Vessel wall reactivity and 13-hydroxy-octadecadienoic acid synthesis.
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Blood Platelets↗
Biomedical subjects
T A Haas
Publications and source records attributed to T A Haas.
Automated high-performance liquid chromatographic extraction and quantification procedure for lipoxygenase metabolites.
A number of methods have been used to measure various lipoxygenase metabolites in aqueous samples. These methods, however, suffer from three major limitations: first, they require extensive extraction and isolation from protein-containing media; second, mainly due to the first limitation, they have poor recoveries; and third, these methods usually require a two-step procedure, one for the actual extraction and the other for the quantification of the lipoxygenase metabolites. We have developed a fully automated high-performance liquid chromatographic method which circumvents these limitations. As a result, we are able to obtain high recoveries of various lipoxygenase metabolites from protein-containing samples (i.e. biological samples) while simultaneously quantifying each metabolite. The method employs a column venting technique, whereby the fatty acids are extracted by a pre-column and the proteins are vented to waste. The pre-column eluate is then directed through the analytical column which separates the lipoxygenase metabolites. The described method is reproducible and minimizes both the time and the cost involved in assaying a sample.
Acetonitriles↗
Binding of 13-HODE and 5-, 12- and 15-HETE to endothelial cells and subsequent platelet, neutrophil and tumor cell adhesion.
Some studies report that endothelial cells preferentially take up the lipoxygenase-derived arachidonic acid metabolite, 5-hydroxyeicosatetraenoic acid (5-HETE), released from stimulated leukocytes (polymorphonuclear leukocytes, PMNs), whereas others report that endothelial cells preferentially take up 12-HETE released from platelets. The biological relevance of these observations, however, is unknown. Recently, we and others have found that, under basal conditions, endothelial cells, PMNs and tumor cells metabolize linoleic acid via the lipoxygenase enzyme to 13-hydroxyoctadecadienoic acid (13-HODE). We propose that endogenous levels of these metabolites regulate blood-vessel wall cell adhesion. In this study, we have measured (1) the relative binding of 5-, 12- and 15-HETE, and 13-HODE to endothelial cell monolayers, and (2) their effects on endothelial cell adhesivity with platelets, PMNs and tumor cells. There was a dose-related and specific binding of 5-[3H]HETE to endothelial cells but no binding of 12- or 15-HETE or 13-HODE. Platelet or PMN adhesion to endothelial cells was unaffected by the 5-HETE binding, but tumor cell adhesion was blocked by 40% (P less than 0.01). Interestingly, preincubation of endothelial cells with 13-HODE, 12-HETE or 15-HETE decreased platelet adhesion to endothelial cells (P less than 0.05), even though these metabolites did not bind to the endothelial cells. We conclude that 5-HETE preferentially binds to endothelial cells and interferes with a specific receptor for tumor cells, whereas the other metabolites neither bind to cells nor affect cell adhesion.
12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
Generation of lipid neutrophil chemoattractant by irradiated bovine aortic endothelial cells.
Radiation injury to blood vessels is associated with an acute inflammatory process. We investigated the capacity of cultured bovine aortic endothelial cells (BAEC) to produce chemotactic factors after radiation injury. BAEC in serum-free media were irradiated with a cobalt-60 Gammacell 220 and the cell supernatants were assayed for chemotactic activity for human neutrophils in a Boyden chamber. There was a rapid release of chemotactic activity into the BAEC supernatants which was dependent both on the dose of radiation (5 to 40 Gy) and the time between irradiation and sample collection. In contrast, isolation of BAEC lysates by freeze-thawing was not associated with the presence of similar chemotactic activity. The chemotactic activity released from the irradiated BAEC was not destroyed by boiling nor by treatment with trypsin. The release of the chemotactic activity was, however, inhibited by the addition of a lipoxygenase inhibitor but not by the addition of a cyclooxygenase inhibitor before the irradiation. The chemotactic activity was recovered from the cell supernatants in the lipid phase after extraction with chloroform/methanol. Furthermore, the chloroform/methanol extracts co-eluted with authentic leukotriene B4 when the BAEC were prelabeled with [14C] arachidonic acid. However, we were unable to detect endogenous leukotriene B4 with RIA. Instead, the only detectable endogenous lipid present in the supernatants was 13-hydroxyoctadecadienoic acid which is derived from linoleic acid via the lipoxygenase pathway. 13-Hydroxyoctadecadienoic acid, however, had no chemotactic activity. These findings suggest that endothelial cells rapidly release a chemotactic agent after irradiation, the release of which is associated with a lipoxygenase pathway. The release of this chemotactic activity may account in part for the acute inflammatory response that is observed after ionizing irradiation.
Animals↗
Fatty acid metabolism and the vascular endothelial cell. New thoughts about old data.
Fatty acid metabolism by vascular endothelial cells occurs both under basal conditions and following endothelial cell stimulation or injury. Under basal conditions, endothelial cells are metabolically very active and rapidly turn over triglycerides and synthesize 13-hydroxyoctadecadienoic acid from linoleic acid via the cytosolic enzyme, omega 6-lipoxygenase. When endothelial cells are stimulated (or injured), these pathways are turned off, and, instead, arachidonic acid is liberated from the membrane phospholipids and metabolized into prostacyclin via the cyclooxygenase enzyme. The biological relevance of these two fatty acid metabolites is discussed in respect to the regulation of cell/cell interactions during both 'homeostasis' and 'injury'.
Arachidonic Acid↗
The basement membrane underlying the vascular endothelium is not thrombogenic: in vivo and in vitro studies with rabbit and human tissue.
Studies examining the interaction of platelets with exposed subendothelium in vivo have reported conflicting results. To examine possible explanations for the apparently discrepant findings, we measured the platelet reactivity of subendothelium prepared by a number of methods both in vivo and in vitro. In addition, we examined the possibility that 13-hydroxyoctadecadinoic acid (13-HODE), an endothelial cell-derived chemorepellant, modulates the reactivity of the subendothelium to platelets. In vivo, the subendothelium of segments of rabbit carotid arteries was exposed by removing the endothelial cells by air perfusion or by balloon catheter stripping. Platelet accumulation onto the de-endothelialized segments was assessed by 3H-radioactivity uptake, using 3H-adenine-labelled platelets, and by scanning electron microscopy. In vitro, 3H-adenine-labelled platelet adhesion was measured onto plain plastic discs and onto plastic discs coated with the following purified basement membrane components: collagens type I, III, IV, V, laminin, or fibronectin. In addition, 3H-adenine-labelled platelet adhesion was measured onto plastic discs covered with human endothelial cells or onto the basement membrane underlying the endothelial cells. In vivo, there was marked 3H-platelet accumulation onto the balloon catheter carotid arteries one hour after injury. In contrast, there was no platelet accumulation onto the subendothelium of carotid arteries de-endothelialized by air perfusion. These differences were confirmed by scanning electron microscopy. Transmission electron microscopic examination demonstrated that the extracellular matrix was intact following the air perfusion injury whereas the majority of it was removed by the balloon catheter injury.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
13-Hydroxyoctadecadienoic acid is the vessel wall chemorepellant factor, LOX.
We have previously reported that endothelial cells synthesize a cytosol-associated, lipoxygenase-derived metabolite, LOX, which acts as a chemorepellant and, in so doing, maintains the vessel wall thromboresistance. In this study we demonstrate that LOX is a 13-hydroxylinoleic acid (13-OH-18:2) derived from linoleic acid and identical to 13-hydroxy-9-cis,11-trans-octadecadienoic acid, as measured by both reverse phase high pressure liquid chromatography and gas chromatography/mass spectrometry. In addition, we demonstrate that 13-OH-18:2 is produced in significantly greater quantities by endothelial cells than by smooth muscle cels or by fibroblasts. Furthermore, we demonstrate that 13-OH-18:2 is produced in microgram amounts under basal conditions and is decreased by thrombin, calcium ionophore, and trypsin stimulation. And finally, we demonstrate that endothelial cells do not synthesize any significant amounts of lipoxygenase-derived arachidonic acid metabolites either under basal or stimulated conditions unless exogenous arachidonic acid is added. These observations indicate that the major lipoxygenase-derived, chemorepellant metabolite produced by the endothelial is 13-hydroxy-9-cis,11-trans-octadecadienoic acid.
Antithrombins↗
Interconvertible kinetic and physical forms of human erythrocyte pyruvate kinase.
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Adenine Nucleotides↗
Platelet adhesion to exposed endothelial cell extracellular matrixes is influenced by the method of preparation.
The relative thrombogenicity of extracellular matrixes (ECMs) produced by cultured human umbilical endothelial cells (ECs) was studied under flow conditions. ECMs were prepared using a number of physical and chemical methods, and their reactivity toward platelets was morphometrically evaluated. von Willebrand factor (vWF), fibronectin (FN), and 13-hydroxy-9-cis,11-trans-octadecadienoic acid (13-HODE) were also determined. We found that platelet adhesion to ECMs differed significantly, both quantitatively and qualitatively, with the method of ECM preparation. Mechanically prepared ECM exposed a less thrombogenic surface compared with ECM prepared by chemical methods (platelet-covered surface of 20% and 50%, respectively). Evaluation of the ECM components vWF, FN, and 13-HODE showed significant changes, both in their concentrations and distribution patterns, depending on the method of ECM preparation. The decrease measured in the levels of ECM-associated vWF (from 108 to 9.2 ng/10(4) cells) and the minor changes observed in the distribution pattern of subendothelial FN did not appear to be sufficient to explain the altered platelet adhesion observed in our model. This suggests that the amount of 13-HODE probably associated to the remaining ECs present in the mechanically exposed ECM could be one factor that specifically contributed to the nonthrombogenic state of these preparations. We conclude that the degree of ECM reactivity toward platelets is dependent on the method of ECM preparation and that this is related to the removal of specific EC/ECM components that modulate their thromboresistant/thrombogenic properties. This fact should be taken into account when ECMs produced by cultured ECs are used in platelet adhesion studies.
Antithrombins↗
Regulation of tumor cell adhesion by intracellular 13-HODE: 15-HETE ratio.
We performed studies to determine whether tumor cells (TCs) produce 13-hydroxyoctadecadienoic acid (13-HODE) and 15-hydroxyeicosatetraenoic acid (15-HETE), and to determine the relationship between TC and endothelial cell (EC) 13-HODE and 15-HETE synthesis, and TC adhesion to ECs and their underlying extracellular matrix (ECM). We measured (1) the amounts and ratios of 13-HODE: 15-HETE in three different human TC lines and in three different murine TC lines under basal and stimulated conditions; and (2) the relationship between 13-HODE synthesis and cAMP levels in TCs and ECs. Under basal conditions, TCs produced both 13-HODE and 15-HETE, the intracellular ratios of which correlated with TC adhesivity. Stimulation of the TCs with the chemotactic tripeptide, N-formyl-methionyl-leucyl-phenylalanine, decreased 13-HODE synthesis, and increased 15-HETE synthesis and TC adhesion to ECs and to their ECM. Alternatively, enhancing 13-HODE synthesis in either TCs or ECs (by elevating the resting levels of intracellular cAMP) was associated with decreased TC adhesion to ECs and ECM. These results suggest that intracellular 13-HODE: 15-HETE ratio in TCs regulates TC adhesivity, and that an alteration in 13-HODE: 15-HETE ratio will markedly influence TC adhesion.
Animals↗