Purification, molecular cloning and mechanism of action of graminelysin I, a snake-venom-derived metalloproteinase that induces apoptosis of human endothelial cells

2001 ◽  
Vol 357 (3) ◽  
pp. 719-728 ◽  
Author(s):  
Wen Bin WU ◽  
Shin C. CHANG ◽  
Ming-Yi LIAU ◽  
Tur-Fu HUANG
Keyword(s):  
Endothelial Cells ◽  
Umbilical Vein ◽  
Matrix Proteins ◽  
Dependent Manner ◽  
Single Chain ◽  
Human Endothelial Cells ◽  
Putative Precursor ◽  
Induced Apoptosis ◽  
Umbilical Vein Endothelial Cells ◽  
Dose Dependent

Apoptosis, a programmed, physiological mode of cell death, is important in tissue homoeostasis. Here we report that a new metalloproteinase, graminelysin I, purified from Trimeresurus gramineus venom, induced apoptosis of human endothelial cells as examined by electrophoresis and flow cytometry. Graminelysin I contains only a metalloproteinase domain. It is a single-chain proteinase with a molecular mass of 27020Da. cDNA sequence analysis revealed that the disintegrin-like and cysteine-rich domains of the putative precursor protein of graminelysin I are likely to be processed post-translationally, producing the proteinase domain (graminelysin I). Graminelysin I cleaved the α chain of fibrinogen preferentially and cleaved the β chain either on longer incubation or at higher concentration. Graminelysin I inhibited the adhesion of human umbilical-vein endothelial cells (HUVECs) to immobilized fibrinogen and induced HUVECs detachment in a dose-dependent manner. These effects on HUVECs were abolished when graminelysin I was pretreated with EDTA. However, graminelysin I did not inhibit the adhesion of HUVECs to immobilized collagen. HUVECs were susceptible to death after treatment with graminelysin I when they were cultured on immobilized fibrinogen. In contrast, HUVECs were rather resistant to treatment with graminelysin I if they were cultured on immobilized collagen. Furthermore, graminelysin I induced apoptosis of HUVECs in a dose-dependent manner. Similarly, its apoptosis-inducing activity was blocked if it was treated with EDTA. These results suggest that the catalytic activity of graminelysin I on matrix proteins contributes to its apoptosis-inducing activity.

Defibrotide Stimulates Expression of Thrombomodulin in Human Endothelial Cells

1994 ◽  
Vol 71 (04) ◽  
pp. 507-510 ◽  
Author(s):  
Quansheng Zhou ◽  
Xiaohong Chu ◽  
Changgeng Ruan
Keyword(s):  
Endothelial Cells ◽  
Conditioned Medium ◽  
Umbilical Vein ◽  
Dependent Manner ◽  
Human Endothelial Cells ◽  
Antithrombotic Activity ◽  
Beneficial Effects ◽  
Chromogenic Assay ◽  
Umbilical Vein Endothelial Cells ◽  
Dose Dependent

SummaryCultured human umbilical vein endothelial cells were incubated with defibrotide at concentrations of 0, 5, 50 and 500 jxg/ml for 4 and 24 h respectively. Thrombomodulin activity and molecules on the surface of the cells were determined by chromogenic assay and radioimmunoassay, thrombomodulin antigen in endothelial cells and in conditioned medium of the cells was measured by immunoradioassay. Thrombomodulin mRNA within the cells was analysed by slot blot. After 24 h of incubation, the activity and molecules of thrombomodulin on the surface of endothelial cells, as well as the antigen and mRNA of thrombomodulin in the cells were significantly increased in a dose dependent manner. However, the level of thrombomodulin antigen in conditioned medium was about equal to that of the control. Our data indicate that defibrotide stimulates expression of thrombomodulin in human endothelial cells. These beneficial effects may play a role in antithrombotic activity of defibrotide.

Single-Chain and Two-Chain Tissue-Type Plasminogen Activator (t-PA) Bind Differently to Cultured Human Endothelial Cells

1989 ◽  
Vol 62 (02) ◽  
pp. 699-703 ◽  
Author(s):  
Rob J Aerts ◽  
Karin Gillis ◽  
Hans Pannekoek
Keyword(s):  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Binding Sites ◽  
Umbilical Vein ◽  
Tissue Type ◽  
Single Chain ◽  
Human Endothelial Cells ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Umbilical Vein Endothelial Cells

SummaryIt has recently been shown that the fibrinolytic components plasminogen and tissue-type plasminogen activator (t-PA) both bind to cultured human umbilical vein endothelial cells (HUVEC). After cleavage of t-PA by plasmin, “single-chain” t-PA (sct-PA) is converted into “two-chain” t-PA (tct-PA), which differs from the former in a number of respects. We compared binding of sct-PA and tct-PA to the surface of HUVEC. Removal of t-PA bound to HUVEC by a mild treatment with acid and a subsequent quantification of eluted t-PA both by activity- and immunoradiometric assays revealed that, at concentrations between 10 and 500 nM, HUVEC bind about 3-4 times more sct-PA than tct-PA. At these concentrations, both sct-PA and tct-PA remain active when bound to HUVEC. Mutual competition experiments showed that sct-PA and tct-PA can virtually fully inhibit binding of each other to HUVEC, but that an about twofold higher concentration of tct-PA is required to prevent halfmaximal binding of sct-PA than visa versa. These results demonstrate that sct-PA and tct-PA bind with different affinities to the same binding sites on HUVEC.

scholarly journals Ethyl Acetate Fraction of Teucrium Polium Extract Abolishes Human Umbilical Vein Endothelial Cells (HUVEC) Tubulogenesis in Collagen Bed through Suppression of Cell Proliferation/VEGF Secretion

2019 ◽  
Author(s):  
Vahide Askari ◽  
Somayeh Shamlou ◽  
Ali Mostafaie ◽  
Sara Khaleqi
Keyword(s):  
Endothelial Cells ◽  
Ethyl Acetate ◽  
Umbilical Vein ◽  
Ethyl Acetate Fraction ◽  
Dependent Manner ◽  
Angiogenic Effect ◽  
Teucrium Polium ◽  
Umbilical Vein Endothelial Cells ◽  
Dose Dependent

Angiogenesis has essential role in growth and metastasis of tumors. Development of therapies aimed to suppress angiogenesis using medicinal plants is one of the effective approaches for prevention/treatment of cancer. The current study was performed to investigate in vitro anti-angiogenic effect of Teucrium Polium (TP) extract and its fractions. The aerial part of Teucrium Polium was powdered and extracted with 50% ethanol. The extract was fractionated in to aqueous (AQ), n-butanol (BU), ethyl acetate (EA) and n-hexane (HE) fractions. Anti-angiogenic effect of TP was examined on human umbilical vein endothelial cells (HUVECs) in three-dimensional collagen matrix. The endothelial cells form capillary-like branches that can be visualized using phase contrast microscope and the number of tube-like structures can be quantified as a measure of in vitro angiogenesis. Furthermore, anti-proliferative and vascular endothelial growth factor(VEGF )suppressive effect of TP as important factors in the process of angiogenesis were assessed using3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)and quantitative ELISA, respectively. Based on our findings, among the TP fractions, EA fraction showed the highest inhibitory activity on angiogenesis. This fraction with IC50: 68 µg/mL, inhibited angiogenesis at 60 µg/mL. The crude extract and other fractions of TP inhibited angiogenesis in a dose-dependent manner at doses higher concentrations than EA fraction, significantly.TP extract and EA fraction were able to inhibit proliferation of HUVEC and inhibited VEGF secretion in a dose dependent manner. The ethyl acetate fraction at 60 µg/ml inhibited VEGF secretion perfectly. Our data indicated that ethyl acetate fraction of Teucrium Polium could be a potential candidate for the prevention of angiogenesis in cancer and other related disorders. However, this suggestion needs more quantitative and in vivo investigations for confirmation.

Decrease of tetrahydrobiopterin and NO generation in endothelial cells exposed to simulated diving

2019 ◽  
pp. 159-169
Author(s):  
Ronja Hesthammer ◽  
◽  
Torunn Eide ◽  
Eimar Thorsen ◽  
Asbjørn M. Svardal ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Decompression Sickness ◽  
Synergistic Effects ◽  
Umbilical Vein ◽  
Bubble Formation ◽  
No Production ◽  
Dependent Manner ◽  
Hyperoxic Exposure ◽  
Umbilical Vein Endothelial Cells ◽  
Dose Dependent

Purpose: Nitric oxide (NO) has been shown to protect against bubble formation and the risk of decompression sickness. We hypothesize that oxidation of tetrahydrobiopterin (BH4) leads to a decreased production of NO during simulated diving. Methods: Human umbilical vein endothelial cells (HUVEC) were exposed to hyperoxia or simulated diving for 24 hours. The levels of biopterins (BH4, BH2 and B) were determined by LC-MS/MS, and the production of NO by monitoring the conversion of L-arginine to L-citrulline. Results: Exposure to hyperoxia decreased BH4 in a dose-dependent manner; by 48 ± 15% following exposure to 40 kPa O2 (P < 0.001 vs. control at 20 kPa O2), and 70 ± 16% following exposure to 60 kPa O2. Exposure to 40 kPa O2 decreased NO production by 25 ± 9%, but there was no further decrease when increasing oxygen exposure to 60 kPa (25 ± 10%). No additional effects of simulated diving were observed, indicating no additive or synergistic effects of hyperbaria and hyperoxia on the BH4 level or NO generation. Conclusion: NO generation in intact human endothelial cells was decreased by simulated diving, as well as by hyperoxic exposure, while BH4 levels seem to be affected only by hyperoxia. Hence, the results suggest that BH4 is not the sole determinant of NO generation in HUVEC.

scholarly journals Orosomucoid has a cAMP-dependent effect on human endothelial cells and inhibits the action of histamine

2000 ◽  
Vol 278 (5) ◽  
pp. H1725-H1731 ◽  
Author(s):  
Jenny Sörensson ◽  
Maria Ohlson ◽  
Anna Björnson ◽  
Börje Haraldsson
Keyword(s):  
Endothelial Cells ◽  
Inflammatory Mediator ◽  
Umbilical Vein ◽  
Primary Cultures ◽  
Microvascular Endothelial Cells ◽  
Capillary Barrier ◽  
Human Endothelial Cells ◽  
Anti Inflammatory ◽  
Umbilical Vein Endothelial Cells ◽  
Dose Dependent

The plasma protein orosomucoid (α1-acid glycoprotein) has previously been shown to constitute a critical component of the capillary barrier. The protein has also been suggested to act as an anti-inflammatory mediator in a diversity of experimental situations. Recently we reported that orosomucoid is synthesized by the microvascular endothelial cells per se. In the present study, the effects of orosomucoid on primary cultures of human umbilical vein endothelial cells (HUVEC) were studied using the Cytosensor microphysiometer. We found that 1) orosomucoid (0.01 g/l) increased the metabolic activity of HUVEC as reflected by the increased acidification rate of +14 ± 1%; 2) pretreatment with 0.5 mM 8-bromo-cAMP for 20 min markedly and reversibly inhibited the effect of orosomucoid, whereas 8-bromo-cGMP did not; 3) histamine elicited a dose-dependent response that was abolished by pretreatment with either cAMP or cGMP; and finally, 4) pretreatment of HUVEC for 6 min with orosomucoid (0.01 g/l) inhibited the action of histamine. In summary, this is the first report demonstrating that orosomucoid affects human endothelial cells and that it does so by using cAMP as a second messenger. This provides an explanation for previous findings of anti-inflammatory effects of the protein and shows that orosomucoid affects the endothelium during both normal and pathophysiological conditions.

The Protective Effect of Tetrahydroxystilbene Glucoside on High Glucose-Induced Injury in Human Umbilical Vein Endothelial Cells through the PI3K/Akt/eNOS Pathway and Regulation of Bcl-2/Bax

2021 ◽  
pp. 1-10
Author(s):  
Jiankun Cui ◽  
Bo Zhang ◽  
Min Gao ◽  
Baohai Liu ◽  
Cong Dai ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Protective Effect ◽  
High Glucose ◽  
Umbilical Vein ◽  
Vascular Complications ◽  
Dependent Manner ◽  
Human Umbilical Vein ◽  
Induced Apoptosis ◽  
Umbilical Vein Endothelial Cells

Endothelial dysfunction plays a central role in the patho­genesis of diabetic vascular complications. 2,3,5,4′-tetra­hydroxystilbene-2-O-β-D-glucoside (TSG), an active component extracted from the roots of Polygonum multiflorum Thunb, has been shown to have strong antioxidant and antiapoptotic activities. In the present study, we investigated the protective effect of TSG on apoptosis induced by high glucose in human umbilical vein endothelial cells (HUVECs) and the possible mechanisms. Our data demonstrated that TSG significantly reversed the high glucose-induced decrease in cell viability, suppressed high glucose-induced generation of intracellular reactive oxygen species (ROS), the activity of caspase-3, and decreased the percentage of apoptotic cells in a dose-dependent manner. In addition, we found that TSG not only increased the expression of Bcl-2, while decreasing Bax expression, but also activated phosphorylation of Akt and endothelial nitric oxide synthase (eNOS) with subsequent nitric oxide production and ultimately reduced high glucose-induced apoptosis. However, the antiapoptotic effects of TSG were abrogated by pretreatment of the cells with PI3K inhibitor (LY294002) or eNOS inhibitor N<sup>G</sup>-L-nitro-arginine methyl ester, respectively. These results suggest that TSG inhibits high glucose-induced apoptosis in HUVECs through inhibition of ROS production, activation of the PI3K/Akt/eNOS pathway, and upregulation of the Bcl-2/Bax ratio, and thus may demonstrate significant potential for preventing diabetic cardiovascular complications.

ROLE OF BIOLOGICAL RESPONSE MODIFIERSIN THE REGULATION OF THROMBOPLASTIN SYNTHESIS IN MONOCYTES AND ENDOTHELIAL CELLS

1987 ◽  
Author(s):  
E Carlsen ◽  
H Prydz
Keyword(s):  
Endothelial Cells ◽  
Human Brain ◽  
Umbilical Vein ◽  
Biological Response ◽  
Cdna Libraries ◽  
Dependent Manner ◽  
Cellular Interactions ◽  
Human Umbilical Vein ◽  
Umbilical Vein Endothelial Cells ◽  
Dose Dependent

A rabbit polyclonal antibody which was monospecific for thromboplastin (TP) apoprotein from human brain according to a number of criteria was used to screen two human placenta cDNA libraries in the expression vector Agtll, one randomly primed and one oligo-dT primed, by the method of Young and Davis. 23 positive clones expressing TP related antigen were isolated and plaque purified. DNA from the different clones was isolated and the TPcDNA inserts released by EcoRl digestion. The inserts could be classified into11 size classes ranging from approx. 300-1100 base pairs.The largest insert (1100 bp) was subcloned intothe plasmid vector pGEM-1. When the nick-translated plasmid (pTP4-l) was used as a probe to screen the phage clones by slot blot hybridization all the 23clones hybridized to the 1100 bp insert. A XgtllTP4 lysogen expressed B-galactosidase- TP4 fusion peptide upon IPTG induction as shown by immunobinging studied using two different antibodies to TP apoprotein: the rabbit antibody originally used to screen the libraries and an antibody raised in goat against human brain TP purified by affinity chromatography on a Factor Vll-antiVII-agarose column.Cytokines mediate many of the cellular interactions in the inflammatory and immune response systems and have a variety of actions. We have investigated the effect of rIL-1a/$,rIL-2, rlFNa/y and rTNFa on thromboplastin synthesis (TPL) in monocytes (M) and human umbilical vein endothelial cells (HUVEC). Recombinant IL- 1a and IL-16 both induced a dose dependent increase in TPL activity of monocyte (8-fold) and HUVEC cultures (15-fold) at 6h. The increase levelled off at interleukinconcentrations of 50-100u/ml. Recombinant IL-2 at 50u/ml induced a 5-fold rise in monocytes TPL. The effect of rIL-2 on HUVEC TPL synthesis at 6 h was smaller than on monocytes but still clearly significant at dose dependent. Recombinant IFN-Y (10 -10 u/ml) increased.TPL activity in HUVEC at 6h and 16 h in adose dependent manner, whereas no effect of rIFN-Y and IFN-a (1-10 u/ml) on M TPL was seen. When LPS (5pg/ml) was used to induce TPL synthesis, additional stimulation with rIFN-Y further enhanced HUVEC TPL activity, but decreased M TPL activity. Recombinant IFNa also decreased LPS induced TPL synthesis in M andhad no effect on HUVEC TPL. Recombinant TNFa (0.3x104u/ml) increased HUVEC TPL 7-fold at 6h. There wasno effect on M TPL synthesis. No endotoxin was detected in any of these preparations. CONCLUSIONS: Some biological response modifiers induced thromboplastin synthesis in monocytes (IL-ia, IL-13, IL-2) and in human umbilical vein endothelial cells (IL-ia, IL-18, IL-2, TNFa and IFNY). Some had no direct effect on TPL synthesis but inhibited the response to monocytes to other thromboplastin-inducing agents like LPS (IFNa and IFNY).

scholarly journals The antioxidant effects of quercetin metabolites on the prevention of high glucose-induced apoptosis of human umbilical vein endothelial cells

2008 ◽  
Vol 101 (8) ◽  
pp. 1165-1170 ◽  
Author(s):  
Chia-Lun Chao ◽  
Yu-Chi Hou ◽  
Pei-Dawn Lee Chao ◽  
Ching-Sung Weng ◽  
Feng-Ming Ho
Keyword(s):  
Endothelial Cells ◽  
Ascorbic Acid ◽  
High Glucose ◽  
Caspase 3 ◽  
Umbilical Vein ◽  
Dependent Manner ◽  
Human Umbilical Vein ◽  
Micromolar Concentration ◽  
Induced Apoptosis ◽  
Umbilical Vein Endothelial Cells

Diabetes mellitus is an important risk factor for CVD. A previous study showed that high glucose induced the apoptosis of human umbilical vein endothelial cells (HUVEC) via the sequential activation of reactive oxygen species, Jun N-terminal kinase (JNK) and caspase-3. The apoptosis cascade could be blocked by ascorbic acid at the micromolar concentration (100 μm). In addition to ascorbic acid, quercetin, the most abundant dietary flavonol, has been recently actively studied in vascular protection effects due to its antioxidant effect at low micromolar concentrations (10–50 μm). Quercetin sulfate/glucuronide, the metabolite of quercetin in blood, however, has been rarely evaluated. In the present study, we investigated the effect of quercetin sulfate/glucuronide on the prevention of high glucose-induced apoptosis of HUVEC. HUVEC were treated with media containing high glucose (33 mm) in the presence or absence of ascorbic acid (100 μm) or quercetin sulfate/glucuronide (100 nm, 300 nm and 1 μm). For the detection of apoptosis, a cell death detection ELISA assay was used. The level of intracellular H2O2 was measured by flow cytometry. JNK and caspase-3 were evaluated by a kinase activity assay and Western blot analysis. The results showed that high glucose-induced apoptosis was inhibited by quercetin sulfate/glucuronide in a dose-dependent manner. The effect of quercetin sulfate/glucuronide on H2O2 quenching, inhibition of JNK and caspase-3 activity at the nanomolar concentration (300 nm) was similar to that of ascorbic acid at the micromolar concentration (100 μm). The findings of the present study may shed light on the pharmacological application of quercetin in CVD.

Verotoxin-1 Induces Tissue Factor Expression in Human Umbilical Vein Endothelial Cells through Activation of NF-κB/Rel and AP-1

2000 ◽  
Vol 84 (10) ◽  
pp. 712-721 ◽  
Author(s):  
Kimihiko Takada ◽  
Toshiyuki Higuchi ◽  
Junichi Sugiyama ◽  
Hidemi Ishii
Keyword(s):  
Endothelial Cells ◽  
Tissue Factor ◽  
Binding Sites ◽  
Umbilical Vein ◽  
Nuclear Proteins ◽  
Binding Motif ◽  
Dependent Manner ◽  
Human Umbilical Vein ◽  
Umbilical Vein Endothelial Cells ◽  
Dose Dependent

SummaryThis study examined the effect of verotoxin-1 (VT-1), which is released from Escherichia coli O157:H7, on endothelial expression of tissue factor (TF), a cofactor required to initiate blood coagulation. In order to elucidate the molecular basis for development of hemolytic uremic syndrome (HUS) in patients infected with E. coli O157:H7, human umbilical vein endothelial cells (HUVECs) were exposed to purified VT-1. VT-1 increased both TF activity and TF mRNA in HUVECs without loss of cell viability in a time-and dose-dependent manner from 0.1 to 10 ng/ml VT-1. Nuclear proteins extracted from VT-1-stimulated HUVECs bound to the consensus NF-κB/Rel and AP-1 binding oligonucleotides in a dose-dependent manner within 2 h after the stimulation in electrophoretic mobility shift assays (EMSA). Nuclear proteins from VT-1-stimulated HUVECs formed two complexes with the NF-κB/Rel binding motif in the human TF promoter (TF-κB motif). The supershift assays, using antibodies for human p65, p50 or c-Rel, indicated that the lower complex was composed of p65/p50 and the higher complex was a p65 homo-or hetero-dimer with the Rel family, except c-Rel. The human TF promoter contains two AP-1 binding sites, the proximal and distal AP-1 binding sites. The supershift assays indicated that AP-1 containing mainly c-Jun and JunD, positively bound to the proximal AP-1 motif of TF (TF-AP-1). The distal TF-AP-1 motif did not show positive binding with nuclear proteins from VT-1-stimulated HUVECs. Pretreatment of HUVECs with curcumin, an inhibitor of NF-κB/Rel activation, synthesis of c-Jun mRNA and binding of activated AP-1 with AP-binding oligonucleotide, prevented the VT-1 induced increase in TF mRNA and activity in VT-1-stimulated HUVECs. Curcumin also inhibited NF-κB and AP-1 binding to TF-κB and proximal TF-AP-1 oligonucleotides, respectively, in a dose-dependent manner. The present work suggests that both the NF-κB/Rel and AP-1 activated in endothelial cells by stimulation with VT-1 binds to the TF-κB and proximal AP-1 binding sites, respectively, of the TF promoter.

Assembly and Activation of the Intrinsic Fibrinolytic Pathway on the Surface of Human Endothelial Cells in Culture

1995 ◽  
Vol 74 (02) ◽  
pp. 698-703 ◽  
Author(s):  
Catherine Lenich ◽  
Ralph Pannell ◽  
Victor Gurewich
Keyword(s):  
Endothelial Cells ◽  
Umbilical Vein ◽  
Factor Xii ◽  
Plasminogen Activation ◽  
High Molecular Weight Kininogen ◽  
Human Endothelial Cells ◽  
Intrinsic Pathway ◽  
Local Fibrinolysis ◽  
Umbilical Vein Endothelial Cells ◽  
And Function

SummaryFactor XII has long been implicated in the intrinsic pathway of fibrinolysis, but the mechanism by which it triggers plasminogen activation and targets fibrinolysis has not been established. In the present study, the assembly and function of activated Factor XII (F.XIIa), prourokinase (pro-u-PA), high molecular weight kininogen (H-kininogen), and prekallikrein on human umbilical vein endothelial cells (HUVEC) was investigated. 125I-prekallikrein was shown to bind to HUVEC via receptor-bound H-kininogen in the presence of 50 μM ZnCl2. After the addition of F.XIIa, 78% of the 125I-prekallikrein initially bound to HUVEC was converted to 125I-kallikrein. However, only 6% of the HUVEC-bound 125I-pro-u-PA was thereby activated. This discrepancy was shown to be related to rapid dissociation (>50% within 15 min) of prekallikrein/kallikrein, but not pro-u-PA, from HUVEC. Increasing the level of cell-bound kallikrein increased the portion of cell-bound pro-u-PA activated, indicating that their co-localization was important for this pathway. Finally, F.XIIa was shown to trigger plasminogen activation on HUVEC via this pathway. This assembly of reactants on the endothelium suggests a mechanism whereby local fibrinolysis may be triggered by blood coagulation.

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