Neutrophil-mediated increased permeability of microcarrier-cultured endothelial monolayers: a model for the in vitro study of neutrophil-dependent mediators of vasopermeability.

Changes in the permeability of human endothelial monolayers in response to activated human neutrophils were examined in a novel, in vitro model of vasopermeability changes. Microcarrier-cultured human umbilical vein endothelial monolayers were used in a system that responds to histamine. Human neutrophils did not increase Evans Blue staining of the endothelium-covered microcarriers if added alone or if added with the neutrophil-dependent mediator of vasopermeability, formyl-methionyl-leucyl-phenylalanine (FMLP, 0.1 microM). In contrast, neutrophils, added to the endothelial cells in a ratio as low as 2.5:1 caused time-dependent increases in microcarrier staining if pretreated with cytochalasin B (5 micrograms/mL) before addition with FMLP. Neutrophil cell-free releasate and purified human sputum elastase also caused concentration-related increases in Evans Blue staining of the endothelial-covered microcarriers and these effects were inhibited by the elastase inhibitor methoxysuccinyl-alanyl-alanyl-prolyl-valyl chloromethyl ketone. This compound also inhibited neutrophil-mediated endothelial permeability increases. The microcarrier-cultured human endothelial monolayer system rapidly detects permeability alterations of endothelial monolayers in response to activated human neutrophils. This model is a potentially useful screening assay for the development of therapeutic agents, directed at neutrophil degranulation or degranulation products, for the control of inflammatory vasopermeability abnormalities.

A new class of mechanism-based inhibitors of transglutaminase enzymes inhibits the formation of cross-linked envelopes by human malignant keratinocytes.

A series of tyrosinamidomethyl dihydrohaloisoxazole compounds, designed as mechanism-based inhibitors of bovine epidermal transglutaminase enzyme, was examined for effects on the formation of cross-linked envelopes by human SCC-9 malignant keratinocytes. Compounds inhibited ionophore-induced envelope formation in a manner that reflected their capacity to inhibit transglutaminase activity. Preincubation and inhibitor wash-out studies indicated that the inhibitor must be present at the time of cell activation by ionophore in order to inhibit envelope formation. The stereospecific nature of the inhibitory activity of these compounds on both transglutaminase activity and cross-linked envelope formation makes this class of compounds an important tool in the study of transglutaminase-mediated events at the cellular level.

Increased permeability of microcarrier-cultured endothelial monolayers in response to histamine and thrombin. A model for the in vitro study of increased vasopermeability.

The permeability response of endothelial monolayers to some "direct-action" type mediators of vasopermeability were studied in vitro. Endothelial cells, cultured to confluence on denatured collagen-coated dextran microcarriers or gelatin microcarriers, prevented staining of the microcarriers with Evans blue dye. Increases in staining, as determined by the spectrophotometric quantitation of the dye after extraction from the microcarriers with formamide, occurred after treatment of human umbilical vein endothelium with histamine (10(-5) M) or thrombin (0.1 U/ml). These increases in monolayer permeability were reversible. Neither bradykinin nor serotonin had any effect in this system. Endothelial monolayers cultured this way consistently stained with silver nitrate at the cell junction areas. Monolayer response to histamine was characterized morphologically by small openings which occurred randomly along the cell junctions; while with thrombin, the spaces, which had developed at junctions, occurred to a greater extent. Prostaglandin E1 (30 microM) and isoproterenol (10 microM), in the presence of 3-isobutyl-1-methylxanthine (1 mM), partially inhibited histamine- and thrombin-mediated changes in permeability. This model responds to certain vasopermeability-altering agents in a manner similar to that of the microcirculation. These studies support the concept that the vasopermeability enhancing effect of histamine in vivo results, in part, from a direct effect on the endothelium.

Cyclic nucleotide phosphodiesterase inhibition by a benzoic acid derivative.

Acetylsalicylic acid (ASA) and three structurally related benzoic acid derivatives, 2-acetylbenzoic acid (ABA), 3-methylphthalide (3-MP), and 3-hydroperoxy-3-methylphthalide (3-HMP), were tested for inhibitory effects on three human blood platelet cyclic nucleotide phosphodiesterase (PDE) activities. 3-MP caused a dose-dependent inhibition of the high and low affinity cyclic AMP PDE activities and the cyclic GMP PDE activity. 3-HMP had some inhibitory effects but only on the low affinity cyclic AMP PDE activity. ASA and ABA had no effects. This study shows that progressive structural changes in the ASA molecule can shift the pharmacological profile from a cyclooxygenase inhibitor (ASA) to an inactive compound (ABA) to a PDE inhibitor (3-MP) and back again to a cyclooxygenase inhibitor (3-HMP). It is proposed that the potent anti-inflammatory effects of 3-MP, which differ from those of ASA, are mediated through the inhibition of the cyclic nucleotide PDE system.

The effects of some benzoic acid derivatives on polymorphonuclear leukocyte accumulation in vivo.

Previous studies, aimed at establishing a relationship between the inhibition of prostaglandin (PG) biosynthesis and the suppression of carrageenin-induced rat paw edema, indicated that, in a series of acetylsalicylic acid (ASA)-like compounds, there is not a good correlation between ability to inhibit platelet PG biosynthesis and anti-inflammatory activity. Some of the compounds tested had good anti-edema properties compared to ASA, but did not inhibit platelet lysate conversion of 14C-arachidonic acid (AA) to PGs. The effects of these compounds on polymorphonuclear (PMN) leukocyte accumulation in urethane-anesthetized rats were examined to extend the pharmacological profile of these agents in a search for other mechanisms of anti-inflammatory activity. 3-Methylphthalide (3-MP), an inhibitor of rat paw edema, suppressed PMN leukocyte accumulation although it is a poor inhibitor of PG cyclo-oxygenase activity. 3-Propionyloxybenzoic acid (3-PBA), an agent which increases primary platelet aggregation and arterial PGI2 production, caused increases in edema formation but decreases in PMN leukocyte accumulation. 2-Propionyloxybenzoic acid (2-PBA), which is similar to ASA in many effects, resulted in a trend towards decreased PMN leukocyte accumulation, while ASA did not. 2-Acetylbenzoic acid (ABA), an agent with anti-edema properties similar to ASA in the rat paw model but without any effect on PG biosynthesis, also caused a trend towards inhibition of PMN leukocyte accumulation. In addition to drug effects on prostaglandin biosythesis, this study indicates that drug effects on PMN leukocyte accumulation is an important determinant of anti-inflammatory potential.

Antiinflammatory properties of a hydroperoxide compound, structurally related to acetylsalicylic acid.

3-Hydroperoxy-3-methylphthalide (3-HMP), a structural analog of acetylsalicylic acid (ASA), was found to have some antiinflammatory properties which are distinct from those of ASA. 3-HMP inhibits human platelet aggregation and ATP release in response to low concentrations of collagen but is less effective than ASA. 3-HMP inhibits prostaglandin and thromboxane production from exogenous [14C]arachidonic acid by human platelet lysates in vitro and does so at lower concentrations than ASA (3-HMP IC50 = 10 microM; ASA IC50 = 50 microM). 3-HMP is also more effective than ASA as an inhibitor of prostacyclin-like activity production by rings of rabbit aorta. Human polymorphonuclear (PMN) leukocyte [14C]arachidonic acid metabolism is inhibited by 3-HMP but not ASA. In urethane-anesthetized rats, 3-HMP (10 mg/kg intravenously) is effective in inhibiting PMN leukocyte accumulation in response to intrapleural carrageenan administration whereas ASA is ineffective (100 mg/kg intravenously). This hydroperoxy analog of ASA has antiinflammatory activity which may result from a combination of the ASA-like and hydroperoxide-related pharmacological properties.

The effects of an ASA-like hydroperoxide compound on adenosine diphosphate induced platelet aggregation.

A hydroperoxide compound structurally related to acetylsalicylic acid, 3-hydroperoxy-3-methylphthalide, inhibits both the first and second phases of adenosine diphosphate induced, biphasic, human platelet aggregation. This occurs over the same concentration range (0.05-0.5 mM) that acetylsalicylic acid inhibits second phase aggregation and the release reaction only. The complete inhibition of adenosine diphosphate induced aggregation is a unique pharmacological property for an acetylsalicylic-acid-like compound.

The effects of high doses of aspirin and related benzoic acid derivatives on arterial thrombosis in male rats.

The antithrombotic effects of four compounds structurally related to aspirin (acetylsalicylic acid, ASA) were examined in a rat model of arterial thrombosis and compared to ASA. ASA had antithrombotic activity, but only at high doses (200 mg/kg), when carotid artery thrombosis was induced 15 min after intravenous drug administration. Lower doses were associated with augmented thrombus formation in some animals. 2-Propionyloxybenzoic acid, which has in vitro activities similar to ASA, caused similar in vivo effects, but was antithrombotic at 100 mg/kg. 3-Propionyloxybenzoic acid, which augments platelet function in vitro, and 3-methylphthalide, which inhibits biphasic adenosine diphosphate-induced platelet aggregation, had no statistically significant effects. 2-Acetoxybenzoic acid, which is a weak platelet aggregation and prostaglandin biosynthesis inhibitor, had antithrombotic activity at 100 and 200 mg/kg and was not associated with augmented thrombosis at lower doses as found with ASA. The pattern of antithrombotic activity of this series of compounds does not reflect in vitro effects on prostaglandin biosynthesis and indicates alternative mechanisms of antithrombotic activity.

Proteinase inhibitors of human articular cartilage.

Extracts of human articular cartilage contain a variety of inhibitors to serine, cysteine and metallo-proteinases. By gel filtration chromatography, inhibitory activity towards serine proteinases was resolved into two components of apparent molecular weights 62,000 and 12,000 daltons; whereas inhibitory activity towards cysteine proteinases eluted with an apparent molecular weight of 13,000 daltons. In both cases the low molecular weight inhibitors were further resolved into two components by ion-exchange chromatography. Inhibitory activity towards metallo-proteinases resolved into two components of apparent molecular weights 35,000 and 25,000 daltons. No inhibitor of aspartic proteinases was detected. Although most of the inhibitory activities to serine and cysteine proteinases could be extracted from cartilage with 1 M NaCl, the complete removal of metalloproteinase inhibitory activities required extraction with 4 M guanidinium chloride. This suggests that they are more strongly associated with the cartilage.

The influence of dose, time of administration, body temperature and salicylate kinetics on the antithrombotic action of acetylsalicylic acid in male rats.

Acetylsalicylic acid (ASA), given i.v. to male rats 10 min before electrical injury to the carotid artery, was found to reduce rate and extent of thrombosis at 3.3 and 10.0 mg/kg but not at 1.7, 20 or 100 mg/kg, indicating a narrow, low-dose window for antithrombotic effect. ASA was more effective in rats in which body temperature was allowed to fall greater than 0.5 degrees C but protection was lost if injury was delayed 15 min or more after ASA administration. Serum salicylate studies did not support the view that loss of protection was due to competition between salicylate and ASA for cyclooxygenase-binding sites. ASA was also protective at 200 mg/kg i.v., possibly through non-specific toxic effects.

Structure-activity studies of aspirin and related compounds on platelet aggregation, arachidonic acid metabolism in platelets and artery, and arterial prostacyclin activity.

A series of benzoic acid derivatives was tested for specificity of action on human platelet function and platelet prostaglandin (PG) synthesis versus prostacyclin (PGI2) production by rat and rabbit aorta rings. None of the agents tested was more specific for one system than the other. ASA was more potent than 2-propionyloxybenzoic acid (2-PBA) in inhibiting platelet function and platelet PG synthesis although the potencies of these agents were comparable in inhibiting PGI2 synthesis. 3-Propionyloxybenzoic acid (3-PBA) caused increased activity in both systems while 2-acetylbenzoic acid (ABA) had only minor effects. A cyclical derivative, 3-methylphthalide (3-MP), inhibited both platelet function and PGI2 synthesis although it did not inhibit cyclo-oxygenase activity, suggesting a novel mechanism of action. Thus only minor changes in the ASA molecule could be effected without significant changes in pharmacological activity. The investigation of novel agents such as 3-MP may lead to a better understanding of arachidonate metabolism in different tissues and possibly to the development of more tissue-specific drugs.