Endotoxin-Mediated Endothelial Injury In Vitro

1981 ◽  
Author(s):  
J M Harlan ◽  
L A Harker ◽  
G E Striker ◽  
R B Counts
Keyword(s):  
Cell Surface ◽  
Lipid A ◽  
Umbilical Vein ◽  
Blue Dye ◽  
Methyl Prednisolone ◽  
Sds Page ◽  
Substrate Conversion ◽  
Direct Cell ◽  
Significant Release

Endotoxin (ET) infusion into animals produces endothelial cell (EC) injury. It is not clear whether EC injury is produced by ET directly or by an ET-generated mediator, or whether ET-mediated EC injury also occurs in man and non-human primates. To answer these questions we examined the effects of ET on human umbilical vein (HUVEC) and bovine aortic (BAEC) endothelial cells in culture. ET (100 ug/ml) ± complement ± neutrophils produced no significant HUVEC Cr-release or cell detachment at 4 hours. HUVEC proliferation by direct cell count, angiotensin converting enzyme activity by labeled substrate conversion, VIII-Ag release by RIA and fibronectin release by ELISA were not affected by ET (100 ug/ml). ET alone did not induce significant release of PGI2 measured by bioassay or 6-keto-PGF. I α measured by RIA. In contrast to the lack of effect of ET on HUVEC, ET produced a time (3 hours: 22.0 ± 4.0%; 6 hours: 40.0 ± 6.0%; 24 hours: 71.0 ± 5.0%) and dose (100 pg/ml: 25.4 ± 2.5%; I ug/ml: 83.0± 4.0%; 10 ug/ml: 92.0 ± 3.0%) dependent BAEC detachment which was initially sublethal without 51 Cr-release or trypan blue dye uptake (40.6 ± 6.0% detachment, <1% Cr-release at 6 hours with ET 10 ug/ml). BAEC detachment was seen with all ET preparations including lipid A. ET-mediated BAEC detachment was inhibited by incubation at 4° but not by indomethacin, methyl prednisolone, or chi or promazine and was not dependent on serum or complement. Examination of ET-detached BAEC by SDS-PAGE after 125I- surface labeling or by immunofluorescence using anti-fibronectin antibody revealed complete loss of cell surface fibronectin. ET- mediated detachment was also seen with bovine mesenteric EC but not with bovine aortic smooth muscle cells or monkey aortic EC. These studies demonstrate the importance of studying EC from appropriate species and vascular sites. Although ET has no cytotoxic effect on HUVEC, it does induce selective, sublethal BAEC detachment associated with loss of cell surface fibronectin. If a similar effect occurs in human adult arterial or microvascular EC, it would have profound pathophysiologic effects accounting for the permeability changes and thrombosis seen in sepsis in man.

Models Of Granulocyte-Mediated Endothelial Injury In Vitro

1981 ◽  
Author(s):  
G Vercellotti ◽  
P Flynn ◽  
D Weisdorf ◽  
C J Lammi-Keefe ◽  
H Jacob ◽  
...  
Keyword(s):  
Lipid A ◽  
Umbilical Vein ◽  
Endothelial Damage ◽  
Endothelial Injury ◽  
Free Radical Scavengers ◽  
Methyl Prednisolone ◽  
Vitro Model ◽  
Umbilical Vein Endothelial Cells

To determine the role of neutrophil (PMN) induced vascular injury during inflammation an in vitro model of endothelial damage was investigated. Injury to human umbilical vein endothelial cells (EC) labeled with 51Cr or 14c sodium arachidonate was monitored by specific release of these labels or their products. Several agents were capable of triggering PMN to induce significant EC injury: these include activated serum complement (C') opsonized particles, serotonin , phorbol myristate acetate, and the lipid A moiety of endotoxin. PMN must adhere closely to the EC for effective cytotoxicity, since agents which retard PMN adherence (cytochalasin B, methyl prednisolone) inhibit 51Cr release.Lyscsorcal proteases did not mediate PMN induced endothelial injury since there was no correlation between release and injury, and soluble stimuli which did not release lysosomal contents induced injury. Free radical scavengers such as SOD/catalase, and a-tocopherol significantly reduced PMN mediated endothelial injury implying that PMN generated reactive oxygen species were responsible for this damage.To further study PMN mediated endothelial injury, other inflammatory agents were also utilized. C' activated PMN’s exposed to Ibuprofen (I) (50 μg/ml) but not Aspirin (ASA) (200 μg/ml) induced no EC injury (51cr releasey Furthermore, it was shown that I inhibits O- 2 production, blocks release of PMN lysozyme and glucuronidase, and inhibits aggregation of C' stimulated PMN’s. ASA at doses of 500 μg/ml (clinically toxic), failed to inhibit these in vitro activities. This data suggests that I’s anti-inflammatory affect may be expressed through inhibition of PMN functions. Since I and ASA both inhibit cyclooxygenase, but only I modulated PMN induced endothelial injury, these agents may provide useful probes to elucidate PMN-endothelial interactions in vivo.

scholarly journals Preparation and In Vitro Characterization of Gels Based on Bromelain, Whey and Quince Extract

Gels ◽  
2021 ◽  
Vol 7 (4) ◽  
pp. 191
Author(s):  
Amalia Mazilu (Moldovan) ◽  
Violeta Popescu ◽  
Codruta Sarosi ◽  
Radu Dumitrescu ◽  
Andrea Maria Chisnoiu ◽  
...  
Keyword(s):  
Tooth Enamel ◽  
Whey Proteins ◽  
Antibacterial Properties ◽  
Blue Dye ◽  
Sem Images ◽  
Sds Page ◽  
Wide Range ◽  
Vitro Analysis ◽  
In Vitro Analysis

The growing interest in the appearance and color of teeth has led to the emergence of a wide range of teeth whitening methods, both in dental offices and in patients’ homes. Concerns about the possible side effects or toxic effects of peroxide-based whitening gels leads to the identification of alternative whitening methods, based on natural compounds with mild action on tooth enamel and remineralizing effect. In this context, this study describes the preparation and in vitro analysis of whitening gels based on natural active agents—bromelain, quince and whey—using organic (polyacrylate, polyethylene glycol) and/or inorganic (silicate) excipients. Five natural products gels were prepared, containing bromelain extract, quince extract and whey, in various proportions. Two supplementary gels, one containing Lubrizol and another containing SiO2, were prepared. All gels were submitted for multiple in vitro analysis such as: SDS-PAGE analysis, UV-vis and FTIR spectroscopy, SEM microscopy, antibacterial activity on Streptococcus mutans ATCC 25175, Porphyromonas gingivalis ATCC 33277, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923. The quince extract sample was the only one which completely discolored the blue dye on SDS-PAGE analysis. On the UV-vis spectra, the 303 nm band is assigned to an in situ modified form of bromelain. SEM images of gels containing SiO2 particles show evident marks of these particles, while the rest of the gels containing Lubrizol or whey are more uniform. Regarding antibacterial tests, the SiO2 gel samples did not show inhibition in any strains, but the other tested samples varied in the size of the inhibition diameter depending on the amicrobial strain tested; the protease activity of bromelain modulates the composition of the added whey proteins. Bromelain added as a nanoencapsulated assembly better preserves its integrity. The prepared gels showed antibacterial properties.

scholarly journals Pengaruh Induksi Bakteri Pseudomonas aeruginosa Terhadap Human Umbilical Vein Endothelial Cells (HUVECS) Culture

2019 ◽  
Vol 1 (1) ◽  
pp. 1
Author(s):  
Dwi Yuni Nur Hidayati
Keyword(s):  
Cell Culture ◽  
Pseudomonas Aeruginosa ◽  
Receptor Cell ◽  
Umbilical Vein ◽  
Main Study ◽  
Endothelial Cell Culture ◽  
Sds Page ◽  
Result Show ◽  
Umbilical Vein Endothelial Cells

Abstract One of the cause of negative gram bacteria is Pseudomonas aeruginosa (Ps. aeruginosa). It continuing become sepsis on the first step of patogenesa happen sticking between aeruginosa with endhotel cell. This sticking is mediated by adhesi molecul which have characterize same with hemaglutin protein. The main study of this research is to know the profile of endothelial cell culture within induction of Ps. aeruginosa. The methods are Ps. aeruginosa (9064) which have been isolated then processes on TCBS Media and continued with isolation terracely. The profile of weight molecule protein obtained by SDS PAGE and then conducted electroelution. The test of hemaglutinin using eritrosit mencit. The receptor cell used is endhotel cell (HUVECs) culture. The result show that dosage of hemaglutin protein of Aeruginosa bring effect to profile of HUVECs culture determined by index adhesion of Ps. aeruginosa to HUVECs culture. We conclude that protein 38,19 kDa give effect to profile of HUVECs culture in vitro.

Adenosine prevents permeability increase in oxidant-injured endothelial monolayers

1998 ◽  
Vol 274 (1) ◽  
pp. H35-H42 ◽  
Author(s):  
Lois F. Richard ◽  
Thomas E. Dahms ◽  
Robert O. Webster
Keyword(s):  
Endothelial Cells ◽  
Protective Effect ◽  
Umbilical Vein ◽  
Direct Action ◽  
Blue Dye ◽  
Oxidant Injury ◽  
Cyclic Monophosphate ◽  
Endothelial Monolayers ◽  
Permeability Increase

Adenosine is thought to prevent or reduce the increase in permeability, which is a hallmark of oxidant injury to endothelium. However, the effect of adenosine on endothelial cells directly exposed to oxidant species has not been demonstrated in vitro. By measuring the passage of Evan’s blue dye-labeled albumin across confluent monolayers, we demonstrated the ability of adenosine (0.1–100 μM) to lower basal permeability of human umbilical vein endothelial cells in a concentration-dependent fashion and prevent the permeability increase induced by exposure of the cells to xanthine plus xanthine oxidase (X/XO). Whereas pretreatment of monolayers for 10 min with adenosine (10 and 100 μM) prevented the X/XO-induced permeability increase, these same concentrations of adenosine failed to increase intracellular adenosine 3′,5′-cyclic monophosphate in X/XO-exposed cells. The protective effect of adenosine on endothelial monolayers was mimicked by adenosine amine congener and 5′-( N-ethylcarboxamido)adenosine but not by other agonists examined. Hence, the protective effect of adenosine against oxidant injury may include an adenosine 3′,5′-cyclic monophosphate-independent mechanism by direct action of adenosine at A1receptors on endothelial cells.

scholarly journals Chlamydia pneumoniae GroEL1 Protein Is Cell Surface Associated and Required for Infection of HEp-2 Cells

2008 ◽  
Vol 190 (10) ◽  
pp. 3757-3767 ◽  
Author(s):  
Frederik N. Wuppermann ◽  
Katja Mölleken ◽  
Marion Julien ◽  
Christian A. Jantos ◽  
Johannes H. Hegemann
Keyword(s):  
Cell Surface ◽  
Chlamydial Infection ◽  
Chlamydia Pneumoniae ◽  
Umbilical Vein ◽  
Surface Proteins ◽  
Eukaryotic Cells ◽  
Dependent Manner ◽  
Adhesive Properties ◽  
Latex Beads

ABSTRACT Chlamydia pneumoniae is an important obligate intracellular pathogen that replicates within an inclusion in the eukaryotic cell. The initial event of a chlamydial infection is the adherence to and subsequent uptake of the infectious elementary bodies (EBs) by the human cell. These processes require yet-unidentified bacterial and eukaryotic surface proteins. The GroEL1 protein, which exhibits a very strong antigenicity and in vitro can activate various eukaryotic cells, is a potential pathogenicity factor. We localized the protein during the infection process and found it in the inclusion but outside the chlamydial particles. GroEL1 was also localized on the surface of EBs, and the protein could be washed off the EBs. Latex beads coated with recombinantly produced GroEL1 (rGroEL1) bound in a dose-dependent manner to HEp-2 cells. Likewise, GroEL1, when expressed and displayed on the yeast cell surface, mediated adhesion to HEp-2 cells. Interestingly, the homologous GroEL2 and GroEL3 proteins showed no adhesive properties. Incubation of primary umbilical vein endothelial cells with soluble GroEL1 and GroEL1-coated latex beads activated the translocation of the general transcription factor NF-κB into the nucleus. Finally, preincubation of HEp-2 cells with rGroEL1 significantly reduced subsequent infection with C. pneumoniae, although adhesion of infectious bacteria to eukaryotic cells was not affected. Taken together, these data support a role for extracellular GroEL1 in the establishment of the chlamydial infection.

scholarly journals Retinoic acid stimulates expression of thrombomodulin, a cell surface anticoagulant glycoprotein, on human endothelial cells. Differences between up-regulation of thrombomodulin by retinoic acid and cyclic AMP

1992 ◽  
Vol 281 (1) ◽  
pp. 149-154 ◽  
Author(s):  
S Horie ◽  
K Kizaki ◽  
H Ishii ◽  
M Kazama
Keyword(s):  
Endothelial Cells ◽  
Retinoic Acid ◽  
Cyclic Amp ◽  
Cell Surface ◽  
Untreated Control ◽  
Umbilical Vein ◽  
Phosphodiesterase Inhibitor ◽  
Mrna Levels ◽  
Significant Difference

Thrombomodulin (TM) is a surface protein on endothelial cells, and represents one of the most valuable regulatory factors in the anticoagulant system. In this paper, we demonstrate that retinoic acid (RA) causes an increase in TM antigen on human umbilical vein endothelial cells (HUVECs) in vitro. The effect of RA on the surface TM level of HUVECs was dose-dependent in the range from 0.01 to 10 microM-RA. Antigen levels began to increase 3 h after addition of 10 microM-RA, and plateaued at a maximum level of approx. 2.5 times that of the untreated control at 24 h. TM levels remained at a maximum for a further 12 h, and then gradually decreased. The effects of RA on cell surface TM activity and antigen levels were parallel in all experiments. TM expression was also increased by treatment with 10 microM-retinal or 10 microM-retinol for 24 h, though the increases were approx. 70% and 30% respectively of that produced by 10 microM-RA. Pretreatment of HUVECs with cycloheximide inhibited the effect of RA. When HUVECs were incubated with both 10 microM-RA and 5 mM-8-bromo cyclic AMP (or 1 mM-3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor), the increase in TM antigen was greater than that observed with either compound alone. Northern blot analysis showed that treatment of HUVECs with 8-bromo cyclic AMP, RA or RA plus 8-bromo cyclic AMP increased TM mRNA levels by 2.2-, 4.5- and 5.5-fold respectively compared with the untreated control. Furthermore, no significant difference in cellular cyclic AMP levels was observed between RA-treated and control cells. These results indicate that the expression of TM is not only controlled by the intracellular cyclic AMP level but is also affected by RA, and suggest that RA-induced up-regulation of TM on HUVECs is independent of cyclic AMP regulation.

Ultrastructural observations on the in vitro cytoadherence of Plasmodium falciparum infected red blood cells

1990 ◽  
Vol 48 (3) ◽  
pp. 914-915
Author(s):  
D.J.P. Ferguson ◽  
A.R. Berendt ◽  
J. Tansey ◽  
K. Marsh ◽  
C.I. Newbold
Keyword(s):  
Plasmodium Falciparum ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Umbilical Vein ◽  
Cell Types ◽  
Amelanotic Melanoma ◽  
Cytoplasmic Process ◽  
Umbilical Vein Endothelial Cells

In human malaria, the most serious clinical manifestation is cerebral malaria (CM) due to infection with Plasmodium falciparum. The pathology of CM is thought to relate to the fact that red blood cells containing mature forms of the parasite (PRBC) cytoadhere or sequester to post capillary venules of various tissues including the brain. This in vivo phenomenon has been studied in vitro by examining the cytoadherence of PRBCs to various cell types and purified proteins. To date, three Ijiost receptor molecules have been identified; CD36, ICAM-1 and thrombospondin. The specific changes in the PRBC membrane which mediate cytoadherence are less well understood, but they include the sub-membranous deposition of electron-dense material resulting in surface deformations called knobs. Knobs were thought to be essential for cytoadherence, lput recent work has shown that certain knob-negative (K-) lines can cytoadhere. In the present study, we have used electron microscopy to re-examine the interactions between K+ PRBCs and both C32 amelanotic melanoma cells and human umbilical vein endothelial cells (HUVEC).We confirm previous data demonstrating that C32 cells possess numerous microvilli which adhere to the PRBC, mainly via the knobs (Fig. 1). In contrast, the HUVEC were relatively smooth and the PRBCs appeared partially flattened onto the cell surface (Fig. 2). Furthermore, many of the PRBCs exhibited an invagination of the limiting membrane in the attachment zone, often containing a cytoplasmic process from the endothelial cell (Fig. 2).

The Anticoagulant Properties of a Modified Form of Protein S

1988 ◽  
Vol 60 (02) ◽  
pp. 298-304 ◽  
Author(s):  
C A Mitchell ◽  
S M Kelemen ◽  
H H Salem
Keyword(s):  
Monoclonal Antibody ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Factor Xa ◽  
Protein S ◽  
Human Neutrophil Elastase ◽  
Factor X ◽  
Sds Page

SummaryProtein S (PS) is a vitamin K-dependent anticoagulant that acts as a cofactor to activated protein C (APC). To date PS has not been shown to possess anticoagulant activity in the absence of APC.In this study, we have developed monoclonal antibody to protein S and used to purify the protein to homogeneity from plasma. Affinity purified protein S (PSM), although identical to the conventionally purified protein as judged by SDS-PAGE, had significant anticoagulant activity in the absence of APC when measured in a factor Xa recalcification time. Using SDS-PAGE we have demonstrated that prothrombin cleavage by factor X awas inhibited in the presence of PSM. Kinetic analysis of the reaction revealed that PSM competitively inhibited factor X amediated cleavage of prothrombin. PS preincubated with the monoclonal antibody, acquired similar anticoagulant properties. These results suggest that the interaction of the monoclonal antibody with PS results in an alteration in the protein exposing sites that mediate the observed anticoagulant effect. Support that the protein was altered was derived from the observation that PSM was eight fold more sensitive to cleavage by thrombin and human neutrophil elastase than conventionally purified protein S.These observations suggest that PS can be modified in vitro to a protein with APC-independent anticoagulant activity and raise the possibility that a similar alteration could occur in vivo through the binding protein S to a cellular or plasma protein.

In Vitro Stability of a Tissue-Type Plasminogen Activator Mutant, BM 06.022, in Human Plasma

1994 ◽  
Vol 72 (06) ◽  
pp. 906-911 ◽  
Author(s):  
D C Rijken ◽  
E Groeneveld ◽  
M M Barrett-Bergshoeff
Keyword(s):  
Plasminogen Activator ◽  
Half Life ◽  
Polyacrylamide Gel Electrophoresis ◽  
Tissue Type ◽  
Single Chain ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Sds Page ◽  
Chain Form

SummaryBM 06.022 is a non-glycosylated mutant of human tissue-type plasminogen activator (t-PA) comprising only the kringle-2 and proteinase domains. The in vivo half-life of BM 06.022 antigen is 4- to 5-fold longer than that of t-PA antigen. The in vitro half-life of the activity of BM 06.022 at therapeutic concentrations in plasma is shorter than that of t-PA. In this study the inactivation of BM 06.022 in plasma was further investigated.Varying concentrations of BM 06.022 were incubated in plasma for 0-150 min. Activity assays on serial samples showed a dose-dependent decline of BM 06.022 activity with a half-life from 72 min at 0.3 μg/ml to 38 min at 10 μg/ml. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by fibrin autography showed the generation of several BM 06.022-complexes. These complexes could be completely precipitated with antibodies against Cl-inactivator, α2-antiplasmin and α1-antitrypsin.During the incubation of BM 06.022 in plasma, plasmin was generated dose-dependently as revealed by varying degrees of a2-anti-plasmin consumption and fibrinogen degradation. SDS-PAGE and immunoblotting showed that single-chain BM 06.022 was rapidly (i. e. within 45 min) converted into its two-chain form at concentrations of 5 μg/ml BM 06.022 and higher.In conclusion, BM 06.022 at therapeutic concentrations in plasma was inactivated by Cl-inactivator, a2-antiplasmin and a j-antitrypsin. The half-life of the activity decreased at increasing BM 06.022 concentrations, probably as a result of the generation of two-chain BM 06.022 which may be inactivated faster than the single-chain form.

Induction of Endothelial Tissue Factor by Endotoxin and Its Precursors

1993 ◽  
Vol 70 (04) ◽  
pp. 702-706 ◽  
Author(s):  
Charles F Moldow ◽  
Ronald R Bach ◽  
Katherine Staskus ◽  
Paul D Rick
Keyword(s):  
Tissue Factor ◽  
Lipid A ◽  
Umbilical Vein ◽  
Structural Determinants ◽  
Monophosphoryl Lipid A ◽  
Maximal Induction ◽  
Umbilical Vein Endothelial Cells ◽  
Acyloxyacyl Hydrolase ◽  
Endothelial Tissue ◽  
Specific Mrna

SummaryThe structural determinants of lipopolysaccharide required for the induction of tissue factor in human umbilical vein endothelial cells were studied. Intact lipid A was essential for the induction of tissue factor whereas the incomplete lipid A precursors lipid IVA and lipid X, as well as monophosphoryl lipid A and acyloxyacyl hydrolase-treated lipopolysaccharide, were unable to induce tissue factor and tissue factor specific mRNA. However, the lipid A precursor, lipid IVA, was able to inhibit LPS-mediated induction of tissue factor; structural determinants distal to lipid A were found to be required for maximal induction of tissue factor activity and tissue factor mRNA. The presence of serum in the assay was found to amplify but was not obligate for tissue factor induction by LPS.

Sign in / Sign up

Export Citation Format

Share Document