E. Dejana

Endothelial adherens junctions control tight junctions by VE-cadherin-mediated upregulation of claudin-5.

Intercellular junctions mediate adhesion and communication between adjoining cells. Although formed by different molecules, tight junctions (TJs) and adherens junctions (AJs) are functionally and structurally linked, but the signalling pathways behind this interaction are unknown. Here we describe a cell-specific mechanism of crosstalk between these two types of structure. We show that endothelial VE-cadherin at AJs upregulates the gene encoding the TJ adhesive protein claudin-5. This effect requires the release of the inhibitory activity of forkhead box factor FoxO1 and the Tcf-4–β-catenin transcriptional repressor complex. Vascular endothelial (VE)-cadherin acts by inducing the phosphorylation of FoxO1 through Akt activation and by limiting the translocation of β-catenin to the nucleus. These results offer a molecular basis for the link between AJs and TJs and explain why VE-cadherin inhibition may cause a marked increase in permeability.

Interendothelial junctions: structure, signalling and functional roles

Endothelial cell-cell adhesive junctions are formed by transmembrane adhesive proteins linked to a complex cytoskeletal network. These structures are important not only for maintaining adhesion between endothelial cells and, as a consequence, for the control of vascular permeability, but also for intracellular signalling properties. The establishment of intercellular junctions might affect the endothelial functional phenotype by the downregulation or upregulation of endothelial-specific activities.

Salicylate-aspirin interaction in the rat. Evidence that salicylate accumulating during aspirin administration may protect vascular prostacyclin from aspirin-induced inhibition.

Aspirin inhibits cyclooxygenase, thus preventing thromboxane A2 production in blood platelets and prostacyclin in vascular cells. Aspirin is rapidly hydrolyzed to salicylate in the circulation. The objectives of this study were (a) to evaluate whether administration of salicylate, though ineffective by itself, prevents the inhibitory effect of aspirin on platelet and/or vascular cyclooxygenase activity; (b) to verify whether salicylate accumulating in blood after aspirin administration interferes with the pharmacological activity of further doses of aspirin. Pretreatment of rats with sodium salicylate (25-100 mg/kg i.p.) resulted in dose-related prevention of the effect of a subsequent dose of aspirin (2.5-10 mg/kg i.v.) on both platelet and vascular cells. Sodium salicylate appeared to amplify the greater response of platelets to aspirin compared with vessel wall. Pretreatment of rats with repeated high doses of aspirin (200 mg/kg) resulted after 24 h in blood salicylate levels (150-200 microgram/ml) that significantly prevented the inhibitory effect of a subsequent dose of aspirin on newly synthesized vascular prostacyclin. Blood salicylate levels obtained after 36 or 48 h (less than 50 microgram/ml) were too low to blunt aspirins effect. The interference with aspirin of its major endogenous metabolite should be borne in mind when interpreting results obtained with high dose aspirin or during repeated administration of this drug.

Prostacyclin production by human endothelial and bovine smooth muscle cells in culture: Effect of repeated stimulation with arachidonic acid, thrombin and ionophore A23187

Prostacyclin (prostaglandin I2) is the major product of arachidonic acid metabolism in vascular cells. Its physiological role may be linked to the ability of the cells to respond continuously with prostaglandin I2 production to a variety of stimuli. We report that human endothelial cells or bovine smooth muscle cells in culture respond with prostaglandin I2 synthesis to a first but not to a second stimulation with arachidonic acid. The development of this refractoriness was independent of the arachidonic acid concentration used (6.6-25 microM) and lasted for about 6 h. The same time was required for the cells to recover completely after inhibition of cyclooxygenase activity by aspirin. Neither cis-polyunsaturated fatty acids (linoleic or oleic acids) nor stearic acid (a long-chain saturated fatty acid) prevented the generation of prostaglandin I2 by arachidonic acid. Similarly to arachidonic acid, thrombin and ionophore A23187 could elicit vascular prostaglandin I2 synthesis only once. Pretreatment of the cells with arachidonic acid rendered the cells unresponsive to any other stimulus. These results indicate that the mechanism of the refractoriness induced by arachidonic acid was different from that induced by the other stimuli. It is proposed that vascular cells cannot be stimulated continuously to produce prostaglandin I2, but this process is regulated by different feedback mechanisms.

Bidirectional modulation of platelet and polymorphonuclear leukocyte activities

The first event in the pathogenesis of thrombosis has long been considered the activation of circulating platelets, as well as their adhesion to the subendothelial matrix of damaged vessels. Although this concept has emphasized the primary role of platelets in vascular occlusion, several observations indicate that also polymorphonuclear leukocytes (PMN) may participate m the development of arterial thrombosis [46, 73, 165]. Moreover, a large amount of experimental data indicates that platelet activation elicits PMN responses and that PMN in turn modulate platelet functions. With the increasing awareness of the possible relevance of these interactions, the concept of thrombosis as a multicellular process has emerged [74, 95]. On the other hand, the interaction between platelets and PMN is potentially relevant also in inflammatory diseases. Although leukocytes are the main cellular effectors of inflammation, platelets, too, although primarily involved in hemostasis and thrombosis, may act as inflammatory cells by releasing phlogistic substances [116, 117] and by interacting with circulating leukocytes [162] and vascular endothelium [34]. Platelet responsiveness to leukocyte-derived products and, conversely, platelet ability to modulate the functional activation of leukocytes may therefore condition the course of the inflammatory process. In this review we first describe the cellular interactions particularly relevant in thrombosis, considering that primary platelet activation may cause secondary PMN responses. In the second part we examine the interactions potentially relevant in acute inflammation, with particular regard to the hypothesis that primary PMN activation may subsequently stimulate platelets.

Adhesive Proteins At Endothelial Cell-To-Cell Junctions And Leukocyte Extravasation

Endothelial cell junctions are complex structures formed by transmembrane adhesive molecules linked to a network of cytoplasmic/cytoskeletal proteins. At least four different types of endothelial junctions have been described (tight junctions, gap junctions, adherence junctions and syndesmos or complexus adhaerentes). Leukocytes adhesion to endothelial cells is frequently followed by their extravasation. The mechanisms which regulate the passage of leukocytes through endothelial clefts remain to be clarified. Many indirect data suggest that leukocytes might transfer signals to endothelial cells both through the release of active agents and adhesion to the endothelial cell surface. These signals could induce the disorganization of interendothelial junctions and facilitate leukocyte transmigration.

AD 6, a coronary dilating agent, stimulates PGI2 production in rat aorta ex vivo and in human endothelial cells in culture

Summary AD6(8-monochloro-3-beta-diethylaminoethyl-4-methyl-7-ethoxy-carbonyl-methoxy-coumarin) reportedly exerts specific coronary vasodilatory activity and reduces platelet aggregation in laboratory animals. We investigated whether AD6 stimulates prostacyclin (PGI2) synthesis by vascular cells. AD6 treatment of rats induced a dose dependent enhancement of prostacyclin (PGI2) synthesis by rings of thoracic aorta measured by bioassay and confirmed by thin layer radio-chromatography of the products of 14C arachidonic acid(14CAA) metabolism. In vitro incubation of the drug with cultured human endothelial cells caused a concentration dependent stimulation of PGI2 production measured by radioimmunoassay of 6keto PGF1α. It is proposed that the previously described vasodilatory activity of the drug might be mediated through the stimulation of PGI2 release.

Inhibition by AD6 (8-monochloro-3-beta-diethylaminoethyl-4-methyl-7-ethoxycarbonylmetoxy coumarin) of platelet aggregation in dog stenosed coronary artery

The action of AD6 as an anti-thrombotic agent was studied in a model of coronary artery thrombosis and on platelet aggregation in the dog. AD6 (10-100 microM) in vitro inhibited aggregation induced by ADP, epinephrine, collagen and PAF (platelet aggregating factor) used at their threshold concentration for maximal aggregation. Arterial thrombosis was induced in a coronary vessel by critically reducing (about 70%) the vessel lumen. Thrombus formation was estimated by measuring coronary flow in the stenosed vessel. Using this procedure on the left descending coronary artery (LAD), we obtained reproducible blood flow changes in 18 dogs. AD6 was given i.v. at three different doses. At 0.25 mg/kg two out of four dogs showed decreased thrombus formation at the stenosis site. Seven out of eleven dogs treated with 0.5 mg/kg and two out of three treated with 1.5 mg/kg showed decreased thrombus formation. Major decreases in coronary resistance, evaluated by measuring blood flow in the unstenosed left circumflex artery (LCX), were evident only after the highest dose. We conclude that AD6 has an inhibitory action on dog platelet aggregation and reduces thrombus formation in a stenosed coronary vessel.

Effects of dipyridamole and low-dose aspirin therapy on platelet adhesion to vascular subendothelium

The effect on platelet function of low-dose aspirin (ASA) and dipyridamole alone or in combination was evaluated after repeated dosing in 5 healthy volunteers. The subjects were treated according to a randomized, single-blind, crossover design with 150 mg of dipyridamole, 25 mg of ASA, the 2 drugs together or placebo twice a day for 3 days. Platelet adhesin was evaluated using an experimental model of adhesion to rat aorta subendothelium under controlled hemodynamic conditions in the presence of red blood cells. Dipyridamole significantly reduced platelet adhesion both alone and in combination with ASA. ASA by itself did not significantly modify platelet adhesion, but completely blocked serum thromboxane production and platelet aggregation by arachidonic acid. Thus, low-dose ASA and dipyridamole may have a complementary action, modifying at the same time 2 platelet functions, adhesion and aggregation, both relevant in the pathogenesis of thrombosis.

Platelet aggregation induced by the endoperoxide analogue U46619 is inhibited by polymorphonuclear leukocyte ADPase activity.

Platelet activation by the stable endoperoxide analogue U46619 is mediated largely by ADP released from platelet-dense granules. Polymorphonuclear leukocytes (PMNs) endowed with ecto-ADPase activity may operate as antiaggregatory cells in platelet aggregation induced by U46619. Unstimulated PMNs were effective in reducing aggregation when platelets were stimulated by threshold concentrations of U46619, whereas at higher concentrations of the stimulus, PMN activation is required. Evidence that the inhibition was mediated by PMN ecto-ADPase activity was obtained by high-performance liquid chromatography analysis, indicating that PMNs were able to efficiently metabolize platelet-active ADP into AMP. Moreover, PMN-derived supernatants were able to inhibit platelet aggregation, suggesting that under this circumstance the inhibition was exerted by an uncharacterized, releasable ADPase activity. This study supports the hypothesis that, besides nitric oxide and hydrogen peroxide, ADPase activity may represent another PMN-mediated pathway capable of regulating platelet activity in areas of reduced blood flow, such as those found in conditions of myocardial ischemia.