ELIDA ADALGISA NERI
Projetos de Pesquisa
Unidades Organizacionais
Instituto do Coração, Hospital das Clínicas, Faculdade de Medicina
LIM/13 - Laboratório de Genética e Cardiologia Molecular, Hospital das Clínicas, Faculdade de Medicina
LIM/13 - Laboratório de Genética e Cardiologia Molecular, Hospital das Clínicas, Faculdade de Medicina
12 resultados
Resultados de Busca
Agora exibindo 1 - 10 de 12
- Time-regulated transcripts with the potential to modulate human pluripotent stem cell-derived cardiomyocyte differentiation(2022) MUNOZ, Juan J. A. M.; DARIOLLI, Rafael; SILVA, Caio Mateus da; NERI, Elida A.; VALADAO, Iuri C.; TURACA, Lauro Thiago; LIMA, Vanessa M.; CARVALHO, Mariana Lombardi Peres de; VELHO, Mariliza R.; SOBIE, Eric A.; KRIEGER, Jose E.Background Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are a promising disease model, even though hiPSC-CMs cultured for extended periods display an undifferentiated transcriptional landscape. MiRNA-target gene interactions contribute to fine-tuning the genetic program governing cardiac maturation and may uncover critical pathways to be targeted. Methods We analyzed a hiPSC-CM public dataset to identify time-regulated miRNA-target gene interactions based on three logical steps of filtering. We validated this process in silico using 14 human and mouse public datasets, and further confirmed the findings by sampling seven time points over a 30-day protocol with a hiPSC-CM clone developed in our laboratory. We then added miRNA mimics from the top eight miRNAs candidates in three cell clones in two different moments of cardiac specification and maturation to assess their impact on differentiation characteristics including proliferation, sarcomere structure, contractility, and calcium handling. Results We uncovered 324 interactions among 29 differentially expressed genes and 51 miRNAs from 20,543 transcripts through 120 days of hiPSC-CM differentiation and selected 16 genes and 25 miRNAs based on the inverse pattern of expression (Pearson R-values < - 0.5) and consistency in different datasets. We validated 16 inverse interactions among eight genes and 12 miRNAs (Person R-values < - 0.5) during hiPSC-CMs differentiation and used miRNAs mimics to verify proliferation, structural and functional features related to maturation. We also demonstrated that miR-124 affects Ca2+ handling altering features associated with hiPSC-CMs maturation. Conclusion We uncovered time-regulated transcripts influencing pathways affecting cardiac differentiation/maturation axis and showed that the top-scoring miRNAs indeed affect primarily structural features highlighting their role in the hiPSC-CM maturation.
- Beneficial effects of IL-4 and IL-6 on rat neonatal target cardiac cells(2020) ZOGBI, Camila; OLIVEIRA, Nathalia C.; LEVY, Debora; BYDLOWSKI, Sergio P.; BASSANEZE, Vinicius; NERI, Elida A.; KRIEGER, Jose E.The nature of the early post-natal immune response in rodents appears to influence cardiac regeneration even though the underlying molecules remain poorly understood. Consistent with this idea, we show now significant changes in the expression of immune and cell movement gene pathways in heart samples from 1- and 7-day-old rats with ventricle resection. We then tested whether conditioned media from adult M2 anti-inflammatory macrophages target neonatal cardiac cells to a pro-regenerative like phenotype compared to the M1 pro-inflammatory macrophages. We found that M2 compared to M1 macrophage-conditioned media upregulates neonatal cardiomyocyte proliferation, suppresses myofibroblast-induced differentiation and stimulates endothelial cell tube formation. Using a cytokine array, we selected four candidate cytokine molecules uniquely expressed in M2 macrophage-conditioned media and showed that two of them (IL-4 and IL-6) induce endothelial cell proliferation whilst IL-4 promotes proliferation in neonatal cardiomyocytes and prevents myofibroblast-induced collagen type I secretion. Altogether, we provided evidence that adult M2 macrophage-conditioned media displays a paracrine beneficial pro-regenerative response in target cardiac cells and that IL-4 and IL-6 recapitulate, at least in part, these pleiotropic effects. Further characterization of macrophage immune phenotypes and their secreted molecules may give rise to novel therapeutic approaches for post-natal cardiac repair.
- Fructose Acutely Stimulates NHE3 Activity in Kidney Proximal Tubule(2012) QUEIROZ-LEITE, Gabriella D.; CRAJOINAS, Renato O.; NERI, Elida A.; BEZERRA, Camila N. A.; GIRARDI, Adriana C. C.; REBOUCAS, Nancy Amaral; MALNIC, GerhardBackground/Aims: Fructose causes a sodium-sensitive hypertension and acutely reduces the urinary Na+ excretion, suggesting that it may regulate the activity of renal tubular sodium transporters. NHE3 is highly expressed in proximal tubule (PT), along with proteins that mediate fructose transport and metabolism. The present work was outlined to investigate whether fructose modulates proximal NHE3 activity and to elucidate the molecular mechanisms underlying this modulation. Methods/Results: Using in vivo stationary microperfusion, we observed that fructose stimulates NHE3 mediated JHCO(3)(-) reabsorption. The MAPK pathway is not involved in this activation, as demonstrated by using of MEK/MAPK inhibitors, whereas experiments using a PKA inhibitor suggest that PKA inhibition plays a role in this response. These results were confirmed in vitro by measuring the cell pH recovery rate after NH4Cl pulse in LLC-PK1, a pig PT cell line, which showed reduced cAMP levels and NHE3 phosphorylation at serine-552 (PKA consensus site) after fructose treatment. Conclusions: NHE3 activity is stimulated by fructose, which increases proximal tubule Na+ reabsorption. The molecular mechanisms involved in this process are mediated, at least in part, by downregulation of the PKA signaling pathway. Future studies are needed to address whether fructose-stimulated NHE3 activity may contribute to renal injury and hypertension.
- The fungicide Tebuconazole induces electromechanical cardiotoxicity in murine heart and human cardiomyocytes derived from induced pluripotent stem cells(2022) SANTOS-MIRANDA, Artur; JOVIANO-SANTOS, V. Julliane; CRUZ-NASCIMENTO, Taynara; NERI, Elida Adalgisa; SOUZA, Diego Santos; MARQUES, Leisiane Pereira; KRIEGER, Jose E.; ROMAN-CAMPOS, DaniloTebuconazole (TEB) is an important fungicide that belongs to the triazole family. It is widely used in agriculture and its use has experienced a tremendous increase in the last decade. The long-term exposure of humans to this pesticide is a real threat because it is stable in water and soil. The association between long-term exposure to TEB and damage of several biological systems, including hepatotoxicity and cardiotoxicity is evident, however, acute toxicological studies to reveal the toxicity of TEB are limited. This research paper addressed the acute exposure of TEB in murine hearts, cardiomyocytes, and human cardiomyocytes derived from an induced pluripotent stem cell (hiPSC-CMs), spelling out TEB's impact on electromechanical properties of the cardiac tissue. In ex vivo experiments, TEB dose dependently, caused significant electrocardiogram (ECG) remodeling with prolonged PR and QTc interval duration. The TEB was also able to change the action potential waveform in murine cardiomyocytes and hiPSC-CMs. These effects were associated with the ability of the compound to block the L-type calcium current (IC50 = 33.2 +/- 7.4 mu mol.l(-1)) and total outward potassium current (IC50 = 5.7 +/- 1.5 mu mol.l(-1)). TEB also increased the sodium/calcium exchanger current in its forward and reverse modes. Additionally, sarcomere shortening and calcium transient in isolated cardiomyocytes were enhanced when cells were exposed to TEB at 30 mu mol.l(-1). Combined, our results demonstrated that acute TEB exposure affects the cardiomyocyte's electrocontractile properties and triggers the appearance of ECG abnormalities.
- Tebuconazole fungicide interacts with human Nav1.5 channels and alters the cellular excitability of cardiomyocytes derived from human induced pluripotent stem cells(2022) MARQUES, Leisiane; SANTOS-MIRANDA, Artur; JOVIANO-SANTOS, Julliane; NERI, Elida A.; SARMENTO, Jaqueline O.; KRIEGER, Jose Eduardo; CAMPOS, Danilo Roman
- Electrical stimulation applied during differentiation drives the hiPSC-CMs towards a mature cardiac conduction-like cells(2020) CRESTANI, Thayane; STEICHEN, Clara; NERI, Elida; RODRIGUES, Mariliza; FONSECA-ALANIZ, Miriam Helena; ORMROD, Beth; HOLT, Mark R.; PANDEY, Pragati; HARDING, Sian; EHLER, Elisabeth; KRIEGER, Jose E.Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) resemble fetal cardiomyocytes and electrical stimulation (ES) has been explored to mature the differentiated cells. Here, we hypothesize that ES applied at the beginning of the differentiation process, triggers both differentiation of the hiPSC-CMs into a specialized conduction system (CS) phenotype and cell maturation. We applied ES for 15 days starting on day 0 of the differentiation process and found an increased expression of transcription factors and proteins associated with the development and function of CS including Irx3, Nkx2.5 and contactin 2, Hcn4 and Scn5a, respectively. We also found activation of intercalated disc proteins (Nrap and beta-catenin). We detected ES-induced CM maturation as indicated by increased Tnnil and Tnni3 expression. Confocal micrographs showed a shift towards expression of the gap junction protein connexin 40 in ES hiPSC-CM compared to the more dominant expression of connexin 43 in controls. Finally, analysis of functional parameters revealed that ES hiPSC-CMs exhibited faster action potential (AP) depolarization, longer intracellular Ca2+ transients, and slower AP duration at 90% of repolarization, resembling fast conducting fibers. Altogether, we provided evidence that ES during the differentiation of hiPSC to cardiomyocytes lead to development of cardiac conduction-like cells with more mature cytoarchitecture. Thus, hiPSC-CMs exposed to ES during differentiation can be instrumental to develop CS cells for cardiac disease modelling, screening individual drugs on a precison medicine type platform and support the development of novel therapeutics for arrhythmias.
- Multicellular regulation of miR-196a-5p and miR-425-5 from adipose stem cell-derived exosomes and cardiac repair(2022) OLIVEIRA, N. C. de Almeida; NERI, E. A.; SILVA, C. M.; VALADãO, I. C.; FONSECA-ALANIZ, M. H.; ZOGBI, C.; LEVY, D.; BYDLOWSKI, S. P.; KRIEGER, J. E.Cardiac transplantation of adipose-derived stem cells (ASC) modulates the post-myocardial infarction (post-MI) repair response. Biomolecules secreted or shuttled within extracellular vesicles, such as exosomes, may participate in the concerted response. We investigated the exosome’s microRNAs due to their capacity to fine-tune gene expression, potentially affecting the multicellular repair response. We profiled and quantified rat ASC-exosome miRNAs and used bioinformatics to select uncharacterized miRNAs down-regulated in post-MI related to cardiac repair. We selected and validated miR-196a-5p and miR-425-5p as candidates for the concerted response in neonatal cardiomyocytes, cardiac fibroblasts, endothelial cells, and macrophages using a high-content screening platform. Both miRNAs prevented cardiomyocyte ischemia-induced mitochondrial dysfunction and reactive oxygen species production, increased angiogenesis, and polarized macrophages toward the anti-inflammatory M2 immunophenotype. Moreover, miR-196a-5p reduced and reversed myofibroblast activation and decreased collagen expression. Our data provide evidence that the exosome-derived miR-196a-5p and miR-425-5p influence biological processes critical to the concerted multicellular repair response post-MI. © 2022 The Author(s).
- A modified hydrogel production protocol to decrease cellular content(2022) BRAGA, Gabriela Catao Diniz; CAMARGO, Cristina Pires; HARMSEN, Martin Conrad; CORREIA, Aristides Tadeu; SOUZA, Sonia; SEELAENDER, Marilia; NUNES, Viviane Araujo; SANTOS, Jeniffer Farias dos; NERI, Elida Adalgisa; VALADAO, Iuri Cordeiro; MOREIRA, Luiz Felipe Pinho; GEMPERLI, RolfPurpose: To analyze the cytotoxicity and cell in porcine-derived decellularized skin matrix. Methods: We analyzed the effect of multiple decellularization processes by histological analysis, DNA quantification, and flow cytometry. Subsequently, we analyzed the most appropriate hydrogel concentration to minimize cytotoxicity on fibroblast culture and to maximize cell proliferation. Results: After the fourth decellularization, the DNA quantification showed the lowest DNA concentration (< 50 ng/mg). Histological analysis showed no cell components in the hydrogel. Moreover, hematoxylin and eosin showed a heterogeneous structure of collagen fibers. The best hydrogel concentration ranged from 3 to 25%, and there was no significant difference between the 24 hours and seven days. Conclusion: The process of hydrogel production was effective for removing cells and DNA elements. The best hydrogel concentration ranged from 3 to 25%.
- SCN5A compound heterozygosity mutation in Brugada syndrome: Functional consequences and the implication for pharmacological treatment(2021) V, J. Joviano-Santos; SANTOS-MIRANDA, A.; NERI, E. A.; FONSECA-ALANIZ, M. H.; KRIEGER, J. E.; PEREIRA, A. C.; ROMAN-CAMPOS, D.Aims: SCN5A gene encodes the alpha-subunit of Nav1.5, mainly found in the human heart. SCN5A variants are the most common genetic alterations associated with Brugada syndrome (BrS). In rare cases, compound heterozygosity is observed; however, its functional consequences are poorly understood. We aimed to analyze the functional impact of de novo Nav1.5 mutations in compound heterozygosity in distinct alleles (G400R and T1461S positions) previously found in a patient with BrS. Moreover, we evaluated the potential benefits of quinidine to improve the phenotype of mutant Na+ channels in vitro. Materials and methods: The functional properties of human wild-type and Nav1.5 variants were evaluated using whole-cell patch-clamp and immunofluorescence techniques in transiently expressed human embryonic kidney (HEK293) cells. K ey findings: Both variants occur in the highly conservative positions of SCN5A. Although all variants were expressed in the cell membrane, a significant reduction in the Na+ current density (except for G400R alone, which was undetected) was observed along with abnormal biophysical properties, once the variants were expressed in homozygosis and heterozygosis. Interestingly, the incubation of transfected cells with quinidine partially rescued the biophysical properties of the mutant Na+ channel. Significance: De novo compound heterozygosis mutations in SNC5A disrupt the Na+ macroscopic current. Quinidine could partially reverse the in vitro loss-of-function phenotype of Na+ current. Thus, our data provide, for the first time, a detailed biophysical characterization of dysfunctional Na+ channels linked to compound heterozygosity in BrS as well as the benefits of the pharmacological treatment using quinidine on the biophysical properties of Nav1.5.
- NOTCH1 is critical for fibroblast-mediated induction of cardiomyocyte specialization into ventricular conduction system-like cells in vitro(2020) SILVA, Agatha Ribeiro da; NERI, Elide A.; TURACA, Lauro Thiago; DARIOLLI, Rafael; FONSECA-ALANIZ, Miriam H.; SANTOS-MIRANDA, Artur; ROMAN-CAMPOS, Danilo; VENTURINI, Gabriela; KRIEGER, Jose E.Cardiac fibroblasts are present throughout the myocardium and are enriched in the microenvironment surrounding the ventricular conduction system (VCS). Several forms of arrhythmias are linked to VCS abnormalities, but it is still unclear whether VCS malformations are cardiomyocyte autonomous or could be linked to crosstalk between different cell types. We reasoned that fibroblasts influence cardiomyocyte specialization in VCS cells. We developed 2D and 3D culture models of neonatal rat cardiac cells to assess the influence of cardiac fibroblasts on cardiomyocytes. Cardiomyocytes adjacent to cardiac fibroblasts showed a two-fold increase in expression of VCS markers (NAV1.5 and CONTACTIN 2) and calcium transient duration, displaying a Purkinje-like profile. Fibroblast-conditioned media (fCM) was sufficient to activate VCS-related genes (Irx3, Scn5a, Connexin 40) and to induce action potential prolongation, a hallmark of Purkinge phenotype. fCM-mediated response seemed to be spatially-dependent as cardiomyocyte organoids treated with fCM had increased expression of connexin 40 and NAV1.5 primarily on its outer surface. Finally, NOTCH1 activation in both cardiomyocytes and fibroblasts was required for connexin 40 up-regulation (a proxy of VCS phenotype). Altogether, we provide evidence that cardiac fibroblasts influence cardiomyocyte specialization into VCS-like cells via NOTCH1 signaling in vitro.