GABRIELA VENTURINI DA SILVA
Projetos de Pesquisa
Unidades Organizacionais
LIM/13 - Laboratório de Genética e Cardiologia Molecular, Hospital das Clínicas, Faculdade de Medicina
6 resultados
Resultados de Busca
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- Proteome analysis of acute kidney injury - Discovery of new predominantly renal candidates for biomarker of kidney disease(2017) MALAGRINO, Pamella Araujo; VENTURINI, Gabriela; YOGI, Patricia Schneider; DARIOLLI, Rafael; PADILHA, Kallyandra; KIERS, Bianca; GOIS, Tamiris Carneiro; CARDOZO, Karina Helena Morais; CARVALHO, Valdemir Melechco; SALGUEIRO, Jessica Silva; GIRARDI, Adriana Castello Costa; TITAN, Silvia Maria de Oliveira; KRIEGER, Jose Eduardo; PEREIRA, Alexandre CostaThe main bottleneck in studies aiming to identify novel biomarkers in acute kidney injury (AKI) has been the identification of markers that are organ and process specific. Here, we have used different tissues from a controlled porcine renal ischemia/reperfusion (I/R) model to identify new, predominantly renal biomarker candidates for kidney disease. Urine and serum samples were analyzed in pre-ischemia, ischemia (60 min) and 4, 11 and 16 h post-reperfusion, and renal cortex samples after 24 h of reperfusion. Peptides were analyzed on the Q-Exactive (TM). In renal cortex proteome, we observed an increase in the synthesis of proteins in the ischemic kidney compared to the contralateral, highlighted by transcription factors and epithelial adherens junction proteins. Intersecting the set of proteins up- or down-regulated in the ischemic tissue with both serum and urine proteomes, we identified 6 proteins in the serum that may provide a set of targets for kidney injury. Additionally, we identified 49, being 4 predominantly renal, proteins in urine. As prove of concept, we validated one of the identified biomarkers, dipeptidyl peptidase IV, in a set of patients with diabetic nephropathy. In conclusion, we identified 55 systemic proteins, some of them predominantly renal, candidates for biomarkers of renal disease. Biological significance: The main bottleneck in studies aiming to identify novel biomarkers in acute kidney injury (AKI) has been the identification of markers that are predominantly renal. In fact, putative biomarkers for this condition have also been identified in a number of other clinical scenarios, such as cardiovascular diseases, chronic kidney failure or in patients being treated in intensive care units from a number of conditions. Here we propose a comprehensive, sequential screening procedure able to identify and validate potential biomarkers for kidney disease, using kidney ischemia/reperfusion as a paradigm for a kidney pathological event.
- 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.
- mTOR signaling inhibition decreases lysosome migration and impairs the success of Trypanosoma cruzi infection and replication in cardiomyocytes(2023) ALVIM, Juliana M.; VENTURINI, Gabriela; OLIVEIRA, Theo G. M.; SEIDMAN, Jonathan G.; SEIDMAN, Christine E.; KRIEGER, Jose E.; PEREIRA, Alexandre C.Chagas disease is caused by the parasite Trypanosoma cruzi (T. cruzi) and, among all the chronic manifestations of the disease, Chronic Chagas Cardiomyopathy (CCC) is the most severe outcome. Despite high burden and public health importance in Latin America, there is a gap in understanding the molecular mechanisms that results in CCC development. Previous studies showed that T. cruzi uses the host machinery for infection and replication, including the repurposing of the responses to intracellular infection such as mitochondrial activity, vacuolar membrane, and lysosomal activation in benefit of parasite infection and replication. One common signaling upstream to many responses to parasite infection is mTOR pathway, previous associated to several downstream cellular mechanisms including autophagy, mitophagy and lysosomal activation. Here, using human iPSC derived cardiomyocytes (hiPSC-CM), we show the mTOR pathway is activated in hiPSC-CM after T. cruzi infection, and the inhibition of mTOR with rapamycin reduced number of T. cruzi 48 h post infection (hpi). Rapamycin treatment also reduced lysosome migration from nuclei region to cell periphery resulting in less T. cruzi inside the parasitophorous vacuole (PV) in the first hour of infection. In addition, the number of parasites leaving the PV to the cytoplasm to replicate in later times of infection was also lower after rapamycin treatment. Altogether, our data suggest that host's mTOR activation concomitant with parasite infection modulates lysosome migration and that T. cruzi uses this mechanism to achieve infection and replication. Modulating this mechanism with rapamycin impaired the success of T. cruzi life cycle independent of mitophagy.
- Vascular smooth muscle cells exhibit a progressive loss of rigidity with serial culture passaging(2012) DINARDO, Carla Luana; VENTURINI, Gabriela; OMAE, Samantha Vieira; ZHOU, Enhua H.; MOTTA-LEAL-FILHO, Joaquim Maurcio da; DARIOLLI, Rafael; KRIEGER, Jose Eduardo; ALENCAR, Adriano Mesquita; PEREIRA, Alexandre CostaOne drawback of in vitro cell culturing is the dedifferentiation process that cells experience. Smooth muscle cells (SMC) also change molecularly and morphologically with long term culture. The main objective of this study was to evaluate if culture passages interfere in vascular SMC mechanical behavior. SMC were obtained from five different porcine arterial beds. Optical magnetic twisting cytometry (OMTC) was used to characterize mechanically vascular SMC from different cultures in distinct passages and confocal microscopy/western blotting, to evaluate cytoskeleton and extracellular matrix proteins. We found that vascular SMC rigidity or viscoelastic complex modulus (G) decreases with progression of passages. A statistically significant negative correlation between G and passage was found in four of our five cultures studied. Phalloidin-stained SMC from higher passages exhibited lower mean signal intensity per cell (confocal microscopy) and quantitative western blotting analysis showed a decrease in collagen I content throughout passages. We concluded that vascular SMC progressively lose their stiffness with serial culture passaging. Thus, limiting the number of passages is essential for any experiment measuring viscoelastic properties of SMC in culture.
- Cardiomyocyte infection by Trypanosoma cruzi promotes innate immune response and glycolysis activation(2023) VENTURINI, Gabriela; ALVIM, Juliana M.; PADILHA, Kallyandra; TOEPFER, Christopher N.; GORHAM, Joshua M.; WASSON, Lauren K.; BIAGI, Diogo; SCHENKMAN, Sergio; CARVALHO, Valdemir M.; SALGUEIRO, Jessica S.; CARDOZO, Karina H. M.; KRIEGER, Jose E.; PEREIRA, Alexandre C.; SEIDMAN, Jonathan G.; SEIDMAN, Christine E.IntroductionChagas cardiomyopathy, a disease caused by Trypanosoma cruzi (T. cruzi) infection, is a major contributor to heart failure in Latin America. There are significant gaps in our understanding of the mechanism for infection of human cardiomyocytes, the pathways activated during the acute phase of the disease, and the molecular changes that lead to the progression of cardiomyopathy. MethodsTo investigate the effects of T. cruzi on human cardiomyocytes during infection, we infected induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) with the parasite and analyzed cellular, molecular, and metabolic responses at 3 hours, 24 hours, and 48 hours post infection (hpi) using transcriptomics (RNAseq), proteomics (LC-MS), and metabolomics (GC-MS and Seahorse) analyses. ResultsAnalyses of multiomic data revealed that cardiomyocyte infection caused a rapid increase in genes and proteins related to activation innate and adaptive immune systems and pathways, including alpha and gamma interferons, HIF-1 alpha signaling, and glycolysis. These responses resemble prototypic responses observed in pathogen-activated immune cells. Infection also caused an activation of glycolysis that was dependent on HIF-1 alpha signaling. Using gene editing and pharmacological inhibitors, we found that T. cruzi uptake was mediated in part by the glucose-facilitated transporter GLUT4 and that the attenuation of glycolysis, HIF-1 alpha activation, or GLUT4 expression decreased T. cruzi infection. In contrast, pre-activation of pro-inflammatory immune responses with LPS resulted in increased infection rates. ConclusionThese findings suggest that T. cruzi exploits a HIF-1 alpha-dependent, cardiomyocyte-intrinsic stress-response activation of glycolysis to promote intracellular infection and replication. These chronic immuno-metabolic responses by cardiomyocytes promote dysfunction, cell death, and the emergence of cardiomyopathy.
- Variation of mechanical properties and quantitative proteomics of VSMC along the arterial tree(2014) DINARDO, Carla Luana; VENTURINI, Gabriela; ZHOU, Enhua H.; WATANABE, Ii Sei; CAMPOS, Luciene Cristina Gastalho; DARIOLLI, Rafael; MOTTA-LEAL-FILHO, Joaquim Mauricio da; CARVALHO, Valdemir Melechco; CARDOZO, Karina Helena Morais; KRIEGER, Jose Eduardo; ALENCAR, Adriano Mesquita; PEREIRA, Alexandre CostaVascular smooth muscle cells (VSMCs) are thought to assume a quiescent and homogeneous mechanical behavior after arterial tree development phase. However, VSMCs are known to be molecularly heterogeneous in other aspects and their mechanics may play a role in pathological situations. Our aim was to evaluate VSMCs from different arterial beds in terms of mechanics and proteomics, as well as investigate factors that may influence this phenotype. VSMCs obtained from seven arteries were studied using optical magnetic twisting cytometry (both in static state and after stretching) and shotgun proteomics. VSMC mechanical data were correlated with anatomical parameters and ultrastructural images of their vessels of origin. Femoral, renal, abdominal aorta, carotid, mammary, and thoracic aorta exhibited descending order of stiffness (G, P < 0.001). VSMC mechanical data correlated with the vessel percentage of elastin and amount of surrounding extracellular matrix (ECM), which decreased with the distance from the heart. After 48 h of stretching simulating regional blood flow of elastic arteries, VSMCs exhibited a reduction in basal rigidity. VSMCs from the thoracic aorta expressed a significantly higher amount of proteins related to cytoskeleton structure and organization vs. VSMCs from the femoral artery. VSMCs are heterogeneous in terms of mechanical properties and expression/organization of cytoskeleton proteins along the arterial tree. The mechanical phenotype correlates with the composition of ECM and can be modulated by cyclic stretching imposed on VSMCs by blood flow circumferential stress.