Thomas A. Haas

Interleukin 1-induced cancer cell/endothelial cell adhesion in vitro and its relationship to metastasis in vivo: role of vessel wall 13-HODE synthesis and integrin expression

Previously, we have demonstrated that stimulation of endothelial cells (ECs) with interleukin-la (IL-lα) 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-lα, 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.

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.

Relationship between vessel wall 13-HODE synthesis and vessel wall thrombogenecity following injury: Influence of salicylate and dipyridamole treatment

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.

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

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.

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.

Fatty Acid Metabolism and the Vascular Endothelial Cell

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

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.

Reduction in the free radical status and clinical benefit of repeated intrathecal triamcinolone acetonide application in patients with progressive multiple sclerosis.

BackgroundPrevious open trials performed repeated intrathecal application of the sustained release steroid triamcinolone acetonide every third day in patients with progressive multiple sclerosis and described enhanced walking abilities. ObjectivesThe objectives of this study were to demonstrate the efficacy of 5 triamcinolone administrations every other day and to describe their effects on the amount of inducible free radicals in cerebrospinal fluid. Subjects/MethodsClinical ratings, determinations of maximum walking distance, and execution of an instrumental peg insertion test were performed at baseline and on each day after a triamcinolone injection in 21 patients with progressive multiple sclerosis. Induction of free radicals was assessed in cerebrospinal fluid before each triamcinolone application by electron spin resonance spectroscopy. ResultsScores for multiple sclerosis improved, walking distance increased, and necessary intervals for the peg insertion procedure were shortened. The amount of inducible free radicals decreased. ConclusionsRepeat triamcinolone application improves dysfunction of upper and lower extremities even when administered 5 times only and in series every other day. The declined potential for free radical synthesis may be caused by the anti-inflammatory effect of triamcinolone. It may contribute to suppress the smoldering, chronic inflammation, particularly in spinal lesions of patients with progressive multiple sclerosis. The enhanced arm function hypothetically reflects the effect on cervical and brain lesions due to the hypobaric features of triamcinolone.

Eicosanoid Metabolism and Endothelial Cell Adhesion Molecule Expression: Effects on Platelet/Vessel Wall Interactions

Platelet interactions with the blood vessel wall following injury influence both the acute thrombotic event (platelet/vessel wall adhesion) and the chronic pathogenesis of arteriosclerosis (chronic vessel wall thickening and occlusion). Platelets are thought not to interact with the healthy intact endothelium, but only to adhere to vessel wall components exposed following endothelial cell damage and vessel wall injury (1–3). When platelets adhere to the injured vessel wall, they release a number of constituents which facilitate further platelet activation (3), activation of the coagulation cascade (1), and/or are mitogenic, facilitating smooth muscle cell proliferation and hyperplasia (4). Endothelial cells also synthesize and release a number of constituents which counterbalance the platelet responses, thereby enhancing fibrinolysis, inhibiting coagulation and preventing platelet aggregation and adhesion (5–7). Recently, with the development of endothelial cell culture techniques and a number of molecular biologic tools, we have learned that platelets and other blood cells actively interact not only with the injured vessel wall, but also with the intact endothelium itself. In this chapter, we will focus on the latter interaction and specifically on endothelial cell fatty acid metabolism and its possible consequence on the reactivity of adhesion molecule receptors on the surface of the intact endothelium which in turn may influence platelet/vessel wall interactions.