An alternate mechanism for regulation of leukotriene production in leukocytes: studies with an anti-inflammatory drug, sodium diclofenac.

Sodium diclofenac, a potent cyclooxygenase inhibitor, was recently shown to inhibit arachidonic acid conversion to leukotriene products in human leukocytes. This activity was confirmed by radioimmunoassay in calcium ionophore A 23187-stimulated leukocytes isolated from the rat peritoneal cavity and human peripheral blood. Studies with rat peritoneal leukocytes revealed that this effect was not mediated by inhibition of 5-lipoxygenase or phospholipase A2, but rather through modulation of arachidonic acid uptake and release. The potency of this effect was dependent upon cell type; macrophages being more sensitive to the drug than neutrophils. In leukocytes treated with sodium diclofenac, arachidonic acid released from phospholipids in response to A 23187 challenge was reincorporated into triacylglycerols. The drug enhanced the spontaneous uptake of arachidonic acid into the cellular triacylglycerol pool and, in this manner, decreased the availability of intracellular arachidonic acid. Therefore, sodium diclofenac, in addition to inhibition of cyclooxygenase, regulates leukotriene production of inflammatory cells by a mechanism mediated in part through the redistribution of arachidonic acid in lipid pools.

Aortic cholesteryl esterase. Influence of bile salts.

The in vitro activity of cholesteryl ester hydrolase preparations of rat and rabbit aortas was assayed in the presence of the taurine and glycine conjugates of cholic, chenodeoxycholic, deoxycholic and lithocholic acids or in the presence of Triton X-100 and Tween-20. Maximum activity was obtained with tauro- or glycocholic acids. As in the case of pancreatic cholesteryl esterase, trihydroxycholanoic acid derivatives may serve an obligatory function.

Inhibition of cholesterol ester transfer protein CGS 25159 and changes in lipoproteins in hamsters.

As a result of screening, several isoflavans were identified to be antagonists of cholesterol ester transfer protein (CETP) activity. The present study evaluates CGS 25159, a synthetic isoflavan, as a putative inhibitor of CETP activity of human and hamster plasma. Determined by [3]CE transfer from HDL to VLDL + LDL fraction or by fluorescent-CE transfer assay, CGS 25159 inhibited CETP in both human plasma bottom fraction (d = 1.21 g/ml) and in plasma from Golden Syrian Hamsters with an IC50 < 10 microM. The compound also inhibited (IC 50 approximately equal to 15 microM) the reciprocal transfer of triglycerides in the incubated whole plasma from normal and hyperlipidemic hamsters. When orally administered to normolipidemic hamsters, CGS 25159 (10 mg/kg, 4 days) reduced plasma transfer activity by 35-60%. Treatment with CGS 25159 (10 and 30 mg/kg, p.o.) resulted in dose dependent and time dependent changes in CETP activity. After two weeks of treatment at 10 mg/kg, the changes in VLDL + LDL cholesterol, total triglycerides and HDL cholesterol were -22 +/- 4.6*, -23 +/- 7.5 and +10 +/- 2.8%, respectively. The corresponding changes at 30 mg/kg were -28 +/- 5.5*, -38 +/- 6.8* and +29 +/-4.4.*%, (*, P, 0.05; mean +/- S.E.M., n = 6). A single spin gradient density ultracentrifugation of plasma lipoproteins and treated animals showed an increase in HDL cholesterol and a redistribution to larger HDL particles. These data support the contention that pharmacological down regulation of CETP activity could result in favorable changes in lipoprotein profile.

Identification of potential antiatherosclerotic/hypolipidemic agents by their effect on hepatic conversion of androst-4-ene-3,17-dione to etiocholanolone and androsterone.

Clinical observations have shown that hypercholesterolemia is associated with abnormal androgen metabolism, viz. an increased excretion of etiocholanolone (E) relative to androsterone (A). Substances which restore the A/E ratio to normal likewise lower serum cholesterol. Postulating that the abnormal steroid and sterol metabolism may be either causally related or dependent on the same metabolic defect, we have developed in vitro and in vivo models to select drugs which favorably effect the ratio of A to E produced from [4-14C]androst-4-ene-3,17-dione [4-14C]A-dione). The in vitro model employs a mixture of rat liver microsomal delta 4-3-ketosteroid-5 alpha-reductase and cytosolic 3 alpha-hydroxysteroid dehydrogenase and delta 4-3-ketosteroid-5 beta-reductase. Kinetic and mechanistic studies have been performed on active compounds using this in vitro assay. The in vivo model employs i.v. injection of [4-14C]A-dione followed by collection of bile in anesthetized, hypophysectomized female rats. Many compounds preselected in the in vitro assay likewise reduced the A/E ratio in vivo. One of these compounds (CGS 10614A) also lowered serum cholesterol and reduced the incidence and severity of atherosclerotic lesions in aortas of cholesterol-fed rabbits.

Effects of altered endocrine function on biliary metabolites of [4-14C]androst-4-ene-3,17-dione in rats. Possible utility as a model for identifying anti-atherosclerotic agents.

Atherosclerosis, coronary artery disease and elevated serum cholesterol are frequently associated with an abnormal pattern of androgen metabolites, especially an elevation of etiocholanolone (E) and/or epiandrosterone (EA) relative to androsterone (A). Therapeutic correction of these metabolic defects may lower serum cholesterol. We have attempted to reproduce this metabolic syndrome in rats by altering their endocrine status. Intact male rats excreted very little A or E in their bile; more than 80% of the [4-14C]A-dione was excreted as unknown polar compounds. Adrenalectomy, thyroidectomy or streptozotocin diabetes induced little or no change in the excretion of both E and A and did not alter the A/E ratio. Hypophysectomy (hypox), however, resulted in a huge increase in E excretion and a 10-fold decrease in the A/E ratio. Treatment of hypophysectomized males with bovine growth hormone (bGH) but not testosterone or thyroxine restored the pattern of androgen metabolites to that of intact male rats. Intact female rats excreted mainly A, and this was decreased by ovariectomy. Hypophysectomy, however, resulted in a marked increase in E and a corresponding large decrease in A excretion. Treatment of hypox females with estradiol or triiodothyronine did not correct the metabolic defects in A and E production, whereas GH resulted in a pattern of A-dione metabolism resembling that of intact males; i.e., primarily polar metabolites with low A and E. Hypophysectomy thus results in a dramatic increase in 5 beta-reductase activity in male and female rats. GH therapy restores the metabolic pathway to that seen in intact males. Our objective had been to find a model capable of detecting substances which would increase A and decrease E production. The male rat (regardless of endocrine status) has little 5 alpha-reductase activity. The intact female rat, however, has high 5 alpha-reductase activity, and retains significant 5 alpha-reductase in the absence of the ovaries. In hypox females, 5 alpha-reductase was much reduced while 5 beta-reductase was increased. Furthermore, serum cholesterol was elevated in hypox females but could be lowered by exogenous androsterone. Thus the hypox female rat appears to offer the best model for identifying non-hormonal agents which could enhance the production of A and/or decrease the production of E. Such agents might favorably influence cholesterol metabolism.

Effect of a novel series of macrocyclic hypolipidemic agents on plasma lipid and lipoprotein levels of four non-primate species.

The ansamycins are structurally novel hypolipidemic agents derived from rifampicin, but lacking antibacterial activity. Oral or intravenous administration resulted in rapid lowering of plasma cholesterol in rats, hamsters, guinea pigs and dogs. In the chow-fed rat, three related compounds (CGP 43371, CGS 23810 and CGS 24565) exhibited ED50 values of 13.7, 3.1 and 0.18 mg/kg, respectively. A feature common to the lipid lowering documented in these four species was the concomitant reduction of low density lipoprotein (LDL) and high density lipoprotein (HDL) cholesterol. In the chow-fed rat, however, apolipoprotein AI (apo AI) levels were much less affected than were those of HDL cholesterol. CGP 43371 at 3 and 10 mg/kg, lowered HDL cholesterol by 20% and 39%, respectively, whereas plasma apo AI was reduced by only 1% and 12%. Similarly, in lipoprotein fractions separated by ultracentrifugation, apo AI was unchanged in the d = 1.019-1.21 g/ml fraction after treatment with 3 or 10 mg/kg of CGP 43371, but HDL cholesterol was reduced 12% and 26% in this fraction at the two dose levels. Plasma and lipoprotein apo B levels, on the other hand, were reduced to a level equivalent to that of the reduction in cholesterol. The ansamycins thus represent a new structural series which may possess a novel mechanism of action as well, involving differential effects on HDL cholesterol and protein.

Effect of diclofenac sodium on the arachidonic acid cascade.

The anti-inflammatory activity of nonsteroidal anti-inflammatory drugs is primarily attributed to inhibition of distinct steps in the arachidonic acid cascade, particularly, the cyclo-oxygenase pathway. Diclofenac sodium, a compound of this class of drugs, appears to have a dual effect since it also regulates the lipoxygenase pathway. Study of appropriate cell systems (leukocytes and whole blood in rats) demonstrates that diclofenac's potent inhibition of cyclo-oxygenase activity causes a sharp reduction in the formation of prostaglandin, prostacyclin, and thromboxane products, all key mediators of inflammation. Recent work discloses that at higher concentrations, diclofenac sodium also reduces the formation of products of the lipoxygenase pathway (5-hydroxyeicosatetraenoic acid, leukotrienes). The mechanism by which this evolves, however, appears to be unrelated to direct inhibition of lipoxygenase. Instead, by enhancing its reincorporation into triglycerides, diclofenac sodium reduces the intracellular level of free arachidonic acid.

The effects of diclofenac sodium on arachidonic acid metabolism.

Evidence has been presented that inhibition by diclofenac sodium of the production of leukotrienes by cells participating in the inflammatory process is due to a decreased availability of intracellular arachidonic acid which results from enhanced uptake of the substrate into triglyceride pools. The diminished leukotriene production does not result from direct inhibition of 5-lipoxygenase or phospholipase A2. Reduced availability of arachidonic acid would also limit production of prostaglandins, although in this case manifestation is obscured by the potent inhibitory effect of diclofenac sodium on cyclooxygenase. This recently discovered action of diclofenac sodium, which has been characterized by studies on isolated leukocytes, appears to be operative in vivo. Consistent with this mechanism, and not explainable by classical cyclooxygenase inhibition, diclofenac sodium inhibited leukotriene production in whole blood from drug-treated animals and also suppressed leukocyte infiltration of subcutaneously implanted sponges. The latter effect contrasts with increased infiltration frequently obtained with other NSAIDs and thought to reflect enhanced production of leukotrienes. In conclusion, the findings suggest that patient acceptance or preference for diclofenac sodium is not merely subjective but has a logical scientific basis.

Purification and properties of aortic cholesteryl ester hydrolase.

The enzyme(s) present in acetonedried powder of rat and rabbit aortas, which catalyzes the synthesis and hydrolysis of cholesteryl ester, was purified partially by acid precipitation, acetone fractionation, O-(diethylaminoethyl) cellulose chromatography, and Sephadex G-100 filtration. The synthetic activity was purified by 120-fold (rat) and 140-fold (rabbit). Purification of hydrolytic activity was 90-fold (rat) and 103-fold (rabbit). Cholesteryl ester hydrolase activity was separated from nonspecific esterase by column chromatography. Both synthetic and the hydrolytic activities are apparently the functions of one enzyme. The mol wt of the enzyme was estimated to be 140,000 dalton as determined by Sephadex G-200 gel filtration. The extracts of the acetone-dried powders of aortas of both species contained an inhibitor of synthetic activity. The inhibitor was nondialyzable and was precipitated at pH 5.7. Both activities were found to be fairly nonspecifc with regard to sterol and fatty acids. With oleic acid, the relative rates of sterol ester synthesis were: cholesterol, 100; cholestanol, 94; desmosterol, 35; coprostanol, 24; ergosterol, 20; and beta-sitosterol, 19. Epicholesterol was not esterified. Oleic acid was most active in cholesteryl ester synthesis, the relative rates being: oleic greater than linoleic greater than arachidonic greater than palmitic greater than stearic greater than butyric. The rate of hydrolysis was maximum with cholesteryl linoleate followed by oleate, linolenate, palmitate, stearate, and laurate in decreasing order.

Analysis of tissue factor expression in various cell model systems: cryptic vs. active.

BACKGROUND: Tissue factor (TF) encryption plays an important role in regulating TF coagulant activity. Potential differences in experimental cell model systems and strategies hampered our understanding of the TF encryption mechanisms. OBJECTIVE: To characterize the procoagulant activity status of TF in different cell types, and to determine whether increased TF procoagulant activity following the activation stems from transformation of the cryptic TF to the active form. METHODS: Simultaneous kinetic analyses of TF-FVIIa activation of FX and FVIIa binding to cell surface TF were performed under identical experimental conditions in fibroblast (WI-38), cancer cell (MDA-231), endothelial cell (HUVEC) and monocytic cell (THP-1) model systems. These data were then utilized to estimate TF coagulant-specific activity and percentages of active and cryptic TF present in these cell types. RESULTS: MDA-231 and WI-38 cells express 10 to 100 times more TF on their cell surfaces compared with perturbed HUVEC and THP-1 cells. TF-specific activity on cell surfaces of MDA-231, WI-38 and THP-1 cells was very similar. Nearly 80-90% of the TF in MDA-231, WI-38 and THP-1 cells was cryptic. A plasma concentration of FVII would be sufficient to bind both active and cryptic TF on cell surfaces. Increased TF activity following cell activation stems from decryption of cryptic TF rather than increasing the coagulant activity of the active TF. CONCLUSIONS: Our data demonstrate that TF encryption is not limited to a specific cell type, and unlike previously thought, the majority of the TF expressed in cancer cells is not constitutively procoagulant.

Glycosylation of tissue factor is not essential for its transport or functions.

BACKGROUND: Glycosylation plays an important role in protein function. The importance of glycosylation for tissue factor (TF) function is unclear. OBJECTIVE: The aim of the present study is to investigate the importance of TF glycosylation in transport to the cell surface and its coagulant and signaling functions. METHODS: Endothelial cells and peripheral blood mononuclear cells (PBMC) were treated with tunicamycin to inhibit N-linked glycosylation. Site-specific mutagenesis of one or more potential N-linked glycosylation sites in TF was used to generate TF mutants lacking glycans. TF expression at the cell surface was determined in binding assays using (125)I-FVIIa or (125)I-TF mAb and confocal microscopy. TF coagulant activity was measured by factor (F) Xa generation assay, and TF signaling function was assessed by measuring cleavage of protease activated receptor 2 (PAR2) and activation of p44/42 MAPK. RESULTS: Tunicamycin treatment reduced TF activity at the endothelial cell surface; however, this reduction was found to be the result of decreased TF protein production in tunicamycin-treated cells. Tunicamycin treatment had no significant effect on TF activity or antigen levels in PBMC. No significant differences were observed in TF protein expression and procoagulant activity among cells transfected to express either wild-type TF or TF mutants. A fully non-glycosylated TF is shown to bind FVIIa and interact with FX with the same efficiency as that of wild-type TF. Non-glycosylated TF is also capable of supporting FVIIa cleavage of PAR2 and PAR2-dependent p44/42 MAPK activation. CONCLUSIONS: Glycosylation is not essential for TF transport and coagulant or signaling functions.

Plasmin enhances cell surface tissue factor activity in mesothelial and endothelial cells.

BACKGROUND: Mesothelial cells that line the thoracic cavity play an important role in maintaining the local balance between procoagulant and fibrinolytic activity, a role akin to the endothelial cells in blood vessels. The mechanism(s) responsible for increased tissue factor (TF) expression in mesothelial cells in response to injury are at present unclear. OBJECTIVE: To investigate whether plasmin or thrombin, two major proteases that may be generated on the pleural surface upon injury, induce TF expression in human pleural mesothelial cells (HMC) and elucidate the underlying mechanism(s). METHODS: Confluent monolayers of HMC and human umbilical vein endothelial cells (HUVEC) were exposed to plasmin or thrombin for varying time periods and TF expression was analyzed by measuring its activity in a factor Xa generation assay, TF antigen levels by immunoblot analysis and TF mRNA by Northern blot analysis. RESULTS: Both plasmin and thrombin treatments increased cell surface TF activity in HMC by 3- to 4-fold. In contrast to thrombin, plasmin-induced TF activity is not dependent on the de novo synthesis of TF. In HUVEC, plasmin had a minimal effect on unperturbed HUVEC whereas it markedly increased TF activity of activated HUVEC. Plasmin treatment neither affected anionic phospholipid levels at the cell surface nor released protein disulfide isomerase, an oxidoreductase protein that was newly described to play a role in TF activation. Plasmin cleaved cell-associated TFPI. CONCLUSION: Thrombin up-regulates TF activity in HMC through the transcriptional activation of TF whereas plasmin increases TF activity by inactivating the cell-associated TFPI by a limited proteolysis.