scholarly journals Invasion and Killing of Human Endothelial Cells by Viridans Group Streptococci

2003 ◽  
Vol 71 (5) ◽  
pp. 2365-2372 ◽  
Author(s):  
Murray W. Stinson ◽  
Susan Alder ◽  
Sarmishtha Kumar
Keyword(s):  
Endothelial Cells ◽  
Infective Endocarditis ◽  
Culture Medium ◽  
Umbilical Vein ◽  
Bovine Liver ◽  
Surface Proteins ◽  
Oral Streptococci ◽  
Bovine Liver Catalase ◽  
Umbilical Vein Endothelial Cells ◽  
Viridans Group Streptococci

ABSTRACT Colonization of the cardiovascular endothelium by viridans group streptococci can result in infective endocarditis and possibly atherosclerosis; however, the mechanisms of pathogenesis are poorly understood. We investigated the ability of selected oral streptococci to infect monolayers of human umbilical vein endothelial cells (HUVEC) in 50% human plasma and to produce cytotoxicity. Planktonic Streptococcus gordonii CH1 killed HUVEC over a 5-h period by peroxidogenesis (alpha-hemolysin) and by acidogenesis but not by production of protein exotoxins. HUVEC were protected fully by addition of supplemental buffers and bovine liver catalase to the culture medium. Streptococci were also found to invade HUVEC by an endocytic mechanism that was dependent on polymerization of actin microfilaments and on a functional cytoskeleton, as indicated by inhibition with cytochalasin D and nocodazole. Electron microscopy revealed streptococci attached to HUVEC surfaces via numerous fibrillar structures and bacteria in membrane-encased cytoplasmic vacuoles. Following invasion by S. gordonii CH1, HUVEC monolayers showed 63% cell lysis over 4 h, releasing 64% of the total intracellular bacteria into the culture medium; however, the bacteria did not multiply during this time. The ability to invade HUVEC was exhibited by selected strains of S. gordonii, S. sanguis, S. mutans, S. mitis, and S. oralis but only weakly by S. salivarius. Comparison of isogenic pairs of S. gordonii revealed a requirement for several surface proteins for maximum host cell invasion: glucosyltransferase, the sialic acid-binding protein Hsa, and the hydrophobicity/coaggregation proteins CshA and CshB. Deletion of genes for the antigen I/II adhesins, SspA and SspB, did not affect invasion. We hypothesize that peroxidogenesis and invasion of the cardiovascular endothelium by viridans group streptococci are integral events in the pathogenesis of infective endocarditis and atherosclerosis.

Factor VIII Related Antigen (FVIIIRAG) In Porcine Endothelial Cells

1981 ◽  
Author(s):  
J C Giddings ◽  
A L Jarvis ◽  
A L Bloom
Keyword(s):  
Endothelial Cells ◽  
Factor Viii ◽  
Culture Medium ◽  
Umbilical Vein ◽  
Aortic Endothelial Cells ◽  
Factor Viii Related Antigen ◽  
Umbilical Vein Endothelial Cells ◽  
Porcine Endothelial Cells ◽  
Willebrand Factor ◽  
Porcine Aortic Endothelial Cells

Porcine aortic and umbilical vein endothelial cells were maintained in culture and examined for their ability to synthesise or release factor VIII related antigen (Willebrand factor, FVIIIRAG). Two different antisera raised against purified porcine FVIIIRAG were used in conventional immunodiffusion, immunoelectrophoresis and immunofluorescence techniques. Both antisera were adsorbed with porcine von Willebrand's disease (vWd) plasma and gave identical monospecific immunoprecipitin reactions when tested against normal porcine plasma or concentrated porcine FVIIIRAG and failed to show any precipitin reaction against vWd pig plasma. Primary and extended cultures of umbilical vein endothelial cells showed positive intracellular immunofluorescence with either antiserum and supernatant culture medium from these cells contained FVIIIRAG when tested in immunoprecipitin tests. However, cultures derived from aorta demonstrated a different pattern of results. Antiserum one showed homogenous intracellular immunofluorescence but antiserum two gave an immunofluorescence pattern which was extracellular and fibrillar, similar to that seen with human endothelial cells stained with anti-fibronectin. Culture supernatant and lysedaortic endothelial cells did not contain FVIIIRAG in immunoprecipitin tests with either antiserum. The results indicate either that the antisera were not specific and that the aortic cells were not endothelial or that porcine endothelial cells derived from aorta may be different from those obtained from umbilical vein in their ability to synthesise FVIIIRAG. Immunohistological demonstration of “FVIIIRAG” cannot therefore be used as the only criterion for identifying porcine aortic endothelial cells.

The effect of cultured endothelial cells on factor VIII procoagulant activity

Blood ◽  
1979 ◽  
Vol 54 (3) ◽  
pp. 560-572
Author(s):  
NW Stead ◽  
PA McKee
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Protease Activity ◽  
Culture Medium ◽  
Umbilical Vein ◽  
Procoagulant Activity ◽  
Cultured Endothelial Cells ◽  
Umbilical Vein Endothelial Cells ◽  
Von Willebrand ◽  
Willebrand Factor

Cultured human umbilical vein endothelial cells produce a protein that has von Willebrand factor activity and forms immunoprecipitates with rabbit antibody to purified plasma factor VIII/von Willebrand factor (FVIII/vWF) protein, but it has no FVIII procoagulant activity. Of the three characteristics of plasma FVIII/vWF protein, only FVIII procoagulant activity is readily destroyed by trace proteases. A previous report from this laboratory demonstrated protease activity in culture medium under conditions that had been used by others to show that endothelial cells do not synthesize protein with FVIII procoagulant activity. However, even if cultured endothelial cells are placed in protease-free culture medium, no FVIII procoagulant activity can be detected, despite an increase in the level of protein with vWF activity from 0 to 0.57 microgram/ml by 48 hr. This observation and the lack of protease activity in medium left in contact with the cells for 48 hr led to the hypothesis that proteases exist on the surface of cultured umbilical vein endothelial cells. Protease activity was quantitated by the hydrolysis of p-nitroaniline from the substrate, N- benzoyl-phenylalanyl-valyl-arginyl-p-nitroanilide and by degradation of the procoagulant activity of added purified plasma FVIII/vWF protein. In the absence of endothelial cells, no protease activity was present in protease-free culture medium whether or not it had previously overlaid cultured cells. This medium did not cause cleavage of p- nitroaniline from the tripeptide substrate, and 83% of added FVIII procoagulant activity remained after 48 hr. When the synthetic tripeptide was incubated in contact with cultured endothelial cells, 7.3 +/- 0.8 X 10(-10) moles of p-nitroaniline/hr was released; moreover, only 47% of the added FVIII procoagulant activity remained after 48 hr. Given this rate of destruction, it can be calculated that sufficient protease activity exists on the surface of cultured endothelial cells to degrade the procoagulant activity of approximately 1.6 microgram FVIII/vWF protein/hr. This degradation rate is 45 times the rate of release of FVIII/vWF protein from cultured endothelial cells when assessed by the generation of protein with vWF activity. Hence, the detection of FVIII procoagulant activity, if in fact synthesized by cultured endothelial cells, will be most difficult.

scholarly journals Mesenchymal Stromal Cell Derived Membrane Particles Are Internalized by Macrophages and Endothelial Cells Through Receptor-Mediated Endocytosis and Phagocytosis

2021 ◽  
Vol 12 ◽  
Author(s):  
Fabiany da Costa Gonçalves ◽  
Sander S. Korevaar ◽  
Maitane Ortiz Virumbrales ◽  
Carla C. Baan ◽  
Marlies E. J. Reinders ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Inflammatory Responses ◽  
Inflammatory Diseases ◽  
Umbilical Vein ◽  
Surface Proteins ◽  
Dependent Manner ◽  
Membrane Particles ◽  
Dose Time ◽  
Inflammatory Conditions ◽  
Umbilical Vein Endothelial Cells

Mesenchymal stromal cells (MSC) are a promising therapy for inflammatory diseases. However, MSC are large and become trapped in the lungs after intravenous infusion, where they have a short survival time. To steer MSC immunoregulatory therapy beyond the lungs, we generated nm-sized particles from MSC membranes (membrane particles, MP), which have immunomodulatory properties, and investigated their internalization and mode of interaction in macrophages subtypes and human umbilical vein endothelial cells (HUVEC) under control and inflammatory conditions. We found that macrophages and HUVEC take up MP in a dose, time, and temperature-dependent manner. Specific inhibitors for endocytotic pathways revealed that MP internalization depends on heparan sulfate proteoglycan-, dynamin-, and clathrin-mediated endocytosis but does not involve caveolin-mediated endocytosis. MP uptake also involved the actin cytoskeleton and phosphoinositide 3-kinase, which are implicated in macropinocytosis and phagocytosis. Anti-inflammatory M2 macrophages take up more MP than pro-inflammatory M1 macrophages. In contrast, inflammatory conditions did not affect the MP uptake by HUVEC. Moreover, MP induced both anti- and pro-inflammatory responses in macrophages and HUVEC by affecting gene expression and cell surface proteins. Our findings on the mechanisms of uptake of MP under different conditions help the development of target-cell specific MP therapy to modulate immune responses.

scholarly journals Effects of the Antioxidant α-Lipoic Acid on Human Umbilical Vein Endothelial Cells Infected with Rickettsia rickettsii

1998 ◽  
Vol 66 (5) ◽  
pp. 2290-2299 ◽  
Author(s):  
Marina E. Eremeeva ◽  
David J. Silverman
Keyword(s):  
Cell Culture ◽  
Endothelial Cells ◽  
Lipoic Acid ◽  
Culture Medium ◽  
Umbilical Vein ◽  
Vero Cells ◽  
Cell Culture Medium ◽  
Rickettsia Rickettsii ◽  
Infected Cells ◽  
Umbilical Vein Endothelial Cells

ABSTRACT Rickettsia rickettsii infection of endothelial cells is manifested in very distinctive changes in cell morphology, consisting of extensive dilatation of the membranes of the endoplasmic reticulum and outer nuclear envelope and blebbing of the plasma membrane, as seen by transmission electron microscopy (D. J. Silverman, Infect. Immun. 44:545–553, 1984). These changes in cellular architecture are thought to be due to oxidant-mediated cell injury, since their occurrence correlates with dramatic alterations in cellular metabolism, particularly with regard to antioxidant systems. In this study, it was shown that R. rickettsii infection of human umbilical vein endothelial cells resulted in a significant depletion of intracellular reduced glutathione (thiol) content at 72 and 96 h and decreased glutathione peroxidase activity at 72 h postinfection. Infected cells displayed a dramatic increase in the concentration of intracellular peroxides by 72 h. Supplementation of the cell culture medium with 100, 200, or 500 μM α-lipoic acid, a metabolic antioxidant, after inoculation with R. rickettsii restored the intracellular levels of thiols and glutathione peroxidase and reduced the intracellular peroxide levels in infected cells. These effects were dose dependent. Treated infected monolayers maintained better viability at 96 h after inoculation with R. rickettsii than did untreated infected cells. Moreover, supplementation of the cell culture medium with 100 μM α-lipoic acid for 72 h after infection prevented the occurrence of morphological changes in the infected cells. The presence of 100 or 200 μM α-lipoic acid did not influence rickettsial growth in endothelial cells, nor did it affect the ability of R. rickettsii to form lytic plaques in Vero cells. Treatment with 500 μM α-lipoic acid decreased by 50% both the number and size of lytic plaques in Vero cells, and it also decreased the recovery of viable rickettsiae from endothelial cells. However, under all treatment conditions, a significant number of rickettsiae could be detected microscopically. Furthermore, the rickettsiae apparently retained their capacity for intracellular movement, since they possessed long polymerized actin tails after 72 and 96 h of treatment regardless of the concentration of α-lipoic acid used. Since α-lipoic acid does not seem to exhibit direct antirickettsial activity except with long-term exposure at very high concentrations, the mechanism of its protective activity for endothelial cells infected with rickettsiae may involve complex changes in cellular metabolism that only indirectly affect rickettsiae.

Estradiol reduces F2α-isoprostane production in cultured human endothelial cells

2002 ◽  
Vol 283 (6) ◽  
pp. H2644-H2649 ◽  
Author(s):  
Carlos Hermenegildo ◽  
Marı́a Cinta Garcı́a-Martı́nez ◽  
Juan J. Tarı́n ◽  
Antonio Cano
Keyword(s):  
Endothelial Cells ◽  
Estrogen Receptor ◽  
Free Radical ◽  
Medroxyprogesterone Acetate ◽  
Time Course ◽  
Culture Medium ◽  
Umbilical Vein ◽  
Human Endothelial Cells ◽  
Human Umbilical Vein ◽  
Umbilical Vein Endothelial Cells

Free radical-generated F2α-isoprostanes are a group of compounds with vasoconstrictor properties. To investigate whether estradiol exerts antioxidant actions modifying F2α-isoprostane production, cultured human umbilical vein endothelial cells were exposed to estradiol and other compounds and F2α-isoprostanes were measured in culture medium. Exposure to 1 and 10 nM estradiol for 24 h reduced F2α-isoprostane production by 36 and 49%, respectively ( P < 0.001 vs. control). Exposure to antiestrogens alone (ICI-182780 or EM-652) slightly reduced F2α-isoprostanes ( P < 0.05 vs. control), but much less than exposure to estradiol ( P < 0.05). ICI-182780 reversed the estradiol-induced reduction of F2α-isoprostane concentration ( P < 0.05). Along with time-course analysis, these results suggest that estradiol effects were mediated through estrogen receptor-dependent and -independent mechanisms. Progestogens alone (progesterone or medroxyprogesterone acetate) did not modify F2α-isoprostane production at any of the tested concentrations (1, 10, and 100 nM). Progesterone completely reversed estradiol-induced reduction of F2α-isoprostane production ( P < 0.05 vs. control and estradiol), but medroxyprogesterone acetate did not ( P < 0.05 vs. control).

scholarly journals The effect of cultured endothelial cells on factor VIII procoagulant activity

Blood ◽  
1979 ◽  
Vol 54 (3) ◽  
pp. 560-572 ◽  
Author(s):  
NW Stead ◽  
PA McKee
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Protease Activity ◽  
Culture Medium ◽  
Umbilical Vein ◽  
Procoagulant Activity ◽  
Cultured Endothelial Cells ◽  
Umbilical Vein Endothelial Cells ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Cultured human umbilical vein endothelial cells produce a protein that has von Willebrand factor activity and forms immunoprecipitates with rabbit antibody to purified plasma factor VIII/von Willebrand factor (FVIII/vWF) protein, but it has no FVIII procoagulant activity. Of the three characteristics of plasma FVIII/vWF protein, only FVIII procoagulant activity is readily destroyed by trace proteases. A previous report from this laboratory demonstrated protease activity in culture medium under conditions that had been used by others to show that endothelial cells do not synthesize protein with FVIII procoagulant activity. However, even if cultured endothelial cells are placed in protease-free culture medium, no FVIII procoagulant activity can be detected, despite an increase in the level of protein with vWF activity from 0 to 0.57 microgram/ml by 48 hr. This observation and the lack of protease activity in medium left in contact with the cells for 48 hr led to the hypothesis that proteases exist on the surface of cultured umbilical vein endothelial cells. Protease activity was quantitated by the hydrolysis of p-nitroaniline from the substrate, N- benzoyl-phenylalanyl-valyl-arginyl-p-nitroanilide and by degradation of the procoagulant activity of added purified plasma FVIII/vWF protein. In the absence of endothelial cells, no protease activity was present in protease-free culture medium whether or not it had previously overlaid cultured cells. This medium did not cause cleavage of p- nitroaniline from the tripeptide substrate, and 83% of added FVIII procoagulant activity remained after 48 hr. When the synthetic tripeptide was incubated in contact with cultured endothelial cells, 7.3 +/- 0.8 X 10(-10) moles of p-nitroaniline/hr was released; moreover, only 47% of the added FVIII procoagulant activity remained after 48 hr. Given this rate of destruction, it can be calculated that sufficient protease activity exists on the surface of cultured endothelial cells to degrade the procoagulant activity of approximately 1.6 microgram FVIII/vWF protein/hr. This degradation rate is 45 times the rate of release of FVIII/vWF protein from cultured endothelial cells when assessed by the generation of protein with vWF activity. Hence, the detection of FVIII procoagulant activity, if in fact synthesized by cultured endothelial cells, will be most difficult.

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