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.

scholarly journals Superoxide Dismutase-Dependent, Catalase-Sensitive Peroxides in Human Endothelial Cells Infected by Rickettsia rickettsii

1998 ◽  
Vol 66 (4) ◽  
pp. 1293-1298 ◽  
Author(s):  
June E. Hong ◽  
Lisa A. Santucci ◽  
Xiaojiang Tian ◽  
David J. Silverman
Keyword(s):  
Endothelial Cells ◽  
Superoxide Dismutase ◽  
Cell Injury ◽  
Umbilical Vein ◽  
Sod Activity ◽  
Rickettsia Rickettsii ◽  
Diethyldithiocarbamic Acid ◽  
Infected Cells ◽  
Enzyme Superoxide Dismutase ◽  
Umbilical Vein Endothelial Cells

ABSTRACT The generation and intracellular accumulation of reactive oxygen species have been shown to be associated with the infection of human umbilical vein endothelial cells (HUVEC) by Rickettsia rickettsii. In response to the oxidant superoxide, the activity of the enzyme superoxide dismutase (SOD) increases following infection by this obligate intracellular bacterium. Other oxidants which are capable of oxidizing the fluorescent probe 2′,7′-dichlorofluorescin (DCFH) also accumulate intracellularly within infected cells. In the study reported here, we show that (i) an inhibitor of SOD, diethyldithiocarbamic acid, reduces the observed rise in SOD activity in infected cells by 40 to 60% and at the same time reduces the degree of intracellular oxidation of DCFH; (ii) catalase-sensitive peroxides can be detected in supernatants of R. rickettsii-infected cells shortly after rickettsial exposure; and (iii) fluorescence-activated cell sorter analysis demonstrates significant intracellular oxidant activity in infected cells within 5 h after exposure to R. rickettsii. The results of these experiments indicate that hydrogen peroxide is a major oxidant associated with infection of HUVEC by R. rickettsii and that intracellular oxidant activity sensitive to SOD inhibition is detectable early and prior to significant rickettsial multiplication and much earlier than the ultrastructural manifestations of cell injury seen by electron microscopy.

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.

Endothelial cell cystine uptake and glutathione increase with N,N-bis(2-chloroethyl)-N-nitrosourea exposure

1992 ◽  
Vol 262 (3) ◽  
pp. L301-L304 ◽  
Author(s):  
S. M. Deneke ◽  
R. A. Lawrence ◽  
S. G. Jenkinson
Keyword(s):  
Cell Culture ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Culture Medium ◽  
Cultured Cells ◽  
Potent Inhibitor ◽  
Cell Types ◽  
Cell Culture Medium ◽  
Intracellular Accumulation ◽  
Pulmonary Endothelial Cells

Glutathione (gamma-glutamylcysteinylglycine, GSH) is an important cellular antioxidant. In typical cultured cell preparations GSH synthesis is limited by the availability of intracellular cysteine. Because extracellular cystine is the chief source of intracellular cysteine in cultured cells, increasing cystine transport can result in increased intracellular GSH. Depletion of GSH or exposure to oxidants has been shown to stimulate cystine transport in bovine pulmonary endothelial cells and other cell types. BCNU [N,N-bis(2-chloroethyl)-N-nitrosourea] is a potent inhibitor of glutathione reductase (GSSG-Red). We examined the effects of BCNU on cystine uptake by bovine pulmonary artery endothelial cells (BPAEC). We hypothesized that blocking GSSG-Red could result in increased cellular uptake of cystine to replenish decreases in GSH caused by oxidation. Levels of BCNU between 0.005 and 0.05 mM added to the cell culture medium inhibited GSSG-Red at 2, 4, and 24 h after addition. BCNU treatment resulted in concentration-dependent increases in both cystine uptake and GSH levels after 24 h of exposure. The increases in uptake were specific for cystine and glutamate and were sodium independent, suggesting induction of a xc(-)-like transport system. No intracellular accumulation of GSSG was measured nor was any significant depletion of GSH noted at any time of BCNU exposure.

scholarly journals HEPES inhibits the conversion of prion protein in cell culture

2011 ◽  
Vol 92 (5) ◽  
pp. 1244-1250 ◽  
Author(s):  
Karine Delmouly ◽  
Maxime Belondrade ◽  
Danielle Casanova ◽  
Ollivier Milhavet ◽  
Sylvain Lehmann
Keyword(s):  
Cell Culture ◽  
Prion Protein ◽  
Cell Biology ◽  
Culture Medium ◽  
Inhibitory Effect ◽  
Live Cells ◽  
Cell Culture Medium ◽  
Dependent Inhibition ◽  
Infected Cells ◽  
Significant Concentration

HEPES is a well-known buffering reagent used in cell-culture medium. Interestingly, this compound is also responsible for significant modifications of biological parameters such as uptake of organic molecules, alteration of oxidative stress mechanisms or inhibition of ion channels. While using cell-culture medium supplemented with HEPES on prion-infected cells, it was noticed that there was a significant concentration-dependent inhibition of accumulation of the abnormal isoform of the prion protein (PrPSc). This effect was present only in live cells and was thought to be related to modification of the PrP environment or biology. These results could modify the interpretation of cell-culture assays of prion therapeutic agents, as well as of previous cell biology results obtained in the field using HEPES buffers. This inhibitory effect of HEPES could also be exploited to prevent contamination or propagation of prions in cell culture.

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.

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