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Intimal hyperplasia after percutaneous transluminal coronary angioplasty (PTCA) or vascular surgical procedures remains a significant problem despite current antithrombotic therapy. The use of the current antithrombotic drugs, namely heparin + chronic aspirin (ASA) +/- oral anticoagulants, is based upon the assumptions that: i) heparin blocks thrombin generation and/or accelerates thrombin inhibition by antithrombin III (ATIII); ii) aspirin acetylates platelet cyclooxygenase, thereby preventing thromboxane A2 (TxA2) synthesis; and iii) oral anticoagulants reduce the availability of vitamin K-dependent procoagulants, thereby reducing the risk of thrombus formation. Albeit beneficial, this approach has a number of shortcomings and limitations: i) when thrombin binds to an injured vessel wall, it becomes resistant to inhibition by heparin/ATIII; thus, surface-bound thrombin remains active, stimulating further thrombus formation, smooth muscle cell proliferation and subsequent hyperplasia; ii) while TxA2 inhibition reduces platelet reactivity, platelets are able to respond to multiple stimuli generated at the time of, or after, vessel wall injury; and iii) heparin, aspirin and the oral anticoagulants all render the patient hemostatically defective and at risk of bleeding. Recent studies suggest that alternate therapeutic approaches can inhibit thrombogenesis more effectively at the time of injury, thereby not only inhibiting hyperplasia more effectively than the currently used drugs, but also reducing (or eliminating) the need for long-term therapy. For example, we suggest that the heparin cofactor II (HCII) catalysts, dermatan sulfate and Intimatan, inhibit surface-bound thrombin more effectively than heparin/ATIII, thereby inhibiting intimal hyperplasia effectively. Their effects are achieved when the drug is given only at the time of injury; i.e. with no further antithrombotic therapy. Other studies indicate that injured vessel wall thrombogenicity can be reduced by pretreatment with Persantine (dipyridamole) or with certain fatty acid supplements which either increase vessel wall cAMP and/or 13HODE synthesis. These increases are associated with decreased vessel wall thrombogenicity, which, in turn, is associated with decreased intimal hyperplasia. Such results suggest that vessel wall repair is achieved more effectively by targeting antithrombotic drugs directly at the vessel wall thrombogenicity per se rather than indirectly by altering the circulating blood cells and systemic coagulant system.

Depletion of internal Ca2+ stores causes capacitative Ca2+ entry which occurs through non-selective cation channels sensitive to blockade by SK&F 96365. Recently, alkaloids of Chinese herbal medicinal origin, tetrandrine and hernandezine, have been shown to possess actions including inhibition of Ca2+ channels in non-excitable cell types. In this study, we compared the actions of these novel inhibitors to those of SK&F 96365 in fura-2-loaded endothelial cells from human umbilical vein and bovine pulmonary artery. Depletion of Ca2+ from the internal stores was accomplished in Ca(2+)-free medium using an endoplasmic reticulum Ca2+ pump inhibitor, cyclopiazonic acid (CPA) or receptor agonists, histamine and bradykinin. Stimulation with histamine or bradykinin caused a marked and rapid transient increase in Ca2+ signal whereas CPA caused a smaller amplitude increase of longer duration. Restoring Ca2+ to the medium caused marked and sustained increases in the fluorescence indicating movement of Ca2+ into the cytosol presumably stimulated by the emptied Ca2+ stores. SK&F 96365 as well as tetrandrine and hernandezine antagonized depletion-induced Ca2+ entry. The results suggest that these putative inhibitors interact with Ca2+ entry triggered by depletion of the internal Ca2+ stores and their action is presumed to be on the non-selective cation channels. Their effectiveness may be enhanced by the mechanisms which lead to the opening of the Ca2+ influx channel.

A non-specific cation channel in cultured human umbilical vein endothelial cells was obtained by cell-attached patch-clamp study. This channel showed a conductance of 28 pS when both pipette and bath contained 140 mM potassium chloride. when pipette solution was changed into 140 sodium chloride with 5 mM calcium chloride, the conductance was 26 pS. when 120 mM calcium chloride was used as the only cation in the pipette, a conductance of 6 pS was obtained. Bath application of cyclopiazonic acid, an inhibitor of the sarcoplasmic reticulum Ca2+ pump in smooth muscle and other tissues, dose dependently activates this non-specific cation channel. It is assumed that cyclopiazonic acid by blockade of the refilling of Ca2+ stores depletes the rapidly exchanging intracellular Ca2+ stores and this action stimulates Ca2+ influx through the non-specific cation channels in human umbilical vein endothelial cells.

Blood/vessel wall cell interactions depend, in part, on the expression of adhesion receptors on cell surfaces, such as expression of the vitronectin receptor (VnR) on the apical surface of endothelial cells (ECs) for platelet/EC adhesion. However, it is unclear how receptor expression is regulated from within cells. In previous studies, we found that ECs metabolize linoleic acid into the lipoxygenase monohydroxide, 13-hydroxyoctadecadienoic acid (13-HODE), and that the intracellular level of 13-HODE correlates inversely with VnR expression and platelet adhesion to the EC apical surface. In this study, we determined the physical associations of 13-HODE and VnR in unstimulated and stimulated ECs, ie, at times when ECs were and were not adhesive for specific ligands and platelets, using double antibody immunofluorescent staining techniques and binding assays. 13-HODE and the VnR were colocalized within unstimulated ECs. When ECs were stimulated, 13-HODE was no longer detectable, either in or outside the ECs, and the VnR was detected on the apical surface of the ECs. These changes were paralleled by increased vitronectin binding and increased platelet adhesion to the ECs. We suggest that colocalization of 13-HODE with VnR reflects a 13-HODE/VnR interaction, confining the VnR in a nonadhesive form inside unstimulated ECs, and, as a result, the ECs are nonadhesive. When the ECs are stimulated, 13-HODE and VnR dissociate, allowing the VnR to relocate on the EC surface, where the VnR undergoes a conformational change resulting in increased EC adhesivity.

Previously, we have demonstrated that stimulation of endothelial cells (ECs) with interleukin-1 alpha (IL-1 alpha) enhances the synthesis and expression of the vitronectin receptor (VnR), promotes VnR-dependent adhesion of human A549 adenocarcinoma cells to ECs, and is associated with decreased EC 13-hydroxyoctadecadienoic acid (13-HODE) synthesis in vitro. To determine whether these observations are relevant in vivo, we examined the acute retention and subsequent metastasis of intravenously-injected B16F10 melanoma cells in murine lungs, in relation to vessel wall 13-HODE. In C57BL/6 mice pretreated with IL-1 alpha, vessel wall 13-HODE was decreased and B16F10 lung entrapment and metastasis were increased. The latter two events were blocked by pretreating the animals with the GRGDS peptide. These data suggest a relationship between vessel wall 13-HODE synthesis, adhesion molecule expression, and adhesion of B16F10 cells to the endothelium.

The strategies used to prevent and treat thrombosis are based on our understanding of how to ameliorate the pathophysiological responses to injury, such as using inhibitors of platelet activation and inhibitors of coagulation. Both platelets and the coagulation pathway are activated in response to injury. An alternative strategy which has not been investigated to the same extent, is to use substances which contribute to the biocompatibility of blood and vessel wall which predominate under normal conditions. This latter strategy exploits the concept of restoring blood/vessel wall compatibility without risk of bleeding such as can occur when platelets as rendered haemostatically defective (antiplatelet therapies) or maintaining the patient hypocoagulated (anticoagulant therapies). Recent studies suggest that by better understanding the biocompatibility of the healthy endothelium with blood, we may be able to identify those substances which contribute to blood/vessel wall biocompatibility in the healthy environment, and subsequently which may achieve effective antithrombotic therapy with minimal risk of bleeding side-effects. In this review, we identify three such substances all of which are produced by the blood vessel wall. These agents are 13-hydroxyoctadecadienoic acid (13-HODE), a lipoxygenase metabolite of linoleic acid, and two glycosaminoglycans, heparan sulfate and dermatan sulfate.

The transport of cancer cells from blood vessels to extravascular tissue is a critical step in metastasis, where endothelial cells and the vascular basement membrane act as barriers to cell traffic. Because endothelial injury can facilitate the metastasis of intravascular cancer cells in vivo, the authors have studied in vitro the free-radical-mediated endothelial damage caused by the rat Walker 256 carcinosarcoma (W256) cell after stimulation with 10(-6) mol/l (molar) phorbol ester. Here the authors have examined the hypothesis that W256 cell-mediated endothelial injury is dependent on adhesion between the effector and target cells. Attachment of phorbol 12-myristate, 13-acetate (PMA)-stimulated W256 cells to endothelial monolayers was increased 1.8 +/- 0.1-fold and damage (3H-2-deoxyglucose release from labeled endothelium) 1.4 +/- 0.1-fold after 4-hour pretreatment of the endothelium with 10 ng/ml recombinant human interleukin-1 alpha (rIL-1 alpha). Under various assay conditions, the release of 3H-2-deoxyglucose correlated directly with tumor cell adhesion (r = 0.98, P less than 0.005). In the presence of a polyclonal anti-vitronectin receptor antiserum, adhesion of stimulated W256 cells to rIL-1 alpha-treated monolayers was inhibited by 39% +/- 2%, and 3H-2-deoxyglucose release was inhibited by 53% +/- 13%. Immunoblot analysis and immunofluorescence flow cytometry demonstrated that the endothelial cells but not the W256 cells expressed vitronectin receptor (VnR) on their cell surface. The surface expression of VnR by endothelial cells was increased 1.9 +/- 0.1-fold after 4 hours' incubation with rIL-1 alpha. The authors conclude that W256 cell-mediated endothelial damage is dependent on cell adhesion, which, in turn, is partly regulated by the expression of VnR on the endothelial cell surface.

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.

Adhesion of tumor cells to vascular endothelial surfaces is one of the key steps in metastatic dissemination. Several factors are believed to be implicated in the regulation of the adhesive properties of tumor cells. We show that the adhesion of five different tumor cell lines, all of them of human origin, to human umbilical vascular endothelial cells (ECs) significantly increases following pretreatment of ECs with the cytokines interleukin-1 and tumor necrosis factor, whereas tumor cell/EC interactions remained unchanged after incubation with interferon-gamma. Significant augmentation in tumor cell adhesion was also observed when ECs were treated with the lipoxygenase inhibitors salicylate and the compound BW755C. In all cases, increased tumor cell adhesion was concomitant with significant decreases in the EC levels of linoleic acid, lipoxygenase-derived metabolite 13-hydroxy-octadecadienoic acid (13-HODE). On the contrary, pretreatment of the EC monolayers with aspirin did not result in any changes towards tumor cell adhesion. These results suggest that tumor cell/EC interaction is modulated, at least in part, by intracellular levels of 13-HODE and is independent of prostacyclin (PGI2) production by the ECs.

Endothelial cells synthesize two important fatty acid metabolites, PGI2, which is synthesized from arachidonic acid via the cyclooxygenase pathway, and 13-HODE, which is synthesized from linoleic acid via the lipoxygenase pathway. PGI2 is synthesized following cell activation or injury while 13-HODE is synthesized in the unstimulated cell. While the role of PGI2 in platelet vessel wall interactions has been studied extensively, the role of 13-HODE in platelet vessel wall interactions is just now being understood. The present evidence suggests that 13-HODE is continuously synthesized in "resting" vessel wall cells and is in close juxtaposition with the ubiquous integrin adhesion molecule, the vitronectin receptor. The observation that the endothelial cell is not adhesive when 13-HODE and the vitronectin receptor are in close association and becomes adhesive when these two moieties dissociate and the vitronectin receptor relocates on the surface of the cell, provides further evidence that 13-HODE may induce conformational changes in the vitronectin receptor to reduce its ability to recognize its adhesive ligands. The additional observations that 13-HODE levels in both human and animal vessel walls are inversely related with vessel wall adhesivity, and that this adhesivity can be altered by altering 13-HODE synthesis, provides evidence that 13-HODE down-regulates the thrombogenecity of the injured vessel wall surface.

The response to air-dry injury to the carotid artery of the normolipemic rabbit was compared with the response to de-endothelialization with a balloon catheter. Air drying induced an inflammatory response that resembled arteritis rather than atherosclerosis. There was medial damage, neutrophil but not macrophage infiltration, and fibrin formation, limited smooth muscle proliferation, which regressed after 3 months, and no lipid deposition. Within 1 week the smooth muscle cells were mainly of the secretory phenotype, and a neointima had formed. At 4 weeks the neointimal proliferation continued, but most cells showed a contractile phenotype. By 3 months, the lesion consisted of fibromuscular thickening with few small smooth muscle cells. Balloon injury induced minimal medial damage and continuing intimal proliferation with no evidence of regression by 3 months. It is concluded that air drying the carotid artery induces smooth muscle damage as well as endothelial cell loss, and this stimulates a wound-healing mechanism that is different from the response to selective intimal injury.

Recent studies indicate that chemotherapy is a cause for thrombosis in breast cancer patients. We performed experiments to determine whether the enhanced thrombosis was due, in part, to an effect of chemotherapy on endothelial cell reactivity. Heparinized blood samples were obtained from stage II breast cancer patients receiving monthly adjuvant chemotherapy consisting of cyclophosphamide, epirubicin and 5-fluorouracil. Cultured human endothelial cells were incubated with the plasmas for 2 h, and then the reactivity of the endothelial cells to normal donor platelets was determined isotopically. Endothelial cell reactivity was increased when the endothelial cells were incubated with the post-chemotherapy plasmas. The plasma effect persisted after the chemotherapy drugs were cleared from the circulation, but this plasma effect was abolished when the plasmas were heat-inactivated. Furthermore, the increase in endothelial cell reactivity correlated with the level of interleukin-1 present in the post-chemotherapy plasmas. Finally, the increased endothelial cell reactivity was inhibited by the GRGDS peptide, or by an antibody to the endothelial cell vitronectin receptor. These observations suggest that chemotherapeutic drugs alter endothelial cell reactivity to platelets by inducing the release of interleukin-1 which, in turn, facilitates adhesion molecule expression on the endothelial cell surface. If so, these observations provide a possible explanation for one mechanism which may contribute to the thrombogenic effect seen in breast cancer patients undergoing chemotherapy.

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.

We performed studies to determine the relationship between injured vessel wall thrombogenicity, vessel wall 13-hydroxyoctadecadienoic acid (13-HODE) synthesis and cAMP levels in rabbit treated with salicylate or dipyridamole. Injured vessel wall thrombogenicity was measured as the number of 3H-adenine labelled platelets adhered to the subendothelial basement membrane exposed by air injury in carotid arteries of rabbits treated orally with salicylate or dipyridamole. Vessel wall 13-HODE was measured by HPLC and vessel wall cAMP was measured by RIA. Vessel wall thrombogenicity was increased two-fold in rabbits treated with salicylate and decreased by half in rabbits treated with dipyridamole. The levels of vessel wall cAMP levels were correlated both with the plasma dipyridamole levels and increases in 13-HODE synthesis. cAMP levels were unaffected by salicylate treatment, but 13-HODE synthesis was decreased. We conclude that there is a significant relationship between vessel wall cAMP levels and 13-HODE synthesis, which in turn, influences subsequent vessel wall thrombogenicity.

The effects of human recombinant interleukin-1 alpha and beta (rIL-1 alpha; rIL-1 beta) on the adhesion of human A549 lung carcinoma cells and M6 melanoma cells (TC) to human endothelial cells (HECs) in vitro were studied, and on TC/lung entrapment in vivo. In vitro, there was a significant increase in TC/HEC adhesion to HECs pretreated for 4 h with rIL-1 alpha or rIL-1 beta. The effects of rIL-1 alpha and beta on TC/HEC adhesion were time dependent and reached a plateau within 4-6 h. TC/HEC adhesion was not blocked when measured in the presence of antibodies to either fibronectin, glycoprotein IIb/IIIa, anti-ICAM, or anti-LFA. However, enhanced TC/HEC adhesion was completely blocked in the presence of the peptide, GRGDS. In vivo, pretreatment of nude mice for 4 h with rIL-1 alpha (given i.p. before i.v. injection of TCs) enhanced TC retention in the lung 24 h later. Our data demonstrate that IL-1 enhances TC adhesion to the vascular surface both in vitro and in vivo, suggesting that IL-1 can facilitate the metastatic process.

Von Willebrand factor (vWF), fibronectin (FN) and 13-hydroxy-octadecadienoic acid (13-HODE) are known to influence the regulation of the adhesive properties of vascular surfaces. In the present study vWF, FN and 13-HODE were comparatively localized in endothelial cells (EC) and in the extracellular matrix (ECM) produced by EC. An indirect immunofluorescent technique was applied to coverslips containing human EC cultures previously fixed and permeabilized following different procedures: A. Alcohol/acetone; B. Paraformaldehyde alone and C. Paraformaldehyde followed by Triton X-100. vWF was observed inside EC (A), on the ECM produced by EC (B) or in EC and ECM (C) depending on the fixation procedures used. FN was mainly localized in the ECM despite the fixation procedures employed. FN was only seen in relation to cell bodies after strong permeabilization (A). Under our experimental conditions 13-HODE was never found in ECM. This latter antigen was observed randomly dispersed in those preparations fixed with alcohol/acetone, indicating that it is probably extracted by this fixative. 13-HODE was detected in granular shaped structures in EC after permeabilization with detergent (C). These results suggest that the cellular localization of vWF and FN is compatible with an adhesive role related to the abluminal side of ECs. 13-HODE was readily observed after mild permeabilization. This finding would be morphologically consistent with its contribution to the regulation of the vessel wall thromboresistance.

We investigated the effects of formylmethionylleucylphenylalanine (FMLP), interleukin-1 alpha (IL1 alpha) and interleukin-1 beta (IL1 beta) on tumor cell chemotaxis and tumor cell/endothelial cell adhesion. Chemotaxis of A549 human lung carcinoma cells was measured as the number of tumor cells which migrated across a nitrocellulose filter in a Boyden chamber. Tumor cell/endothelial cell adhesion was measured as the number of 125IUdR tumor cells adherent to monolayers of endothelial cells. Confluent monolayers of human umbilical endothelial cells were incubated from 10 to 240 minutes with FMLP, monocyte-derived interleukin-1, or recombinant IL1 alpha or IL1 beta. The endothelial cells were washed and then incubated with 125IUdR-tumor cells. Thirty minutes later the number of adherent tumor cells was assessed isotopically. Our results demonstrate that (a) interleukin-1 but not FMLP, has chemotactic activity for tumor cells, and (b) both FMLP and interleukin-1 enhance tumor cell adhesion to the endothelium independent of any chemotactic activity. Furthermore, we demonstrate that IL1 alpha and IL1 beta have different effects on tumor cell/endothelial cell adhesion, and raise the possibility that IL1 alpha but not IL1 beta is continuously synthesized and stored within the endothelium. We postulate that IL1 alpha and IL1 beta influence tumor cell/endothelial cell adhesion independent of chemotaxis through the expression of adhesive receptors on the endothelial cell surface.

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'.

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)

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.

The passage of circulating tumor cells across vessel walls is an important step in cancer metastasis and is promoted by endothelial injury. Because Walker carcinosarcoma 256 (W256) cells generate oxygen-derived free radicals after cellular activation, the authors tested the hypothesis that these cancer cells can damage endothelial monolayers by producing such reactive oxygen species. To confirm that oxygen-derived radicals can damage endothelial cells, 3H-2-deoxyglucose-labeled human endothelial cell monolayers were exposed to xanthine oxidase in the presence of 0.2 mmol/l xanthine. 3H-2-deoxyglucose release was observed after the addition of xanthine oxidase in concentrations ranging from 6.5 x 10(-3) to 52 x 10(-3) units/ml. The extent of damage correlated with xanthine oxidase-dependent chemiluminescence (r = 0.91). Chemiluminescence assays in the presence of 5 x 10(-5) M luminol confirmed activation of the W256 cells by 1 x 10(-6) M chemotactic peptide fMLP. When fMLP-activated activated W256 cells were incubated with endothelial monolayers, concentrations of 2 x 10(6) to 6 x 10(6) W256 cells/ml were found to cause a 27% increase in the specific release of 2-deoxyglucose after a 90-minute incubation. A small but significant increase in 3H-2-deoxyglucose release also was observed in the absence of fMLP. Detection of 3H-2-deoxyglucose release in the presence of activated or unactivated tumor cells was dependent on preincubating the endothelial cell monolayer with 1 mM buthionine sulfoximine, an inhibitor of glutathione synthesis. Under these conditions, the specific release of 3H-2-deoxyglucose was increased from nondetectable levels to 21%, in the presence of 6.5 x 10(-3) units of the oxidase. Cultured W256 cells promoted isotope release from endothelial cell monolayers when activated with phorbol myristate acetate. Catalase (1000 units/ml) inhibited the tumor cell-induced release of 3H-2-deoxyglucose by 84% whereas superoxide dismutase, even at concentrations of 1 mg/ml, had no effect. A requirement for cell contact was shown because addition of cell-free supernatants from fMLP activated tumor cells did not cause 3H-2-deoxyglucose release and because pretreatment of W256 cells with 1 microM cytochalasin B inhibited their ability to promote isotope release even while increasing tumor cell-generated chemiluminescence threefold. Electron microscopy revealed that fewer cytochalasin B-treated W256 cells were attached to the endothelial cell monolayer than in untreated controls. It is concluded that the W256 tumor cells can damage endothelial cells directly via a mechanism involving production of reactive oxygen species.

We hypothesize that the ratio of intracellular 13-hydroxy-octadeca-dienoic acid (13 HODE) and hydroxy-eicosatetraenoic acid (5-, 12- and/or 15-HETE) influences the expression or presentation of adhesive moieties on platelets, leukocytes, malignant cells and endothelial cells, thereby influencing their subsequent adhesive interactions. Thus, we demonstrate that under unstimulated conditions, these cells preferentially synthesize linoleic acid via their lipoxygenase enzymes into 13-HODE, the intracellular level of which is associated with limited or no cell adhesion, while following stimulation, the same cells preferentially metabolize arachidonic acid via the lipoxygenase enzyme into HETEs, the production of which is associated with enhanced adhesion. Which metabolite is synthesized by these cells and the subsequent adhesivity of these cells appear to be dependent upon both the intracellular level of cAMP and the ratio of linoleic and arachidonic acid substrates. This suggests that manipulation of this ratio will have significant effects on the adhesive events involved in the pathogenesis of thrombosis, inflammation and metastasis.

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.