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Biomedical subjects

G Folco

Publications and source records attributed to G Folco.

At least 19 recordsLinked to original sources
PubMed2000

Monoclonal anti-CD18 antibody prevents transcellular biosynthesis of cysteinyl leukotrienes in vitro and in vivo and protects against leukotriene-dependent increase in coronary vascular resistance and myocardial stiffness.

BACKGROUND: Cysteinyl leukotrienes (cys-LT) can constrict small and large vessels and increase vascular permeability. Formation of cys-LT arising from polymorphonuclear leukocytes (PMNL) and endothelial cell cooperation (transcellular synthesis) led to the hypothesis that PMNL-endothelial cell adhesion may represent a key step toward the formation of vasoactive cys-LT. METHODS AND RESULTS: We studied the effect of pretreatment with a monoclonal antibody directed against the CD18 subunit of PMNL beta(2)-integrin on the synthesis of cys-LT in a PMNL-perfused isolated rabbit heart in vitro and in a model of permanent ligature of the left descending coronary artery in the rabbit in vivo. Challenge of PMNL-perfused rabbit hearts with formyl-met-leu-phe (0.3 micromol/L) caused synthesis of cys-LT and increase in coronary perfusion pressure that were prevented by the anti-CD18 antibody. Similar results were obtained with the use of A-23187 (0.5 micromol/L) as a challenge. Persistence of PMNL-associated myeloperoxidase activity in the perfusion buffer was observed in the presence of the anti-CD18 antibody, indicating decreased PMNL infiltration. Coronary artery ligature in vivo increased urinary excretion of leukotriene E(4), supporting the activation of the 5-lipoxygenase pathway during experimental acute myocardial infarction. Pretreatment with the anti-CD18 antibody (1 mg/kg) prevented the increase in leukotriene E(4) excretion. CONCLUSIONS: These data support the importance of adhesion in promoting cys-LT formation, originating from PMNL-endothelial cell cooperation, and contributing to myocardial stiffness and increased coronary resistance.

Animals↗
PubMed1999

Differential metabolism of exogenous and endogenous arachidonic acid in human neutrophils.

Leukotrienes can be produced by cooperative interactions between cells in which, for example, arachidonate derived from one cell is oxidized to leukotriene A(4) (LTA(4)) by another and this can then be exported for conversion to LTB(4) or cysteinyl leukotrienes (cys-LTs) by yet another. Neutrophils do not contain LTC(4) synthase but are known to cooperate with endothelial cells or platelets (which do have this enzyme) to generate cys-LTs. Stimulation of human neutrophils perfusing isolated rabbit hearts resulted in production of cys-LTs, whereas these were not seen with perfused hearts alone or isolated neutrophils. In addition, the stimulated, neutrophil-perfused hearts generated much greater amounts of total LTA(4) products, suggesting that the hearts were supplying arachidonate to the neutrophils and, in addition, that this externally derived arachidonate was preferentially used for exported LTA(4) that could be metabolized to cys-LTs by the coronary endothelium. Stable isotope-labeled arachidonate and electrospray tandem mass spectrometry were used to differentially follow metabolism of exogenous and endogenous arachidonate. Isolated, adherent neutrophils at low concentrations (to minimize transcellular metabolism between them) were shown to generate higher proportions of nonenzymatic LTA(4) products from exogenous arachidonate (deuterium-labeled) than from endogenous (unlabeled) sources. The endogenous arachidonate, on the other hand, was preferentially used for conversion to LTB(4) by the LTA(4) hydrolase. This result was not because of saturation of the LTA(4) hydrolase, because it occurred at widely differing concentrations of exogenous arachidonate. Finally, in the presence of platelets (which contain LTC(4) synthase), the LTA(4) synthesized from exogenous deuterium-labeled arachidonate was converted to cys-LTs to a greater degree than that from endogenous sources. These experiments suggest that exogenous arachidonate is preferentially converted to LTA(4) for export (not intracellular conversion) and raises the likelihood that there are different intracellular pathways for arachidonate metabolism.

Animals↗
PubMed1999

Interleukin-4 enhances 15-lipoxygenase activity and incorporation of 15(S)-HETE into cellular phospholipids in cultured pulmonary epithelial cells.

15(S)-Hydroxyeicosatetraenoic acid (15[S]-HETE) is a 15-lipoxygenase (15-LO) metabolite that may play an important role in different pulmonary diseases. 15-HETE is synthesized by different epithelial cells and may be subsequently incorporated into cellular phospholipids. We studied the role of interleukin-4 (IL-4) on 15-LO activity and on 15(S)-HETE incorporation into cellular phospholipids by WI-26 pulmonary epithelial cells. 15-LO activity was evaluated by measuring 15(S)-HETE production, through combined reverse-phase-high-pressure liquid chromatography (RP-HPLC) separation and specific radioimmunoassay (RIA), after incubation with arachidonic acid (AA). We also studied 15-LO messenger RNA (mRNA) expression, using primed in situ (PRINS) labeling. IL-4 (10 ng/ml) markedly increased the percentage of 15-LO mRNA-bearing cells as well as 15-LO activity after 24, 48, and 72 h, with a maximal response at 48 h. Uptake and incorporation into cellular phospholipid was studied with [3H]15(S)-HETE, which showed that IL-4 was able to increase significantly 15(S)-HETE incorporation into WI-26 cells, with a maximal effect observed at 72 h. Cellular-lipid-associated [3H]15(S)-HETE, evaluated with RP-HPLC after base-catalyzed hydrolysis, increased concomitantly with disappearance of the radiolabel from the supernatant. Class separation of cellular lipids with normal-phase HPLC (NP-HPLC) showed that IL-4 increased [3H]15(S)- HETE incorporation mainly in the phosphatidylinositol (PI) fraction. The ability of IL-4 to promote 15-LO activity and incorporation into cellular phospholipids of human lung epithelial cells may be important in airway inflammation and in modulation of the potential autocrine function of 15(S)-HETE.

Arachidonate 15-Lipoxygenase↗
PubMed1998

Delapril slows the progression of atherosclerosis and maintains endothelial function in cholesterol-fed rabbits.

The renin-angiotensin system is an important modulator of arterial blood pressure and inhibitors of the angiotensin-converting enzyme (ACE-Is) and are currently used in the treatment of hypertension. The pleiotropic actions exerted by angiotensin II (AngII) on the functionality of the vessel wall may have pro-atherosclerotic outcomes; evidence for an anti-atherosclerotic effect of ACE-Is has been presented and an antioxidant effect has been attributed to thiol-containing ACE-Is, like Captopril. The present study has been undertaken to investigate the effect of Delapril, a lipophilic ACE-I, on the development of atherosclerosis in cholesterol-fed rabbits. While it did not correct hyperlipidemia, Delapril dose dependently inhibited the development of atherosclerosis, expressed as aortic area covered by lesions (23.3+/-4.1, 21.3+/-2.4 and 18.5+/-3.3% with Delapril at the daily dose of 5, 10 and 20 mg/kg, respectively, versus 38.2%+/-6.4 for control animals) and its effect was similar to that of Captopril (14.5+/-5.1% at the daily dose of 25 mg/kg). Furthermore, Delapril partially and dose dependently restored endothelium-dependent relaxation, which is impaired in vessels from hypercholesterolemic animals (51.80+/-12.18, 59.74+/-5.16, 69.13+/-8.70 maximal percent relaxation versus 48.26+/-3.05% for the untreated control and 67.67+/-6.72% for Captopril-treated animals). An antioxidant mechanism is unlikely to explain this data, since Delapril does not contain thiol groups. These observations suggest that Delapril may represent an effective pharmacological approach for the treatment of atherosclerosis during its early phases.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1998

Simultaneous investigation of the neuronal and vascular compartments in the guinea pig brain isolated in vitro.

We describe a new method for studying the interactions between vascular tone changes and neuronal activity in the arterially perfused isolated brain of the adult guinea pig maintained in vitro. Electrophysiological recordings were performed in the piriform and entorhinal cortices with the entire arterial bed preserved or after vascular restriction to the territories of median and posterior cerebral arteries of one hemisphere. The changes in vascular tone were measured by means of a pressure transducer. The arterial pressure was 53.77+/-12.74 mmHg in control conditions at 30 degreesC. Intraluminal application of vasoactive drugs, such as the tromboxane A2 receptor agonist U46619 (0.1 microM) and 5-HT (3 microM), induced an increase in the resistance to perfusion pressure that was prevented by the selective antagonists. The preservation of the endothelial function was verified by inducing the release of endogenous endothelial relaxant factor after intraluminal application of 1 microM acetylcholine. The study of the reciprocal interactions between neuronal activity and vascular tone modifications demonstrated that evoked responses in the piriform and entorhinal cortices were not modulated by rapid changes of the vascular tone. A sustained and elevated plateau of vasoconstriction maintained for several minutes determined a cortical spreading depression. Epileptiform discharges induced in limbic cortices by GABAa receptor blockade were consistently associated with a vasodilation (8.26+/-2.8 mmHg). The results demonstrate that the in vitro isolated guinea pig brain preparation can be exploited for studying simultaneously neuronal activity and cerebrovascular motility.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1997

14,15-Dehydroleukotriene A4: a specific substrate for leukotriene C4 synthase.

We studied the metabolism of 14,15-dehydro-leukotriene A4 (14, 15-dehydro-LTA4) by human platelet leukotriene C4 (LTC4) synthase and polymorphonuclear leucocyte (PMNL) leukotriene A4 (LTA4) hydrolase. Metabolites were separated and identified using reversed-phase HPLC coupled to diode-array UV detection. Human platelets metabolize 14,15-dehydro-LTA4 to 14,15-dehydro-LTC4 with apparent kinetics identical with authentic LTA4. Metabolism to 14, 15-dehydro-LTC4 is inhibited by MK-886, a reported LTC4 synthase inhibitor in human platelets, with a potency comparable with that shown by LTA4. In contrast, neither human red-blood-cell lysates nor human PMNL enzymically convert 14,15-dehydro-LTA4 into 14, 15-dehydro-leukotriene B4. Minor amounts of 14,15-dehydro-LTC4, observed in some PMNL preparations, result from variable eosinophil contamination, as confirmed using highly purified neutrophil and eosinophil-enriched preparations. In addition, 14,15-dehydro-LTA4 irreversibly inhibits PMNL LTA4 hydrolase with an IC50 of 0.73 microM. The geometry of the methyl terminus of LTA4 does not influence the metabolism by human platelet LTC4 synthase. The double bond at C-14,15 is essential for the catalytic activity of LTA4 hydrolase but not for binding to this enzyme.

Binding Sites↗
PubMed1997

Comparative analysis of isolated human bronchi contraction and biosynthesis of cysteinyl leukotrienes using a direct 5-lipoxygenase inhibitor.

Quantitation of cysteinyl leukotriene production and smooth muscle contraction upon immunological challenge of isolated human bronchi was evaluated. Analysis of picomole amounts of leukotriene C4, D4, and E4 was achieved using HPLC separation and enzyme immunoassay quantitative determination. The aim of the study was to correlate the contraction of airway smooth muscle and cysteinyl leukotriene production with and without 5-lipoxygenase inhibition. In human isolated bronchial tissue treated with indomethacin and pyrilamine to make their contractile responses leukotriene dependent only, the novel 5-lipoxygenase inhibitor 5,6-Dihydroxy-2-(N,N-Dimethylhydrazino)-1,2,3,4-tetrahydro-naph talene bromide (CHF 1909) caused a concentration-dependent inhibition of the immunologically induced contraction, showing an IC50 value of 13 +/- 2.2 microM (mean +/- CV). At the concentration of 30 microM, this compound caused more than 90% inhibition of the maximal bronchoconstriction in vitro, and inhibited cysteinyl leukotriene production by 90% as well. Contemporary measurement of immunologically induced contraction and production of cysteinyl leukotrienes in isolated human bronchi provided a direct correlation between smooth muscle contraction and synthesis of leukotriene C4, D4, and E4.

Bronchi↗
PubMed1997

Pharmacological modulation of human platelet leukotriene C4-synthase.

The aim of this study was to test if human platelet leukotriene C4-synthase (LTC4-S) is pharmacologically different from cloned and expressed LTC4-S and, in light of the significant homologies between 5-lipoxygenase activating protein (FLAP) and LTC4-S, if different potencies of leukotriene synthesis inhibitors acting through binding with FLAP (FLAP inhibitors) reflect in different potencies as LTC4-S inhibitors. Leukotriene C4 (LTC4) synthesis by washed human platelets supplemented with synthetic leukotriene A4 (LTA4) was studied in the absence and presence of two different, structurally unrelated FLAP inhibitors (MK-886 and BAY-X1005) as well as a direct 5-lipoxygenase inhibitor (zileuton). LTC4 production was analyzed by RP-HPLC coupled to diode array detection. We report that human platelet LTC4-S was inhibited by MK-886 and BAY-X1005 (IC50 of 4.7 microM and 91.2 microM, respectively), but not by zileuton (inactive up to 300 microM); all 3 compounds were able to inhibit 5-lipoxygenase metabolite biosynthesis in intact human polymorphonuclear leukocytes (IC50 of 0.044 microM, 0.85 microM, and 1.5 microM, respectively). Platelet LTC4-S does not appear pharmacologically different from expression cloned LTC4-S. LTC4-S inhibition by FLAP inhibitors is in agreement with the significant homology reported for expression-cloned LTC4-S with FLAP, Furthermore, functional homology of the binding sites for inhibitors on LTC4-S and FLAP is suggested by the conservation of the relative potencies of MK-886 and BAY-X1005 vs FLAP-dependent 5-lipoxygenase activity and LTC4-S inhibition: MK-886 was 19.3-fold more potent than BAY-X1005 as FLAP inhibitor and 19.6-fold more potent than BAY-X1005 as LTC4-S inhibitor.

Blood Platelets↗
PubMed1997

Nitric oxide modulation of transcellular biosynthesis of cys-leukotrienes in rabbit leukocyte-perfused heart.

1. We have studied the role of nitric oxide (NO) in the regulation of the transcellular biosynthesis of sulphidopeptide leukotrienes (cys-LT) generated upon neutrophil-vascular wall interactions and their functional consequences, in the spontaneously beating, cell-perfused, heart of the rabbit. 2. Hearts were perfused under recirculating conditions (50 ml) with 5 x 10(6) purified human neutrophils (PMNL), and challenged with 0.5 microM A-23187 for 30 min. Coronary perfusion pressure (CPP) and left-ventricular end-diastolic pressure (LVEDP) were monitored. Cys-LT formation was measured by reversed phase high performance liquid chromatography (h.p.l.c.) and u.v. spectral analysis. Myeloperoxidase (MPO) enzyme activity, assayed in aliquots of the recirculating buffer, was used as a marker of PMNL, adhesion to the coronary endothelium. 3. Basal CPP and LVEDP values averaged 45 +/- 1.4 mmHg and 5 +/- 0.1 mmHg, respectively; A-23187 triggered an increase in CPP (134 +/- 9 mmHg, at 30 min) which was significantly attenuated by pretreatment with L-arginine, 100 microM (90 +/- 3 mmHg, at 30 min). Pretreatment with NG-monomethyl-L-arginine, 10 microM (L-NMMA), induced a marked increase in CPP (290 +/- 40 mmHg, at 20 min) and in LVEDP (47 +/- 16 mmHg), so pronounced that it caused cardiac arrest in systole in 5 out of 6 hearts and these were prevented by L-arginine, 100 microM, (CPP 115 +/- 10 mmHg, LVEDP 6 +/- 1.1 mmHg, at 30 min). 4. The increase in CPP was accompanied by the release of cys-LT in the circulating buffer, which was reduced significantly by L-arginine. Pretreatment with L-NMMA, caused a marked rise in cys-LT concentrations which was prevented by L-arginine. 5. Neither L-arginine nor L-NMMA affected directly the A-23187-induced arachidonic acid (AA) metabolism in isolated PMNL alone. 6. Pretreatment with L-NMMA caused a prompt drop in myeloperoxidase (MPO), activity, suggesting rapid adhesion of PMNL to the coronary wall; this effect was significantly blunted by L-arginine. 7. This study suggests that NO provides cardioprotection in an organ model of transcellular metabolism of cys-LT by preventing PMNL adhesion to the coronary intima.

Animals↗
PubMed1997

Release of prostaglandin E2 and leukotriene B4 by alveolar macrophages from patients with sarcoidosis.

BACKGROUND: Mediators released by alveolar macrophages, as well as by T cells, play an important part in modulating local immune processes in sarcoidosis. Among alveolar macrophage secretory products, arachidonic acid metabolites are known to regulate inflammatory and immune reactions. It has been suggested that cyclo-oxygenase and lipoxygenase pathway metabolites of arachidonic acid modulate the evolution of the granulomatous inflammatory response in the lung differently. METHODS: Alveolar macrophages recovered from the bronchoalveolar lavage (BAL) fluid of 32 patients with sarcoidosis in different states of disease activity and 10 normal subjects were evaluated for their ability to release prostaglandin E2 (PGE2) and leukotriene B4 (LTB4). Alveolar macrophages were cultured in the presence or absence of opsonised zymosan (500 micrograms/ml), and PGE2 and LTB4 levels in the culture supernatants were determined by enzyme immunoassay (EIA). RESULTS: Stimulated alveolar macrophages from patients with active sarcoidosis released higher LTB4 levels than those from normal subjects, but no differences in PGE2 release were observed between the two groups. The time course of LTB4 release by activated alveolar macrophages showed that normal cells produced similar levels of the hydroxyacid during the early and late times of culture while LTB4 release by activated cells from patients with sarcoidosis increased markedly after 60 minutes of culture, remaining elevated until 24 hours. Indomethacin (3 x 10(6) M) caused the expected inhibition of PGE2 formation without affecting LTB4 release. CONCLUSIONS: These results suggest that alveolar macrophages from the BAL fluid of patients with active sarcoidosis are primed to release LTB4, which may contribute to the locally heightened immune response.

Adult↗
PubMed1997

Cyclooxygenase-2 and synthesis of PGE2 in human bronchial smooth-muscle cells.

The purpose of this study was to determine the mechanism of enhanced prostaglandin synthesis in cultured human bronchial smooth-muscle cells challenged with interleukin-1 beta (IL-1 beta). Cells were incubated with IL-1 beta (10 to 50 U/ml) for 0 to 24 h. Prostaglandin E2 (PGE2) production was evaluated through the conversion of exogenous (14C)-arachidonic acid and specific enzyme immunoassay of endogenous products. IL-1 beta enhanced PGE2 formation in a concentration- and time-dependent manner, reaching its peak at 6 to 8 h and fading at 18 to 24 h. Immunoblot analysis showed that the inducible cyclooxygenase enzyme (COX-2) was expressed only in IL-1 beta treated cells, whereas the constitutive isoform of cyclooxygenase (COX-1) remained unaltered. COX-2 expression and PGE2 formation were inhibited by dexamethasone (2 microM), cycloheximide (10 microM), and IL-1-receptor antagonist (IL-1 ra) (250 ng/ml), independently. PGE2 synthesis was significantly reduced by compound SC-58125, a specific COX-2 inhibitor. The close parallelism between the kinetics of COX-2 protein expression and PGE2 accumulation, as well as the constitutive nature of COX-1 isoform, indicate that IL-1 beta-driven PGE2 formation in human bronchial smooth-muscle cells is mediated by de novo expression of COX-2 enzyme.

Arachidonic Acid↗
PubMed1997

Transcellular metabolism of leukotriene A4 by rabbit blood cells: lack of relevant LTC4-synthase activity in rabbit platelets.

The objective of this study was to determine the transcellular metabolism of leukotriene A4 by rabbit blood cells. mixed peripheral blood leukocyte preparations with and without platelets in a ratio of 1:40 were challenged with the Ca(2+)-ionophore A23187. 5-Lipoxygenase metabolites production was assessed by RP-HPLC coupled with diode-array UV detection. In light of the observation that leukotriene C4 production in leukocyte-platelet coincubation was the same as with leukocytes alone, mixed coincubation of human and rabbit blood cells was tested. Rabbit leukocytes with human platelets resulted in a significant increase of leukotriene C4 production, while no changes were observed in human leukocytes with or without rabbit platelets. In agreement with these results, intact rabbit platelets or rabbit platelet lysates, unlike human platelets, were not able to convert synthetic leukotriene A4 free acid to leukotriene C4. These data provide evidence that rabbit leukocytes are able to make a significant amount of leukotriene A4 available for transcellular metabolism, while rabbit platelets, unlike human platelets, lack leukotriene C4-synthase activity.

Animals↗
PubMed1996

Release of leukotriene A4 versus leukotriene B4 from human polymorphonuclear leukocytes.

The reactive intermediate formed by 5-lipoxygenase metabolism of arachidonic acid, leukotriene A4, is known to be released from cells and subsequently taken up by other cells for biochemical processing. The objective of this study was to determine the relative amount of leukotriene A4 synthesized by human polymorphonuclear leukocytes (PMNL) that is available for transcellular biosynthetic processes. This was accomplished by diluting cell suspensions and measuring the relative amounts of enzymatic versus nonenzymatic leukotriene A4-derived metabolites after challenge with the Ca2+ ionophore A23187. Nonenzymatic leukotriene A4-derived metabolites were used as a quantitative index of the amount of leukotriene A4 released into the extracellular milieu. The results obtained demonstrated that in human PMNL, the relative amounts of nonenzymatic versus enzymatic leukotriene A4-derived metabolites increased with decreasing cell concentrations. After a 20-fold dilution of PMNL in cell preparations, a doubling in the amount of nonenzymatic leukotriene A4-derived metabolites was observed following challenge (from 53.9 +/- 1.3 to 110.4 +/- 8.9 pmol/10(6) PMNL, p < 0.01). Reduction of possible cell-cell interactions by dilution suggested that over 50% of leukotriene A4 synthesized is released from the PMNL. These data provide evidence that, in human PMNL preparations, transfer of leukotriene A4 to neighboring PMNL is taking place, resulting in additional formation of leukotriene B4 and its omega-oxidized metabolites 20-hydroxy- and 20-carboxy-leukotriene B4. Neutrophil reuptake of extracellular leukotriene A4 leads to an underestimation of the fraction of leukotriene A4 that is in fact available for transcellular metabolism when tight cell-cell interactions occur, such as during PMNL adhesion to the microvascular endothelium and diapedesis.

Biological Transport↗