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.     

Toxoplasma gondii stimulates the release of 13- and 9-hydroxyoctadecadienoic acids by human platelets.

We have recently demonstrated a novel cytotoxic effect of human platelets against Toxoplasma gondii and a role for thromboxane (TX) in this process (Yong et al., 1991). We now report on the spectrum of lipid mediators released by human platelets after interaction with T. gondii. In addition to TXB2, human platelets after incubation with T. gondii for 90 min released 12-hydroxyheptadecatrienoic acid (12-HHT), 12-hydroxyeicosatetraenoic acid (12-HETE), and an unidentified peak (UVmax 234 nm) as determined by reverse-phase high-performance liquid chromatography. Thermospray-liquid chromatography/mass spectrometry analysis and straight-phase HPLC identified the unknown peak as a mixture of 13-hydroxyoctadecadienoic acid (HODE) and 9-HODE. Radiolabeling studies with [14C]linoleic acid indicated that the platelets were the cellular source of the octadecanoids with 13-HODE (87.7%) greater than 9-HODE (12.3%). Inhibitor studies with indomethacin indicated that 13-HODE was a lipoxygenase product and 9-HODE was a cyclooxygenase product of linoleic acid. Thus, Toxoplasma-stimulated platelets release oxygenated products of both arachidonic acid and linoleic acid which may be important in the host response to T. gondii infection.     

Preferential formation of 13-hydroxylinoleic acid by human peripheral blood eosinophils

Lipid mediators released by inflammatory and immune cells play an important role in inflammatory and immune processes. Most attention has been focussed on arachidonic-derived mediators, including prostaglandins, thromboxanes, leukotrienes, and lipoxins. Literature data, however, suggest that also metabolites of the unsaturated fatty acid linoleic acid may be important in this respect. We have studied the formation and release of 9-hydroxy- and 13-hydroxy-linoleic acid (9-HODE and 13-HODE) by enriched populations of human peripheral blood neutrophils, eosinophils, basophils, monocytes, and lymphocytes. We demonstrate that the eosinophil preferentially produces 13-HODE, whereas the other cell types produce equal amounts of 9-HODE and 13-NODE. The biological significance of these findings is discussed.     

9- and 13-hydroxy-linoleic acid possess chemotactic activity for bovine and human polymorphonuclear leukocytes

The presence of polymorphonuclear leukocytes (PMNs) within the airways is a characteristic feature of a variety of lung diseases. Pulmonary alveolar macrophages (PAMs) and epithelial cells release many different factors which contribute to the recruitment of inflammatory cells into infected airways. PAMs and tracheal epithelial cells are able to produce linoleic acid metabolites (9-HODE and 13-HODE) besides arachidonic acid metabolites. The objective of the present study was to determine whether 9-HODE and 13-HODE possess chemotactic activity for isolated PMNs. It was found that 9-HODE and 13-HODE induced a chemotactic response of both human and bovine PMNs in vitro. The HODEs evoked chemotaxis with a linear dose response from 10(-10) to 10(-6) M to the same extent as the arachidonic acid metabolite 15-HETE. At 10(-8) M, 9-HODE and 13-HODE were approximately half as potent in inducing chemotaxis as compared to LTB4.     

Cyclic AMP regulation of endothelial cell triacylglycerol turnover, 13-hydroxyoctadecadienoic acid (13-HODE) synthesis and endothelial cell thrombogenicity

The 15-omega-lipoxygenase enzyme in endothelial cells metabolizes endogenous linoleic acid (18:2) into 13-hydroxyoctadecadienoic acid (13-HODE) under basal conditions, i.e., in unstimulated endothelial cells. 13-HODE is thought to regulate the non-adhesivity of the endothelium, contributing to vessel wall/blood cell biocompatibility. We performed experiments, therefore, to determine the relationship between basal levels of cAMP, 13-HODE synthesis, and platelet/endothelial cell adhesion. We found that 13-HODE synthesis increased with elevated cAMP levels and that the elevated 13-HODE levels correlated with increased 18:2 turnover in the triacylglycerol pool. In contrast, neither 18:2 nor arachidonic acid (20:4) turnover in the phospholipid nor prostacyclin (PGI2) production were changed with elevated cAMP levels. Platelet/endothelial cell adhesion was inversely proportional to 13-HODE synthesis. We conclude that intracellular 13-HODE influences platelet/vessel wall interactions, is synthesized from 18:2 released from the endogenous triacylglycerol pool, and that this pathway is modulated by intracellular cAMP levels.     

13-Hydroxyoctadeca-9,11-dienoic acid (13-HODE) inhibits thromboxane A2 synthesis, and stimulates 12-HETE production in human platelets

The effect of 13-hydroxyoctadeca-9,11-dienoic acid (13-HODE), a major lipoxygenase product of endothelial cell linoleic acid metabolism on thrombin-induced platelet thromboxane B2 (TxB2), and 12-hydroxyeico-satetraenoic acid (12-HETE) production was evaluated. 13-HODE inhibited thrombin-induced TxB2 production in human platelets in a concentration-dependent manner. At concentrations of 10 and 30 microM, 13-HODE inhibited TxB2 production by 28 +/- 8% (1SE, n = 5; P less than 0.05) and 48 +/- 6% (P less than 0.01) respectively. 13-HODE (30 microM) also inhibited the production of platelet hydroxyheptadecatrienoic acid (38 +/- 5%, P less than 0.01). A concomitant stimulation of 12-HETE production by 13-HODE was observed (25 +/- 5% and 49 +/- 22% over control values at 10 and 30 microM respectively, P less than 0.01). Our results demonstrate a differential effect of 13-HODE on thrombin stimulated platelet cyclooxygenase and lipoxygenase metabolites.     

Oxidative metabolism of linoleic acid by human leukocytes

Upon incubation with human leukocytes, [1-14C] linoleic acid is almost exclusively transformed into 13-hydroxy-9Z, 11E-octadecadienoic acid (13-HODE) if the linoleic acid concentration is lower than 50 microM. Identification of 13-HODE was done by GLC-MS at the level of its methyl ester, trimethylsilyl ether and by comparison with authentic 13-HODE in two different HPLC systems. Analysis of the products by chiral phase HPLC shows that 13(S)-hydroxy-9Z, 11E-octadecadienoic acid is by far the major metabolite formed by human leukocytes. Comparison of reactions performed with intact or lyzed cells suggests that the formation of 13(S)-HODE by human leukocytes occurs in two steps, a dioxygenation catalyzed by a 15-lipoxygenase and a reduction of intermediate 13-HPODE by a glutathione-dependent peroxidase.     

13-(S)-hydroxyoctadecadienoic acid (13-HODE) incorporation and conversion to novel products by endothelial cells

13(S)-Hydroxy-[12,13-3H]octadecadienoic acid (13-HODE), a linoleic acid oxidation product that has vasoactive properties, was rapidly taken up by bovine aortic endothelial cells. Most of the 13-HODE was incorporated into phosphatidylcholine, and 80% was present in the sn -2 position. The amount of 13-HODE retained in the cells gradually decreased, and radiolabeled metabolites with shorter reverse-phase high-performance liquid chromatography retention times (RT) than 13-HODE accumulated in the extracellular fluid. The three major metabolites were identified by gas chromatography combined with mass spectrometry as 11-hydroxyhexadecadienoic acid (11-OH-16:2), 9-hydroxytetradecadienoic acid (9-OH-14:2), and 7-hydroxydodecadienoic acid (7-OH-12:2). Most of the radioactivity contained in the cell lipids remained as 13-HODE. However, some 11-OH-16:2 and several unidentified products with longer RT than 13-HODE were detected in the cell lipids. Normal human skin fibroblasts also converted 13-HODE to the three major chain-shortened metabolites, but Zellweger syndrome fibroblasts produced only a very small amount of 11-OH-16:2. Therefore, the chain-shortened products probably are formed primarily by peroxisomal beta-oxidation. These findings suggest that peroxisomal beta-oxidation may constitute a mechanism for the inactivation and removal of 13-HODE from the vascular wall. Because this is a gradual process, some 13-HODE that is initially incorporated remains in endothelial phospholipids, especially phosphatidylcholine. This may be the cause of some of the functional perturbations produced by 13-HODE in the vascular wall.     

Formation of 9-hydroxyoctadecadienoic acid from linoleic acid in endothelial cells

Human umbilical vein endothelial cells convert linoleic acid to two monohydroxyoctadecadienoic (HODE) acids, 9- and 13-HODE. More 9-HODE than 13-HODE is formed under most conditions. The production of these metabolites is reduced substantially by acetylsalicylic acid, ibuprofen, or arachidonic acid, suggesting that cyclooxygenase may be involved in endothelial HODE synthesis. Incubations lasting up to 4 h indicate that the endothelial cells can convert [U-14C] linoleic acid into at least four additional products, some of which may be derived from the HODE that is formed initially. Radioactive 9- and 13-HODE are produced when the endothelial cells are labeled with linoleic acid and then exposed to thrombin, suggesting that these metabolites also may be formed when the endothelium is activated. If endothelial monolayers grown on micropore filters are incubated with linoleic acid, a substantial amount of the HODE formed accumulates in the basolateral fluid. This suggests that HODE may have extracellular effects, especially within the vascular wall. Furthermore, when 9- or 13-HODE are added, endothelial cultures produce less prostaglandin I2 and convert less 12-hydroxyeicosatetraenoic acid to its main metabolite, 8-hydroxyhexadecatrienoic acid. Therefore, in addition to extracellular actions, HODE also may have functional effects within the endothelium.     

Phorbol ester, 1,2-diacylglycerol, and collagen induce inhibition of arachidonic acid incorporation into phospholipids in human platelets

We have shown that phorbol myristate acetate (PMA) enhanced A-23187-induced arachidonate release and thromboxane synthesis in human platelets (Mobley, A., and Tai, H. H. (1985) Biochem. Biophys. Res. Commun. 130, 717-723). The mechanism of enhancement by PMA was not elucidated. In the present study, we have shown that PMA-treated platelets exhibited significantly less [1-14C]arachidonate incorporation than did control platelets. However, no significant change in uptake of labeled linoleate or oleate was observed by PMA treatment. Examination of the two enzyme activities involved in arachidonate incorporation into phospholipids indicated that both arachidonoyl-coenzyme A (CoA) synthase and arachidonoyl-CoA lysophosphatide acyltransferase were inactivated following treatment with PMA or 1-oleoyl-2-acetyl glycerol. When platelets were stimulated with A-23187 plus PMA which produced a significant synergism in thromboxane synthesis, both enzyme activities were substantially less than those in platelets treated with A-23187 alone. In addition to PMA and 1-oleoyl-2-acetyl glycerol induced decreases in both enzyme activities, collagen, a platelet agonist which can activate protein kinase C (Ca2+/phospholipid-dependent enzyme), was also found to cause a concentration-dependent attenuation of both enzyme activities. These results suggest that protein kinase C activation induced by PMA or collagen may cause inactivation of both arachidonoyl-CoA synthase and arachidonoyl-CoA lysophosphatide acyltransferase resulting in inhibition of the reincorporation of arachidonate released by A-23187 and, consequently, greater availability of arachidonate for thromboxane synthesis.     

Metabolism of oxidized linoleic acid by glutathione transferases: peroxidase activity toward 13-hydroperoxyoctadecadienoic acid

The oxidation of linoleic acid produces several products with biological activity including the hydroperoxy fatty acid 13-hydroperoxyoctadecadienoic acid (13-HPODE), the hydroxy fatty acid 13-hydroxyoctadecadienoic acid (13-HODE), and the 2,4-dienone 13-oxooctadecadienoic acid (13-OXO). In the present work, the peroxidase activity of glutathione transferases (GST) A1-1, M1-1, M2-2, and P1-1(Val 105) toward 13-HPODE has been examined. The alpha class enzyme is the most efficient peroxidase while the two enzymes from the mu class exhibit weak peroxidase activity toward 13-HPODE. It was also determined that the conjugated diene 13-HODE is not a substrate for GST from the alpha and mu classes but that 13-HODE does inhibit the GST-catalyzed conjugation of CDNB by enzymes from the alpha, mu, and pi classes. Finally, both 13-HODE and 13-OXO were shown to be inducers of GST activity in HT-29 and HCT-116 colon tumor cells. These data help to clarify the role of GST in the metabolic disposition of linoleic acid oxidation products.     

Specific protein targets of 13-oxooctadecadienoic acid (13-OXO) and export of the 13-OXO-glutathione conjugate in HT-29 cells.

The linoleic acid metabolite, 13-oxooctadecadienoic acid (13-OXO), is reactive with cellular thiols. In the present report, incubations of HT-29 or CaCo-2 homogenates with 13-OXO and GSH indicate that HT-29 cell homogenates produce a 13-OXO-GSH conjugate. The conjugate formed was likely of enzymatic origin as chiral-phase HPLC showed the major product consisted of only one of two possible diastereomers. The glutathione transferase activity (GST), using chlorodinitrobenzene, was found to be 126 nmol/mg/min in HT-29 cells and 21 nmol/mg/min in CaCo-2 cells. These levels of activity are consistent with the relative ability of the two cell lines to conjugate GSH to 13-OXO. Incubation of intact HT-29 cells with either 13-OXO, or the metabolic precursor 13-hydroxyoctadecadienoic acid (13-HODE), showed detectable 13-OXO-GSH conjugate in the media, but none in the cells. The stereochemistry of the extracellular conjugate suggested an enzymatic origin. In additional experiments, the labeling of cellular protein by 13-HODE was much more specific than the labeling of protein by 13-OXO suggesting that in situ generation of 13-OXO from 13-HODE confers selectivity on the reactions between cellular thiols and 13-OXO. These results demonstrate that in HT-29 cells, 13-HODE is converted to 13-OXO which then either reacts with cellular protein or is conjugated to GSH by GST. The 13-OXO-GSH conjugate is then exported from the cell.     

Nonradiometric HPLC measurement of 13(S)-hydroxyoctadecadienoic acid from rat tissues

A major bioactive metabolite of linoleic acid formed by the action of 15-lipoxygenase-1 is 13(S)-hydroxy-cis-9, trans-11-octadecadienoic acid (13(S)-HODE). 13(S)-HODE is an important intracellular signal agent and is involved in cell proliferation and differentiation in various biological systems. Separation and quantification of 13(S)-HODE from biological materials has previously been achieved only by using radiolabeled linoleic acid as the substrate and two serially connected or two separate HPLC columns to achieve separation of 13(S)-HODE. In the current method, separation and quantification of 13(S)-HODE was achieved by use of a normal-phase HPLC and a solvent system containing hexane/isopropanol/acetonitrile/acetic acid (800/8/30/1, v/v) using isocratic elution with detection at 235 nm. With the currently described method, good separation from unreacted interfering compounds and quantification for 13(S)-HODE were achieved within 35 min with a minimum detection limit of 0.5 ng per injection.     

Synthesis of hydroxy fatty acids from linoleic acid by human blood platelets

The metabolism of linoleic acid by washed human platelets was investigated. [1.14C] linoleic acid was converted to [1.14C] hydroxy octadecadienoic acids (HODEs) at about the same rate with which [1.14C] 12-HETE was produced from [1.14C] arachidonic acid. The total radioactivity in HODEs was distributed among two isomers: 13-HODE (85%) and 9-HODE (15%) as defined by CG-MS. The production of HODEs by intact washed platelets was inhibited by indomethacin (IC50:5 x 10(-7) M) which suggest that hydroxy fatty acids were produced by PGH-synthase. By contrast, the production of HODEs by platelet cytosolic fractions was not modified under indomethacin treatment but completely abolished by NDGA (10(-3) M) and inhibited by the platelet lipoxygenase inhibitors 15-HETE (2.10(-5) M) and baicalein (10(-5) M). Platelets thus contain two different active systems which may convert linoleic acid to hydroxy fatty acids. Since these compounds remained essentially associated with the platelets, their presence may significantly participate in the mechanisms of platelet activation.     

Stereospecificity of the hydroxyeicosatetraenoic and hydroxyoctadecadienoic acids produced by cultured bovine endothelial cells

Characterization of the stereospecificity of the derivatives of arachidonic acid and linoleic acid produced by endothelial cells is needed to define the enzymatic origin of these compounds and their role in vascular physiology. In studies utilizing two bovine endothelial cell lines (CPAE and AG04762), both free 15-hydroxyeicosatetraenoic acid (15-HETE) and 11-hydroxyeicosatetraenoic acid (11-HETE) were generated during incubations with exogenous arachidonic acid and both free 9-hydroxyoctadecadienoic acid (9-HODE) and 13-hydroxyoctadecadienoic acid (13-HODE) were generated during incubations with exogenous linoleic acid. Esterification of 15-HETE, 9-HODE and 13-HODE during these incubations was demonstrated. The analyses included reversed-phase high performance liquid chromatography of the free acid and its methyl ester and chiral separation of the methyl ester on straight phase chiral columns. The ratio of 9-HODE/13-HODE averaged 2.7 in the chromatographic analyses of the extracts of the incubations with linoleic acid. The combined production of 13-HODE and 9-HODE from linoleic acid was four times greater than that of 15-HETE and 11-HETE from arachidonic acid. With regard to the products of the CPAE endothelial cell line, the S/R ratio of the stereoisomers averaged 1.5 for free 15-HETE, 5.7 for free 13-HODE and 0.2 for free 9-HODE. The 11-HETE had strict (R) stereospecificity. The products from the AG04762 endothelial cell line had similar stereochemistry. All these stereochemical findings point to the activity of a cyclooxygenase rather than that of a lipoxygenase.     

Acetyltransferase activity in human platelet microsomes and washed platelets

It has been demonstrated that human platelets form platelet-activating factor (PAF) when stimulated by thrombin, collagen and ionophore A-23187, but the mechanism of its formation has not been elucidated. In this study we demonstrated increased acetyltransferase activity (i.e., transfer of the acetyl moiety of [3H]acetyl-CoA to lyso-PAF (1-alkyl-sn-glycero-3-phosphocholine) to form PAF) occurring in human platelet microsomes made from platelets stimulated by thrombin or ionophore A-23187. This stimulation resulted in a 2-4-fold increase in acetyltransferase activity over unstimulated platelets. Acetyltransferase activity was also demonstrated by incubating [3H]acetate with whole platelets and stimulating with thrombin or ionophore A-23187. Radioactive PAF was detected when the platelets were stimulated. None was formed without stimulation. These findings indicate that acetyltransferase may play a role in the biosynthesis of PAF by human platelets.     

Arachidonic acid metabolism by erythrocytes

Rabbit, chicken, rat, and dog erythrocytes (10(9) cells/ml) synthesized immunologically active 12-hydroxyeicosatetraenoic acid (12-HETE) when stimulated by the Ca2+ ionophore, A-23187. The levels of immunologically active hydroxyeicosatetraenoic acid were independent of the number of white blood cells and platelets in the erythrocyte suspensions. Two products were resolved by high performance liquid chromatography; one product was identified as 12-HETE, while a second product appeared to be a dihydroxyeicosatetraenoic acid. Radiolabeled arachidonic acid was incorporated into phospholipids. Phosphatidylcholine and phosphatidylethanolamine were primary sources of the 12-HETE and dihydroxyeicosatetraenoic acid, all of which were released from the cells.     

Inhibition of protein kinase C by H-7 potentiates the release of oleic, linoleic and arachidonic acids in A23187-stimulated human neutrophils

This present report describes the effect of H-7, a protein kinase C inhibitor, on the release of oleic, linoleic and arachidonic acids in A23187-stimulated neutrophils. Surprisingly, the inhibitor potentiated the release of all three unsaturated fatty acids in neutrophils stimulated with A23187 alone. In contrast, released oleic acid, linoleic acid and arachidonic acid in phorbol 12-myristate 13-acetate-primed neutrophils were attenuated by 35, 47 and 33%, respectively, in the presence of H-7 (300 microM). Phorbol 12-myristate 13-acetate (PMA) had no effect on A23187-stimulated release of saturated fatty acids. Both PMA and H-7 when used alone had no effect on the release of saturated or unsaturated fatty acids. We, therefore, conclude that H-7 may have effects other than inhibiting PMA-primed responses including superoxide generation, degranulation and arachidonic acid release in human neutrophils.     

Lipoxygenase metabolites of arachidonic and linoleic acids modulate the adhesion of tumor cells to endothelium via regulation of protein kinase C.

12(S)-hydroxyeicosatetraenoic acid (12[S]-HETE) and 13(S)-hydroxyoctadecadienoic acid (13[S]-HODE), lipoxygenase metabolites of arachidonic acid and linoleic acid, respectively, previously have been suggested to regulate tumor cell adhesion to endothelium during metastasis. Adhesion of rat Walker carcinosarcoma (W256) cells to a rat endothelial cell monolayer was enhanced after treatment with 12(S)-HETE and this 12(S)-HETE enhanced adhesion was blocked by 13(S)-HODE. Protein kinase inhibitors, staurosporine, calphostin C, and 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine, inhibited the 12(S)-HETE enhanced W256 cell adhesion. Depleting W256 cells of protein kinase C (PKC) with phorbol 12-myristate-13-acetate abolished their ability to respond to 12(S)-HETE. Treatment of W256 cells with 12(S)-HETE induced a 100% increase in membrane-associated PKC activity whereas 13(S)-HODE inhibited the effect of 12(S)-HETE on PKC translocation. High-performance liquid chromatographic analysis revealed that in W256 cells 12-HETE and 13-HODE were two of the major lipoxygenase metabilites of arachidonic acid and linoleic acid, respectively. Therefore, these two metabolites may provide an alternative signaling pathway for the regulation of PKC. Further, these findings suggest that the regulation of tumor cell adhesion to endothelium by 12(S)-HETE and 13(S)-HODE may be a PKC-dependent process.     

Relationship of vascular 15-lipoxygenase induction to atherosclerotic plaque formation in rabbits

Aortas from atherosclerotic rabbits have increased levels of 15-lipoxygenase, but the relationship between induction of this enzyme and the atherosclerotic process has not been defined. We found that dietary administration of cortisone acetate significantly suppressed atherosclerotic plaque formation in both Watanabe Heritable Hyperlipidemic (WHHL) and cholesterol-fed WHHL/NZW heterozygous rabbits. There was, however, no corresponding decrease in the elevated 15-lipoxygenase activity. In addition, the elevated 15-lipoxygenase activity in atherosclerotic rabbit aortas was uniformly distributed throughout the aorta, and was not preferentially localized in the lesions. These results indicate that induction of the 15-lipoxygenase is not necessarily causally related to plaque development, and that plaques are not the major source of the increased enzyme activity. However, the results confirm that hypercholesterolemia is a necessary condition for both atherosclerosis and 15-lipoxygenase induction, suggesting that perhaps the 15-lipoxygenase may represent a protective response to the hyperlipidemic stress. This possibility is supported by the finding that the induced 15-lipoxygenase converts linoleic acid, which is the predominant essential fatty acid in aorta, to 13-hydroxyoctadecadienoic acid (13-HODE). This compound is a chemorepellant factor for platelets, inhibits platelet thromboxane synthesis, and stimulates prostacyclin synthesis by endothelial cells.