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
Resultados de Busca
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- Time-Dependent Effects of Training on Cardiovascular Control in Spontaneously Hypertensive Rats: Role for Brain Oxidative Stress and Inflammation and Baroreflex Sensitivity(2014) MASSON, Gustavo S.; COSTA, Tassia S. R.; YSHII, Lidia; FERNANDES, Denise C.; SOARES, Pedro Paulo Silva; LAURINDO, Francisco R.; SCAVONE, Cristoforo; MICHELINI, Lisete C.Baroreflex dysfunction, oxidative stress and inflammation, important hallmarks of hypertension, are attenuated by exercise training. In this study, we investigated the relationships and time-course changes of cardiovascular parameters, pro-inflammatory cytokines and pro-oxidant profiles within the hypothalamic paraventricular nucleus of the spontaneously hypertensive rats (SHR). Basal values and variability of arterial pressure and heart rate and baroreflex sensitivity were measured in trained (T, low-intensity treadmill training) and sedentary (S) SHR at weeks 0, 1, 2, 4 and 8. Paraventricular nucleus was used to determine reactive oxygen species (dihydroethidium oxidation products, HPLC), NADPH oxidase subunits and pro-inflammatory cytokines expression (Real time PCR), p38 MAPK and ERK1/2 expression (Western blotting), NF-kappa B content (electrophoretic mobility shift assay) and cytokines immunofluorescence. SHR-S vs. WKY-S (Wistar Kyoto rats as time control) showed increased mean arterial pressure (172 +/- 3 mmHg), pressure variability and heart rate (358 +/- 7 b/min), decreased baroreflex sensitivity and heart rate variability, increased p47(phox) and reactive oxygen species production, elevated NF-kappa B activity and increased TNF-alpha and IL-6 expression within the paraventricular nucleus of hypothalamus. Two weeks of training reversed all hypothalamic changes, reduced ERK1/2 phosphorylation and normalized baroreflex sensitivity (4.04 +/- 0.31 vs. 2.31 +/- 0.19 b/min/mmHg in SHR-S). These responses were followed by increased vagal component of heart rate variability (1.9-fold) and resting bradycardia (-13%) at the 4th week, and, by reduced vasomotor component of pressure variability (-28%) and decreased mean arterial pressure (-7%) only at the 8th week of training. Our findings indicate that independent of the high pressure levels in SHR, training promptly restores baroreflex function by disrupting the positive feedback between high oxidative stress and increased pro-inflammatory cytokines secretion within the hypothalamic paraventricular nucleus. These early adaptive responses precede the occurrence of training-induced resting bradycardia and blood pressure fall.
- Oxidation, inactivation and aggregation of protein disulfide isomerase promoted by the bicarbonate-dependent peroxidase activity of human superoxide dismutase(2014) IQBAL, Asif; PAVIANI, Veronica; MORETTI, Ana Iochabel; LAURINDO, Francisco R. M.; AUGUSTO, OharaProtein disulfide isomerase (PDI) is a dithiol-disulfide oxidoreductase that has essential roles in redox protein folding. PDI has been associated with protective roles against protein aggregation, a hallmark of neurodegenerative diseases. Intriguingly, PDI has been detected in the protein inclusions found in the central nervous system of patients of neurodegenerative diseases. Oxidized proteins are also consistently detected in such patients, but the agents that promote these oxidations remain undefined. A potential trigger of protein oxidation is the bicarbonate-dependent peroxidase activity of the human enzyme superoxide dismutase 1 (hSOD1). Therefore, we examined the effects of this activity on PDI structure and activity. The results showed that PDI was oxidized to radicals that lead to PDI inactivation and aggregation. The aggregates are huge and apparently produced by covalent cross-links. Spin trapping experiments coupled with MS analysis indicated that at least 3 residues of PDI are oxidized to tyrosyl radicals (Y-63, Y-116 and Y-327) Parallel experiments showed that PDI is also oxidized to radicals, inactivated and aggregated by the action of photolytically generated carbonate radical and by UV light. PDI is prone to inactivation and aggregation by one-electron oxidants and UV light probably because of its high content of aromatic amino acids.
- Peri/epicellular Protein Disulfide Isomerase Reshapes Vascular Architecture to Counteracts Constrictive Remodeling(2014) TANAKA, Leonardo Yuji; ARAUJO, Haniel Alves; HIRONAKA, Gustavo Ken; ARAUJO, Thais Larissa; RODRIGUEZ, Andres Ignacio; CASAGRANDE, Annelise Silva; TAKIMURA, Celso Kiyoshi; LAURINDO, Francisco Rafael
- Cardioprotective mechanism of S-nitroso-N-acetylcysteine via S-nitrosated betadrenoceptor-2 in the LDLr-/- mice(2014) WANSCHEL, Amarylis Claudine Bonito Azeredo; CACERES, Viviane Menezes; MORETTI, Ana Iochabel Soares; BRUNI-CARDOSO, Alexandre; CARVALHO, Hernandes Faustino de; SOUZA, Heraldo Possolo de; LAURINDO, Francisco Rafael Martins; SPADARI, Regina Celia; KRIEGER, Marta HelenaPrevious studies from our group have demonstrated the protective effect of S-nitroso-N-acetylcysteine (SNAC) on the cardiovascular system in dyslipidemic LDLr-/- mice that develop atheroma and left ventricular hypertrophy after 15 days on a high fat diet. We have shown that SNAC treatment attenuates plaque development via the suppression of vascular oxidative stress and protects the heart from structural and functional myocardial alterations, such as heart arrhythmia, by reducing cardiomyocyte sensitivity to catecholamines. Here we investigate the ability of SNAC to modulate oxidative stress and cell survival in cardiomyocytes during remodeling and correlation with beta(2)-AR signaling in mediating this protection. Ventricular superoxide (O-2(-)) and hydrogen peroxide (H2O2) generation was measured by HPLC methods to allow quantification of dihydroethidium (DHE) products. Ventricular histological sections were stained using terminal dUTP nick-end labeling (TUNEL) to identify nuclei with DNA degradation (apoptosis) and this was confirmed by Western blot for cleaved caspase-3 and caspase-7 protein expression. The findings show that O-2(-) and H2O2 production and also cell apoptosis were increased during left ventricular hypertrophy (LVH). SNAC treatment reduced oxidative stress during on cardiac remodeling, measured by decreased H2O2 and O-2(-) production (65% and 52%, respectively), and a decrease in the ratio of p-Ser1177 eNOS/total eNOS. Left ventricle (LV) from SNAC-treated mice revealed a 4-fold increase in beta(2)-AR expression associated with coupling change to Gi; beta(2)-ARs-S-nitrosation (beta(2)-AR-SNO) increased 61%, while apoptosis decreased by 70%. These results suggest that the cardio-protective effect of SNAC treatment is primarily through its anti-oxidant role and is associated with beta(2)-ARs overexpression and beta(2)-AR-SNO via an anti-apoptotic pathway.
- RedoxModulation of FAK Controls Melanoma Survival - Role of NOX4(2014) RIBEIRO-PEREIRA, Cristiane; MORAES, Joao Alfredo; SOUZA, Mariele de Jesus; LAURINDO, Francisco R.; ARRUDA, Maria Augusta; BARJA-FIDALGO, ChristinaStudies have demonstrated that reactive oxygen species (ROS) generated by NADPH oxidase are essential for melanoma proliferation and survival. However, the mechanisms by which NADPH oxidase regulates these effects are still unclear. In this work, we investigate the role of NADPH oxidase-derived ROS in the signaling events that coordinate melanoma cell survival. Using the highly metastatic human melanoma cell line MV3, we observed that pharmacological NADPH oxidase inhibition reduced melanoma viability and induced dramatic cellular shape changes. These effects were accompanied by actin cytoskeleton rearrangement, diminished FAK(Y397) phosphorylation, and decrease of FAK-actin and FAK-cSrc association, indicating disassembly of focal adhesion processes, a phenomenon that often results in anoikis. Accordingly, NADPH oxidase inhibition also enhanced hypodiploid DNA content, and caspase-3 activation, suggesting activation of the apoptotic machinery. NOX4 is likely to be involved in these effects, since silencing of NOX4 significantly inhibited basal ROS production, reduced FAK(Y397) phosphorylation and decreased tumor cell viability. Altogether, the results suggest that intracellular ROS generated by the NADPH oxidase, most likely NOX4, transmits cell survival signals on melanoma cells through the FAK pathway, maintaining adhesion contacts and cell viability.
- Nox NADPH Oxidases and the Endoplasmic Reticulum(2014) LAURINDO, Francisco R. M.; ARAUJO, Thais L. S.; ABRAHAO, Thalita B.Significance: Understanding isoform- and context-specific subcellular Nox reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase compartmentalization allows relevant functional inferences. This review addresses the interplay between Nox NADPH oxidases and the endoplasmic reticulum (ER), an increasingly evident player in redox pathophysiology given its role in redox protein folding and stress responses. Recent Advances: Catalytic/regulatory transmembrane subunits are synthesized in the ER and their processing includes folding, N-glycosylation, heme insertion, p22phox heterodimerization, as shown for phagocyte Nox2. Dual oxidase (Duox) maturation also involves the regulation by ER-resident Duoxa2. The ER is the activation site for some isoforms, typically Nox4, but potentially other isoforms. Such location influences redox/Nox-mediated calcium signaling regulation via ER targets, such as sarcoendoplasmic reticulum calcium ATPase (SERCA). Growing evidence suggests that Noxes are integral signaling elements of the unfolded protein response during ER stress, with Nox4 playing a dual prosurvival/proapoptotic role in this setting, whereas Nox2 enhances proapoptotic signaling. ER chaperones such as protein disulfide isomerase (PDI) closely interact with Noxes. PDI supports growth factor-dependent Nox1 activation and mRNA expression, as well as migration in smooth muscle cells, and PDI overexpression induces acute spontaneous Nox activation. Critical Issues: Mechanisms of PDI effects include possible support of complex formation and RhoGTPase activation. In phagocytes, PDI supports phagocytosis, Nox activation, and redox-dependent interactions with p47phox. Together, the results implicate PDI as possible Nox organizer. Future Directions: We propose that convergence between Noxes and ER may have evolutive roots given ER-related functional contexts, which paved Nox evolution, namely calcium signaling and pathogen killing. Overall, the interplay between Noxes and the ER may provide relevant insights in Nox-related (patho)physiology.
- Coronary artery plaque burden and calcium scores in healthy men adhering to long-term wine drinking or alcohol abstinence(2014) LUZ, P. L. da; COIMBRA, S.; FAVARATO, D.; ALBUQUERQUE, C.; MOICHIDUKI, R. I.; ROCHITTE, C. E.; HOJAIJ, E.; GONSALVES, C. R. L.; LAURINDO, F. R.Observational studies suggest there are clinical benefits to moderate red wine (RW) consumption. However, the effects on coronary vasculature and overall lifestyle are unclear. We investigated whether a lifestyle of regular long-term RW consumption is associated with changes in coronary plaque burden, calcium score, carotid intima/media thickness, endothelial function, and metabolic variables, compared with alcohol abstinence. Healthy volunteers were evaluated by coronary computed tomography angiography (CTA) as well as carotid and brachial artery ultrasound. Nutritional status, psychological status, and metabolic variables were assessed. The study included 101 drinkers [aged 58.9 +/- 7.3 years (means +/- SD)], from wine brotherhoods, and 104 abstainers, from Anglican, Evangelical and Catholic churches both in the city of Sao Paulo, Brazil. No significant differences in demographics were noted. Lesion prevalence per patient assessed by coronary CTA and classified as absent (0), 1-25, 26-49, and >= 50% stenosis was similar between groups. When analyzed by individual arteries, i.e., left anterior descending, circumflex, and right coronary, prevalence was also not different. On the other hand, calcium scores were higher among drinkers than abstainers (144.4 +/- 362.2 vs 122.0 +/- 370.3; P < 0.01). However, drinkers reported less history of diabetes and exercised more. RW drinkers consumed 2127.9 +/- 387.7 kcal/day while abstainers consumed 1836.0 +/- 305.0 (P < 0.0001). HDL cholesterol was significantly higher among drinkers compared to abstainers (46.9 +/- 10.9 vs 39.5 +/- 9.0 mg/dL; P < 0.001), while fasting plasma glucose was lower (97.6 +/- 18.2 vs 118.4 +/- 29.6 mg/dL; P < 0.02). Liver enzymes were normal in both groups. In conclusion, long-term wine drinkers displayed a similar plaque burden but greater calcium score than abstainers, despite a more atherogenic diet, and the mechanisms for the increased calcium scores in the former remain speculative.
- Methods of measuring protein disulfide isomerase activity: a critical overview(2014) WATANABE, Monica M.; LAURINDO, Francisco R. M.; FERNANDES, Denise C.Protein disulfide isomerase is an essential redox chaperone from the endoplasmic reticulum (ER) and is responsible for correct disulfide bond formation in nascent proteins. PDI is also found in other cellular locations in the cell, particularly the cell surface. Overall, PDI contributes to ER and global cell redox homeostasis and signaling. The knowledge about PDI structure and function progressed substantially based on in vitro studies using recombinant PDI and chimeric proteins. In these experimental scenarios, PDI reductase and chaperone activities are readily approachable. In contrast, assays to measure PDI isomerase activity, the hallmark of PDI family, are more complex. Assessment of PDI roles in cells and tissues mainly relies on gain- or loss-of-function studies. However, there is limited information regarding correlation of experimental readouts with the distinct types of PDI activities. In this mini-review, we evaluate the main methods described for measuring the different kinds of PDI activity: thiol reductase, thiol oxidase, thiol isomerase and chaperone. We emphasize the need to use appropriate controls and the role of critical interferents (e.g., detergent, presence of reducing agents). We also discuss the translation of results from in vitro studies with purified recombinant PDI to cellular and tissue samples, with critical comments on the interpretation of results.
- Estudo Pré-Clínico de Stent com Polímero Biodegradável e Liberação Abluminal de Sirolimus(2014) TAKIMURA, Celso Kiyochi; CAMPOS, Carlos Augusto Homem M.; MELO, Pedro Henrique Magualhães Craveiro; CAMPOS, Julliana Carvalho; GUTIERREZ, Paulo Sampaio; BORGES, Thiago Francisco Costa; CURADO, Luciano; MORATO, Spero Penha; LAURINDO, Francisco Rafael Martins; LEMOS NETO, Pedro AlvesBackground: Bioabsorbable polymer stents with drug elution only on the abluminal surface may be safer than durable polymer drug-eluting stents. Objective: To report the experimental findings with the Inspiron(tm) stent - a bioabsorbable polymer-coated stent with sirolimus release from the abluminal surface only, recently approved for clinical use. Methods: 45 stents were implanted in the coronary arteries of 15 pigs. On day 28 after implantation, angiographic, intracoronary ultrasonographic and histomorphological data were collected. Five groups were analyzed: Group I (nine bare-metal stents); Group II (nine coated with bioabsorbable polymer on the luminal and abluminal surfaces); Group III (eight stents coated with bioabsorbable polymer on the abluminal surface); Group IV (nine stents with bioabsorbable polymer and sirolimus on the luminal and abluminal surfaces); and Group V (ten stents with bioabsorbable polymer and sirolimus only on the abluminal surface). Results: The following results were observed for Groups I, II, III, IV and V, respectively: percentage stenosis of 29 ± 20; 36 ± 14; 33 ± 19; 22 ± 13 and 26 ± 15 (p = 0.443); late lumen loss (in mm) of 1.02 ± 0.60; 1.24 ± 0.48; 1.11 ± 0.54; 0.72 ± 0.44 and 0.78 ± 0.39 (p = 0.253); neointimal area (in mm2)) of 2.60 ± 1.99; 2.74 ± 1.51; 2.74 ± 1.30; 1.30 ± 1.14 and 0.97 ± 0.84 (p = 0.001; Groups IV and V versus Groups I, II and III); and percentage neointimal area of 35 ± 25; 38 ± 18; 39 ± 19; 19 ± 18 and 15 ± 12 (p = 0.001; Groups IV and V versus Groups I, II and III). Injury and inflammation scores were low and with no differences between the groups. Conclusion: The Inspiron(tm) stent proved to be safe and was able to significantly inhibit the neointimal hyperplasia observed on day 28 after implantation in porcine coronary arteries.
- Mechanisms underlying hypertriglyceridemia in rats with monosodium L-glutamate-induced obesity: Evidence of XBP-1/PDI/MTP axis activation(2014) FRANCA, Lucas Martins; FREITAS, Larissa Nara Costa; CHAGAS, Vinicyus Teles; COELHO, Caio Fernando Ferreira; BARROSO, Wermerson Assuncao; COSTA, Graciomar Conceicao; SILVA, Lucilene Amorim; DEBBAS, Victor; LAURINDO, Francisco Rafael Martins; PAES, Antonio Marcus de AndradeNon-alcoholic fatty liver disease (NAFLD) is intimately associated with insulin resistance and hypertriglyceridemia, whereas many of the mechanisms underlying this association are still poorly understood. In the present study, we investigated the relationship between microsomal triglyceride transfer protein (MTP) and markers of endoplasmic reticulum (ER) stress in the liver of rats subjected to neonatal monosodium L-glutamate (MSG)-induced obesity. At age 120 days old, the MSG-obese animals exhibited hyperglycemia, hypertriglyceridemia, insulin resistance, and liver steatosis, while the control (CTR) group did not. Analysis using fast protein liquid chromatography of the serum lipoproteins revealed that the triacylglycerol content of the very low-density lipoprotein (VLDL) particles was twice as high in the MSG animals compared with the CTR animals. The expression of ER stress markers, GRP76 and GRP94, was increased in the MSG rats, promoting a higher expression of X-box binding protein 1 (XBP-1), protein disulfide isomerase (PDI), and MTP. As the XBP-1/PDI/MTP axis has been suggested to represent a significant lipogenic mechanism in the liver response to ER stress, our data indicate that hypertriglyceridemia and liver steatosis occurring in the MSG rats are associated with increased MTP expression.