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1.
Int J Mol Sci ; 22(13)2021 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-34199089

RESUMO

The meniscus possesses low self-healing properties. A perfect regenerative technique for this tissue has not yet been developed. This work aims to evaluate the role of hypoxia in meniscal development in vitro. Menisci from neonatal pigs (day 0) were harvested and cultured under two different atmospheric conditions: hypoxia (1% O2) and normoxia (21% O2) for up to 14 days. Samples were analysed at 0, 7 and 14 days by histochemical (Safranin-O staining), immunofluorescence and RT-PCR (in both methods for SOX-9, HIF-1α, collagen I and II), and biochemical (DNA, GAGs, DNA/GAGs ratio) techniques to record any possible differences in the maturation of meniscal cells. Safranin-O staining showed increments in matrix deposition and round-shape "fibro-chondrocytic" cells in hypoxia-cultured menisci compared with controls under normal atmospheric conditions. The same maturation shifting was observed by immunofluorescence and RT-PCR analysis: SOX-9 and collagen II increased from day zero up to 14 days under a hypoxic environment. An increment of DNA/GAGs ratio typical of mature meniscal tissue (characterized by fewer cells and more GAGs) was observed by biochemical analysis. This study shows that hypoxia can be considered as a booster to achieve meniscal cell maturation, and opens new opportunities in the field of meniscus tissue engineering.


Assuntos
Diferenciação Celular , Hipóxia/metabolismo , Menisco/citologia , Menisco/metabolismo , Animais , Biomarcadores , Células Cultivadas , Condrócitos/metabolismo , Expressão Gênica , Glicosaminoglicanos/metabolismo , Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Imuno-Histoquímica , Suínos , Engenharia Tecidual/métodos
2.
Int J Mol Sci ; 22(9)2021 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-33946750

RESUMO

Genetic testing in Brugada syndrome (BrS) is still not considered to be useful for clinical management of patients in the majority of cases, due to the current lack of understanding about the effect of specific variants. Additionally, family history of sudden death is generally not considered useful for arrhythmic risk stratification. We sought to demonstrate the usefulness of genetic testing and family history in diagnosis and risk stratification. The family history was collected for a proband who presented with a personal history of aborted cardiac arrest and in whom a novel variant in the SCN5A gene was found. Living family members underwent ajmaline testing, electrophysiological study, and genetic testing to determine genotype-phenotype segregation, if any. Patch-clamp experiments on transfected human embryonic kidney 293 cells enabled the functional characterization of the SCN5A novel variant in vitro. In this study, we provide crucial human data on the novel heterozygous variant NM_198056.2:c.5000T>A (p.Val1667Asp) in the SCN5A gene, and demonstrate its segregation with a severe form of BrS and multiple sudden deaths. Functional data revealed a loss of function of the protein affected by the variant. These results provide the first disease association with this variant and demonstrate the usefulness of genetic testing for diagnosis and risk stratification in certain patients. This study also demonstrates the usefulness of collecting the family history, which can assist in understanding the severity of the disease in certain situations and confirm the importance of the functional studies to distinguish between pathogenic mutations and harmless genetic variants.


Assuntos
Síndrome de Brugada/genética , Mutação de Sentido Incorreto , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Adolescente , Adulto , Idoso , Ajmalina/farmacologia , Substituição de Aminoácidos , Síndrome de Brugada/complicações , Síndrome de Brugada/metabolismo , Morte Súbita Cardíaca/etiologia , Eletrocardiografia , Feminino , Testes Genéticos , Células HEK293 , Heterozigoto , Humanos , Mutação com Perda de Função , Masculino , Pessoa de Meia-Idade , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Técnicas de Patch-Clamp , Linhagem , Polimorfismo de Nucleotídeo Único , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
3.
J Cell Physiol ; 236(7): 4857-4873, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33432663

RESUMO

Cardiovascular diseases (CVDs) are the leading cause of death globally and the number of cardiovascular patients, which is estimated to be over 30 million in 2018, represent a challenging issue for the healthcare systems worldwide. Therefore, the identification of novel molecular targets to develop new treatments is an ongoing challenge for the scientific community. In this context, sphingolipids (SLs) have been progressively recognized as potent bioactive compounds that play crucial roles in the modulation of several key biological processes, such as proliferation, differentiation, and apoptosis. Furthermore, SLs involvement in cardiac physiology and pathophysiology attracted much attention, since these molecules could be crucial in the development of CVDs. Among SLs, ceramide and sphingosine-1-phosphate (S1P) represent the most studied bioactive lipid mediators, which are characterized by opposing activities in the regulation of the fate of cardiac cells. In particular, maintaining the balance of the so-called ceramide/S1P rheostat emerged as an important novel therapeutical target to counteract CVDs. Thus, this review aims at critically summarizing the current knowledge about the antithetic roles of ceramide and S1P in cardiomyocytes dysfunctions, highlighting how the modulation of their metabolism through specific molecules, such as myriocin and FTY720, could represent a novel and interesting therapeutic approach to improve the management of CVDs.


Assuntos
Ceramidas/metabolismo , Transtornos Cerebrovasculares/patologia , Lisofosfolipídeos/metabolismo , Esfingolipídeos/metabolismo , Esfingosina/análogos & derivados , Idoso , Animais , Transtornos Cerebrovasculares/epidemiologia , Transtornos Cerebrovasculares/mortalidade , Doença das Coronárias/patologia , Humanos , Camundongos , Doença Arterial Periférica/patologia , Embolia Pulmonar/patologia , Cardiopatia Reumática/patologia , Esfingosina/metabolismo , Trombose Venosa/patologia
4.
Int J Mol Sci ; 22(2)2021 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-33445410

RESUMO

Dilated cardiomyopathy (DCM) is the leading indication for heart transplantation. TTN gene truncating mutations account for about 25% of familial DCM cases and for 18% of sporadic DCM cases. The clinical relevance of specific variants in TTN has been difficult to determine because of the sheer size of the protein for which TTN encodes, as well as existing extensive genetic variation. Clinicians should communicate novel clinically-relevant variants and genotype-phenotype associations, so that animal studies evaluating the molecular mechanisms are always conducted with a focus on clinical significance. In the present study, we report for the first time the novel truncating heterozygous variant NM_001256850.1:c.72777_72783del (p.Phe24259Leufs*51) in the TTN gene and its association with DCM in a family with sudden death. This variant occurs in the A-band region of the sarcomere, in a known mutational hotspot of the gene. Truncating titin variants that occur in this region are the most common cause of DCM and have been rarely reported in asymptomatic individuals, differently from other pathogenic TTN gene variants. Further studies are warranted to better understand this particular clinically-relevant variant.


Assuntos
Cardiomiopatia Dilatada/complicações , Cardiomiopatia Dilatada/genética , Conectina/genética , Morte Súbita Cardíaca/etiologia , Mutação da Fase de Leitura , Biomarcadores , Cardiomiopatia Dilatada/diagnóstico , Análise Mutacional de DNA , Diagnóstico por Imagem , Eletrocardiografia , Feminino , Estudos de Associação Genética/métodos , Predisposição Genética para Doença , Testes de Função Cardíaca , Humanos , Masculino , Pessoa de Meia-Idade
5.
Eur Heart J ; 42(11): 1082-1090, 2021 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-33221895

RESUMO

AIMS: Brugada syndrome (BrS) is associated with an increased risk of sudden cardiac death due to ventricular tachycardia/fibrillation (VT/VF) in young, otherwise healthy individuals. Despite SCN5A being the most commonly known mutated gene to date, the genotype-phenotype relationship is poorly understood and remains uncertain. This study aimed to elucidate the genotype-phenotype correlation in BrS. METHODS AND RESULTS: Brugada syndrome probands deemed at high risk of future arrhythmic events underwent genetic testing and phenotype characterization by the means of epicardial arrhythmogenic substrate (AS) mapping, and were divided into two groups according to the presence or absence of SCN5A mutation. Two-hundred probands (160 males, 80%; mean age 42.6 ± 12.2 years) were included in this study. Patients harbouring SCN5A mutations exhibited a spontaneous type 1 pattern and experienced aborted cardiac arrest or spontaneous VT/VF more frequently than the other subjects. SCN5A-positive patients exhibited a larger epicardial AS area, more prolonged electrograms and more frequently observed non-invasive late potentials. The presence of an SCN5A mutation explained >26% of the variation in the epicardial AS area and was the strongest predictor of a large epicardial area. CONCLUSION: In BrS, the genetic background is the main determinant for the extent of the electrophysiological abnormalities. SCN5A mutation carriers exhibit more pronounced epicardial electrical abnormalities and a more aggressive clinical presentation. These results contribute to the understanding of the genetic determinants of the BrS phenotypic expression and provide possible explanations for the varying degrees of disease expression.


Assuntos
Síndrome de Brugada , Taquicardia Ventricular , Adulto , Síndrome de Brugada/genética , Eletrocardiografia , Mapeamento Epicárdico , Humanos , Masculino , Pessoa de Meia-Idade , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Fenótipo , Taquicardia Ventricular/genética , Fibrilação Ventricular
6.
Front Cell Dev Biol ; 8: 593508, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33262987

RESUMO

Herein we unveil that Hypoxia-inducible factor-1α (HIF-1α) directly regulates WNT7A expression during myogenesis. In fact, chromatin immunoprecipitation (ChiP) and site-directed mutagenesis experiments revealed two distinct hypoxia response elements (HREs) that are specific HIF-1α binding sites on the WNT7A promoter. Remarkably, a pharmacological activation of HIF-1α induced WNT7A expression and enhanced muscle differentiation. On the other hand, silencing of WNT7A using CRISPR/Cas9 genome editing blocked the effects of HIF-1α activation on myogenesis. Finally, treatment with prolyl hydroxylases (PHDs) inhibitors improved muscle regeneration in vitro and in vivo in a cardiotoxin (CTX)-induced muscle injury mouse model, paving the way for further studies to test its efficacy on acute and chronic muscular pathologies.

7.
Int J Mol Sci ; 21(21)2020 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-33143039

RESUMO

The so-called "sialo-chemical-biology" has become an attractive research area, as an increasing number of natural products containing a sialic acid moiety have been shown to play important roles in biological, pathological, and immunological processes. The intramolecular lactones of sialic acids are a subclass from this crucial family that could have central functions in the discrimination of physiological and pathological conditions. In this review, we report an in-depth analysis of the synthetic achievements in the preparation of the intramolecular lactones of sialic acids (1,4-, 1,7- and γ-lactones), in their free and/or protected form. In particular, recent advances in the synthesis of the 1,7-lactones have allowed the preparation of key sialic acid derivatives. These compounds could be used as authentic reference standards for their correct determination in biological samples, thus overcoming some of the limitations of the previous analytical procedures.


Assuntos
Lactonas/síntese química , Ácidos Siálicos/química
8.
Biochem J ; 477(17): 3401-3415, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32869836

RESUMO

Cardiac fibrosis is a key physiological response to cardiac tissue injury to protect the heart from wall rupture. However, its progression increases heart stiffness, eventually causing a decrease in heart contractility. Unfortunately, to date, no efficient antifibrotic therapies are available to the clinic. This is primarily due to the complexity of the process, which involves several cell types and signaling pathways. For instance, the transforming growth factor beta (TGF-ß) signaling pathway has been recognized to be vital for myofibroblasts activation and fibrosis progression. In this context, complex sphingolipids, such as ganglioside GM3, have been shown to be directly involved in TGF-ß receptor 1 (TGF-R1) activation. In this work, we report that an induced up-regulation of sialidase Neu3, a glycohydrolytic enzyme involved in ganglioside cell homeostasis, can significantly reduce cardiac fibrosis in primary cultures of human cardiac fibroblasts by inhibiting the TGF-ß signaling pathway, ultimately decreasing collagen I deposition. These results support the notion that modulating ganglioside GM3 cell content could represent a novel therapeutic approach for cardiac fibrosis, warranting for further investigations.


Assuntos
Fibroblastos/metabolismo , Gangliosídeo G(M3)/metabolismo , Regulação Enzimológica da Expressão Gênica , Miocárdio/metabolismo , Neuraminidase/biossíntese , Regulação para Cima , Fibroblastos/patologia , Fibrose , Humanos , Miocárdio/patologia , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo
9.
Circ Arrhythm Electrophysiol ; 13(9): e008524, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32755392

RESUMO

BACKGROUND: In Brugada syndrome (BrS), diagnosed in presence of a spontaneous or ajmaline-induced type-1 pattern, ventricular arrhythmias originate from the right ventricle outflow tract (RVOT). We developed a novel CineECG method, obtained by inverse electrocardiogram (ECG) from standard 12-lead ECG, to localize the electrical activity pathway in patients with BrS. METHODS: The CineECG enabled the temporospatial localization of the ECG waveforms, deriving the mean temporospatial isochrone from standard 12-lead ECG. The study sample included (1) 15 patients with spontaneous type-1 Brugada pattern, and (2) 18 patients with ajmaline-induced BrS (at baseline and after ajmaline), in whom epicardial potential duration maps were available; (3) 17 type-3 BrS pattern patients not showing type-1 BrS pattern after ajmaline (ajmaline-negative); (4) 47 normal subjects; (5) 18 patients with right bundle branch block (RBBB). According to CineECG algorithm, each ECG was classified as Normal, Brugada, RBBB, or Undetermined. RESULTS: In patients with spontaneous or ajmaline-induced BrS, CineECG localized the terminal mean temporospatial isochrone forces in the RVOT, congruent with the arrhythmogenic substrate location detected by epicardial potential duration maps. The RVOT location was never observed in normal, RBBB, or ajmaline-negative patients. In most patients with ajmaline-induced BrS (78%), the RVOT location was already evident at baseline. The CineECG classified all normal subjects and ajmaline-negative patients at baseline as Normal or Undetermined, all patients with RBBB as RBBB, whereas all patients with spontaneous and ajmaline-induced BrS as Brugada. Compared with standard 12-lead ECG, CineECG at baseline had a 100% positive predictive value and 81% negative predictive value in predicting ajmaline test results. CONCLUSIONS: In patients with spontaneous and ajmaline-induced BrS, the CineECG localized the late QRS activity in the RVOT, a phenomenon never observed in normal, RBBB, or ajmaline-negative patients. The possibility to identify the RVOT as the location of the arrhythmogenic substrate by the noninvasive CineECG, based on the standard 12-lead ECG, opens new prospective for diagnosing patients with BrS.


Assuntos
Síndrome de Brugada/diagnóstico , Bloqueio de Ramo/diagnóstico , Eletrocardiografia , Frequência Cardíaca , Ventrículos do Coração/fisiopatologia , Processamento de Sinais Assistido por Computador , Vetorcardiografia , Potenciais de Ação , Adulto , Algoritmos , Síndrome de Brugada/fisiopatologia , Bloqueio de Ramo/fisiopatologia , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Sistema de Registros , Adulto Jovem
10.
Int J Mol Sci ; 21(16)2020 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-32824506

RESUMO

Brugada syndrome (BrS) is diagnosed by the presence of an elevated ST-segment and can result in sudden cardiac death. The most commonly found mutated gene is SCN5A, which some argue is the only gene that has been definitively confirmed to cause BrS, while the potential causative effect of other genes is still under debate. While the issue of BrS genetics is currently a hot topic, current knowledge is not able to result in molecular confirmation of over half of BrS cases. Therefore, it is difficult to develop research models with wide potential. Instead, the clinical genetics first need to be better understood. In this study, we provide crucial human data on the novel heterozygous variant NM_198056.2:c.4285G>A (p.Val1429Met) in the SCN5A gene, and demonstrate its segregation with BrS, suggesting a pathogenic effect. These results provide the first disease association with this variant and are crucial clinical data to communicate to basic scientists, who could perform functional studies to better understand the molecular effects of this clinically-relevant variant in BrS.


Assuntos
Síndrome de Brugada/genética , Mutação , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Adulto , Idoso , Síndrome de Brugada/diagnóstico , Feminino , Heterozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem
11.
Bioorg Med Chem ; 28(14): 115563, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32616179

RESUMO

The optimization of the synthetic protocol to obtain the 3,4-unsaturated sialic acid derivatives, through the fine-tuning of both the Ferrier glycosylation conditions and the subsequent hydrolysis work-up, is herein reported. The accomplishment of the desired ß-anomers and some selected α-ones, in pure form, led us to evaluate their specific inhibitory activity towards NDV-HN and human sialidase NEU3. Importantly, the resulting data allowed the identification, for the first time, of three active 3,4-unsaturated sialic acid analogs, showing IC50 values against NDV-HN in the micromolar range.


Assuntos
Antivirais/farmacologia , Inibidores Enzimáticos/farmacologia , Hemaglutininas/efeitos dos fármacos , Neuraminidase/antagonistas & inibidores , Vírus da Doença de Newcastle/efeitos dos fármacos , Ácidos Siálicos/farmacologia , Antivirais/síntese química , Antivirais/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Hemaglutininas/metabolismo , Humanos , Estrutura Molecular , Neuraminidase/metabolismo , Vírus da Doença de Newcastle/enzimologia , Ácidos Siálicos/síntese química , Ácidos Siálicos/química , Relação Estrutura-Atividade
12.
Cells ; 9(3)2020 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-32204578

RESUMO

Bone is an active tissue where bone mineralization and resorption occur simultaneously. In the case of fracture, there are numerous factors required to facilitate bone healing including precursor cells and blood vessels. To evaluate the interaction between bone marrow-derived mesenchymal stem cells (BMSC)-the precursor cells able to differentiate into bone-forming cells and human umbilical vein endothelial cells (HUVEC)-a cell source widely used for the study of blood vessels. We performed direct coculture of BMSC and HUVEC in normoxia and chemically induced hypoxia using Cobalt(II) chloride and Dimethyloxaloylglycine and in the condition where oxygen level was maintained at 1% as well. Cell proliferation was analyzed by crystal violet staining. Osteogenesis was examined by Alizarin Red and Collagen type I staining. Expression of angiogenic factor-vascular endothelial growth factor (VEGF) and endothelial marker-von Willebrand factor (VWF) were demonstrated by immunohistochemistry and enzyme-linked immunosorbent assay. The quantitative polymerase chain reaction was also used to evaluate gene expression. The results showed that coculture in normoxia could retain both osteogenic differentiation and endothelial markers while hypoxic condition limits cell proliferation and osteogenesis but favors the angiogenic function even after 1 of day treatment.


Assuntos
Diferenciação Celular , Células Endoteliais da Veia Umbilical Humana/citologia , Células-Tronco Mesenquimais/citologia , Neovascularização Fisiológica , Osteogênese , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/metabolismo , Hipóxia Celular , Proliferação de Células , Técnicas de Cocultura , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Células-Tronco Mesenquimais/metabolismo , Pessoa de Meia-Idade , Fator de von Willebrand/metabolismo
13.
Magn Reson Imaging ; 68: 127-135, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32004712

RESUMO

Preclinical cardiac MR is challenging and time-consuming. A fast and comprehensive acquisition protocol and standardized image post-processing may improve preclinical research, reducing acquisition time, costs and variability of results. In the present study, we evaluated the feasibility of a contrast-enhanced 3D IntraGate steady-state cine sequence (ce-3D-IG-cine) with short acquisition time (11 min) for a single-shot combined characterization of left ventricle (LV) remodeling and infarct size (IS) in a mouse model of acute ischemia-reperfusion injury. Sixteen male C57BL/6N mice underwent 7T cardiac MR (Bruker, BioSpec 70/30) including optimized ce-3D-IG-cine (total scan time 11 min) at day 1, 5 and 28 after surgery. LV end-diastolic volume (EDVMR) and ejection fraction (EFMR) extracted from MR were compared to ones from short-axis (SA-EDVecho, SA-EFecho) and parasternal long-axis (LA-EDVecho, LA-EFecho) echocardiography. IS was manually and semiautomatically segmented from ce-3D-IG-cine using different standard deviation (SD +2, +3, +4, +5, +6 in respect to a reference tissue). Mice were sacrificed at day 28, immediately after imaging. IS at day 28 was compared to injury burden at histology. MR and echocardiographic morpho-functional parameters were compared, as IS from MR and histology. Bland-Altman plots were used to assess the agreement in ischemic burden segmentation. Volumetric and functional parameters measured on ce-3D-IG-cine correlated to the correspondent echocardiographic parameter (EDVMR vs SA-EDVecho: ρ = 0.813; EDVMR vs LA-EDVecho: ρ = 0.845; EFMR vs SA-EFecho ρ = 0.612; EFMR vs LA-EFecho ρ = 0.791; p < 0.001 in all cases). Manually segmented IS strongly correlated with the scar at histology (ρ = 0.904, p < 0.001). A threshold of +3SD showed the highest performance for semiautomatic assessment of IS compared to manual segmentation (ρ = 0.965, p < 0.001), with an overall reproducibility of 73%, and a peak reproducibility of 80% at day 1. The ce-3D-IG-cine sequence, manually or semiautomatically segmented using 3SD threshold, allows fast and comprehensive LV morpho-functional and structural characterization in myocardial ischemia-reperfusion injury model.


Assuntos
Ecocardiografia , Ventrículos do Coração/diagnóstico por imagem , Imagem Cinética por Ressonância Magnética , Traumatismo por Reperfusão/diagnóstico por imagem , Animais , Imageamento Tridimensional , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Reprodutibilidade dos Testes
14.
Cell Physiol Biochem ; 54(1): 110-125, 2020 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-31999897

RESUMO

BACKGROUND/AIMS: Cystic Fibrosis (CF) is an inherited disease associated with a variety of mutations affecting the CFTR gene. A deletion of phenylalanine 508 (F508) affects more than 70% of patients and results in unfolded proteins accumulation, originating a proteinopathy responsible for inflammation, impaired trafficking, altered metabolism, cholesterol and lipids accumulation, impaired autophagy at the cellular level. Lung inflammation has been extensively related to the accumulation of the lipotoxin ceramide. We recently proved that inhibition of ceramide synthesis by Myriocin reduces inflammation and ameliorates the defence response against pathogens infection, which is downregulated in CF. Here, we aim at demonstrating the mechanisms of Myriocin therapeutic effects in Cystic Fibrosis broncho-epithelial cells. METHODS: The effect of Myriocin treatment, on F508-CFTR bronchial epithelial cell line IB3-1 cells, was studied by evaluating the expression of key proteins and genes involved in autophagy and lipid metabolism, by western blotting and real time PCR. Moreover, the amount of glycerol-phospholipids, triglycerides, and cholesterols, sphingomyelins and ceramides were measured in treated and untreated cells by LC-MS. Finally, Sptlc1 was transiently silenced and the effect on ceramide content, autophagy and transcriptional activities was evaluated as above mentioned. RESULTS: We demonstrate that Myriocin tightly regulates metabolic function and cell resilience to stress. Myriocin moves a transcriptional program that activates TFEB, major lipid metabolism and autophagy regulator, and FOXOs, central lipid metabolism and anti-inflammatory/anti-oxidant regulators. The activity of these transcriptional factors is associated with the induction of PPARs nuclear receptors activity, whose targets are genes involved in lipid transport compartmentalization and oxidation. Transient silencing of SPTCL1 recapitulates the effects induced by Myriocin. CONCLUSION: Cystic Fibrosis bronchial epithelia accumulate lipids, exacerbating inflammation. Myriocin administration: i) activates the transcriptions of genes involved in enhancing autophagy-mediated stress clearance; ii) reduces the content of several lipid species and, at the same time, iii) enhances mitochondrial lipid oxidation. Silencing the expression of Sptlc1 reproduces Myriocin induced autophagy and transcriptional activities, demonstrating that the inhibition of sphingolipid synthesis drives a transcriptional program aimed at addressing cell metabolism towards lipid oxidation and at exploiting autophagy mediated clearance of stress. We speculate that regulating sphingolipid de novo synthesis can relieve from chronic inflammation, improving energy supply and anti-oxidant responses, indicating an innovative therapeutic strategy for CF.


Assuntos
Ácidos Graxos Monoinsaturados/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Esfingolipídeos/metabolismo , Autofagia/efeitos dos fármacos , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Linhagem Celular , Colesterol/análise , Cromatografia Líquida de Alta Pressão , Fibrose Cística/metabolismo , Fibrose Cística/patologia , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Humanos , Espectrometria de Massas , PPAR gama/genética , PPAR gama/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Serina C-Palmitoiltransferase/antagonistas & inibidores , Serina C-Palmitoiltransferase/genética , Serina C-Palmitoiltransferase/metabolismo , Esfingolipídeos/análise , Esfingomielinas/análise
15.
Curr Med Chem ; 27(21): 3448-3462, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-30605049

RESUMO

The development of new therapeutic applications for adult and embryonic stem cells has dominated regenerative medicine and tissue engineering for several decades. However, since 2006, induced Pluripotent Stem Cells (iPSCs) have taken center stage in the field, as they promised to overcome several limitations of the other stem cell types. Nonetheless, other promising approaches for adult cell reprogramming have been attempted over the years, even before the generation of iPSCs. In particular, two years before the discovery of iPSCs, the possibility of synthesizing libraries of large organic compounds, as well as the development of high-throughput screenings to quickly test their biological activity, enabled the identification of a 2,6-disubstituted purine, named reversine, which was shown to be able to reprogram adult cells to a progenitor-like state. Since its discovery, the effect of reversine has been confirmed on different cell types, and several studies on its mechanism of action have revealed its central role in inhibitory activity on several kinases implicated in cell cycle regulation and cytokinesis. These key features, together with its chemical nature, suggested a possible use of the molecule as an anti-cancer drug. Remarkably, reversine exhibited potent cytotoxic activity against several tumor cell lines in vitro and a significant effect in decreasing tumor progression and metastatization in vivo. Thus, 15 years since its discovery, this review aims at critically summarizing the current knowledge to clarify the dual role of reversine as a dedifferentiating agent and anti-cancer drug.


Assuntos
Antineoplásicos/farmacologia , Pontos de Checagem do Ciclo Celular , Diferenciação Celular , Células-Tronco Pluripotentes Induzidas , Morfolinas , Purinas
16.
Heart Rhythm ; 17(4): 637-645, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31756528

RESUMO

BACKGROUND: The relationship between the typical electrocardiographic pattern and electromechanical abnormalities has never been systematically explored in Brugada syndrome (BrS). OBJECTIVES: The aims of this study were to characterize the electromechanical substrate in patients with BrS and to evaluate the relationship between electrical and mechanical abnormalities. METHODS: We enrolled 50 consecutive high-risk patients with BrS (mean age 42 ± 7.2 years), with implantable cardioverter-defibrillator implantation for primary or secondary prevention of ventricular tachyarrhythmias (ventricular tachycardia/ventricular fibrillation [VT/VF]), undergoing substrate mapping and ablation. Patients underwent 3-dimensional (3D) echocardiography with 3D wall motion/deformation quantification and electroanatomic mapping before and after ajmaline administration (1 mg/kg in 5 minutes); 3D mechanical changes were compared with 50 age- and sex-matched controls. The effect of substrate ablation on electromechanical abnormalities was also assessed. RESULTS: In all patients, ajmaline administration induced Brugada type 1 pattern, with a significant increase in the electrical substrate (P < .001), particularly in patients with previous spontaneous VT/VF (P = .007). Induction of Brugada pattern was associated with lowering of right ventricular (RV) ejection fraction (P < .001) and worsening of 3D RV mechanical function (P < .001), particularly in the anterior free wall of the RV outflow tract, without changes in controls. RV electrical and mechanical abnormalities were highly correlated (r = 0.728, P < .001). By multivariate analysis, only the area of RV dysfunction was an independent predictor of spontaneous VT/VF (odds ratio 1.480; 95% confidence interval 1.159-1.889; P = .002). Substrate ablation abolished both BrS-electrocardiographic pattern and mechanical abnormalities, despite ajmaline rechallenge. CONCLUSION: BrS is an electromechanical disease affecting the RV. The typical BrS pattern reflects an extensive RV arrhythmic substrate, driving consistent RV mechanical abnormalities. Substrate ablation abolished both Brugada pattern and mechanical abnormalities.


Assuntos
Síndrome de Brugada/fisiopatologia , Eletrocardiografia/métodos , Mapeamento Epicárdico/métodos , Ventrículos do Coração/fisiopatologia , Adulto , Ecocardiografia , Feminino , Ventrículos do Coração/diagnóstico por imagem , Humanos , Masculino , Pessoa de Meia-Idade
17.
Int J Mol Sci ; 20(22)2019 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-31698696

RESUMO

Brugada syndrome (BrS) is marked by coved ST-segment elevation and increased risk of sudden cardiac death. The genetics of this syndrome are elusive in over half of the cases. Variants in the SCN5A gene are the single most common known genetic unifier, accounting for about a third of cases. Research models, such as animal models and cell lines, are limited. In the present study, we report the novel NM_198056.2:c.1111C>T (p.Gln371*) heterozygous variant in the SCN5A gene, as well as its segregation with BrS in a large family. The results herein suggest a pathogenic effect of this variant. Functional studies are certainly warranted to characterize the molecular effects of this variant.


Assuntos
Síndrome de Brugada/genética , Códon sem Sentido/genética , Estudos de Associação Genética , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Adulto , Sequência de Bases , Síndrome de Brugada/diagnóstico por imagem , Simulação por Computador , Família , Feminino , Heterozigoto , Humanos , Masculino , Linhagem
18.
Int J Mol Sci ; 20(19)2019 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-31590245

RESUMO

Brugada syndrome (BrS) is marked by an elevated ST-segment elevation and increased risk of sudden cardiac death. Variants in the SCN5A gene are considered to be molecular confirmation of the syndrome in about one third of cases, while the genetics remain a mystery in about half of the cases, with the remaining cases being attributed to variants in any of a number of genes. Before research models can be developed, it is imperative to understand the genetics in patients. Even data from humans is complicated, since variants in the most common gene in BrS, SCN5A, are associated with a number of pathologies, or could even be considered benign, depending on the variant. Here, we provide crucial human data on a novel NM_198056.2:c.2091G>A (p.Trp697X) point-nonsense heterozygous variant in the SCN5A gene, as well as its segregation with BrS. The results herein suggest a pathogenic effect of this variant. These results could be used as a stepping stone for functional studies to better understand the molecular effects of this variant in BrS.


Assuntos
Síndrome de Brugada/genética , Códon sem Sentido , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Adulto , Síndrome de Brugada/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem
19.
Europace ; 21(12): 1900-1910, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31647530

RESUMO

AIMS: Brugada syndrome (BrS) represents a major cause of sudden cardiac death in young individuals. The risk stratification to forecast future life-threatening events is still controversial. Non-invasive assessment of late potentials (LPs) has been proposed as a risk stratification tool. However, their nature in BrS is still undetermined. The purpose of this study is to assess the electrophysiological determinants of non-invasive LPs. METHODS AND RESULTS: Two hundred and fifty consecutive patients with (Group 1, n = 96) and without (Group 2, n = 154) BrS-related symptoms were prospectively enrolled in the registry. Signal-averaged electrocardiogram (SAECG) was performed in all subjects before undergoing epicardial mapping. Group 1 patients exhibited larger arrhythmogenic substrates (AS; 5.8 ± 2.8 vs. 2.6 ± 2.1 cm2, P < 0.001) with more delayed potentials (220.4 ± 46.0 vs. 186.7 ± 42.3 ms, P < 0.001). Late potentials were present in 82/96 (85.4%) Group 1 and in 31/154 (20.1%) Group 2 individuals (P < 0.001). Patients exhibiting LPs had more frequently a spontaneous Type 1 pattern (30.1% vs. 10.9%, P < 0.001), SCN5A mutation (34.5% vs. 21.2%, P = 0.02), and exhibited a larger AS with longer potentials (5.8 ± 2.7 vs. 2.2 ± 1.7 cm2; 231.2 ± 37.3 vs. 213.8 ± 39.0 ms; P < 0.001, respectively). Arrhythmogenic substrate dimension was the strongest predictor of the presence of LPs (odds ratio 1.9; P < 0.001). An AS area of at least 3.5 cm2 identified patients with LPs (area under the curve 0.88, 95% confidence interval 0.843-0.931; P < 0.001) with a sensitivity of 86%, specificity 88%, positive predictive value 85%, and negative predictive value 89%. CONCLUSION: The results of this study support the role of the epicardial AS as an electrophysiological determinant of non-invasive LPs, which may serve as a tool in the non-invasive assessment of the BrS substrate, as SAECG-LPs could be considered an expression of the abnormal epicardial electrical activity.ClinicalTrials.gov number (NCT02641431; NCT03106701).


Assuntos
Potenciais de Ação , Síndrome de Brugada/fisiopatologia , Morte Súbita Cardíaca/prevenção & controle , Desfibriladores Implantáveis , Eletrocardiografia/métodos , Mapeamento Epicárdico/métodos , Processamento de Sinais Assistido por Computador , Taquicardia Ventricular/fisiopatologia , Fibrilação Ventricular/fisiopatologia , Adolescente , Adulto , Síndrome de Brugada/diagnóstico , Síndrome de Brugada/terapia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Medição de Risco , Taquicardia Ventricular/terapia , Fibrilação Ventricular/terapia , Adulto Jovem
20.
Diagnostics (Basel) ; 9(3)2019 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-31540197

RESUMO

BACKGROUND: Pediatric open-heart surgery with cardiopulmonary bypass (CPB) still remains a risky interventional procedure at high mortality/morbidity. To date, there are no clinical, laboratory, and/or monitoring parameters providing useful information on perioperative stress. We therefore investigated whether blood concentrations of glutathione (GSH), a powerful endogenous antioxidant, changed in the perioperative period. METHODS: We conducted an observational study in 35 congenital heart disease (CHD) children in whom perioperative standard laboratory and monitoring parameters and GSH blood levels were assessed at five monitoring time points. RESULTS: GSH showed a pattern characterized by a progressive increase from pre-surgery up to 24 h after surgery, reaching its highest peak at the end of CPB. GSH measured at the end of CPB correlated with CPB duration, cross-clamping, arterial oxygen partial pressure, and with body core temperature. CONCLUSIONS: The increase in GSH levels in the perioperative period suggests a compensatory mechanism to oxidative damage during surgical procedure. Caution is needed in controlling different CPB phases, especially systemic reoxygenation in a population that is per se more prone to oxidative stress/damage. The findings may point the way to detecting the optimal temperature and oxygenation target by biomarker monitoring.

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