[57] Endothelial cells for studies of platelet-activating factor and arachidonate metabolites

Zimmerman, Guy A.; Whatley, Ralph E.; McIntyre, Thomas M.; Benson, Donelle M.; Prescott, Stephen M.

doi:10.1016/0076-6879(90)87059-c

Cited by 49 publications

(37 citation statements)

References 24 publications

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“…Cell Culture and Monolayer Wounding-Human umbilical vein endothelial cells were isolated and cultured as described (19). These cells were allowed to achieve confluence, typically in 3-5 days, and then were serum-starved by culturing for 24 h in media containing 1% human serum before initiating our experiments.…”

Section: Methodsmentioning

confidence: 99%

Endothelial Cell Confluence Regulates Cyclooxygenase-2 and Prostaglandin E2 Production That Modulate Motility

Jiang

Weyrich

Zimmerman

et al. 2004

Journal of Biological Chemistry

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Endothelial cells line the vasculature and, after mechanical denudation during invasive procedures or cellular loss from natural causes, migrate to reestablish a confluent monolayer. We find confluent monolayers of human umbilical vein endothelial cells were quiescent and expressed low levels of cyclooxygenase-2, but expressed cyclooxygenase-2 at levels comparable with cytokine-stimulated cells when present in a subconfluent culture. Mechanically wounding endothelial cell monolayers stimulated rapid cyclooxygenase-2 expression that increased with the level of wounding. Cyclooxygenase-2 re-expression occurred throughout the culture, suggesting signaling from cells proximal to the wound to distal cells. Media from wounded monolayers stimulated cyclooxygenase-2 expression in confluent monolayers, which correlated with the level of wounding of the donor monolayer. Wounded monolayers and cells in subconfluent cultures secreted enhanced levels of prostaglandin (PG) E 2 that depended on cyclooxygenase-2 activity, and PGE 2 stimulated cyclooxygenase-2 expression in confluent endothelial cell monolayers. Cells from subconfluent monolayers migrated through filters more readily than those from confluent monolayers, and the cyclooxygenase-2-selective inhibitor NS-398 suppressed migration. Adding PGE 2 to NS-398-treated cells augmented migration. Endothelial cells also migrated into mechanically denuded areas of confluent monolayers, and this too was suppressed by NS-398. We conclude that endothelial cells not in contact with neighboring cells express cyclooxygenase-2 that results in enhanced release of PGE 2 , and that this autocrine and paracrine loop enhances endothelial cell migration to cover denuded areas of the endothelium.

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Section: Methodsmentioning

confidence: 99%

Endothelial Cell Confluence Regulates Cyclooxygenase-2 and Prostaglandin E2 Production That Modulate Motility

Jiang

Weyrich

Zimmerman

et al. 2004

Journal of Biological Chemistry

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“…Bovine pulmonary artery endothelial cells were cultured as described (3) in medium 199 containing 25 mM Hepes with 20% fetal bovine serum, 100 U/mI penicillin, and 100 Mg/ml streptomycin. Human umbilical vein endothelial cells were cultured using a modification of the method of Jaffe et al (4,39). We have described the characteristics of both of these cells in culture (3,39).…”

Section: Methodsmentioning

confidence: 99%

Proliferation-dependent changes in release of arachidonic acid from endothelial cells.

Whatley¹,

Satoh²,

Zimmerman³

et al. 1994

J. Clin. Invest.

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Stimulation of endothelial cells resulted in release of arachidonic acid from phospholipids. The magnitude of this response decreased as the cells became confluent and the change coincided with a decrease in the percentage of cells in growth phases (G2 + M); this was not a consequence of time in culture or a factor in the growth medium. Preconfluent cells released 30% of arachidonic acid; confluent cells released only 6%. The decreasing release of arachidonic acid was demonstrated using metabolic labeling, mass measurements of arachidonic acid, and measurement of PGI2. The decrease was not due to a changing pool of arachidonic acid, and mass measurements showed no depletion of arachidonic acid. Release from each phospholipid and from each phospholipid class decreased with confluence. Conversion of confluent cells to the proliferative phenotype by mechanical wounding of the monolayer caused increased release of arachidonic acid. Potential mechanisms for these changes were investigated using assays of phospholipase activity. Phospholipase A2 activity changed in concert with the alteration in release, a consequence of changes in phosphorylation of the enzyme. The increased release of arachidonic acid from preconfluent, actively dividing cells may have important physiologic implications and may help elucidate mechanisms regulating release of arachidonic acid. (J. Clin.

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“…Cell Culture-Primary cultures of human umbilical vein endothelial cells (HUVEC) were plated and maintained as described previously (18). COS-7 cells were maintained in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) containing 10% fetal bovine serum (Hyclone Laboratories), 110 mg/liter pyruvate, 100 units/ml penicillin, and 100 g/ml streptomycin sulfate.…”

Section: Materials-[␥-mentioning

confidence: 99%

Molecular Cloning of a Novel Human Diacylglycerol Kinase Highly Selective for Arachidonate-containing Substrates

Tang

Bunting

Zimmerman

et al. 1996

Journal of Biological Chemistry

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200

152

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Diacylglycerol (DAG) is a second messenger that activates protein kinase C and also occupies a central role in phospholipid biosynthesis. Conversion of DAG to phosphatidic acid by DAG kinase regulates the amount of DAG and the route it takes. We used degenerate primers to amplify polymerase chain reaction products from cDNA derived from human endothelial cells. A product with a novel sequence was identified and used to clone a 2.6-kilobase cDNA from an endothelial cell library. When transfected with a truncated version of this cDNA, COS-7 cells had a marked increase in DAG kinase activity, which demonstrated clear selectivity for arachidonoyl-containing species of diacylglycerol. The open reading frame of this clone has 567 residues with a predicted protein of 64 kDa. This enzyme, which we designated DGK⑀, has two distinctive zinc finger-like structures in its N-terminal region, but does not contain the E-F hand motifs found in several other mammalian DGKs. The catalytic domain of DGK⑀, which is related to other DGKs, contains two ATP-binding motifs. Northern blotting demonstrated that DGK⑀ is expressed predominantly in testis. This unique diacylglycerol kinase may terminate signals transmitted through arachidonoyl-DAG or may contribute to the synthesis of phospholipids with defined fatty acid composition.Diacylglycerol occupies a central position in the biosynthesis of phospholipids and triglycerides. It also is an important intracellular messenger because it can bind to and activate protein kinase C, which, in turn, phosphorylates target proteins (1). This pathway has been implicated in many cellular response including growth, differentiation, and other events such as secretion. The mechanisms by which the signaling pathway and the synthesis of complex lipids are differentially regulated is not clear, but the concentration of DAG 1 within the cell is almost certainly one important component. In response to a variety of signals, the DAG level rises by the activation of one or more phospholipases C and, in some cases, a phospholipase D followed by phosphatidic acid phosphohydrolase. Either pathway causes a rise in the amount of diacylglycerol by degrading phospholipids. The level of DAG also is influenced by the rate at which it is converted into other products. One pathway for decreasing DAG is its conversion to phosphatidic acid, a reaction catalyzed by DAG kinases (EC 2.7.1.107).The stimulated rise in DAG levels is an integral component of the response of cells to a variety of stimuli that lead to growth or differentiation, and the effects of phorbol esters, which are tumor promoters, are through activation of protein kinase C. Thus, the level of DAG may be an important determinant of growth. In support of this, we found that rapidly growing endothelial cells have severalfold higher levels of DAG than quiescent cells, and others observed that transformation of cells by several oncogenes results in an increased content of DAG even in the absence of an additional stimulus (2-4). The conversion of DAG to phospha...

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[57] Endothelial cells for studies of platelet-activating factor and arachidonate metabolites

Cited by 49 publications

References 24 publications

Endothelial Cell Confluence Regulates Cyclooxygenase-2 and Prostaglandin E2 Production That Modulate Motility

Endothelial Cell Confluence Regulates Cyclooxygenase-2 and Prostaglandin E2 Production That Modulate Motility

Proliferation-dependent changes in release of arachidonic acid from endothelial cells.

Molecular Cloning of a Novel Human Diacylglycerol Kinase Highly Selective for Arachidonate-containing Substrates

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