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Biomedical subjects

Adriano G Rossi

Publications and source records attributed to Adriano G Rossi.

At least 19 recordsLinked to original sources
PubMed2006

A novel electron paramagnetic resonance-based assay for prostaglandin H synthase-1 activity.

BACKGROUND: Prostaglandin H2 synthase (PGHS) is the enzyme that catalyses the two-stage conversion of arachidonic acid to prostaglandin H2 (PGH2) prior to formation of prostanoids that are important in inflammation. PGHS isozymes (-1 and -2) are the target for nonsteroidal anti-inflammatory drugs (NSAIDs). Given the rekindled interest in specific anti-inflammatory PGHS inhibitors with reduced unwanted side effects, it is of paramount importance that there are reliable and efficient techniques to test new inhibitors. Here, we describe a novel in vitro electron paramagnetic resonance (EPR)-based assay for measuring the activity of PGHS-1. METHODS: We validated a novel in vitro PGHS-1 activity assay based on the oxidation of spin-trap agent, 1-hydroxy-3-carboxy-pyrrolidine (CPH) to 3-carboxy-proxy (CP) under the action of the peroxidase element of PGHS-1. This quantifiable spin-adduct, CP, yields a characteristic 3-line electron paramagnetic (EPR) spectrum. RESULTS: The assay is simple, reproducible and facilitates rapid screening of inhibitors of PGHS-1. Aspirin (100 microM, 1 mM) caused significant inhibition of spin-adduct formation (72 +/- 11 and 100 +/- 16% inhibition of control respectively; P < 0.05). Indomethacin (100 microM) also abolished the signal (114 +/- 10% inhibition of control; P < 0.01). SA and the PGHS-2-selective inhibitor, NS398, failed to significantly inhibit spin-adduct generation (P > 0.05). CONCLUSION: We have demonstrated and validated a simple, reproducible, quick and specific assay for detecting PGHS-1 activity and inhibition. The EPR-based assay described represents a novel approach to measuring PGHS activity and provides a viable and competitive alternative to existing assays.

Journal Article↗
PubMed2006

Cyclin-dependent kinase inhibitors enhance the resolution of inflammation by promoting inflammatory cell apoptosis.

Apoptosis is essential for clearance of potentially injurious inflammatory cells and subsequent efficient resolution of inflammation. Here we report that human neutrophils contain functionally active cyclin-dependent kinases (CDKs), and that structurally diverse CDK inhibitors induce caspase-dependent apoptosis and override powerful anti-apoptosis signals from survival factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF). We show that the CDK inhibitor R-roscovitine (Seliciclib or CYC202) markedly enhances resolution of established neutrophil-dependent inflammation in carrageenan-elicited acute pleurisy, bleomycin-induced lung injury, and passively induced arthritis in mice. In the pleurisy model, the caspase inhibitor zVAD-fmk prevents R-roscovitine-enhanced resolution of inflammation, indicating that this CDK inhibitor augments inflammatory cell apoptosis. We also provide evidence that R-roscovitine promotes apoptosis by reducing concentrations of the anti-apoptotic protein Mcl-1. Thus, CDK inhibitors enhance the resolution of established inflammation by promoting apoptosis of inflammatory cells, thereby demonstrating a hitherto unrecognized potential for the treatment of inflammatory disorders.

Amino Acid Chloromethyl Ketones↗
PubMed2006

NADPH oxidase-derived ROS: key modulators of heme-induced mitochondrial stability in human neutrophils.

Heme is a proinflammatory molecule able to cause a profound delay of constitutive apoptosis of human neutrophils, an effect that likely contributes to chronic inflammation associated with hemolytic diseases. Herein we show that heme-induced delay of neutrophil apoptosis correlates with the prevention of mitochondrial potential (Deltapsi(m)) dissipation by a mechanism dependent on NADPH oxidase (NADPHox)-generated reactive oxygen species (ROS) and NF-kappaB. Deltapsi(m) maintenance is accompanied by inhibition of Bax insertion into mitochondria and by a decrease in the Bad/Bcl-X(L) ratio. Heme induces Bad degradation in a completely ROS-dependent manner, as well as Bcl-X(L) synthesis, a phenomenon that also requires NF-kappaB activation. These data indicate that heme-induced preservation of mitochondrial integrity is a critical checkpoint controlled by NADPH oxidase generated-ROS and redox-sensitive NF-kappaB activation.

Apoptosis↗
PubMed2006

Mechanism of action of novel NO-releasing furoxan derivatives of aspirin in human platelets.

Incorporation of a nitric oxide (NO)-releasing moiety in aspirin can overcome its gastric side effects. We investigated the NO-release patterns and antiplatelet effects of novel furoxan derivatives of aspirin (B8 and B7) in comparison to existing antiplatelet agents. Cyclooxygenase (COX) activity was investigated in purified enzyme using an electron paramagnetic resonance-based technique. Concentration-response curves for antiplatelet agents +/- the soluble guanylate cyclase inhibitor, ODQ (50 microM) were generated in platelet-rich plasma (PRP) and washed platelets (WP) activated with collagen using turbidometric aggregometry. NO was detected using an isolated NO electrode. The furoxan derivatives of aspirin (B8, B7) and their NO-free furazan equivalents (B16, B15; all 100 microM) significantly inhibited COX activity (P < 0.01; n = 6) in vitro and caused aspirin-independent, cGMP-dependent inhibition of collagen-induced platelet aggregation in WP. B8 was more potent than B7 (PRP IC(50) = 0.62 +/- 0.1 microM for B8; 400 +/- 89 microM for B7; P < 0.0001. WP IC(50)s = 0.6 +/- 0.1 and 62 +/- 10 microM, respectively). The NO-free furazan counterparts were less potent antiplatelet agents (WP IC(50)s = 54 +/- 3 microM and 62 +/- 10 microM, respectively; P < 0.0001, B8 vs B16). Of the hybrids investigated, only B8 retained antiplatelet activity in PRP.NO release from furoxan-aspirin hybrids was undetectable in buffer alone, but was accelerated in the presence of either plasma or plasma components, albumin (4%), glutathione (GSH; 3 microM) and ascorbate (50 microM), the effects of which were additive for B7 but not B8. NO generation from furoxans was greatly enhanced by platelet extract, an effect that could largely be explained by the synergistic effect of intracellular concentrations of GSH (3 mM) and ascorbate (1 mM). We conclude that the decomposition of furoxan-aspirin hybrids to generate biologically active NO is catalysed by endogenous agents which may instil a potential for primarily intracellular delivery of NO. The blunting of the aspirin effects of furoxan hybrids is likely to be due to loss of the acetyl moiety in plasma; the observed antiplatelet effects are thereby primarily mediated via NO release. Compounds of this class might represent a novel means of inhibiting platelet aggregation by a combination of NO generation and COX inhibition.

Adult↗
PubMed2006

E-selectin permits communication between PAF receptors and TRPC channels in human neutrophils.

The selectin family of molecules (L-, P-, and E-selectin) mediates adhesive interactions between leukocytes and endothelial cells required for recruitment of leukocytes to inflammatory sites. Soluble E-selectin levels are elevated in inflammatory diseases and act to promote neutrophil beta(2)-integrin-mediated adhesion by prolonging Ca(2+) mobilization. Although soluble E-selectin alone was unable to initiate Ca(2+) signaling, it allowed a novel "permissive" store-operative calcium entry (SOCE) following the initial platelet-activating factor (PAF)-induced release of Ca(2+) from inositol 1,4,5-trisphosphate (IP(3))-sensitive stores. This induction of permissive SOCE in response to soluble E-selectin and PAF was shown to act through a G protein-coupled receptor (GPCR) coupled to pertussis toxin-insensitive G(q/11). Furthermore, we demonstrated that permissive SOCE was mediated by canonical transient receptor potential channel (TRPC) due to its sensitivity to specific inhibition by MRS1845 and Gd(3+) and that TRPC6 was the principal TRPC family member expressed by human neutrophils. In terms of mechanism, we demonstrated that soluble E-selectin activated Src family tyrosine kinases, an effect that was upstream of phosphatidylinositol 3'-kinase in a signaling pathway that regulates permissive SOCE following exposure of neutrophils to PAF. In summary, this report provides the first evidence for communication between an inflammatory mediator and adhesion receptors at a molecular level, through selectin receptor ligation allowing permissive SOCE to occur following PAF stimulation of human neutrophils.

CD18 Antigens↗
PubMed2006

NO-releasing zeolites and their antithrombotic properties.

Transition metal-exchanged zeolite-A adsorbs and stores nitric oxide in relatively high capacity (up to 1 mmol of NO/g of zeolite). The stored NO is released on contact with an aqueous environment under biologically relevant conditions of temperature and pH. The release of the NO can be tuned by altering the chemical composition of the zeolite, by controlling the amount of water contacting the zeolite, and by blending the zeolite with different polymers. The high capacity of zeolite for NO makes it extremely attractive for use in biological and medical applications, and our experiments indicate that the NO released from Co-exchanged zeolite-A inhibits platelet aggregation and adhesion of human platelets in vitro.

Fibrinolytic Agents↗
PubMed2006

Therapeutic effects of nitric oxide-aspirin hybrid drugs.

This review examines the therapeutic potential and mechanisms of action of drugs known as nitric oxide (NO)-aspirins. Drugs of this class have an NO-releasing moiety joined by ester linkage to the aspirin molecule. NO-aspirins have the capability to release NO in addition to retaining the cyclooxygenase-inhibitory action of aspirin. The protective nature of NO led to the development of NO-aspirins in the hope that they might avoid the gastric side effects associated with aspirin. However, it has become apparent that the drug-derived NO instills potential for a wide range of added beneficial effects over the parent compound. In this review, the authors focus on the analgesic, anti-inflammatory, cardiovascular and chemopreventative actions of compounds of this emerging drug class.

Analgesics↗
PubMed2006

The involvement of the apoptosis-modulating proteins ERK 1/2, Bcl-xL and Bax in the resolution of acute inflammation in vivo.

Inflammatory cell recruitment, activation, and apoptosis are highly regulated processes involving several checkpoints controlling the resolution of inflammation. We investigated the role of the mitogen-activated protein kinase (MAPK) signaling pathway (ie, ERK1/2) and apoptosis-regulating Bcl-2 family members (ie, Bcl-x(L) and Bax) in the resolution of a rat carrageenan-induced pleurisy model. The specific ERK1/2 inhibitor PD98059 enhanced the resolution of inflammation, whereas the MEK1/2 inhibitor U0126 had no effect and the flavonoid apigenin, a nonspecific inhibitor of ERK1/2 and COX-2, augmented inflammation. Specifically, PD98059 significantly decreased the total number of macrophages and neutrophils in the pleural cavity, mainly by increasing the rate of neutrophil apoptosis, as measured by Annexin V labeling and morphological analysis. Conversely, a specific inhibitor of proapoptotic Bax (V5) increased inflammation, indicating that by preventing apoptosis in vivo, resolution of inflammation is delayed. This was associated with a decrease in neutrophil apoptosis and an increase in macrophage and neutrophil numbers perpetuating the inflammatory response. In conclusion, this study shows that ERK1/2, Bax, and Bcl-x(L) play important functional roles in the resolution phase of the acute inflammatory response in vivo by influencing apoptosis. Importantly, these data may provide novel therapeutic targets for the treatment of inflammatory diseases.

Animals↗
PubMed2005

Nitric oxide and the resolution of inflammation: implications for atherosclerosis.

The ubiquitous free radical, nitric oxide (NO), plays an important role in many biological processes including the regulation of the inflammatory response. Alterations in NO synthesis by endogenous systems likely influence inflammatory processes occurring in a wide range of diseases including many in the cardiovascular system (e.g. atherosclerosis). Progression of inflammatory conditions depends not only upon the recruitment and activation of inflammatory cells but also upon their subsequent removal from the inflammatory milieu. Apoptosis, or programmed cell death, is a fundamental process regulating inflammatory cell survival and is critically involved in ensuring the successful resolution of an inflammatory response. Apoptosis results in shutdown of secretory pathways and renders effete, but potentially highly histotoxic, cells instantly recognisable for non-inflammatory clearance by phagocytes (e.g., macrophages). However, dysregulation of apoptosis and phagocytic clearance mechanisms can have drastic consequences for development and resolution of inflammatory processes. In this review we highlight the complexities of NO-mediated regulation of inflammatory cell apoptosis and clearance by phagocytes and discuss the molecular mechanisms controlling these NO mediated effects. We believe that manipulation of pathways involving NO may have previously unrecognised therapeutic potential for limiting or resolving inflammatory and cardiovascular disease.

Apoptosis↗
PubMed2005

Inhibition of NF-kappa B by a cell permeable form of I kappa B alpha induces apoptosis in eosinophils.

An 11 amino acid HIV-TAT peptide can deliver target proteins into a variety of cells in a receptor-independent manner. To generate a highly specific inhibitor of the transcription factor NF-kappa B, we have fused the TAT-peptide to a version of I kappa B alpha that is resistant to signal-induced degradation. TAT-I kappa B alpha(S32A, S36A) inhibited NF-kappa B-dependent transcription in HeLa and A549 cells by retaining NF-kappa B p65 in the cytoplasm. Introduction of TAT-I kappa B alpha(S32A, S36A) into human eosinophils inhibited the nuclear translocation of NF-kappa B and induced apoptosis. Thus, continuous NF-kappa B-dependent transcription is important for eosinophil survival. While eosinophils are normally refractive to standard methods of gene delivery, the ability of TAT fusion proteins to be taken up by these cells should enable a detailed molecular analysis of survival pathways in these cells.

Apoptosis↗
PubMed2005

Interleukin-10 inhibits lipopolysaccharide-induced survival and extracellular signal-regulated kinase activation in human neutrophils.

Lipopolysaccharide (LPS) induces a marked delay in human neutrophil apoptosis that is reversed by the anti-inflammatory cytokine IL-10. The effect of IL-10 is specific since other agents that delay neutrophil apoptosis are not affected. To investigate mechanisms underlying the actions of IL-10, we examined signaling pathways activated by LPS per se and in response to IL-10. The MAPK kinase (MEK) 1 inhibitor PD098059, the protein kinase C (PKC) inhibitor Ro31,8220, and the phosphatidylinositol-3 kinase (PI3-K) inhibitor LY294002 all partially reversed LPS-mediated retardation of neutrophil apoptosis, but the p38 MAPK inhibitor SB203850 did not. LPS activates the transcription factor NF-kappaB, however, IL-10 did not affect the ability of LPS to activate NF-kappaB as assessed by IkappaB-alpha proteolysis. Although IL-10 did not alter activation of ERK by GM-CSF or TNF-alpha, it did inhibit activation induced by LPS. Thus our data illustrate that LPS-induced neutrophil survival is regulated by the MAPK, PKC and PI3-K pathways as well as NF-kappaB, and can be reversed by IL-10, through a mechanism involving inhibition of ERK activation. Because of the specific nature of this inhibition, we conclude that IL-10 interferes with an ERK activation pathway, which is not involved in GM-CSF or TNF-alpha signaling.

Apoptosis↗
PubMed2005

A potential role for extracellular nitric oxide generation in cGMP-independent inhibition of human platelet aggregation: biochemical and pharmacological considerations.

1. Nitric oxide (NO) is a potent inhibitor of platelet activation, that inhibits the agonist-induced increase in cytosolic Ca2+ concentration through both cGMP-dependent and independent pathways. However, the NO-related (NOx) species responsible for cGMP-independent signalling in platelets is unclear. We tested the hypothesis that extracellular NO, but not NO+ or peroxynitrite, generated in the extracellular compartment is responsible for cGMP-independent inhibition of platelet activation via inhibition of Ca2+ signalling. 2. Concentration-response curves for diethylamine diazeniumdiolate (DEA/NO; a spontaneous NO generator), S-nitroso-N-valerylpenicillamine (SNVP; an S-nitrosothiol) and 3-morpholinosydnonomine (SIN-1; a peroxynitrite generator) were generated in platelet-rich plasma (PRP) and washed platelets (WP) in the presence and absence of a supramaximal concentration of the soluble guanylate cyclase inhibitor, ODQ (20 microM). All three NOx donors displayed cGMP-independent inhibition of platelet aggregation in PRP, but only DEA/NO exhibited cGMP-independent inhibition of aggregation in WP. 3. Analysis of NO generation using an isolated NO-electrode revealed that cGMP-independent effects coincided with the generation of substantial levels of extracellular NO (>40 nM) from the NOx donors. 4. Reconstitution of WP with plasma factors indicated that the copper-containing plasma protein, caeruloplasmin (CP), catalysed the release of NO from SNVP, while Cu/Zn superoxide dismutase (SOD) unmasked NO generated from SIN-1. The increased generation of extracellular NO correlated with a switch to cGMP-independent effects with both NOx donors. 5. Analysis of Fura-2 loaded WP revealed that only DEA/NO inhibited Ca2+ signalling in platelets via a cGMP-independent mechanism. However, preincubation of SNVP and SIN-1 with CP and SOD, respectively, induced cGMP-independent inhibition of intraplatelet Ca2+ trafficking by the NOx donors. 6. Taken together, our data suggest that extracellular NO (>40 nM) is required for cGMP-independent inhibition of platelet activation. Plasma constituents may play an important pharmacological role in activating cGMP-independent signalling by S-nitrosothiols or peroxynitrite generators.

Adult↗
PubMed2005

Phosphoinositide-3 kinases critically regulate the recruitment and survival of eosinophils in vivo: importance for the resolution of allergic inflammation.

The phosphatidylinositol-3 kinase (PI3K) family of signaling enzymes plays a crucial role in leukocyte recruitment and activation and hence, likely regulates the induction and propagation phases of inflammation. However, little data have emerged showing a role for these processes in the resolution phase in models of in vivo inflammation. Here, we have evaluated the role of PI3K for the migration and survival of eosinophils in a model of allergic pleurisy in mice. Eosinophil accumulation in PI3Kgamma-deficient mice was inhibited at 48 h, as compared with wild-type mice but not at earlier time-points (6 and 24 h). Experiments with adoptive transfer of bone marrow showed that PI3Kgamma in eosinophils but not in non-bone marrow-derived cells was required for their accumulation. Systemic treatment with PI3K inhibitors before antigen challenge prevented the recruitment of eosinophils. This was associated with decreased Akt phosphorylation, interleukin-5 production, and eosinophil release from the bone marrow. Treatment with PI3K inhibitors 24 h after antigen challenge markedly cleared the accumulated eosinophils, an effect associated with inhibition of Akt phosphorylation and an increased number of apoptotic events. Altogether, our data demonstrate an important role of PI3Kgamma for the maintenance of eosinophilic inflammation in vivo, whereas other isoforms of PI3K may be relevant for the recruitment process.

Androstadienes↗
PubMed2005

Regulation of neutrophil apoptosis and removal of apoptotic cells.

The accumulation of neutrophils during inflammation is essential for the destruction and removal of invading microorganisms. However, for resolution of inflammation to occur, neutrophils must also be removed from the inflammatory site since these cells are capable of releasing tissue toxic molecules. Neutrophil removal has been shown to occur via apoptosis and phagocyte clearance of apoptotic cells. Therefore, manipulation of these processes is likely to be a key therapeutic strategy in the management of inflammatory disease. In this review, we examine mediators of neutrophil survival and apoptosis and the signalling pathways that regulate the balance between life and death in these cells.

Animals↗
PubMed2004

GEA 3162 decomposes to co-generate nitric oxide and superoxide and induces apoptosis in human neutrophils via a peroxynitrite-dependent mechanism.

1. GEA 3162 (1,2,3,4,-oxatriazolium, 5-amino-3-(3,4-dichlorophenyl)-chloride), has powerful effects on neutrophil function and apoptosis, but the underlying mechanisms are unclear, particularly with respect to the possible roles of nitric oxide (NO) and/or peroxynitrite (ONOO(-)). 2. Our hypothesis was that GEA 3162 is a generator of ONOO(-) and that its biological effects on neutrophil apoptosis differ from those of a conventional NO donor. The effects of GEA 3162 were compared to those of the established ONOO(-) donor, 3-morpholinosydnonimine (SIN-1), and the NO donor, diethylamine diazeniumdiolate (DEA/NO) in neutrophils from healthy volunteers. Electrochemical detection and electron paramagnetic resonance were used to define the NO-related species generated from these agents. 3. GEA 3162 and SIN-1 influence neutrophil apoptosis differently from DEA/NO. All three compounds induced morphological neutrophil apoptosis. However, both GEA 3162 and SIN-1 paradoxically inhibited internucleosomal DNA fragmentation, whereas DEA/NO induced fragmentation compared to control. 4. In contrast to DEA/NO, generation of free NO was not detectable in solutions of GEA 3162 or SIN-1 (100 microm). However, Cu/Zn superoxide dismutase (SOD; 50-750 U ml(-1)) unmasked NO generated from these compounds in a concentration-dependent manner. GEA 3162 and SIN-1 oxidised the O(2)(-)- and ONOO(-)-sensitive dye, dihydrorhodamine 123 (DHR 123; 1 microm), suggesting that ONOO(-) released from these compounds is responsible for oxidation of DHR 123. 5. We conclude that GEA 3162 is an ONOO(-) donor with pro-apoptotic properties that more closely resemble SIN-1 than the NO donor, DEA/NO. Moreover, unlike NO, ONOO(-) induces apoptosis in neutrophils via a mechanism that does not require DNA fragmentation.

Apoptosis↗
PubMed2004

Heme inhibits human neutrophil apoptosis: involvement of phosphoinositide 3-kinase, MAPK, and NF-kappaB.

High levels of free heme are found in pathological states of increased hemolysis, such as sickle cell disease, malaria, and ischemia reperfusion. The hemolytic events are often associated with an inflammatory response that usually turns into chronic inflammation. We recently reported that heme is a proinflammatory molecule, able to induce neutrophil migration, reactive oxygen species generation, and IL-8 expression. In this study, we show that heme (1-50 microM) delays human neutrophil spontaneous apoptosis in vitro. This effect requires heme oxygenase activity, and depends on reactive oxygen species production and on de novo protein synthesis. Inhibition of ERK and PI3K pathways abolished heme-protective effects upon human neutrophils, suggesting the involvement of the Ras/Raf/MAPK and PI3K pathway on this effect. Confirming the involvement of these pathways in the modulation of the antiapoptotic effect, heme induces Akt phosphorylation and ERK-2 nuclear translocation in neutrophils. Futhermore, inhibition of NF-kappa B translocation reversed heme antiapoptotic effect. NF-kappa B (p65 subunit) nuclear translocation and I kappa B degradation were also observed in heme-treated cells, indicating that free heme may regulate neutrophil life span modulating signaling pathways involved in cell survival. Our data suggest that free heme associated with hemolytic episodes might play an important role in the development of chronic inflammation by interfering with the longevity of neutrophils.

Active Transport, Cell Nucleus↗
PubMed2004

Macrophage phagocytosis of apoptotic neutrophils is compromised by matrix proteins modified by cigarette smoke and lipid peroxidation products.

Clearance of apoptotic cells by phagocytosis plays an important role in the resolution of an inflammatory response. Macrophages interacting with extracellular matrix (ECM) proteins upregulate their phagocytic capacity. Cigarette smoke contains highly reactive carbonyls that modify proteins which directly/indirectly affects cellular function. We observed, in vitro, that human macrophages interacting with carbonyl or cigarette smoke modified ECM proteins dramatically down regulated their ability to phagocytose apoptotic neutrophils. We also show that this interaction with carbonyl-adduct modified ECM proteins led to increased macrophage adhesion in vitro. We hypothesise that changes in the ECM environment as a result of cigarette smoking affect the ability of macrophages to remove apoptotic cells. Moreover, we postulate that this decreased phagocytic activity was as a result of sequestration of receptors involved in the uptake of apoptotic cells towards that of recognition of carbonyl adducts on the modified ECM proteins leading to increased macrophage adhesion.

Aldehydes↗