Biomedical subjects

Christina Barja-Fidalgo

Publications and source records attributed to Christina Barja-Fidalgo.

16 recordsLinked to original sources

PubMed2007

Aspirin-triggered lipoxin A4 blocks reactive oxygen species generation in endothelial cells: a novel antioxidative mechanism.

Lipoxins and their aspirin-triggered carbon-15 epimers have emerged as mediators of key events in endogenous anti-inflammation and resolution. However, the implication of these novel lipid mediators on cardiovascular diseases such as hypertension, atherosclerosis, and heart failure has not been investigated. One of the major features shared by these pathological conditions is the increased production of reactive oxygen species (ROS) generated by vascular NAD(P)H oxidase activation. In this study, we have examined whether an aspirin-triggered lipoxin A (4) analog (ATL-1) modulates ROS generation in endothelial cells (EC). Pre-treatment of EC with ATL-1 (1 - 100 nM) completely blocked ROS production triggered by different agents, as assessed by dihydrorhodamine 123 and hydroethidine. Furthermore, ATL-1 inhibited the phosphorylation and translocation of the cytosplamic NAD(P)H oxidase subunit p47 (phox) to the cell membrane as well as NAD(P)H oxidase activity. Western blot and immunofluorescence microscopy analyses showed that ATL-1 (100 nM) impaired the redox-sensitive activation of the transcriptional factor NF- kappaB, a critical step in several events associated to vascular pathologies. These results demonstrate that ATL-1 suppresses NAD(P)H oxidase-mediated ROS generation in EC, strongly indicating that lipoxins may play a protective role against the development and progression of cardiovascular diseases.

Antioxidants↗

PubMed2006

Activation of human T lymphocytes via integrin signaling induced by RGD-disintegrins.

Adhesive interactions play important roles in coordinating T cell migration and activation, which are mediated by binding of integrins to RGD motif found on extracellular matrix proteins. Disintegrins, isolated from snake venoms, contain the RGD sequence that confers selectivity to integrin interaction. We have investigated the ability of three RGD-disintegrins, ligands of alpha(5)beta(1) and alpha(v)beta(3), Flavoridin (Fl), Kistrin (Kr) and Echistatin (Ech), in modulating the activation of human T lymphocyte. The disintegrins induced T cell proliferation and CD69 expression. This activation parallels with actin cytoskeleton reorganization and tyrosine phosphorylation. Furthermore, the peptides induced focal adhesion kinase (FAK) and phosphoinositide 3-kinase (PI3K) activation. Finally, RGD-disintegrins were capable of driving NF-kappaB nuclear translocation and c-Fos expression, in a PI3K and ERK1/2 activities dependent manner. This report is the first to show that RGD-disintegrins interact with integrins on human T lymphocyte surface, modulating cell proliferation and activation of specific pathways coupled to integrin receptor.

Actins↗

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

Impaired neutrophil chemotaxis in sepsis associates with GRK expression and inhibition of actin assembly and tyrosine phosphorylation.

The deregulation of inflammatory response during sepsis seems to reflect the overproduction of mediators, which suppress leukocyte functions. We investigated the intracellular mechanisms underlying the inability of neutrophils from severe septic patients to migrate toward chemoattractants. Patients with sepsis (52) and 15 volunteers were prospectively enrolled. Patients presented increased circulating levels of tumor necrosis factor-alpha, interferon-gamma, interleukin (IL)-8, and IL-10. Patients showed reduced neutrophil chemotaxis to formyl-methionyl-leucyl-phenylalanine (FMLP), leukotriene B4 (LTB4) or IL-8. No difference in the transcription or expression of the IL-8 receptor, CXCR1, was detected in neutrophils from controls and patients. However, septic neutrophils failed to increase tyrosine phosphorylation and actin polymerization in response to IL-8 or LTB4. In contrast, septic neutrophils, similar to controls, showed phagocytic activity that induced actin polymerization and augmented phosphotyrosine content. Treatment of control neutrophils with cytokines and lipopolysaccharide (LPS) to mimic endogenous septic environment inhibited actin polymerization and tyrosine phosphorylation in response to IL-8 or LTB4. High expression of G protein-coupled receptor kinase 2 (GRK2) and GRK5 was detected in septic neutrophils and control cells treated with cytokines plus LPS. Data suggest that endogenous mediators produced during sepsis might continually activate circulating neutrophils, leading to GRK activation, which may induce neutrophil desensitization to chemoattractants.

Actins↗

PubMed2006

Heme and innate immunity: new insights for an old molecule.

Hemolytic episodes such as sickle cell disease, malaria and ischemia-reperfusion occurrence are often associated to the statement of an inflammatory response which may develop or not to a chronic inflammatory status. Although these pathological states are triggered by distinct etiological agents, all of them are associated to high levels of free heme in circulation. In this review, we aim to focus the very recent achievements that have led to the statement of free heme as a proinflammatory molecule, which may play a central role during the onset and/or persistence of inflammation during these pathologies.

Acute Disease↗

PubMed2005

Nitric oxide-dependent reduction in soluble guanylate cyclase functionality accounts for early lipopolysaccharide-induced changes in vascular reactivity.

We investigated the role of soluble guanylate cyclase in lipopolysaccharide-induced hyporesponsiveness to phenylephrine. The effects of phenylephrine on the blood pressure of female Wistar rats were evaluated at 2, 8, and 24 h after lipopolysaccharide injection (12.5 mg/kg i.p.). Vasoconstrictive responses to phenylephrine were reduced 40 to 50% in all time periods. Methylene blue, a soluble guanylate cyclase inhibitor (15 micromol/kg i.v.) restored the reactivity to phenylephrine in animals injected with lipopolysaccharide 2 and 24 h earlier. However, it failed to do so in animals injected with lipopolysaccharide 8 h earlier. Incubation with sodium nitroprusside (SNP) increased lung and aorta cGMP levels in control animals and in tissues of rats treated with lipopolysaccharide 24 h earlier. However, SNP failed to increase tissue cGMP in rats injected 8 h earlier. Lipopolysaccharide reduced the vasodilatory response to NO donors 8 h after injection. This effect and the decreased lung cGMP accumulation in response to SNP were reversed by an NO synthase blocker. Guanylate cyclase protein levels were lower than controls in lungs harvested from rats injected 8 h earlier and were back to normal values in lungs of rats injected 24 h earlier with lipopolysaccharide. Thus, data indicate that there is a temporal window of 8 h after lipopolysaccharide injection in which soluble guanylate cyclase is not functional and that this loss of function is NO-dependent. Thus, the putative use of soluble guanylate cyclase inhibitors in the treatment of endotoxemia may be beneficial mainly at early stages of this condition.

Animals↗

PubMed2005

Frutalin, a galactose-binding lectin, induces chemotaxis and rearrangement of actin cytoskeleton in human neutrophils: involvement of tyrosine kinase and phosphoinositide 3-kinase.

Several lectin-like molecules have been shown as potent activators of leukocytes. Galactose-binding lectins are of special interest since they could interact with several endogenous molecules involved in the innate and specific immune responses. The effects of Frutalin (FTL), an alpha-D-galactose (Gal)-binding plant lectin, on the modulation of neutrophil (PMN) functions were investigated. FTL induced a dose-dependent PMN migration in mice pleural cavity. Moreover, FTL was also a potent direct chemotactic for human PMN, in vitro, and triggered oxidative burst in these cells. These effects were accompanied by a rearrangement of the actin cytoskeleton dynamic, activation of tyrosine kinase (TK) pathways, increase in focal adhesion kinase (FAK) phosphorylation, and its subsequent association to phosphoinositide3-kinase (PI3K). All those effects were inhibited in the presence of Gal, suggesting specific carbohydrate recognition for FTL effects. The activations of TK and PI3K pathways are essential events for FTL-induced chemotaxis, since inhibitors of these pathways, genistein and LY294002, inhibited neutrophil migration in vitro. The data indicate that sugar-protein interactions between a soluble lectin and galacto-components on neutrophil surface trigger the TK pathway, inducing FAK and PI3K activation, interfering with cell motility and oxidative response.

Actins↗

PubMed2005

Involvement of phosphatidylinositol-3 kinase-Akt and nuclear factor kappa-B pathways in the effect of frutalin on human lymphocyte.

The mechanisms involved in the mitogenic effect of lectins are not fully understood and are thought to involve a cascade of intracellular signals related to T cell receptor activation. This study shows that frutalin, the alpha-D-galactose-binding lectin from Artocarpus incisa seeds, is a potent mitogenic activator of human lymphocytes. This effect is inhibited by D-galactose and PI3K inhibitors, and is accompanied by an increase in IL-2 receptor expression and by a PI3K-dependent IL-2 gene expression and IL-2 protein synthesis. Frutalin also induces Akt-phosphorylation and activates NF-kappaB, inducing its translocation from the cytosol to the nucleus. Both effects are blocked in the presence of D-galactose or by PI3K inhibitors. In summary, frutalin, interacting with alpha-D-galactose, activates signaling pathways related to TCR, and thereby triggers PI3K/Akt and NF-kappaB pathway, which modulates T cell proliferation, IL-2 synthesis and IL-2R expression. Frutalin might be a useful tool to study intracellular mechanisms following T cell activation that link upstream signaling pathways to downstream events.

Active Transport, Cell Nucleus↗

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

RGD- and MLD-disintegrins, jarastatin and EC3, activate integrin-mediated signaling modulating the human neutrophils chemotaxis, apoptosis and IL-8 gene expression.

The effects of jarastatin (JT), a monomeric RGD-disintegrin, were compared with those of the heterodimeric MLD-disintegrin, EC3, on human neutrophil activation and functions. Both disintegrins inhibited neutrophil chemotaxis induced by fMet-Leu-Phe and were also potent chemotactic agents. These effects were accompanied by an increase in actin polymerization, and both were inhibited by genistein, a tyrosine kinase inhibitor. While JT, but not other RGD-disintegrins, inhibited EC3-induced chemotaxis, EC3 was not able to inhibit JT effect. The chemotactic effect of JT was blocked by anti-alpha(M) antibody whereas anti-alpha(9)beta(1) inhibited EC3 effect. Both JT and EC3 induced focal adhesion kinase (FAK) and phosphoinositide 3-kinase (PI3K) activation. Accordingly, LY294002, a PI3K inhibitor, impaired their chemotactic effect on neutrophils. JT induced Erk-2 translocation to nucleus and a delay of the spontaneous apoptosis of neutrophils in vitro. In contrast, EC3 inhibited Erk-2 activation and had a proapoptotic effect. These effects were reverted by PD98059, an MEK 1/2 inhibitor and blocked by z-VAD-FMK, a caspase inhibitor. In addition, JT, but not EC3, increased the IL-8 mRNA levels in neutrophils. The data indicate that JT and EC3 directly activate an integrin-coupled signaling and modulate the MAPK pathway in different ways, leading the neutrophils to express different functional response.

Active Transport, Cell Nucleus↗

PubMed2003

Up-regulation of phosphatidylinositol 3-kinase and glucose transporter 4 in muscle of rats subjected to maternal undernutrition.

Early postnatal nutrition has been associated with the long-term effects on glucose homeostasis in adulthood. To elucidate the molecular mechanisms by which undernutrition during early life leads to changes in insulin sensitivity, we investigated the insulin signaling in skeletal muscle of rats during development. Offspring of dams fed with either protein-free or normal diets during the first 10 days of lactation were studied from lactation period until adulthood. Early maternal undernutrition impaired secretion of insulin but maintained normal blood glucose levels until adulthood. Insulin receptor (IR) activation after insulin stimulation was decreased during the period of protein restriction. In addition, glucose uptake, insulin receptor substrate 1 (IRS-1) phosphorylation and glucose transporter 4 (GLUT-4) translocation in muscle were reduced in response to insulin during suckling. In contrast, non- or insulin-stimulated glucose uptake and GLUT-4 translocation were found significantly increased in muscle of adult offspring. Finally, basal association of phosphatidylinositol 3-kinase (PI3-kinase) with IRS-1 was increased and was highly stimulated by insulin in muscle from adult rats. Our findings suggest that early postnatal undernutrition increases insulin sensitivity in adulthood, which appears to be directly related to changes in critical steps required for glucose metabolism.

Animals↗

PubMed2003

Role of nitric oxide in the response of Saccharomyces cerevisiae cells to heat shock and high hydrostatic pressure.

Nitric oxide (NO) is a simple and unique molecule that has diverse functions in organisms, including intracellular and intercellular messenger. The influence of NO on cell growth of Saccharomyces cerevisiae and as a signal molecule in stress response was evaluated. Respiring cells were more sensitive to an increase in intracellular NO concentration than fermentatively growing cells. Low levels of NO demonstrated a cytoprotective effect during stress from heat-shock or high hydrostatic pressure. Induction of NO synthase was isoform-specific and dependent on the metabolic state of the cells and the stress response pathway. These results support the hypothesis that an increase in intracellular NO concentration leads to stress protection.

Blotting, Western↗

PubMed2003

Alternagin-C, a nonRGD-disintegrin, induces neutrophil migration via integrin signaling.

Recently, a new protein containing a disintegrin domain, alternagin-C (Alt-C), was purified from Bothrops alternatus venom. Unlike other disintegrins, in Alt-C an ECD amino acid mogif takes the place of the RGD sequence. Most disintegrins contain an RGD/KGD sequence and are very potent inhibitors of platelet aggregation, as well as other cell interactions with the extracellular matrix, including tumor cell metastasis and angiogenesis. The present study investigated the effects of Alt-C on human neutrophil chemotaxis in vitro and the activation of integrin-mediated pathways. Alt-C showed a potent chemotactic effect for human neutrophils when compared to N-formyl-methionyl-leucyl-phenylalanine peptide (fMLP), a classic chemotactic agent. Moreover, preincubation of neutrophils with Alt-C significantly inhibited chemotaxis toward fMLP and itself. In addition, a peptide containing an ECD sequence presented a chemotactic activity and significantly inhibited chemotaxis induced by Alt-C and fMLP. A significant increase of F-actin content was observed in cells treated with Alt-C, showing that the chemotactic activity of Alt-C on neutrophils is driven by actin cytoskeleton dynamic changes. Furthermore, this protein was able to induce an increase of phosphotyrosine content triggering focal adhesion kinase activation and its association with phosphatidylinositol 3-kinase. Alt-C was also able to induce a significant increase in extracellular signal-regulated kinase 2 nuclear translocation. The chemotactic activity of Alt-C was partially inhibited by LY294002, a specific phosphatidylinositol 3-kinase inhibitor, and by PD98056, a Map kinase kinase inhibitor. These findings suggest that Alt-C can trigger human neutrophil chemotaxis modulated by intracellular signals characteristic of integrin-activated pathways and that these effects could be related to the ECD mogif present in disintegrin-like domain.

Actins↗

PubMed2002

Neutrophil activation by heme: implications for inflammatory processes.

Heme, a ubiquitous iron-containing compound, is present in large amounts in many cells and is inherently dangerous, particularly when it escapes from intracellular sites. The release of heme from damaged cells and tissues is supposed to be higher in diseases such as malaria and hemolytic anemia or in trauma and hemorrhage. We investigated here the role of free ferriprotoporphyrin IX (hemin) as a proinflammatory molecule, with particular attention to its ability to activate neutrophil responses. Injecting hemin into the rat pleural cavity resulted in a dose-dependent migration of neutrophils, indicating that hemin is able to promote the recruitment of these cells in vivo. In vitro, hemin induced human neutrophil chemotaxis and cytoskeleton reorganization, as revealed by the increase of neutrophil actin polymerization. Exposure of human neutrophils to 3 microM hemin activated the expression of the chemokine interleukin-8, as demonstrated by quantitative reverse-transcription polymerase chain reaction, indicating a putative molecular mechanism by which hemin induces chemotaxis in vivo. Brief incubation of human neutrophils with micromolar concentrations of hemin (1-20 microM) triggered the oxidative burst, and the production of reactive oxygen species was directly proportional to the concentration of hemin added to the cells. Finally, we observed that human neutrophil protein kinase C was activated by hemin in vitro, with a K(1/2) of 5 microM. Taken together, these results suggest a role for hemin as a proinflammatory agent able to induce polymorphonuclear neutrophil activation in situations of clinical relevance, such as hemolysis or hemoglobinemia.

Actins↗

PubMed2002

Characterization of a monomeric disintegrin, ocellatusin, present in the venom of the Nigerian carpet viper, Echis ocellatus.

Ocellatusin is a new RGD-containing short monomeric disintegrin. It is a better inhibitor of alpha(5)beta(1) integrin and a more potent inducer of the expression of a ligand-induced binding site epitope on beta(1) integrin subunit than echistatin. In further contrast to echistatin, ocellatusin has a direct chemotactic stimulus on human neutrophils in vitro. The distinct effects of these two close evolutionarily related disintegrins might be explained by the presence of methionine-22 and histidine-29 in the RGD loop of ocellatusin, which are arginine and aspartic acid, respectively, in echistatin. These mutations may modulate the conformation and/or recognition properties of the integrin-binding loop of ocellatusin.

Amino Acid Sequence↗

PubMed2002

Expression of inducible nitric oxide synthase in human umbilical vein endothelial cells during primary culture.

The adaptive response of endothelial cells to stress may lead to the upregulation of nitric oxide (NO) production. Herein, we report inducible nitric oxide synthase (iNOS) induction in primary cultures of human umbilical vein endothelial cells (HUVEC). The enzyme expression was earlier observed in 12-h cultures, reaching maximal levels after 3 days and decreasing when cells become confluent. The time course of NO production by HUVEC paralleled iNOS expression during the whole culture period, indicating that enzyme was functionally active. Conversely, iNOS induction could not be further detected in HUVEC subcultures passed once from cells presenting maximal levels of iNOS expression in the primary culture. Induction of iNOS in HUVEC was not related to lipopolysaccharide contamination, since the enzyme expression was not affected in the presence of polymyxin B added to primary cultures. Further analysis showed that aminoguanidine, a specific iNOS inhibitor, did not affect cell proliferation, suggesting that the NO produced by HUVEC may not be directly related to cell growth. Platelet endothelial cell adhesion molecule-1 expression was upregulated during cell confluence, in contrast to the decrease of iNOS expression and activity. The data suggest that iNOS expression may be a molecular mechanism mediating the adaptive response of endothelial cells to culture environment.

Blotting, Western↗