our findings suggest that CRIF1-deleted endothelial cells accumulated more homocysteine, compared with control cells; this was primarily mediated by the disruption of DHFR expression.

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GPR55 Antagonist CID16020046 Protects against Atherosclerosis Development in Mice by Inhibiting Monocyte Adhesion and Mac-1 Expression

International Journal of Molecular Sciences ◽

10.3390/ijms222313084 ◽

2021 ◽

Vol 22 (23) ◽

pp. 13084

Author(s):

Seung-Jin Lee ◽

Dong-Soon Im

Keyword(s):

Endothelial Cells ◽

High Fat Diet ◽

Vascular Endothelial Cells ◽

Umbilical Vein ◽

Vascular Endothelial ◽

Monocyte Adhesion ◽

High Fat ◽

Potent Agonist ◽

Atherosclerosis Development ◽

Umbilical Vein Endothelial Cells

GPR55 recognizes several lipid molecules such as lysophosphatidylinositol. GPR55 expression was reported in human monocytes. However, its role in monocyte adhesion and atherosclerosis development has not been studied. The role of GPR55 in monocyte adhesion and atherosclerosis development was investigated in human THP-1 monocytes and ApoE−/− mice using O-1602 (a potent agonist of GPR55) and CID16020046 (a specific GPR55 antagonist). O-1602 treatment significantly increased monocyte adhesion to human umbilical vein endothelial cells, and the O-1602-induced adhesion was inhibited by treatment with CID16020046. O-1602 induced the expression of Mac-1 adhesion molecules, whereas CID16020046 inhibited this induction. Analysis of the promoter region of Mac-1 elucidated the binding sites of AP-1 and NF-κB between nucleotides −750 and −503 as GPR55 responsive elements. O-1602 induction of Mac-1 was found to be dependent on the signaling components of GPR55, that is, Gq protein, Ca2+, CaMKK, and PI3K. In Apo−/− mice, administration of CID16020046 ameliorated high-fat diet-induced atherosclerosis development. These results suggest that high-fat diet-induced GPR55 activation leads to the adhesion of monocytes to endothelial cells via induction of Mac-1, and CID16020046 blockage of GPR55 could suppress monocyte adhesion to vascular endothelial cells through suppression of Mac-1 expression, leading to protection against the development of atherosclerosis.

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GPR55 antagonist CID16020046 protects against atherosclerosis development in mice by inhibiting monocyte adhesion and Mac-1 expression

10.22541/au.163290912.29610111/v1 ◽

2021 ◽

Author(s):

Seung-Jin Lee ◽

DONG-SOON IM

Keyword(s):

Endothelial Cells ◽

High Fat Diet ◽

Vascular Endothelial Cells ◽

Umbilical Vein ◽

Monocyte Adhesion ◽

High Fat ◽

Atherosclerosis Development ◽

Proinflammatory Role ◽

Umbilical Vein Endothelial Cells

Background and Purpose: GPR55 is a G protein-coupled receptor that recognizes several lipid molecules. GPR55 expression in human monocytes and its proinflammatory role lead us to investigate the role of GPR55 in monocyte adhesion and atherosclerosis development. Experimental Approach: We investigated monocyte adhesion in human THP-1 monocytes and atherosclerosis development in ApoE-/- mice by using O-1602 (a potent agonist of GPR55), CID16020046 (a specific GPR55 antagonist), and a high-fat diet-induced atherosclerosis model. Key Results: In human THP-1 monocytes, treatment with O-1602 signiﬁcantly increased monocyte adhesion to human umbilical vein endothelial cells (HUVECs), and the O-1602-induced adhesion was inhibited by treatment with CID16020046. O-1602 induced the expression of Mac-1 adhesion molecules, whereas CID16020046 inhibited this induction. Analysis of the promoter region of Mac-1 elucidated the binding sites of AP-1 and NF-κB between nucleotides -750 and -503 as GPR55 responsive elements. Furthermore, O-1602 induction of Mac-1 through AP-1 and NF-B was found to be dependent on the signaling components of GPR55, that is, Gq protein, Ca2+, CaMKK, and PI3K. In an in vivo study of high-fat diet-induced atherosclerosis in ApoE-/- mice, administration of CID16020046 ameliorated atherosclerosis development. These results suggest that high-fat diet-induced GPR55 activation leads to adhesion of monocytes to endothelial cells via induction of Mac-1, and CID16020046 blockage of GPR55 could suppress monocyte adhesion to vascular endothelial cells through suppression of Mac-1 expression, leading to protection against the development of atherosclerosis. Conclusions: This report suggests that GPR55 may be a therapeutic target for atherosclerosis development.

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TKI-Resistant Renal Cancer Secretes Low-Level Exosomal miR-549a to Induce Vascular Permeability and Angiogenesis to Promote Tumor Metastasis

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.689947 ◽

2021 ◽

Vol 9 ◽

Author(s):

Zuodong Xuan ◽

Chen Chen ◽

Wenbin Tang ◽

Shaopei Ye ◽

Jianzhong Zheng ◽

...

Keyword(s):

Endothelial Cells ◽

Vascular Permeability ◽

Renal Cancer ◽

Kinase Inhibitors ◽

Vascular Endothelial Cells ◽

Umbilical Vein ◽

Endothelial Permeability ◽

Vascular Endothelial ◽

Cell Renal Cell Carcinoma ◽

Umbilical Vein Endothelial Cells

Tyrosine kinase inhibitors (TKI)-resistant renal cancer is highly susceptible to metastasis, and enhanced vascular permeability promotes the process of metastasis. To evaluate the effect of cancer-derived exosomes on vascular endothelial cells and clarify the mechanism of metastasis in TKI-resistant renal cancer, we studied the crosstalk between clear cell renal cell carcinoma (ccRCC) cells and human umbilical vein endothelial cells (HUVECs). Exosomes from ccRCC cells enhanced the expression of vascular permeability-related proteins. Compared with sensitive strains, exosomes from resistant strains significantly enhanced vascular endothelial permeability, induced tumor angiogenesis and enhanced tumor lung metastasis in nude mice. The expression of miR-549a is lower in TKI-resistant cells and exosomes, which enhanced the expression of HIF1α in endothelial cells. In addition, TKI-resistant RCC cells reduced nuclear output of pre-miR-549a via the VEGFR2-ERK-XPO5 pathway, and reduced enrichment of mature miR-549a in cytoplasm, which in turn promoted HIF1α expression in RCC, leading to increased VEGF secretion and further activated VEGFR2 to form a feedback effect. miR-549a played an important role in the metastasis of renal cancer and might serve as a blood biomarker for ccRCC metastasis and even had the potential of becoming a new drug to inhibit TKI-resistance.

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Thrombin Induces Thromboplastin Synthesis in Cultured Vascular Endothelial Cells

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657742 ◽

1985 ◽

Vol 54 (02) ◽

pp. 373-376 ◽

Cited By ~ 45

Author(s):

K S Galdal ◽

T Lyberg ◽

S A Evensen ◽

E Nilsen ◽

H Prydz

Keyword(s):

Endothelial Cells ◽

De Novo ◽

Vascular Endothelial Cells ◽

Umbilical Vein ◽

Calf Serum ◽

Phosphodiesterase Inhibitors ◽

Fold Increase ◽

Homocysteine Thiolactone ◽

Umbilical Vein Endothelial Cells

SummaryCultured human umbilical vein endothelial cells responded to thrombin (10−2 – 10 NIH u/ml) with a 2-5 fold increase in thromboplastin activity. The maximum response was reached after 4 hr in serum-free medium. The effect of thrombin was fully inhibited by the presence of 50% (v/v) fetal calf serum or more in the medium, by preincubation of thrombin with hirudin or by treatment of thrombin with N-bromosuccinimide or phenylmethylsulfonyl fluoride. The thrombin-induced thromboplastin activity was inhibited by incubation of the cells with cycloheximide (2 μg/ml) or actinomycin D (2 μg/ml) showing that the response depended on de novo protein and RNA synthesis. It was also suppressed by exposure of the cells to two different phosphodiesterase inhibitors, 3-butyl-l-methyl-xanthine (5 · 10−4 M) and rac-4 (3-butoxy-4-methoxybenzyl)-2-imidazole (5 · 10−4 M), to the transmethylation inhibitors 3-deazaadenosine (10−5 M) and 1-homocysteine thiolactone (2 · 10−5 M) in combination and to the intracellular calcium antagonist 8-(N,N-diethylamino)-octyl 3,4,5,-tri-methoxybenzoate hydrochloride (8 · 10−5 M). Our results suggest that small amounts of thrombin can induce thromboplastin synthesis in endothelial cells in vitro and that this synthesis probably is regulated by the intracellular level of cAMP, by cytoplasmic Ca2+ and possibly also by transmethylation reactions.

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