Eva Bastida

Platelet contribution to the formation of metastatic foci: the role of cancer cell-induced platelet activation.

Platelets are thought to be involved in the development of blood borne metastasis. Ultrastructural and experimental studies demonstrate that association between tumor cells and platelets with subsequent activation of the coagulation cascade takes place in malignancy. Several hypotheses have been proposed to explain the mechanisms by which tumor cells activate platelets including generation of thrombin, ADP release and involvement of arachidonate metabolism. Perfusion studies with human homologous systems showed that intact tumor cells and tumor cell microvesicles were able to induce platelet thrombogenicity under defined flow conditions. The presence of divalent cations and plasma factors was necessary for the cancer cells to exert their activating capacity. These results suggest a role for platelets in the development of secondary metastasis as well as in the thrombotic events of malignancy.

Fibronectin is required for platelet adhesion and for thrombus formation on subendothelium and collagen surfaces

Fibronectin (FN) plays a role in several adhesion mediated functions including the interaction of platelets with subendothelium. We investigated the role of plasma FN in platelet adhesion and platelet thrombus formation under flow conditions. We used two different perfusion models: the annular chamber with alpha-chymotrypsin-treated rabbit vessel segments, and the flat chamber with coverslips coated with fibrillar purified human collagen type III. Perfusates consisted of washed platelets and washed RBCs, suspended in normal or FN-depleted plasma. Perfusions were carried out for ten minutes at shear rates of 300 or 1,300 s-1. Platelet deposition and thrombus dimensions were evaluated morphometrically by a computerized system. We found that depletion of plasma fibronectin significantly reduced the percentage of total coverage surface and percentage of platelet thrombus, at both shear rates studied, and in both perfusion systems (P less than .01) (P less than .01). The dimensions of the platelet thrombi formed in perfusions at high shear rate were also significantly reduced in perfusions carried out with FN depleted plasma (P less than .01). Addition of purified FN to FN-depleted perfusates restored all values to those measured in the control perfusions. These results indicate that plasma FN is required for platelet aggregate and thrombus formation following adhesion under flow conditions.

Interleukin-1 increases tumor cell adhesion to endothelial cells through an RGD dependent mechanism: in vitro and in vivo studies.

The effects of human recombinant interleukin-1α and β (rIL-1α; rIL-1β) 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-1α or rIL-1β. The effects of rIL-1α and β on TC/HEC adhesion were time dependent and reached a plateau within 4–6h. 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-1α (given i.p. before ix. 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.

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.

Sex-related differences in the effects of aspirin on the interaction of platelets with subendothelium.

Using the Baumgartner perfusion technique, marked sex-related differences in the extent of platelet-subendothelium interaction and in the effect of aspirin (ASA) have been observed. The administration of ASA (150 mg daily for 15 days) to two groups of healthy volunteers, one composed of males and the other of females, proved to block the generation of TXB2 in both cases. The basic pattern of platelet subendothelium interaction, however, was found to be markedly different in both groups studied. In men, aspirin treatment induced a significant reduction in the percentage of platelet thrombi, whereas in women, post ASA values remained at the same level as in control experiments. These results show that in the Baumgartner perfusion system women display a less thrombogenic tendency than men and that 150 mg of ASA administered daily are effective in reducing the extent of platelet-subendothelium interaction in the male group but not in the female group. These findings could explain the absence of benefit observed for women in clinical trials with aspirin.

Development of a Computer Program to Analyze the Parameters of Platelet-Vessel Wall Interaction

The use of the Baumgartner perfusion system allows the morphometric quantification of platelets interacting with vessel wall, however it presents the basic difficulties of morphometrical measurements. In order to facilitate the procedure of evaluation we developed a semiautomated method to avoid the complexity of the classical evaluation. Our system consists on an optical picture analysis system connected with a specially developed computer program which allows fast quantification. Simultaneously to the outlining of interacting platelets the computer program recognizes, corrects, selects and stores the information, in order to perform the final calculations as previously established. This system has been demonstrated to be as effective as the classical morphometric evaluation in the measure of platelets interacting with subendothelium. Potential sources of error such as subjectivity of the observers in selecting the class of interacting platelets are avoided. The use of this combined method opens the possibility to adapt the Baumgartner perfusion system to clinical routine and to the screening of drugs that modify platelet adherence.

Interaction of platelets with subendothelium in humans treated with aspirin and dipyridamole alone or in combination

Aspirin and dipyridamole have been used separately and in association as antiplatelet drugs. While aspirin is well known as an inhibitor of cyclooxygenase (11, the mechanism of action of dipyridamole is still a subject of discussion (2,3,4). Dipyridamole was shown to be effective in experimental thrombosis (5) and in several clinical conditions in man (6,7,8). However, different attempts to prove its effect in laboratory tests have resulted in relatively controversial findings (3,9,10, 11,121. Previous studies in animals (9) and in humans (10) have demonstrated that dipyridamole alone was unable to reduce platelet interaction with the subendothelium of mechanically damaged aortas. In these experiments aspirin was strongly effective when anticoagulated blood was used, but practically ineffective when using native blood. In the present study, using the perfusion method described by Baumgartner (13) we have studied the effect of aspirin (50 mg t.i.d.1 and dipyridamole (75 mg t.i.d.1 on the interaction of human platelets with fibrillar collagen matrix of chemically digested subendothelium. Due to the current interest in the combination of these two drugs we have also tested the combination of 50 mg of aspirin plus 75 mg of dipyridamole (t.i.d.). The study was carried out in 12 healthy volunteers treated for 15 days.

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.

Fatty acid metabolism and cell/cell interactions

ConclusionsThe majority of studies on fatty acid metabolism, not only via the lipoxygenase pathway, but also via the cyclo-oxygenase pathway, have focused on arachidonic acid. In addition, the majority of the studies have utilized the inhibitor/add-back approach, using exogenous metabolites such as the prostanoids, monohydroxy fatty acids and leukotrienes, and stimulated cells. However, we have explored the possibility that the endothelial cell lipoxygenase metabolite of linoleic acid, specifically, 13 HODE, influences adhesion of a variety of cells implicated in thrombosis, metastasis and inflammation. Albeit, we have until now only demonstrated a correlation between 13 HODE synthesis and maintaining biocompatability between vascular endothelial cells and platelets, tumor cells and leukocytes, our studies provide ample evidence that there is a common relationship between 13 HODE synthesis in vascular wall cells (both endothelial cells and smooth muscle cells), a variety of tumor cells (both human and animal) and leukocytes, and their adhesive interactions with one another. Given that the expression of adhesive sites require an alteration in the cell membrane surface, and, that intracellular 13 HODE inhibition is, in every case, associated with enhanced adhesion, we postulate the intracellular 13 HODE regulates the expression of cell surface adhesive receptors. It is also of interest to note that recent studies in the atherosclerotic-prone Watanabe rabbit are consistant with this hypothesis. Thus, Lawrence et al. [26] found that the atherosclerotic plaque in the Watanabe rabbit wasless, not more, adhesive for platelets than the healthy vessel wall, and Simon et al. [27] found that the same atherosclerotic plaques had increased 15-lipoxygenase activity, consequently, an increased capacity to synthesize linoleic acid into 13 HODE. Studies presently ongoing in our laboratories are focused at identifying the mechanism (s) by which 13 HODE inhibits RGD adhesive site expression and maintains cells less reactive.