Carola Buccellati

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.

The Cycloxygenase-2 inhibitor SC58236 is neuroprotective in an in vivo model of focal ischemia in the rat.

Focal ischemia was induced in the fronto-parietal region of rat brain, by injection of Rose Bengal, followed by light activation. Focal ischemia was accompanied by formation of PGD(2) peaking 60-90 min post irradiation and declining thereafter. Increased Cycloxygenase-2 (COX-2) expression was also observed. Control ischemic rats showed distinct morphological alterations with necrosis of neurons, glial cells and blood vessels, surrounded by a halo with pyknotic cells with cytoplasm swelling and vacuolization. Compound SC58236, a selective COX-2 inhibitor, dose-dependently prevented, ischemia-induced eicosanoid formation (area under the curve (AUC) of controls: 3.11 +/- 0.87; AUC of 20 mg/kg SC58236: 0.39 +/- 0.24), and caused significant reduction of damaged area (30.7 and 18.9% at SC58236 20 and 6.6 mg/kg), suggesting that selective inhibitors of COX-2 are neuroprotective.

Cysteinyl-leukotrienes receptor activation in brain inflammatory reactions and cerebral edema formation: a role for transcellular biosynthesis of cysteinyl-leukotrienes

We studied the effect of intravascular activation of human neutrophils on the synthesis of cysteinyl leukotrienes (cysLT) and the formation of cerebral edema in guinea‐pig brains. Challenge with the chemotactic formylated tripeptide fMLP (0.1 µM) of neutrophil‐perfused brain in vitro resulted in blood‐brain barrier disruption associated with a significant increase of cysLT. Both events were completely prevented by neutrophil pretreatment with a specific 5‐lipoxygenase (5‐LO) inhibitor. Perfusion with the 5‐LO metabolite leukotriene B4 (10 nM), together with neutrophils treated with the 5‐LO inhibitor, did not restore the alteration in permeability observed upon perfusion with untreated and activated neutrophils. The dual cysLT1‐cysLT2 receptor antagonist BAYu9773 was more potent and more effective than a selective cysLT1 antagonist in preventing the brain permeability alteration induced by neutrophil activation. RT‐PCR showed significant expression of cysLT2 receptor mRNA in human umbilical vein endothelial cells. Intravital microscopy in mice showed that inhibition of leukotriene synthesis significantly reduced firm adhesion of neutrophils to cerebral vessels without affecting rolling. These data support the hypothesis that neutrophil and endothelial cells cooperate toward the local synthesis of cysLT within the brain vasculature and, acting via the cysLT2 receptor on endothelial cells, may represent a contributing pathogenic mechanism in the development of cerebral inflammation and edema.

Formation of sulphidopeptide-leukotrienes by cell-cell interaction causes coronary vasoconstriction in isolated, cell-perfused heart of rabbit

1 We have studied the transcellular biosynthesis of bioactive leukotrienes (LTs), generated upon blood cell‐vascular wall interactions and their functional consequences, in the spontaneously beating, cell‐perfused, heart of the rabbit. Rabbit isolated hearts were perfused under recirculating conditions (50 ml) with 5 × 106 cells of unpurified (buffy coat) or purified human neutrophils (PMNL), and challenged with 0.5 μm A23187 for 30 min. Coronary perfusion pressure (CPP), heart rate (HR), left ventricular end‐diastolic pressure (LVEDP) and left ventricular pressure (LVP) were monitored continuously. Leukotriene formation was measured by specific enzyme‐immunoassay and confirmed by reversed phase h.p.l.c. and u.v. spectral analysis. 2 Basal CPP values averaged 44 ± 1.4 mmHg; A23187 triggered a marked increase in CPP both in the presence of buffy coat cells (+ 100% above basal) and PMNL (+ 270% above basal); the latter change in CPP was accompanied by a rise in LVEDP (+ 138% above basal). 3 The increase in CPP was preceded by a statistically significant rise in iLTC4‐D4 concentration in the circulating buffer. Pretreatment with two structurally unrelated LTD4 receptor antagonists, LY171883 and SKF104353 (10 μm), fully prevented the increase in CPP and LVEDP. A similar protection was also observed when the rabbit heart was perfused with PMNL that had been pretreated with MK886 (1 μm), a potent inhibitor of leukotriene biosynthesis. 4 The increased coronary tone was accompanied by a marked release of lactate dehydrogenase (LDH), a marker of ischaemic damage; pretreatment of the heart with the LTD4 receptor antagonists as well as of the PMNL with MK886 resulted in a complete suppression of LDH activity release. 5 Positive identification of LTC4‐D4 in the perfusates was obtained and a significant correlation observed between the CPP values and iLTC4‐D4 concentrations. 6 This study suggests that challenge of PMNL present within the coronary vasculature, causes a LTD4‐dependent coronary vasoconstriction, favoured by an efficient uptake of PMNL‐derived LTA4 by endothelial cells. The activation of the 5‐lipoxygenase pathway in the context of tight interactions between blood cells and coronary vasculature, is suggested to have an important outcome in the alterations of coronary flow and cardiac contractility.

Inhibition of prostanoid synthesis protects against neuronal damage induced by focal ischemia in rat brain

Changes in prostanoids concentration and effects of the non-specific COX inhibitor indomethacin on prostanoids levels and extension of tissue damage were studied following focal ischemia induction in the fronto-parietal region of rat brain. Ischemia was induced in animals bearing a transcerebral microdialysis probe by injection of Rose Bengal, a photosensitive dye, followed by light activation. Prostanoid levels were determined in the dialysate using immunoenzymatic techniques. PGD2 levels rose significantly up to 237+/-22 pg/ml compared to a basal level measured before ischemia induction which was below the detection limit. TXB2 changes were smaller and had a different time course. Treatment with indomethacin abolished the ischemia-induced PGD2 release and reduced the extent of injury to the area by 43+/-3.7%. These results suggest that prostanoid release may play an important role in neurodegenerative processes and that cyclooxygenase inhibitors may contribute to protect against cerebral tissue damage.

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.

Critical role of COX-1 in prostacyclin production by human endothelial cells under modification of hydroperoxide tone

We aimed at evaluating the relative contribution of cyclooxygenase (COX) ‐1 and COX‐2 to the synthesis of prostacyclin in endothelial cells under static conditions in the presence or absence of exogenous arachidonic acid and/or altered intracellular redox balance. Selective inhibitors of either COX‐1 (SC560 and FR122047) or COX‐2 (SC236) concentration dependently (1–300 nM) reduced basal and interleukin (IL) ‐1β‐induced prostacyclin production in human umbilical vein endothelial cells by 70% or more; compound selectivity was confirmed using a whole‐blood assay (IC50 COX‐1/COX‐2: 13 nM/930 nM for SC‐560; 9 μM/457 nM for SC‐236). The observed concomitant formation of isoprostane appeared to be associated with COX enzyme activity, while formation of COX‐1/COX‐2 heterodimers was detected by immunoprecipitation. In the presence of arachidonic acid and 12‐hydroperoxy‐eicosatetraenoic acid, either exogenous or provided by platelet activation, or after glutathione depletion, COX‐1 inhibition but not COX‐2 inhibition concentration dependently decreased prostacyclin production. Both isoforms appear to contribute to basal prostacyclin production by endothelial cells, with COX‐2 providing the hydroperoxide tone required for COX‐1 activity. Conversely, in the case of intracellular glutathione depletion or enhanced availability of arachidonic acid and hydroperoxides, selective COX‐2 inhibition did not significantly affect the production of endothelial prostacyclin. These findings contribute to a better understanding of the effects of cyclooxygenase inhibitors on prostacyclin production.—Bolego, C, Buccellati, C, Prada, A., Gaion, R. M., Folco, G., Sala, A. Critical role of COX‐1 in prostacyclin production by human endothelial cells under modification of hydroperoxide tone. FASEB J. 23, 605–612 (2009)

Tramadol anti‐inflammatory activity is not related to a direct inhibitory action on prostaglandin endoperoxide synthases

The analgesic drug tramadol has been shown to relieve pain in inflammatory conditions, to inhibit the development of experimental inflammation, and to reduce prostaglandin (PG)E2concentrations in the inflammatory exudate. In this study, we evaluated the putative activity of tramadol to suppress prostaglandin endoperoxide synthase‐1 (PGHS‐1), and prostaglandin endoperoxide synthase‐2 (PGHS‐2) activities in human whole blood in vitro. Platelet thromboxane (Tx)B2production and monocyte PGE2production in LPS‐ stimulated blood were measured in samples incubated with different concentrations (300 ng/ml, 3 μ g/ml, 30 μ g/ml) of tramadol or its enantiomers. Neither tramadol nor the enantiomers inhibited the formation of arachidonic acid metabolites. Our results indicate that the anti‐inflammatory effect of tramadol demonstrated in some models is not related to a direct inhibitory effect on the formation of prostanoids.

Antagonism of thromboxane receptors by diclofenac and lumiracoxib.

Non‐steroidal anti‐inflammatory drugs (NSAIDs) are analgesic and anti‐inflammatory by virtue of inhibition of the cyclooxygenase (COX) reaction that initiates biosynthesis of prostaglandins. Findings in a pulmonary pharmacology project gave rise to the hypothesis that certain members of the NSAID class might also be antagonists of the thromboxane (TP) receptor.

CHF6001 I: a novel highly potent and selective phosphodiesterase 4 inhibitor with robust anti-inflammatory activity and suitable for topical pulmonary administration.

This study examined the pharmacologic characterization of CHF6001 [(S)-3,5-dichloro-4-(2-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-2-(3-(cyclopropylmethoxy)-4-(methylsulfonamido)benzoyloxy)ethyl)pyridine 1-oxide], a novel phosphodiesterase (PDE)4 inhibitor designed for treating pulmonary inflammatory diseases via inhaled administration. CHF6001 was 7- and 923-fold more potent than roflumilast and cilomilast, respectively, in inhibiting PDE4 enzymatic activity (IC50 = 0.026 ± 0.006 nM). CHF6001 inhibited PDE4 isoforms A–D with equal potency, showed an elevated ratio of high-affinity rolipram binding site versus low-affinity rolipram binding site (i.e., >40) and displayed >20,000-fold selectivity versus PDE4 compared with a panel of PDEs. CHF6001 effectively inhibited (subnanomolar IC50 values) the release of tumor necrosis factor-α from human peripheral blood mononuclear cells, human acute monocytic leukemia cell line macrophages (THP-1), and rodent macrophages (RAW264.7 and NR8383). Moreover, CHF6001 potently inhibited the activation of oxidative burst in neutrophils and eosinophils, neutrophil chemotaxis, and the release of interferon-γ from CD4+ T cells. In all these functional assays, CHF6001 was more potent than previously described PDE4 inhibitors, including roflumilast, UK-500,001 [2-(3,4-difluorophenoxy)-5-fluoro-N-((1S,4S)-4-(2-hydroxy-5-methylbenzamido)cyclohexyl)nicotinamide], and cilomilast, and it was comparable to GSK256066 [6-((3-(dimethylcarbamoyl)phenyl)sulfonyl)-4-((3-methoxyphenyl)amino)-8-methylquinoline-3-carboxamide]. When administered intratracheally to rats as a micronized dry powder, CHF6001 inhibited liposaccharide-induced pulmonary neutrophilia (ED50 = 0.205 μmol/kg) and leukocyte infiltration (ED50 = 0.188 μmol/kg) with an efficacy comparable to a high dose of budesonide (1 μmol/kg i.p.). In sum, CHF6001 has the potential to be an effective topical treatment of conditions associated with pulmonary inflammation, including asthma and chronic obstructive pulmonary disease.