Biomedical subjects

Mauro Perretti

Publications and source records attributed to Mauro Perretti.

At least 37 records · Page 2Linked to original sources

PubMed2005

Modulation of phagocytosis of apoptotic neutrophils by supernatant from dexamethasone-treated macrophages and annexin-derived peptide Ac(2-26).

Phagocytic clearance of apoptotic leukocytes plays an important role in the resolution of inflammation. The glucocorticoid-inducible protein annexin 1 and annexin 1-derived peptides show potent anti-inflammatory responses in acute and chronic inflammation. In this study, we report that the annexin 1-derived peptide (Ac(2-26)) significantly stimulates nonphlogistic phagocytosis of apoptotic polymorphonuclear leukocytes (PMNs) by human monocyte-derived macrophages (Mphi). Peptide Ac(2-26)-stimulated phagocytosis is accompanied by rearrangement of the Mphi actin cytoskeleton. To investigate the potential role of endogenous annexin on clearance of apoptotic cells, Mphi were cultured for 5 days in the presence of dexamethasone. Supernatants collected from dexamethasone-treated Mphi significantly enhanced the ability of naive Mphi to engulf apoptotic PMNs. This effect was blocked by an annexin blocking Ab, by immunodepletion of the supernatants, and by the formyl peptide receptor/lipoxin receptor antagonist Boc1. In addition, we show that bone marrow-derived Mphi from annexin 1-null mice present a 40% decreased phagocytosis of apoptotic PMNs compared with cells taken from littermate controls. In conclusion, these results emphasize the pivotal role of annexin 1 as mediator for clearance of apoptotic cells and expand its potential therapeutic role in controlling inflammatory diseases.

Actins↗

PubMed2005

Controlling inflammation: a fat chance?

The inflammatory response protects the body against infection and injury but can itself become deregulated with deleterious consequences to the host. It is now clear that several endogenous biochemical pathways activated during defense reactions can counterregulate inflammation. New experimental evidence adds resolvin E1 to this group of endogenous inhibitors and provides further rationale for the beneficial effects of dietary supplementation with fish oils. It also highlights an unexpected twist in the pharmacology of aspirin.

Animals↗

PubMed2005

Inhibitory control of TGF-beta1 on the activation of Rap1, CD11b, and transendothelial migration of leukocytes.

Beta2-integrins are a family of dimeric adhesion molecules expressed on leukocytes. Their capacity to bind ligand is regulated by their state of activation. CD11b, an alphaMbeta2 integrin, is implicated in a number of physiological and pathological events such as inflammation, thrombosis, or atherosclerosis. The GTPase Rap1 is essential for its activation and could therefore play a strategic role in the regulation of leukocyte functioning. Because low levels of circulating TGF-beta have been linked with severe atherosclerosis, we have assessed the role of this cytokine in the regulation of Rap1 and CD11b activation in differentiated U937 cells and in human peripheral blood monocytes. TGF-beta1 caused a significant reduction in the expression of CD11b but not in the expression of other integrins tested. More importantly, TGF-beta1 greatly reduced the capacity of PMA or chemokines to activate CD11b and Rap1, a phenomenon paralleled by a loss of the Epac transcript and a reduction in 8-pCPT-2'-O-Me-cAMP-mediated activation of Rap1. This inhibition diminished the capacity of monocytes to migrate across a monolayer of endothelial cells. The inhibitory effect of TGF-beta1 on Rap1 activity may exert a general protective influence against aberrant transendothelial migration, thereby holding inflammatory responses in check.

CD11b Antigen↗

PubMed2005

In vitro and in vivo induction of heme oxygenase 1 in mouse macrophages following melanocortin receptor activation.

RAW264.7 cell incubation with adrenocorticotrophin (ACTH) led to a time-dependent (4-24 h) and concentration-related (1-100 ng/ml) induction of heme oxygenase (HO)-1, and this was a specific effect, because the pattern of expression of other cellular proteins (HO-2, heat shock proteins 70 and 90) was not modified by ACTH. Combined RT-PCR and Western blot analyses revealed expression of the melanocortin receptor (MC-R) types 1 and 3, but not 4, in these cells. However, use of more selective agonists (including melanotan (MTII)) indicated a predominant role for MC3-R in the induction of HO-1 expression and activity. Relevantly, ACTH and MTII incubation with primary peritoneal macrophages (Mphi) also induced HO-1 expression. The potential link between MC3-R dependent cAMP formation and HO-1 induction was ascertained by the following: 1) ACTH and MTII produced a concentration-dependent accumulation of cAMP in RAW264.7 cells, and 2) whereas a selective inhibitor of cAMP-dependent protein kinase A abrogated ACTH- and MTII-induced HO-1 expression, a soluble cAMP derivative promoted HO-1 induction both in RAW264.7 cells and primary Mphi. HO-1 induction in peritoneal Mphi was also detected following in vivo administration of MTII, and appeared to be functionally related to the antimigratory effect of this melanocortin, as determined with a specific inhibitor (zinc protoporphyrin IX). In conclusion, this study highlights a biochemical link between MC-R activation and HO-1 induction in the Mphi, and proposes that this may be of functional relevance in determining MC-R-dependent control of the host inflammatory response.

Amino Acid Sequence↗

PubMed2005

Aspirin and steroids: new mechanistic findings and avenues for drug discovery.

The inflammatory response is a life-saving protective process mounted by the body to overcome pathogen infection and injury; however, in chronic inflammatory pathologies, this response can become deregulated. Aspirin and glucocorticoids are two examples of drugs developed over the years to rectify deregulated inflammatory reactions. Interestingly, both these prototypes of anti-inflammatory therapeutics have been 'borrowed' from Mother Nature, identified from the plant and animal world, respectively. In the past century, systematic organic chemistry has been the major approach for producing new drugs, and vast quantities of aspirin and prednisolone have been synthesized, packaged and sold. However, the fascination provoked by these often life-saving drugs has not subsided, and recent work into the endogenous control of the host inflammatory response has revitalized these compounds. Thus, epi-lipoxins, produced after aspirin acetylation of inducible cyclooxygenase-2, and glucocorticoid-regulated annexin 1 appear to be important endogenous mediators of their respective anti-inflammatory effects. In addition, aspirin-triggered epi-lipoxins and glucocorticoid-regulated annexin 1 might act on the same G-protein-coupled receptor, thus rendering this shared receptor a more likely and worthwhile target for fruitful drug discovery.

Anti-Inflammatory Agents↗

PubMed2005

Critical protective role for annexin 1 gene expression in the endotoxemic murine microcirculation.

The inflammatory response is a protective process of the body to counteract xenobiotic penetration and injury, although in disease this response can become deregulated. There are endogenous biochemical pathways that operate in the host to keep inflammation under control. Here we demonstrate that the counterregulator annexin 1 (AnxA1) is critical for controlling experimental endotoxemia. Lipopolysaccharide (LPS) markedly activated the AnxA1 gene in epithelial cells, neutrophils, and peritoneal, mesenteric, and alveolar macrophages--cell types known to function in experimental endotoxemia. Administration of LPS to AnxA1-deficient mice produced a toxic response characterized by organ injury and lethality within 48 hours, a phenotype rescued by exogenous application of low doses of the protein. In the absence of AnxA1, LPS generated a deregulated cellular and cytokine response with a marked degree of leukocyte adhesion in the microcirculation. Analysis of LPS receptor expression in AnxA1-null macrophages indicated an aberrant expression of Toll-like receptor 4. In conclusion, this study has detailed cellular and biochemical alterations associated with AnxA1 gene deletion and highlighted the impact of this protective circuit for the correct functioning of the homeostatic response to sublethal doses of LPS.

Animals↗

PubMed2005

Role of the immune system in chronic pain.

During the past two decades, an important focus of pain research has been the study of chronic pain mechanisms, particularly the processes that lead to the abnormal sensitivity - spontaneous pain and hyperalgesia - that is associated with these states. For some time it has been recognized that inflammatory mediators released from immune cells can contribute to these persistent pain states. However, it has only recently become clear that immune cell products might have a crucial role not just in inflammatory pain, but also in neuropathic pain caused by damage to peripheral nerves or to the CNS.

Animals↗

PubMed2004

Formyl-peptide receptor is not involved in the protection afforded by annexin 1 in murine acute myocardial infarct.

Recent interest in the annexin 1 field has come from the notion that specific G-protein-coupled receptors, members of the formyl-peptide receptor (FPR) family, appear to mediate the anti-inflammatory actions of this endogenous mediator. Administration of the annexin 1 N-terminal derived peptide Ac2-26 to mice after 25 min ischemia significantly attenuated the extent of acute myocardial injury as assessed 60 min postreperfusion. Evident at the dose of 1 mg/kg (approximately 9 nmol per animal), peptide Ac2-26 cardioprotection was intact in FPR null mice. Similarly, peptide Ac2-26 inhibition of specific markers of heart injury (specifically myeloperoxidase activity, CXC chemokine KC contents, and endogenous annexin 1 protein expression) was virtually identical in heart samples collected from wild-type and FPR null mice. Mouse myocardium expressed the mRNA for FPR and the structurally related lipoxin A4 receptor, termed ALX; thus, comparable equimolar doses of two ALX agonists (W peptide and a stable lipoxin A4 analog) exerted cardioprotection in wild-type and FPR null mice to an equal extent. Curiously, marked (>95%) blood neutropenia produced by an anti-mouse neutrophil serum did not modify the extent of acute heart injury, whereas it prevented the protection afforded by peptide Ac2-26. Thus, this study sheds light on the receptor mechanism(s) mediating annexin 1-induced cardioprotection and shows a pivotal role for ALX and circulating neutrophil, whereas it excludes any functional involvement of mouse FPR. These mechanistic data can help in developing novel therapeutics for acute cardioprotection.

Amino Acid Sequence↗

PubMed2004

Annexin-1 is an endogenous gastroprotective factor against indomethacin-induced damage.

Adherence of neutrophils to the vascular endothelium is an early and critical event in the pathogenesis of gastric injury induced by NSAIDs. Pretreatment with glucocorticoids has been shown to prevent NSAID-induced neutrophil adherence and, in turn, to protect the stomach from injury. Some of the anti-inflammatory effects of glucocorticoids, including inhibition of neutrophil adherence, are mediated via the release of annexin-1. In this study, we assessed the contribution of annexin-1 to the protective actions of a glucocorticoid (dexamethasone) against indomethacin-induced gastric damage. Dexamethasone pretreatment markedly reduced the extent of indomethacin-induced gastric damage in rats. Immunoneutralization of annexin-1 resulted in a reversal of the gastroprotective actions of dexamethasone. Similarly, pretreatment with either of two antagonists of the formyl peptide receptor family, to which annexin-1 binds, reversed the gastroprotective effects of dexamethasone. The inhibitory effects of dexamethasone on indomethacin-induced leukocyte adherence in the mesenteric microcirculation were abolished by pretreatment with an antibody directed against annexin-1 or with an antagonist of the formyl peptide receptors. These results demonstrate that annexin-1 mediates the gastroprotective effects of a glucocorticoid against NSAID-induced damage. We propose that in some circumstances, annexin-1 plays an important role as an endogenous mediator of mucosal defense.

Animals↗

PubMed2004

MC-3 receptor and the inflammatory mechanisms activated in acute myocardial infarct.

Investigation of the mechanisms activated by endogenous inhibitory pathways can lead to identification of novel targets for cardiovascular inflammatory pathologies. Here we exploited the potential protective role that melanocortin receptor type 3 (MC3-R) activation might play in a myocardial ischemia-reperfusion injury model. In resting conditions, mouse and rat heart extracts expressed MC3-R mRNA and protein, without changes following ischemia-reperfusion. At the cellular level heart macrophages, but not fibroblasts or cardiomyocytes, expressed this receptor, as demonstrated by immunogold labeling. In vivo, administration of the melanocortin agonist MTII (10 microg per mouse equivalent to 9.3 nmol) 30 min prior to ischemia (25 min) attenuated mouse heart 2 h reperfusion injury by approximately 40%, an effect prevented by the mixed MC3/4-R antagonist SHU9119 but not by the selective MC4-R antagonist HS204. Similar results were obtained when the compound was given at the beginning of the reperfusion period. Importantly, delayed myocardial damage as measured 24 h post-reperfusion was equally protected by administration of 10 microg MTII. The focus on MC3-R was also substantiated by analysis of the recessive yellow (e/e) mouse, bearing a mutated (inactive) MC1-R, in which MTII was fully protective. Myocardial protection was associated with reduced markers of systemic and local inflammation, including cytokine contents (interleukin-1 and KC) and myeloperoxidase activity. In conclusion, this study has highlighted a previously unrecognized protective role for MC3-R activation on acute and delayed heart reperfusion injury. These data may open new avenues for therapeutic intervention against heart and possibly other organ ischemia-reperfusion injury.

Acute Disease↗

PubMed2004

Stimulus-specific defect in the phagocytic pathways of annexin 1 null macrophages.

The role of the glucocorticoid-regulated protein annexin 1 during the process of phagocytosis has been studied using annexin 1 null peritoneal macrophages. Wild type and annexin 1 null macrophages were incubated with several distinct phagocytic targets. No differences were observed in rate or the maximal response with respect to IgG complexes or opsonised zymosan phagocytosis, as assessed by monitoring the production of reactive oxygen species. When annexin 1 null macrophages were incubated with non-opsonised zymosan particles, they exhibited impaired generation of reactive oxygen species, which was linked to a defect in binding of cells to the particles, as determined with fluorescent zymosan. This phenomenon was further confirmed by electron microscopy analysis, where annexin 1 null macrophages internalised fewer non-opsonised zymosan particles. Specific alterations in macrophage plasma membrane markers were observed in the annexin 1 null cells. Whereas no differences in dectin-1 and FcgammaR II/III expression were measured between the two genotypes, decreased membrane CD11b and F4/80 levels were measured selectively in macrophages lacking annexin 1. These cells also responded with an enhanced release of PGE(2) and COX-2 protein expression following addition of the soluble stimulants, LPS and heat-activated IgG. In conclusion, these results suggest that participation of endogenous annexin 1 during zymosan phagocytosis is critical and that this protein plays a tonic inhibitory role during macrophage activation.

Animals↗

PubMed2004

The distinct alterations produced in cardiovascular functions by prednisolone and nitro-prednisolone (NCX-1015) in the rat highlight a causal role for endothelin-1.

Daily administration of prednisolone, but not the derivative NCX-1015 (or prednisolone 21-[4'-nitrooxymethyl]benzoate), to rats resulted in a time- and dose-dependent increase in mean arterial blood pressure (MABP), significant after 1 week for the dose of 6.9 micromol/kg i.p. (n = 10; P < 0.05), and 3 weeks for the lower dose of 1.38 micromol/kg. A similar dichotomy of behavior was observed with respect to myocardial contractility and renal vascular resistance, in either case augmented by 3-week treatment with prednisolone but not NCX-1015. In contrast, both NCX-1015 and prednisolone reduced plasma levels of corticosterone in a dose- (dose range of 0.69-6.9 micromol/kg i.p.) and time-dependent (1-3 weeks) manner. Similar profiles were obtained for plasma nitrate values, although they were increased selectively after NCX-1015 administration. In contrast, prednisolone, but not NCX-1015, augmented plasma endothelin 1 (ET-1) with a profile that mirrored the changes observed in MABP and renal blood flow. Supply in the drinking water of the ET-1 receptor type A (ETA) antagonist FR139317 [(R-2-[(R)-2-[(S)-2-[[1-(hexahydro-1H-azepinyl)]-carbonyl]amino-4-methylpentanoyl]-amino-3-(2-pyridil)propionic] or mixed ETA/B, but not of selective ETB, antagonists prevented the changes produced by a 21-day treatment with prednisolone. In conclusion, this study indicates 1) a lack of occurrence of cardiovascular alterations by nitro-releasing derivative of prednisolone (NCX-1015), and 2) a functional link between prednisolone effects and the endogenous endothelin-1 system.

Animals↗

PubMed2004

Neutrophil elastase (NE)-deficient mice demonstrate a nonredundant role for NE in neutrophil migration, generation of proinflammatory mediators, and phagocytosis in response to zymosan particles in vivo.

Neutrophil elastase (NE) remains a controversial player in the process of leukocyte transmigration and much of this controversy stems from conflicting reports on the effects of NE inhibitors. The availability of NE-deficient mice (NE(-/-)) provides a clean and elegant tool for the study of leukocyte migration in vivo. In this study, NE(-/-) mice were used to investigate the role of NE in leukocyte migration through cremasteric venules, as observed by intravital microscopy, induced by locally administered cytokines IL-1beta and TNF-alpha and the particulate stimulus, zymosan. Although no defects in leukocyte responses induced by the cytokines were observed, zymosan-induced leukocyte firm adhesion and transmigration was suppressed in NE(-/-) mice. These responses were also inhibited in wild-type mice when zymosan was coinjected with a specific NE inhibitor. Quantification of inflammatory mediator levels in homogenates of zymosan-stimulated tissues indicated reductions in levels of IL-1beta, KC, and macrophage inflammatory protein-1alpha in NE(-/-) mice. Furthermore, phagocytosis of fluorescent zymosan particles, as observed by intravital microscopy, was diminished in NE-deficient animals. Collectively, the findings of this study indicate a nonredundant role for NE in zymosan-induced leukocyte firm adhesion and transmigration, and that this defect is associated with impaired generation of proinflammatory mediators as well as phagocytosis of zymosan particles in vivo.

Animals↗

PubMed2004

Annexin 1 and the biology of the neutrophil.

This overview will focus on one aspect of neutrophil biology, which is the selective activation of the annexin 1 system in relation to the process of cell extravasation. Besides the current view about the biochemistry of annexin 1 and annexin 1 receptor(s) up-regulation within the microenvironment of the adherent neutrophils, we will also comment on the final result achieved by activation of the system, which is inhibition of neutrophil recruitment. In view of the historical link between annexin 1 and glucocorticoids, the potential for the annexin 1 system in mediating at least some of the anti-inflammatory actions of these powerful drugs is also discussed.

Animals↗

PubMed2004

Synthesis of nitro esters of prednisolone, new compounds combining pharmacological properties of both glucocorticoids and nitric oxide.

Glucocorticoids (GC) are widely used in therapy for their many pharmacological properties including antiinflammatory and immunosuppressive actions. However, their use over long periods is hampered by a number of severe side effects. Given the biological properties of nitric oxide (NO) and previous experience with nonsteroidal antiinflammatory agents, we synthesized new chemical entities combining both NO and GC properties. Here we report the synthesis of nitro esters of prednisolone obtained through the esterification, with different linkers, on the hydroxy group at C-21 position of the corticosteroid structure. The alkyl chain, as of the nitrooxy derivative (2), or aromatic linkers, as of o-, m-, and p-nitrooxymethylbenzoate derivatives (3-5), respectively, furnish stable compounds that release NO and inhibit the GC receptors in biological assays. To improve solubility we introduced a more water-soluble linker such as the nitrooxyalkylpiperidine or -piperazine group (6-9). Also these compounds retained properties of both NO and prednisolone. Compound 5 (NCX 1015) was selected for its better profile: enhanced antiinflammatory properties and reduced side effects compared with prednisolone. NCX 1015 is currently under preclinical development.

Animals↗

PubMed2004

Antiinflammatory activity of soluble guanylate cyclase: cGMP-dependent down-regulation of P-selectin expression and leukocyte recruitment.

Nitric oxide (NO) production by the vascular endothelium maintains an essential antiinflammatory, cytoprotective influence on the blood vessel wall. A key component of this activity is attributed to prevention of leukocyte-endothelial cell interactions, yet the underlying mechanisms remain unclear. The NO receptor, soluble guanylate cyclase (sGC), is expressed in endothelial cells but fulfils an unknown function. Therefore, we used intravital microscopy in mesenteric postcapillary venules from WT and endothelial nitric oxide synthase (eNOS) knockout (eNOS(-/-)) mice, and an sGC activator (BAY 41-2272), to investigate a potential role for sGC in the regulation of adhesion molecule expression and leukocyte recruitment. Leukocyte rolling and adhesion was 6-fold greater in eNOS(-/-) than WT animals. BAY 41-2272 and the NO-donor, diethylamine-NONOate, reduced leukocyte rolling and adhesion in eNOS(-/-) mice to levels observed in WT animals. These effects were blocked by the sGC inhibitor ODQ [1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one], which itself caused a 6-fold increase in leukocyte rolling and adhesion in WT mice. Increased leukocyte rolling and adhesion in IL-1beta-treated mice was also inhibited by BAY 41-2272. Fluorescence-activated cell sorting analysis in vitro and a specific P-selectin neutralizing antibody in vivo revealed that selective down-regulation of P-selectin expression accounted for the antiadhesive effects of sGC activation. These data demonstrate that sGC plays a key antiinflammatory role by inhibiting P-selectin expression and leukocyte recruitment.

Animals↗

PubMed2004

Modulation of inflammation and response to dexamethasone by Annexin 1 in antigen-induced arthritis.

OBJECTIVE: Annexin 1 (Anx-1) is a putative mediator of the antiinflammatory actions of glucocorticoids (GCs). This study investigated the role of Anx-1 in experimental arthritis and in GC-mediated inhibition of inflammation, using antigen-induced arthritis (AIA) in Anx-1 knockout (Anx-1(-/-)) mice. METHODS: Arthritis was induced by intraarticular injection of methylated BSA (mBSA) in mice preimmunized with mBSA. Disease was assessed after 7 days by histologic examination of the knee joints. Serum levels of anti-mBSA IgG were determined by enzyme-linked immunosorbent assay. Cytokine messenger RNA (mRNA) expression was detected by real-time polymerase chain reaction. RESULTS: A significant exacerbation of arthritis was observed in the Anx-1(-/-) mice compared with wild-type (WT) mice. This was associated with increased mRNA expression of synovial interleukin-1 beta, tumor necrosis factor alpha, interleukin-6, and macrophage migration inhibitory factor. Dexamethasone significantly reduced the histologic severity of synovitis and bone damage in the WT mice, but exerted no inhibitory effects in the Anx-1(-/-) mice, and also significantly reduced the serum levels of anti-mBSA IgG and the numbers of peripheral blood neutrophils and lymphocytes in WT mice, but had no such effect in Anx-1(-/-) mice. CONCLUSION: Anx-1 exerts endogenous antiinflammatory effects on AIA via the regulation of cytokine gene expression, and also mediates the antiinflammatory actions of dexamethasone in AIA.

Animals↗

PubMed2004

An isocratic HPLC method for the quantitation of eicosanoids in human platelets.

We describe here a modified protocol for the simultaneous quantification of specific eicosanoids formed during stimulation of human platelets in vitro with adenosine diphosphate. The eicosanoids thromboxane B(2) (TXB(2)), arachidonic acid (AA), 12-R-hydroxyeicosatetraenoic acid (12-R-HETE), 12-S-hydroxyheptadecatrienoic acid (12-S-HHTrE) and the internal standard prostaglandin B(1) (PGB(1)) were extracted from human platelets by liquid-liquid extraction using ethyl acetate. This was followed by derivatization and fluorescent detection prior to analysis by reversed phase liquid chromatography. The high-performance liquid chromatographic method consisted of ODS reversed-phase column (3 microm) and a mobile phase of acetonitrile-water (85:15). TXB(2) and AA plasma calibration curves were linear between 6.25 and 125 ng mL(-1) (r(2) > 0.997), whereas for 12-R-HETE and 12-S-HHTrE the curves were linear between 5.0 and 40 ng mL(-1) (r(2) > 0.998). All calibration curve standards had <15% CV (coefficient of variation) and between-run precision, and the percentage relative deviation for replicate (n = 6) quality controls was less than 5.5%. The method was adapted to allow the screening of drugs that may affect either one or both of the lipoxygenase and cyclo-oxygenase pathways.

Calibration↗