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
5 resultados
Resultados de Busca
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- Transit of H2O2 across the endoplasmic reticulum membrane is not sluggish(2016) APPENZELLER-HERZOG, Christian; BANHEGYI, Gabor; BOGESKI, Ivan; DAVIES, Kelvin J. A.; DELAUNAY-MOISAN, Agnes; FORMAN, Henry Jay; GOERLACH, Agnes; KIETZMANN, Thomas; LAURINDO, Francisco; MARGITTAI, Eva; MEYER, Andreas J.; RIEMER, Jan; RUTZLER, Michael; SIMMEN, Thomas; SITIA, Roberto; TOLEDANO, Michel B.; TOUW, Ivo P.Cellular metabolism provides various sources of hydrogen peroxide (H2O2) in different organelles and compartments. The suitability of H2O2 as an intracellular signaling molecule therefore also depends on its ability to pass cellular membranes. The propensity of the membranous boundary of the endoplasmic reticulum (ER) to let pass H2O2 has been discussed controversially. In this essay, we challenge the recent proposal that the ER membrane constitutes a simple barrier for H2O2 diffusion and support earlier data showing that (i) ample H2O2 permeability of the ER membrane is a prerequisite for signal transduction, (ii) aquaporin channels are crucially involved in the facilitation of H2O2 permeation, and (iii) a proper experimental framework not prone to artifacts is necessary to further unravel the role of H2O2 permeation in signal transduction and organelle biology.
- The flavo-oxidase QSOX1 supports vascular smooth muscle cell migration and proliferation: Evidence for a role in neointima growth(2015) BORGES, Beatriz E.; APPEL, Marcia H.; COFRE, Axel R.; PRADO, Maiara L.; STECLAN, Chelin A.; ESNARD, Frederic; ZANATA, Silvio M.; LAURINDO, Francisco R. M.; NAKAO, Lia S.Quiescin sulfhydryl oxidase 1 (QSOX1) is a flavoenzyme largely present in the extracellular milieu whose physiological functions and substrates are not known. QSOX1 has been implicated in the regulation of tumor cell survival, proliferation and migration, in addition to extracellular matrix (ECM) remodeling. However, data regarding other pathophysiological conditions are still lacking. Arterial injury by balloon catheter is an established model of post-angioplasty restenosis. This technique induces neointima formation due to migration and proliferation of vascular smooth muscle cells (VSMC), followed by ECM synthesis and remodeling. Here, we show that QSOX1 knockdown inhibited VSMC migration and proliferation in vitro. In contrast, QSOX1 overexpression stimulated these processes. While migration could be induced by the incubation of cells with the active recombinant QSOX1, proliferation was induced by addition of the active and also of an inactive mutant QSOX1 protein. The proliferation induced by both recombinants was independent of intracellular hydrogen peroxide and dependent of the MEK/ERK pathway. To recapitulate in vivo VSMC pathophysiology, balloon-induced arterial injury was performed. The expression of QSOX1 in the neointimal layer of balloon-injured rat carotids was high and peaked at 14 days post-injury. In vivo QSOX1 knockdown led to a significant decrease in PCNA expression at day 14 post-injury and a decreased intima/media area ratio at day 21 post-injury, compared with scrambled siRNA transfection. In summary, our findings demonstrate that QSOX1 induces VSMC migration and proliferation in vitro and contributes to neointima thickening in balloon-injured rat carotids. (C) 2015 Published by Elsevier B.V.
- Co-Exposure of Cardiomyocytes to IFN-gamma and TNF-alpha Induces Mitochondrial Dysfunction and Nitro-Oxidative Stress: Implications for the Pathogenesis of Chronic Chagas Disease Cardiomyopathy(2021) NUNES, Joao Paulo Silva; ANDRIEUX, Pauline; BROCHET, Pauline; ALMEIDA, Rafael Ribeiro; KITANO, Eduardo; HONDA, Andre Kenji; IWAI, Leo Kei; ANDRADE-SILVA, Debora; GOUDENEGE, David; SILVA, Karla Deysiree Alcantara; VIEIRA, Raquel de Souza; LEVY, Debora; BYDLOWSKI, Sergio Paulo; GALLARDO, Frederic; TORRES, Magali; BOCCHI, Edimar Alcides; MANO, Miguel; SANTOS, Ronaldo Honorato Barros; BACAL, Fernando; POMERANTZEFF, Pablo; LAURINDO, Francisco Rafael Martins; TEIXEIRA, Priscila Camillo; NAKAYA, Helder I.; KALIL, Jorge; PROCACCIO, Vincent; CHEVILLARD, Christophe; CUNHA-NETO, EdecioInfection by the protozoan Trypanosoma cruzi causes Chagas disease cardiomyopathy (CCC) and can lead to arrhythmia, heart failure and death. Chagas disease affects 8 million people worldwide, and chronic production of the cytokines IFN-gamma and TNF-alpha by T cells together with mitochondrial dysfunction are important players for the poor prognosis of the disease. Mitochondria occupy 40% of the cardiomyocytes volume and produce 95% of cellular ATP that sustain the life-long cycles of heart contraction. As IFN-gamma and TNF-alpha have been described to affect mitochondrial function, we hypothesized that IFN-gamma and TNF-alpha are involved in the myocardial mitochondrial dysfunction observed in CCC patients. In this study, we quantified markers of mitochondrial dysfunction and nitro-oxidative stress in CCC heart tissue and in IFN-gamma/TNF-alpha-stimulated AC-16 human cardiomyocytes. We found that CCC myocardium displayed increased levels of nitro-oxidative stress and reduced mitochondrial DNA as compared with myocardial tissue from patients with dilated cardiomyopathy (DCM). IFN-gamma/TNF-alpha treatment of AC-16 cardiomyocytes induced increased nitro-oxidative stress and decreased the mitochondrial membrane potential (Delta psi m). We found that the STAT1/NF-kappa B/NOS2 axis is involved in the IFN-gamma/TNF-alpha-induced decrease of Delta psi m in AC-16 cardiomyocytes. Furthermore, treatment with mitochondria-sparing agonists of AMPK, NRF2 and SIRT1 rescues Delta psi m in IFN-gamma/TNF-alpha-stimulated cells. Proteomic and gene expression analyses revealed that IFN-gamma/TNF-alpha-treated cells corroborate mitochondrial dysfunction, transmembrane potential of mitochondria, altered fatty acid metabolism and cardiac necrosis/cell death. Functional assays conducted on Seahorse respirometer showed that cytokine-stimulated cells display decreased glycolytic and mitochondrial ATP production, dependency of fatty acid oxidation as well as increased proton leak and non-mitochondrial oxygen consumption. Together, our results suggest that IFN-gamma and TNF-alpha cause direct damage to cardiomyocytes' mitochondria by promoting oxidative and nitrosative stress and impairing energy production pathways. We hypothesize that treatment with agonists of AMPK, NRF2 and SIRT1 might be an approach to ameliorate the progression of Chagas disease cardiomyopathy.
- Subverted regulation of Nox1 NADPH oxidase-dependent oxidant generation by protein disulfide isomerase A1 in colon carcinoma cells with overactivated KRas(2019) BESSA, Tiphany Coralie De; PAGANO, Alessandra; MORETTI, Ana Iochabel Soares; OLIVEIRA, Percillia Victoria Santos; MENDONCA, Samir Andrade; KOVACIC, Herve; LAURINDO, Francisco Rafael MartinsProtein disulfide isomerases including PDIA1 are implicated in cancer progression, but underlying mechanisms are unclear. PDIA1 is known to support vascular Nox1 NADPH oxidase expression/activation. Since deregulated reactive oxygen species (ROS) production underlies tumor growth, we proposed that PDIA1 is an upstream regulator of tumor-associated ROS. We focused on colorectal cancer (CRC) with distinct KRas activation levels. Analysis of RNAseq databanks and direct validation indicated enhanced PDIA1 expression in CRC with constitutive high (HCT116) vs. moderate (HKE3) and basal (Caco2) Ras activity. PDIA1 supported Nox1-dependent superoxide production in CRC; however, we first reported a dual effect correlated with Ras-level activity: in Caco2 and HKE3 cells, loss-of-function experiments indicate that PDIA1 sustains Nox1-dependent superoxide production, while in HCT116 cells PDIA1 restricted superoxide production, a behavior associated with increased Rac1 expression/activity. Transfection of Rac1G12V active mutant into HKE3 cells induced PDIA1 to become restrictive of Nox1-dependent superoxide, while in HCT116 cells treated with Rac1 inhibitor, PDIA1 became supportive of superoxide. PDIA1 silencing promoted diminished cell proliferation and migration in HKE3, not detectable in HCT116 cells. Screening of cell signaling routes affected by PDIA1 silencing highlighted GSK3 beta and Stat3. Also, E-cadherin expression after PDIA1 silencing was decreased in HCT116, consistent with PDIA1 support of epithelial-mesenchymal transition. Thus, Ras overactivation switches the pattern of PDIA1-dependent Rac1/Nox1 regulation, so that Ras-induced PDIA1 bypass can directly activate Rac1. PDIA1 may be a crucial regulator of redox-dependent adaptive processes related to cancer progression.
- Protein Disulfide Isomerase Is Required for Platelet-derived Growth Factor-induced Vascular Smooth Muscle Cell Migration, Nox1 NADPH Oxidase Expression, and RhoGTPase Activation(2012) PESCATORE, Luciana A.; BONATTO, Diego; FORTI, Fabio L.; SADOK, Amine; KOVACIC, Herve; LAURINDO, Francisco R. M.Vascular Smooth Muscle Cell (VSMC) migration into vessel neointima is a therapeutic target for atherosclerosis and postinjury restenosis. Nox1 NADPH oxidase-derived oxidants synergize with growth factors to support VSMC migration. We previously described the interaction between NADPH oxidases and the endoplasmic reticulum redox chaperone protein disulfide isomerase (PDI) in many cell types. However, physiological implications, as well as mechanisms of such association, are yet unclear. We show here that platelet-derived growth factor (PDGF) promoted subcellular redistribution of PDI concomitant to Nox1-dependent reactive oxygen species production and that siRNA-mediated PDI silencing inhibited such reactive oxygen species production, while nearly totally suppressing the increase in Nox1 expression, with no change in Nox4. Furthermore, PDI silencing inhibited PDGF-induced VSMC migration assessed by distinct methods, whereas PDI overexpression increased spontaneous basal VSMC migration. To address possible mechanisms of PDI effects, we searched for PDI interactome by systems biology analysis of physical protein-protein interaction networks, which indicated convergence with small GTPases and their regulator RhoGDI. PDI silencing decreased PDGF-induced Rac1 and RhoA activities, without changing their expression. PDI co-immunoprecipitated with RhoGDI at base line, whereas such association was decreased after PDGF. Also, PDI co-immunoprecipitated with Rac1 and RhoA in a PDGF-independent way and displayed detectable spots of perinuclear co-localization with Rac1 and RhoGDI. Moreover, PDI silencing promoted strong cytoskeletal changes: disorganization of stress fibers, decreased number of focal adhesions, and reduced number of RhoGDI-containing vesicular recycling adhesion structures. Overall, these data suggest that PDI is required to support Nox1/redox and GTPase-dependent VSMC migration.