FRANCISCO RAFAEL MARTINS LAURINDO
Projetos de Pesquisa
Unidades Organizacionais
Instituto do Coração, Hospital das Clínicas, Faculdade de Medicina
LIM/64, Hospital das Clínicas, Faculdade de Medicina - Líder
LIM/64, Hospital das Clínicas, Faculdade de Medicina - Líder
12 resultados
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- Cellular prion protein (PrPC) and superoxide dismutase (SOD) in vascular cells under oxidative stress(2011) SOPRANA, Helen Zocche; SOUZA, Liliete Canes; DEBBAS, Victor; LAURINDO, Francisco Rafael MartinsThe PrPC is expressed in several cell types but its physiological function is unknown. Some studies associate the PrPC with copper metabolism and the antioxidant activity of SOD. Our hypothesis was that changes in PrPC expression lead to abnormal copper regulation and induce SOD downregulation in the vascular wall. Objectives: to study whether the PrPC expression undergoes induction by agents that trigger endoplasmic reticulum stress (ERS) and, in this context, to evaluate the SOD activity. Methods: To trigger ERS, in vitro, rabbit aortic smooth muscle cells were challenged for 4, 8 and 18 hours, with angiotensin-II, tunicamycin and 7-ketocholesterol. For in vivo studies rabbit aortic arteries were subjected to injury by balloon catheter. Results: In vitro baseline SOD activity, determined through inhibition of cytochrome-c reduction, was 13.9 +/- 1.2 U/mg protein, angiotensin-II exposed for 8 hours produced an increase in SOD activity, and cellular copper concentration was about 9 times greater only under these conditions. Western blotting analysis for SOD isoenzymes showed an expression profile that was not correlated with the enzymatic activity. PrPC expression decreased after exposure to all agents after different incubation periods. RT-PCR assay showed increased mRNA expression for PrPC only in cells stimulated for 8 hours with the different stressors. The PrPC mRNA expression in rabbit aortic artery fragments, subjected to balloon catheter injury, showed a pronounced increase immediately after overdistension. The results obtained indicated a PrPC protection factor during the early part of the ERS exposure period, but did not demonstrate a SOD-like profile for the PrPC.
article 36 Citação(ões) na Scopus Tobacco Smoke Induces Ventricular Remodeling Associated with an Increase in NADPH Oxidase Activity(2011) RAFACHO, Bruna P. M.; AZEVEDO, Paula S.; POLEGATO, Bertha F.; FERNANDES, Ana A. H.; BERTOLINE, Maria A.; FERNANDES, Denise C.; CHIUSO-MINICUCCI, Fernanda; ROSCANI, Meliza G.; SANTOS, Priscila P. dos; MATSUBARA, Luiz S.; MATSUBARA, Beatriz B.; LAURINDO, Francisco R. M.; PAIVA, Sergio A. R.; ZORNOFF, Leonardo A. M.; MINICUCCI, Marcos F.Background: Recent studies have assessed the direct effects of smoking on cardiac remodeling and function. However, the mechanisms of these alterations remain unknown. The aim of this study was to investigate de role of cardiac NADPH oxidase and antioxidant enzyme system on ventricular remodeling induced by tobacco smoke. Methods: Male Wistar rats that weighed 200-230 g were divided into a control group (C) and an experimental group that was exposed to tobacco smoke for a period of two months (ETS). After the two-month exposure period, morphological, biochemical and functional analyses were performed. Results: The myocyte cross-sectional area and left ventricle end-diastolic dimension was increased 16.2% and 33.7%, respectively, in the ETS group. The interstitial collagen volume fraction was also higher in ETS group compared to the controls. In addition to these morphological changes, the ejection fraction and fractional shortening were decreased in the ETS group. Importantly, these alterations were related to augmented heart oxidative stress, which was characterized by an increase in NADPH oxidase activity, increased levels of lipid hydroperoxide and depletion of antioxidant enzymes (e.g., catalase, superoxide dismutase and glutathione peroxidase). In addition, cardiac levels of IFN-gamma, TNF-alpha and IL-10 were not different between the groups. Conclusion: Cardiac alterations that are induced by smoking are associated with increased NADPH oxidase activity, suggesting that this pathway plays a role in the ventricular remodeling induced by exposure to tobacco smoke.- Mild Mitochondrial Uncoupling and Calorie Restriction Increase Fasting eNOS, Akt and Mitochondrial Biogenesis(2011) CERQUEIRA, Fernanda M.; LAURINDO, Francisco R. M.; KOWALTOWSKI, Alicia J.Enhanced mitochondrial biogenesis promoted by eNOS activation is believed to play a central role in the beneficial effects of calorie restriction (CR). Since treatment of mice with dinitrophenol (DNP) promotes health and lifespan benefits similar to those observed in CR, we hypothesized that it could also impact biogenesis. We found that DNP and CR increase citrate synthase activity, PGC-1 alpha, cytochrome c oxidase and mitofusin-2 expression, as well as fasting plasma levels of NO(center dot) products. In addition, eNOS and Akt phosphorylation in skeletal muscle and visceral adipose tissue was activated in fasting CR and DNP animals. Overall, our results indicate that systemic mild uncoupling activates eNOS and Akt-dependent pathways leading to mitochondrial biogenesis.
- Antioxidant Activity of Uruguayan Propolis. In Vitro and Cellular Assays(2011) SILVA, Veronica; GENTA, Gonzalo; MOELLER, Matias N.; MASNER, Martin; THOMSON, Leonor; ROMERO, Natalia; RADI, Rafael; FERNANDES, Denise C.; LAURINDO, Francisco R. M.; HEINZEN, Horacio; FIERRO, Walter; DENICOLA, AnaThe antioxidant capacity of propolis from the southern region of Uruguay was evaluated using in vitro as well as cellular assays. Free radical scavenging capacity was assessed by ORAC, obtaining values significantly higher than those of other natural products (8000 mu mol Trolox equiv/g propolis). ORAC values correlated well with total polyphenol content (determined by Folin-Ciocalteu method) and UV absorption. Total polyphenol content (150 mg gallic acid equiv/g propolis) and flavonoids (45 mg quercetin equiv/g propolis) were similar to values reported for southern Brazilian (group 3) and Argentinean propolis. Flavonoid composition determined by RP-HPLC indicates a strong poplar-tree origin. Samples high in polyphenols efficiently inhibit low-density lipoprotein lipoperoxidation and tyrosine nitration. In addition, Uruguayan propolis was found to induce the expression of endothelial nitric oxide synthase and inhibit endothelial NADPH oxidase, suggesting a potential cardiovascular benefit by increasing nitric oxide bioavailability in the endothelium.
- Blood Meal-Derived Heme Decreases ROS Levels in the Midgut of Aedes aegypti and Allows Proliferation of Intestinal Microbiota(2011) OLIVEIRA, Jose Henrique M.; GONCALVES, Renata L. S.; LARA, Flavio A.; DIAS, Felipe A.; GANDARA, Ana Caroline P.; MENNA-BARRETO, Rubem F. S.; EDWARDS, Meredith C.; LAURINDO, Francisco R. M.; SILVA-NETO, Mario A. C.; SORGINE, Marcos H. F.; OLIVEIRA, Pedro L.The presence of bacteria in the midgut of mosquitoes antagonizes infectious agents, such as Dengue and Plasmodium, acting as a negative factor in the vectorial competence of the mosquito. Therefore, knowledge of the molecular mechanisms involved in the control of midgut microbiota could help in the development of new tools to reduce transmission. We hypothesized that toxic reactive oxygen species (ROS) generated by epithelial cells control bacterial growth in the midgut of Aedes aegypti, the vector of Yellow fever and Dengue viruses. We show that ROS are continuously present in the midgut of sugar-fed (SF) mosquitoes and a blood-meal immediately decreased ROS through a mechanism involving heme-mediated activation of PKC. This event occurred in parallel with an expansion of gut bacteria. Treatment of sugar-fed mosquitoes with increased concentrations of heme led to a dose dependent decrease in ROS levels and a consequent increase in midgut endogenous bacteria. In addition, gene silencing of dual oxidase (Duox) reduced ROS levels and also increased gut flora. Using a model of bacterial oral infection in the gut, we show that the absence of ROS resulted in decreased mosquito resistance to infection, increased midgut epithelial damage, transcriptional modulation of immune-related genes and mortality. As heme is a pro-oxidant molecule released in large amounts upon hemoglobin degradation, oxidative killing of bacteria in the gut would represent a burden to the insect, thereby creating an extra oxidative challenge to the mosquito. We propose that a controlled decrease in ROS levels in the midgut of Aedes aegypti is an adaptation to compensate for the ingestion of heme.
- Protein Disulfide Isomerase and Host-Pathogen Interaction(2011) STOLF, Beatriz S.; IOANNIS, Smyrnias; LOPES, Lucia R.; VENDRAMIN, Alcione; GOTO, Hiro; LAURINDO, Francisco R. M.; SHAH, Ajay M.; SANTOS, Celio X. C.Reactive oxygen species (ROS) production by immunological cells is known to cause damage to pathogens. Increasing evidence accumulated in the last decade has shown, however, that ROS (and redox signals) functionally regulate different cellular pathways in the host-pathogen interaction. These especially affect (i) pathogen entry through protein redox switches and redox modification (i. e., intra-and interdisulfide and cysteine oxidation) and (ii) phagocytic ROS production via Nox family NADPH oxidase enzyme and the control of phagolysosome function with key implications for antigen processing. The protein disulfide isomerase (PDI) family of redox chaperones is closely involved in both processes and is also implicated in protein unfolding and trafficking across the endoplasmic reticulum (ER) and towards the cytosol, a thiol-based redox locus for antigen processing. Here, we summarise examples of the cellular association of host PDI with different pathogens and explore the possible roles of pathogen PDIs in infection. A better understanding of these complex regulatory steps will provide insightful information on the redox role and coevolutional biological process, and assist the development of more specific therapeutic strategies in pathogen-mediated infections.
- SvO(2)-GUIDED RESUSCITATION FOR EXPERIMENTAL SEPTIC SHOCK: EFFECTS OF FLUID INFUSION AND DOBUTAMINE ON HEMODYNAMICS, INFLAMMATORY RESPONSE, AND CARDIOVASCULAR OXIDATIVE STRESS(2011) ROSARIO, Andre Loureiro; PARK, Marcelo; BRUNIALTI, Milena Karina; MENDES, Marialice; RAPOZO, Marjorie; FERNANDES, Denise; SALOMAO, Reinaldo; LAURINDO, Francisco Rafael; SCHETTINO, Guilherme Paula; AZEVEDO, Luciano Cesar P.The pathogenetic mechanisms associated to the beneficial effects of mixed venous oxygen saturation (SvO(2))-guided resuscitation during sepsis are unclear. Our purpose was to evaluate the effects of an algorithm of SvO(2)-driven resuscitation including fluids, norepinephrine and dobutamine on hemodynamics, inflammatory response, and cardiovascular oxidative stress during a clinically resembling experimental model of septic shock. Eighteen anesthetized and catheterized pigs (35-45 kg) were submitted to peritonitis by fecal inoculation (0.75 g/kg). After hypotension, antibiotics were administered, and the animals were randomized to two groups: control (n = 9), with hemodynamic support aiming central venous pressure 8 to 12 mmHg, urinary output 0.5 mL/kg per hour, and mean arterial pressure greater than 65 mmHg; and SvO(2) (n = 9), with the goals above, plus SvO(2) greater than 65%. The interventions lasted 12 h, and lactated Ringer's and norepinephrine (both groups) and dobutamine (SvO(2) group) were administered. Inflammatory response was evaluated by plasma concentration of cytokines, neutrophil CD14 expression, oxidant generation, and apoptosis. Oxidative stress was evaluated by plasma and myocardial nitrate concentrations, myocardial and vascular NADP(H) oxidase activity, myocardial glutathione content, and nitrotyrosine expression. Mixed venous oxygen saturation-driven resuscitation was associated with improved systolic index, oxygen delivery, and diuresis. Sepsis induced in both groups a significant increase on IL-6 concentrations and plasma nitrate concentrations and a persistent decrease in neutrophil CD14 expression. Apoptosis rate and neutrophil oxidant generation were not different between groups. Treatment strategies did not significantly modify oxidative stress parameters. Thus, an approach aiming SvO(2) during sepsis improves hemodynamics, without any significant effect on inflammatory response and oxidative stress. The beneficial effects associated with this strategy may be related to other mechanisms.
- Protein disulfide isomerase redox-dependent association with p47(phox): evidence for an organizer role in leukocyte NADPH oxidase activation(2011) PAES, Antonio Marcus de A.; VERISSIMO-FILHO, Sidney; GUIMARAES, Luciana Lopes; SILVA, Ana Carolina B.; TAKIUTI, Julia T.; SANTOS, Celio X. C.; JANISZEWSKI, Mariano; LAURINDO, Francisco R. M.; LOPES, Lucia R.Mechanisms of leukocyte NADPH oxidase regulation remain actively investigated. We showed previously that vascular and macrophage oxidase complexes are regulated by the associated redox chaperone PDI. Here, we investigated the occurrence and possible underlying mechanisms of PDI-mediated regulation of neutrophil NADPH oxidase. In a semirecombinant cell-free system, PDI inhibitors scrRNase (100 mu g/mL) or bacitracin (1 mM) near totally suppressed superoxide generation. Exogenously incubated, oxidized PDI increased (by similar to 40%), whereas PDIred diminished (by similar to 60%) superoxide generation. No change occurred after incubation with PDI serine-mutated in all four redox cysteines. Moreover, a mimetic CxxC PDI inhibited superoxide production by similar to 70%. Thus, oxidized PDI supports, whereas reduced PDI down-regulates, intrinsic membrane NADPH oxidase complex activity. In whole neutrophils, immunoprecipitation and colocalization experiments demonstrated PDI association with membrane complex subunits and prominent thiol-mediated interaction with p47(phox) in the cytosol fraction. Upon PMA stimulation, PDI was mobilized from azurophilic granules to cytosol but did not further accumulate in membranes, contrarily to p47(phox). PDI-p47(phox) association in cytosol increased concomitantly to opposite redox switches of both proteins; there was marked reductive shift of cytosol PDI and maintainance of predominantly oxidized PDI in the membrane. Pulldown assays further indicated predominant association between PDIred and p47(phox) in cytosol. Incubation of purified PDI (> 80% reduced) and p47(phox) in vitro promoted their arachidonate-dependent association. Such PDI behavior is consistent with a novel cytosolic regulatory loop for oxidase complex (re) cycling. Altogether, PDI seems to exhibit a supportive effect on NADPH oxidase activity by acting as a redox-dependent enzyme complex organizer. J. Leukoc. Biol. 90: 799-810; 2011.
- Proteasome Inhibition Represses Unfolded Protein Response and Nox4, Sensitizing Vascular Cells to Endoplasmic Reticulum Stress-Induced Death(2011) AMANSO, Angelica M.; DEBBAS, Victor; LAURINDO, Francisco R. M.Background: Endoplasmic reticulum (ER) stress has pathophysiological relevance in vascular diseases and merges with proteasome function. Proteasome inhibition induces cell stress and may have therapeutic implications. However, whether proteasome inhibition potentiates ER stress-induced apoptosis and the possible mechanisms involved in this process are unclear. Methodology/Principal Findings: Here we show that proteasome inhibition with MG132, per se at non-lethal levels, sensitized vascular smooth muscle cells to caspase-3 activation and cell death during ER stress induced by tunicamycin (Tn). This effect was accompanied by suppression of both proadaptive (KDEL chaperones) and proapoptotic (CHOP/GADD153) unfolded protein response markers, although, intriguingly, the splicing of XBP1 was markedly enhanced and sustained. In parallel, proteasome inhibition completely prevented ER stress-induced increase in NADPH oxidase activity, as well as increases in Nox4 isoform and protein disulfide isomerase mRNA expression. Increased Akt phosphorylation due to proteasome inhibition partially offset the proapoptotic effect of Tn or MG132. Although proteasome inhibition enhanced oxidative stress, reactive oxygen species scavenging had no net effect on sensitization to Tn or MG132-induced cell death. Conclusion/Relevance: These data indicate unfolded protein response-independent pathways whereby proteasome inhibition sensitizes vascular smooth muscle to ER stress-mediated cell death. This may be relevant to understand the therapeutic potential of such compounds in vascular disease associated with increased neointimal hyperplasia.
- Quiescin sulfhydryl oxidase (QSOX) is expressed in the human atheroma core: possible role in apoptosis(2011) ANDRADE, Claudia R. de; STOLF, Beatriz S.; DEBBAS, Victor; ROSA, Daniela S.; KALIL, Jorge; COELHO, Veronica; LAURINDO, Francisco R. M.Quiescin sulfhydryl oxidases (QSOXs) catalyze the formation of disulfide bonds in peptides and proteins, and in vertebrates comprise two proteins: QSOX1 and QSOX2. QSOX1, the most extensively studied type, has been implicated in protein folding, production of extracellular matrix, redox regulation, protection from apoptosis, angiogenesis, and cell differentiation. Atherosclerosis is an immunopathological condition in which redox processes, apoptosis, cell differentiation, and matrix secretion/maturation have critical roles. Considering these data, we hypothesized that QSOX1 could be involved in this disease, possibly reducing apoptosis and angiogenesis inside the plaque. QSOX1 labeling in normal human carotid vessels showed predominant expression by endothelium, subendothelium, and adventitia. In atherosclerotic plaques, however, QSOX1 was highly expressed in macrophages at the lipid core. QSOX1 expression was also studied in terms of mRNA and protein in cell types present in plaques under apoptotic or activating stimuli, emulating conditions found in the atherosclerotic process. QSOX1 mRNA increased in endothelial cells and macrophages after the induction of apoptosis. At the protein level, the correlation between apoptosis and QSOX1 expression was not evident in all cell types, possibly because of a rapid secretion of QSOX1. Our results propose for the first time possible roles for QSOX1 in atherosclerosis, being upregulated in endothelial cells and macrophages by apoptosis and cell activation, and possibly controlling these processes, as well as angiogenesis. The quantitative differences in QSOX1 induction may depend on the cell type and also on local factors.