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Metabolic associated steatotic liver disease (MASLD) is the most common liver condition. It is associated with increased liver-related morbidity and mortality, and also with high risk of cardiovascular events (CVD), representing itself an independent risk factor for it. This makes MASLD a presentation of high interest for internal medicine, also because of its association with metabolic syndrome (MetS). It is crucial to assess its risks in a noninvasive way. With the aim of finding specific risk profiles for CVD development in MASLD by performing a noninvasive assessment of: (1) preclinical signs of endothelial dysfunction (ED); (2) clinical assessment of CVD risk by Framingham Heart Risk Score (FHRs); (3) genomic characterization of MASLD associated polymorphisms; (4) specific untargeted metabolomic profiles, we enrolled 466 MASLD patients non-invasively classified in 4 group of liver fibrosis severity (group-A: low-fibrosis risk, group-B: high-fibrosis risk, group-C: MASLD-cirrhosis, group-D: MASLD-HCC) and 73 healthy controls. FHRs was similar in controls and low-fibrosis group and significantly higher in high-fibrosis patients, cirrhosis, and HCC, increasing among classes. At a multivariable regression, FHRs was associated with liver disease severity and diabetes. 38.2% of patients had altered EndoPAT, resembling ED. Patients with high FHRs (> 40%) and ED had different metabolomics compared to those without ED. Our study reveals that a deep, non-invasive characterization of MASLD patients through precision medicine approaches (untargeted metabolomics, SNPs, ED assessment) was able to show a peculiar pattern in MASLD patients with increased CVD risk, mostly correlated with liver disease severity.
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Portal hypertension (PH) is a complex clinical challenge with severe complications, including variceal bleeding, ascites, hepatic encephalopathy, and hepatorenal syndrome. The gut microbiota (GM) and its interconnectedness with human health have emerged as a captivating field of research. This review explores the intricate connections between the gut and the liver, aiming to elucidate how alterations in GM, intestinal barrier function, and gut-derived molecules impact the development and progression of PH. A systematic literature search, following PRISMA guidelines, identified 12 original articles that suggest a relationship between GM, the gut-liver axis, and PH. Mechanisms such as dysbiosis, bacterial translocation, altered microbial structure, and inflammation appear to orchestrate this relationship. One notable study highlights the pivotal role of the farnesoid X receptor axis in regulating the interplay between the gut and liver and proposes it as a promising therapeutic target. Fecal transplantation experiments further emphasize the pathogenic significance of the GM in modulating liver maladies, including PH. Recent advancements in metagenomics and metabolomics have expanded our understanding of the GM's role in human ailments. The review suggests that addressing the unmet need of identifying gut-liver axis-related metabolic and molecular pathways holds potential for elucidating pathogenesis and directing novel therapeutic interventions.
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Disbiose , Microbioma Gastrointestinal , Hipertensão Portal , Fígado , Humanos , Hipertensão Portal/metabolismo , Hipertensão Portal/fisiopatologia , Hipertensão Portal/etiologia , Microbioma Gastrointestinal/fisiologia , Fígado/metabolismo , Translocação Bacteriana , Animais , Transplante de Microbiota FecalRESUMO
Non-alcoholic fatty liver disease (NAFLD) affects up to a quarter of the adult population in many developed and developing countries. This spectrum of liver disease ranges from simple steatosis to non-alcoholic steatohepatitis (NASH) and cirrhosis. The incidence of NASH is projected to increase by up to 56% over the next 10 years. There is growing epidemiological evidence that NAFLD has become the fastest-growing cause of hepatocellular carcinoma (HCC) in industrialized countries. The annual incidence of HCC varies between patients with NASH cirrhosis and patients with noncirrhotic NAFLD. In this review, NAFLD/NASH-associated HCC will be described, including its epidemiology, risk factors promoting hepatocarcinogenesis, and management of HCC in patients with obesity and associated metabolic comorbidities, including preventive strategies and therapeutic approaches to address this growing problem.
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Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) are commonly used to model arrhythmogenic cardiomyopathy (ACM), a heritable cardiac disease characterized by severe ventricular arrhythmias, fibrofatty myocardial replacement and progressive ventricular dysfunction. Although ACM is inherited as an autosomal dominant disease, incomplete penetrance and variable expressivity are extremely common, resulting in different clinical manifestations. Here, we propose hiPSC-CMs as a powerful in vitro model to study incomplete penetrance in ACM. Six hiPSC lines were generated from blood samples of three ACM patients carrying a heterozygous deletion of exon 4 in the PKP2 gene, two asymptomatic (ASY) carriers of the same mutation and one healthy control (CTR), all belonging to the same family. Whole exome sequencing was performed in all family members and hiPSC-CMs were examined by ddPCR, western blot, Wes™ immunoassay system, patch clamp, immunofluorescence and RNASeq. Our results show molecular and functional differences between ACM and ASY hiPSC-CMs, including a higher amount of mutated PKP2 mRNA, a lower expression of the connexin-43 protein, a lower overall density of sodium current, a higher intracellular lipid accumulation and sarcomere disorganization in ACM compared to ASY hiPSC-CMs. Differentially expressed genes were also found, supporting a predisposition for a fatty phenotype in ACM hiPSC-CMs. These data indicate that hiPSC-CMs are a suitable model to study incomplete penetrance in ACM.
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BACKGROUND: Mucosal-associated invariant T (MAIT) cells are a subset of unconventional T lymphocytes expressing a semi-invariant α/ß T-cell receptor (TCR). The physiological functions of these cells, which are particularly abundant in normal liver and mucosal sites, have become clear only in recent years, but their role in most human diseases is still unknown. Since the cellular origin and etiopathogenesis of most T-lymphomas are still elusive, we decided to explore the presence of MAIT cells in biopsies from these neoplasms. METHODS: Sixteen biopsies obtained from patients with a T-cell lymphoma diagnosis were analyzed via immunofluorescence staining using an anti-Vα7.2 antibody and the MR1-antigen tetramer. Positive cases were subjected to a polymerase chain reaction for the detection of Vα7.2-Jα33, Vα7.2-Jα20, or Vα7.2-Jα12 rearrangements, followed by sequencing of the CDR3α region. RESULTS: CD3+/Vα7.2+ and CD3+/MR1-Ag-tetramer+ cells were found in 4 of 16 samples analyzed. The identification of specific TCR rearrangements confirmed the presence of these cells in all four samples. PCR and sequencing results documented the presence of multiple clones of MAIT cells in each positive sample. CONCLUSIONS: MAIT cells are frequently found in T-cell lymphomas. More in-depth studies and a larger number of samples are needed to better clarify the contribution of MAIT cells to this rare neoplasm.
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Metabolic (dysfunction)-associated fatty liver disease (MAFLD) is the definition recently proposed to better circumscribe the spectrum of conditions long known as non-alcoholic fatty liver disease (NAFLD) that range from simple steatosis without inflammation to more advanced liver diseases. The progression of MAFLD, as well as other chronic liver diseases, toward cirrhosis, is driven by hepatic inflammation and fibrogenesis. The latter, result of a "chronic wound healing reaction," is a dynamic process, and the understanding of its underlying pathophysiological events has increased in recent years. Fibrosis progresses in a microenvironment where it takes part an interplay between fibrogenic cells and many other elements, including some cells of the immune system with an underexplored or still unclear role in liver diseases. Some therapeutic approaches, also acting on the immune system, have been probed over time to evaluate their ability to improve inflammation and fibrosis in NAFLD, but to date no drug has been approved to treat this condition. In this review, we will focus on the contribution of the liver immune system in the progression of NAFLD, and on therapies under study that aim to counter the immune substrate of the disease.
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BACKGROUND AND AIMS: Cardiovascular disease (CVD) is the leading cause of death in patients with non-alcoholic fatty liver disease (NAFLD), both with and without type 2 diabetes mellitus (T2DM). Cardiac autonomic dysfunction is a risk factor for CVD morbidity and mortality. The aim of this pilot study was to assess whether there is an association between NAFLD and impaired cardiac autonomic function. METHODS AND RESULTS: Among the first 4979 participants from the Cooperative Health Research in South Tyrol (CHRIS) study, we randomly recruited 173 individuals with T2DM and 183 age- and sex-matched nondiabetic controls. Participants underwent ultrasonography and vibration-controlled transient elastography (Fibroscan®, Echosens) to assess hepatic steatosis and liver stiffness. The low-to-high-frequency (LF/HF) power ratio and other heart rate variability (HRV) measures were calculated from a 20-min resting electrocardiogram (ECG) to derive a measure of cardiac sympathetic/parasympathetic imbalance. Among the 356 individuals recruited for the study, 117 had NAFLD and T2DM, 56 had T2DM alone, 68 had NAFLD alone, and 115 subjects had neither condition. Individuals with T2DM and NAFLD (adjusted odds ratio [OR] 4.29, 95% confidence intervals [CI] 1.90-10.6) and individuals with NAFLD alone (adjusted OR 3.41, 95% CI 1.59-7.29), but not those with T2DM alone, had a substantially increased risk of having cardiac sympathetic/parasympathetic imbalance, compared with those without NAFLD and T2DM. Logistic regression models were adjusted for age, sex, body mass index (BMI), hypertension, dyslipidemia, insulin resistance, hemoglobin A1c (HbA1c), C-reactive protein (CRP), and Fibroscan®-measured liver stiffness. CONCLUSIONS: NAFLD was associated with cardiac sympathetic/parasympathetic imbalance, regardless of the presence or absence of T2DM, liver stiffness, and other potential confounding factors.
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Doenças Cardiovasculares , Hepatopatia Gordurosa não Alcoólica , Doenças Cardiovasculares/epidemiologia , Estudos de Casos e Controles , Diabetes Mellitus Tipo 2/epidemiologia , Feminino , Fatores de Risco de Doenças Cardíacas , Humanos , Masculino , Hepatopatia Gordurosa não Alcoólica/epidemiologia , Projetos PilotoRESUMO
Arrhythmogenic Cardiomyopathy (ACM) is a genetically based cardiomyopathy associated with ventricular arrhythmias and fibro-fatty substitution of cardiac tissue. It is characterized by incomplete penetrance. We generated human iPSCs by episomal reprogramming of blood cells from three members of the same family: the proband, affected by ACM and carrying the heterozygous plakophillin2 p.N346Lfs*12 mutation, one asymptomatic carrier of the same mutation and one apparently healthy control. hiPSCs were characterized according to standard protocols including karyotyping, pluripotency marker expression and differentiation towards the three germ layers. These hiPSC lines can be used to study the mechanisms of ACM incomplete penetrance in vitro.
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Cardiomiopatias , Células-Tronco Pluripotentes Induzidas , Diferenciação Celular , Heterozigoto , Humanos , MutaçãoRESUMO
BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is characterized by triglyceride accumulation in the hepatocytes in the absence of alcohol overconsumption, commonly associated with insulin resistance and obesity. Both NAFLD and type 2 diabetes (T2D) are characterized by an altered microbiota composition, however the role of the microbiota in NAFLD and T2D is not well understood. To assess the relationship between alteration in the microbiota and NAFLD while dissecting the role of T2D, we established a nested study on T2D and non-T2D individuals within the Cooperative Health Research In South Tyrol (CHRIS) study, called the CHRIS-NAFLD study. Here, we present the study protocol along with baseline and follow-up characteristics of study participants. METHODS: Among the first 4979 CHRIS study participants, 227 individuals with T2D were identified and recalled, along with 227 age- and sex-matched non-T2D individuals. Participants underwent ultrasound and transient elastography examination to evaluate the presence of hepatic steatosis and liver stiffness. Additionally, sampling of saliva and faeces, biochemical measurements and clinical interviews were carried out. RESULTS: We recruited 173 T2D and 183 non-T2D participants (78% overall response rate). Hepatic steatosis was more common in T2D (63.7%) than non-T2D (36.3%) participants. T2D participants also had higher levels of liver stiffness (median 4.8 kPa, interquartile range (IQR) 3.7, 5.9) than non-T2D participants (median 3.9 kPa, IQR 3.3, 5.1). The non-invasive scoring systems like the NAFLD fibrosis score (NFS) suggests an increased liver fibrosis in T2D (mean - 0.55, standard deviation, SD, 1.30) than non-T2D participants (mean - 1.30, SD, 1.17). DISCUSSION: Given the comprehensive biochemical and clinical characterization of study participants, once the bioinformatics classification of the microbiota will be completed, the CHRIS-NAFLD study will become a useful resource to further our understanding of the relationship between microbiota, T2D and NAFLD.
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Diabetes Mellitus Tipo 2/microbiologia , Microbiota , Hepatopatia Gordurosa não Alcoólica/microbiologia , Idoso , Bactérias/metabolismo , Diabetes Mellitus Tipo 2/complicações , Feminino , Humanos , Masculino , Síndrome Metabólica/complicações , Hepatopatia Gordurosa não Alcoólica/complicaçõesRESUMO
BACKGROUND: Heart rate variability (HRV) reflects the autonomous nervous system modulation on heart rate and is associated with several pathologies, including cardiac mortality. While mechanistic studies show that smoking is associated with lower HRV, population-based studies present conflicting results. METHODS: We assessed the mutual effects of active smoking status, cumulative smoking history, and current smoking intensity, on HRV among 4751 adults from the Cooperative Health Research In South Tyrol (CHRIS) study. The HRV metrics standard deviation of normal-to-normal (NN) inter-beat intervals (SDNN), square root of the mean squared differences of consecutive NN intervals (RMSSD), total power (TP), low (LF) and high frequency (HF) power, and their ratio (LF/HF), were derived from 20-minute electrocardiograms. Smoking status, pack-years (PY), and tobacco grams/day from standardized questionnaires were the main exposures. We fitted linear mixed models to account for relatedness, non-linearity, and moderating effects, and including fractional polynomials. RESULTS: Past smokers had higher HRV levels than never smokers, independently of PY. The association of HRV with current smoking became apparent when accounting for the interaction between smoking status and PY. In current smokers, but not in past smokers, we observed HRV reductions between 2.0% (SDNN) and 4.9% (TP) every 5 PY increase. Furthermore, current smokers were characterized by dose-response reductions of 9.8% (SDNN), 8.9% (RMSSD), 20.1% (TP), 17.7% (LF), and 19.1% (HF), respectively, every 10 grams/day of smoked tobacco, independently of common cardiometabolic conditions and HRV-modifying drugs. The LF/HF ratio was not associated with smoking status, history, or intensity. CONCLUSIONS: Smoking cessation was associated with higher HRV levels. In current smokers, heavier smoking intensity appears gradually detrimental on HRV, corroborating previous evidence. By affecting both the sympathetic and parasympathetic nervous system indexes, but not the LF/HF balance, smoking intensity seems to exert a systemic dysautonomic effect.
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Arritmias Cardíacas/epidemiologia , Frequência Cardíaca/fisiologia , Fumantes/estatística & dados numéricos , Fumar/fisiopatologia , Tabagismo/complicações , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Arritmias Cardíacas/etiologia , Feminino , Alemanha/epidemiologia , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Adulto JovemRESUMO
In early diabetes, hyperglycemia and the associated metabolic dysregulation promote early changes in the functional properties of cardiomyocytes, progressively leading to the appearance of the diabetic cardiomyopathy phenotype. Recently, the interplay between histone acetyltransferases (HAT) and histone deacetylases (HDAC) has emerged as a crucial factor in the development of cardiac disorders. The present study evaluates whether HDAC inhibition can prevent the development of cardiomyocyte contractile dysfunction induced by a short period of hyperglycemia, with focus on the potential underlying mechanisms. Cell contractility and calcium dynamics were measured in unloaded ventricular myocytes isolated from the heart of control and diabetic rats. Cardiomyocytes were either untreated or exposed to the pan-HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) for 90 min. Then, a fraction of each group of cells was used to evaluate the expression levels of proteins involved in the excitation-contraction coupling, and the cardiomyocyte metabolic activity, ATP content, and reactive oxygen species levels. SAHA treatment was able to counteract the initial functional derangement in cardiomyocytes by reducing cell oxidative damage. These findings suggest that early HDAC inhibition could be a promising adjuvant approach for preventing diabetes-induced cardiomyocyte oxidative damage, which triggers the pro-inflammatory signal cascade, mitochondrial damage, and ventricular dysfunction.
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Inibidores de Histona Desacetilases/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Vorinostat/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Cálcio/metabolismo , Células Cultivadas , Diabetes Mellitus Experimental/patologia , Histona Desacetilases/química , Histona Desacetilases/metabolismo , Masculino , Miócitos Cardíacos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismoRESUMO
SERCA2a is the Ca2+ ATPase playing the major contribution in cardiomyocyte (CM) calcium removal. Its activity can be regulated by both modulatory proteins and several post-translational modifications. The aim of the present work was to investigate whether the function of SERCA2 can be modulated by treating CMs with the histone deacetylase (HDAC) inhibitor suberanilohydroxamic acid (SAHA). The incubation with SAHA (2.5 µM, 90 min) of CMs isolated from rat adult hearts resulted in an increase of SERCA2 acetylation level and improved ATPase activity. This was associated with a significant improvement of calcium transient recovery time and cell contractility. Previous reports have identified K464 as an acetylation site in human SERCA2. Mutants were generated where K464 was substituted with glutamine (Q) or arginine (R), mimicking constitutive acetylation or deacetylation, respectively. The K464Q mutation ameliorated ATPase activity and calcium transient recovery time, thus indicating that constitutive K464 acetylation has a positive impact on human SERCA2a (hSERCA2a) function. In conclusion, SAHA induced deacetylation inhibition had a positive impact on CM calcium handling, that, at least in part, was due to improved SERCA2 activity. This observation can provide the basis for the development of novel pharmacological approaches to ameliorate SERCA2 efficiency.
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Ácidos Hidroxâmicos/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Processamento de Proteína Pós-Traducional , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Acetilação , Animais , Inibidores de Histona Desacetilases/farmacologia , Humanos , Masculino , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/metabolismo , Ratos , Ratos Wistar , VorinostatRESUMO
Caveolinopathies are a heterogeneous family of genetic pathologies arising from alterations of the caveolin-3 gene (CAV3), encoding for the isoform specifically constituting muscle caveolae. Here, by reprogramming peripheral blood mononuclear cells, we report the generation of induced pluripotent stem cells (iPSCs) from three patients carrying the ΔYTT deletion, T78K and W101C missense mutations in caveolin-3. iPSCs displayed normal karyotypes and all the features of pluripotent stem cells in terms of morphology, specific marker expression and ability to differentiate in vitro into the three germ layers. These lines thus represent a human cellular model to study the molecular basis of caveolinopathies. Resource table.
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Caveolina 3/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Caveolina 3/genética , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Células Cultivadas , Reprogramação Celular/genética , Reprogramação Celular/fisiologia , Citometria de Fluxo , Humanos , Cariótipo , Mutação/genética , Mutação de Sentido Incorreto/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Arrhythmogenic cardiomyopathy (ACM) is an inherited genetic disorder, characterized by the substitution of heart muscle with fibro-fatty tissue and severe ventricular arrhythmias, often leading to heart failure and sudden cardiac death. ACM is considered a monogenic disorder, but the low penetrance of mutations identified in patients suggests the involvement of additional genetic or environmental factors. METHODS: We used whole exome sequencing to investigate digenic inheritance in two ACM families where previous diagnostic tests have revealed a PKP2 mutation in all affected and some healthy individuals. In family members with PKP2 mutations we determined all genes that harbor variants in affected but not in healthy carriers or vice versa. We computationally prioritized the most likely candidates, focusing on known ACM genes and genes related to PKP2 through protein interactions, functional relationships, or shared biological processes. RESULTS: We identified four candidate genes in family 1, namely DAG1, DAB2IP, CTBP2 and TCF25, and eleven candidate genes in family 2. The most promising gene in the second family is TTN, a gene previously associated with ACM, in which the affected individual harbors two rare deleterious-predicted missense variants, one of which is located in the protein's only serine kinase domain. CONCLUSIONS: In this study we report genes that might act as digenic players in ACM pathogenesis, on the basis of co-segregation with PKP2 mutations. Validation in larger cohorts is still required to prove the utility of this model.
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Displasia Arritmogênica Ventricular Direita/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Oxirredutases do Álcool/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas Correpressoras , Conectina/química , Conectina/genética , Distroglicanas/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Mutação , Proteínas do Tecido Nervoso/genética , Linhagem , Placofilinas/genética , Domínios Proteicos , Proteínas Repressoras/genética , Sequenciamento do Exoma , Proteínas Ativadoras de ras GTPase/genéticaRESUMO
Genome-editing technology has emerged as a powerful method that enables the generation of genetically modified cells and organisms necessary to elucidate gene function and mechanisms of human diseases. The clustered regularly interspaced short palindromic repeats- (CRISPR-) associated 9 (Cas9) system has rapidly become one of the most popular approaches for genome editing in basic biomedical research over recent years because of its simplicity and adaptability. CRISPR/Cas9 genome editing has been used to correct DNA mutations ranging from a single base pair to large deletions in both in vitro and in vivo model systems. CRISPR/Cas9 has been used to increase the understanding of many aspects of cardiovascular disorders, including lipid metabolism, electrophysiology and genetic inheritance. The CRISPR/Cas9 technology has been proven to be effective in creating gene knockout (KO) or knockin in human cells and is particularly useful for editing induced pluripotent stem cells (iPSCs). Despite these progresses, some biological, technical, and ethical issues are limiting the therapeutic potential of genome editing in cardiovascular diseases. This review will focus on various applications of CRISPR/Cas9 genome editing in the cardiovascular field, for both disease research and the prospect of in vivo genome-editing therapies in the future.
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Liver fibrosis is a pathological scarring response to chronic hepatocellular injury and hepatic stellate cells (HSCs) are key players in this process. PNPLA3 I148M is a common variant robustly associated with liver fibrosis but the mechanisms underlying this association are unknown. We aimed to examine a) the effect of fibrogenic and proliferative stimuli on PNPLA3 levels in HSCs and b) the role of wild type and mutant PNPLA3 overexpression on markers of HSC activation and fibrosis.Here, we show that PNPLA3 is upregulated by the fibrogenic cytokine transforming growth factor-beta (TGF-ß), but not by platelet-derived growth factor (PDGF), and is involved in the TGF-ß-induced reduction in lipid droplets in primary human HSCs. Furthermore, we show that retinol release from human HSCs ex vivo is lower in cells with the loss-of-function PNPLA3 148M compared with 148I wild type protein. Stable overexpression of PNPLA3 148I wild type, but not 148M mutant, in human HSCs (LX-2 cells) induces a reduction in the secretion of matrix metallopeptidase 2 (MMP2), tissue inhibitor of metalloproteinase 1 and 2 (TIMP1 and TIMP2), which is mediated by retinoid metabolism. In conclusion, we show a role for PNPLA3 in HSC activation in response to fibrogenic stimuli. Moreover, we provide evidence to indicate that PNPLA3-mediated retinol release may protect against liver fibrosis by inducing a specific signature of proteins involved in extracellular matrix remodelling.
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Células Estreladas do Fígado/metabolismo , Lipase/genética , Metabolismo dos Lipídeos/genética , Cirrose Hepática/genética , Proteínas de Membrana/genética , Vitamina A/administração & dosagem , Regulação da Expressão Gênica/genética , Genótipo , Células Estreladas do Fígado/patologia , Humanos , Lipase/biossíntese , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Metaloproteinase 2 da Matriz/biossíntese , Proteínas de Membrana/biossíntese , Fator de Crescimento Derivado de Plaquetas/genética , Fator de Crescimento Derivado de Plaquetas/metabolismo , Cultura Primária de Células , Inibidor Tecidual de Metaloproteinase-1/biossíntese , Inibidor Tecidual de Metaloproteinase-2/biossíntese , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Vitamina A/metabolismoRESUMO
BACKGROUND: Type 1 hyperlipoproteinemia is a rare autosomal recessive disorder most often caused by mutations in the lipoprotein lipase (LPL) gene resulting in severe hypertriglyceridemia and pancreatitis. OBJECTIVES: The aim of this study was to identify novel mutations in the LPL gene causing type 1 hyperlipoproteinemia and to understand the molecular mechanisms underlying the severe hypertriglyceridemia. METHODS: Three patients presenting classical features of type 1 hyperlipoproteinemia were recruited for DNA sequencing of the LPL gene. Pre-heparin and post-heparin plasma of patients were used for protein detection analysis and functional test. Furthermore, in vitro experiments were performed in HEK293 cells. Protein synthesis and secretion were analyzed in lysate and medium fraction, respectively, whereas medium fraction was used for functional assay. RESULTS: We identified two novel mutations in the LPL gene causing type 1 hyperlipoproteinemia: a two base pair deletion (c.765_766delAG) resulting in a frameshift at position 256 of the protein (p.G256TfsX26) and a nucleotide substitution (c.1211 T > G) resulting in a methionine to arginine substitution (p.M404 R). LPL protein and activity were not detected in pre-heparin or post-heparin plasma of the patient with p.G256TfsX26 mutation or in the medium of HEK293 cells over-expressing recombinant p.G256TfsX26 LPL. A relatively small amount of LPL p.M404 R was detected in both pre-heparin and post-heparin plasma and in the medium of the cells, whereas no LPL activity was detected. CONCLUSIONS: We conclude that these two novel mutations cause type 1 hyperlipoproteinemia by inducing a loss or reduction in LPL secretion accompanied by a loss of LPL enzymatic activity.
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Hiperlipoproteinemia Tipo I/enzimologia , Hiperlipoproteinemia Tipo I/genética , Lipase Lipoproteica/genética , Adulto , Análise Mutacional de DNA , Feminino , Células HEK293 , Humanos , Hiperlipoproteinemia Tipo I/sangue , Hiperlipoproteinemia Tipo I/metabolismo , Lipase Lipoproteica/biossíntese , Lipase Lipoproteica/sangue , Lipase Lipoproteica/metabolismo , Masculino , Mutação , Adulto JovemRESUMO
BACKGROUND & AIMS: Nonalcoholic fatty liver disease (NAFLD) is a leading cause of liver damage and is characterized by steatosis. Genetic factors increase risk for progressive NAFLD. A genome-wide association study showed that the rs641738 C>T variant in the locus that contains the membrane bound O-acyltransferase domain-containing 7 gene (MBOAT7, also called LPIAT1) and transmembrane channel-like 4 gene (TMC4) increased the risk for cirrhosis in alcohol abusers. We investigated whether the MBOAT7-TMC4 is a susceptibility locus for the development and progression of NAFLD. METHODS: We genotyped rs641738 in DNA collected from 3854 participants from the Dallas Heart Study (a multi-ethnic population-based probability sample of Dallas County residents) and 1149 European individuals from the Liver Biopsy Cross-Sectional Cohort. Clinical and anthropometric data were collected, and biochemical and lipidomics were measured in plasma samples from participants. A total of 2736 participants from the Dallas Heart Study also underwent proton magnetic resonance spectroscopy to measure hepatic triglyceride content. In the Liver Biopsy Cross-Sectional Cohort, a total of 1149 individuals underwent liver biopsy to diagnose liver disease and disease severity. RESULTS: The genotype rs641738 at the MBOAT7-TMC4 locus associated with increased hepatic fat content in the 2 cohorts, and with more severe liver damage and increased risk of fibrosis compared with subjects without the variant. MBOAT7, but not TMC4, was found to be highly expressed in the liver. The MBOAT7 rs641738 T allele was associated with lower protein expression in the liver and changes in plasma phosphatidylinositol species consistent with decreased MBOAT7 function. CONCLUSIONS: We provide evidence for an association between the MBOAT7 rs641738 variant and the development and severity of NAFLD in individuals of European descent. This association seems to be mediated by changes in the hepatic phosphatidylinositol acyl-chain remodeling.
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Acetiltransferases/genética , Aciltransferases/genética , Cirrose Hepática/genética , Proteínas de Membrana/genética , Hepatopatia Gordurosa não Alcoólica/genética , Polimorfismo Genético , População Branca/genética , Acetiltransferases/metabolismo , Aciltransferases/metabolismo , Biópsia , Estudos de Casos e Controles , Estudos Transversais , Europa (Continente)/epidemiologia , Feminino , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Humanos , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/diagnóstico , Cirrose Hepática/etnologia , Cirrose Hepática/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Hepatopatia Gordurosa não Alcoólica/diagnóstico , Hepatopatia Gordurosa não Alcoólica/etnologia , Hepatopatia Gordurosa não Alcoólica/metabolismo , Fenótipo , Fosfatidilinositóis/metabolismo , Espectroscopia de Prótons por Ressonância Magnética , Fatores de Risco , Índice de Gravidade de Doença , Texas/epidemiologia , Triglicerídeos/metabolismoRESUMO
UNLABELLED: Chronic hepatitis C virus (HCV) infection may progress to cirrhosis and hepatocellular carcinoma (HCC). Recently, two genetic variants, DEPDC5 rs1012068 and MICA rs2596542, were associated with the onset of HCC in Asian subjects with chronic HCV infection. The aim of the present study was to analyze whether DEPDC5 and MICA genetic variants were associated with liver disease progression in European subjects with chronic HCV infection. In a Northern Italian discovery cohort (n = 477), neither DEPDC5 rs1012068 nor MICA rs2596542 were associated with HCC (n = 150). However, DEPDC5 rs1012068 was independently associated with cirrhosis (n = 300; P = 0.049). The association of rs1012068 with moderate to severe fibrosis was confirmed in an independent cross-sectional German cohort (n = 415; P = 0.006). Furthermore, DEPDC5 rs1012068 predicted faster fibrosis progression in a prospective cohort (n = 247; P = 0.027). Next, we examined the distribution of nonsynonymous DEPDC5 variants in the overall cross-sectional cohort (n = 912). The presence of at least one variant increased the risk of moderate/severe fibrosis by 54% (P = 0.040). To understand the molecular mechanism underlying the genetic association of DEPDC5 variants with fibrosis progression, we performed in vitro studies on immortalized hepatic stellate cells (LX-2). In these cells, down-regulation of DEPDC5 resulted in increased expression of ß-catenin and production of its target matrix metallopeptidase 2 (MMP2), a secreted enzyme involved in fibrosis progression. CONCLUSION: DEPDC5 variants increase fibrosis progression in European subjects with chronic HCV infection. Our findings suggest that DEPDC5 down-regulation may contribute to HCV-related fibrosis by increasing MMP2 synthesis through the ß-catenin pathway.
Assuntos
Carcinoma Hepatocelular/etiologia , Progressão da Doença , Hepatite C Crônica/complicações , Hepatite C Crônica/genética , Cirrose Hepática/etiologia , Neoplasias Hepáticas/etiologia , Proteínas Repressoras/genética , Estudos Transversais , Feminino , Proteínas Ativadoras de GTPase , Variação Genética , Alemanha , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Itália , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Suíça , População BrancaRESUMO
UNLABELLED: The patatin-like phosholipase domain-containing 3 (PNPLA3) rs738409 polymorphism (I148M) is a major determinant of hepatic fat and predisposes to the full spectrum of liver damage in nonalcoholic fatty liver disease (NAFLD). The aim of this study was to evaluate whether additional PNPLA3 coding variants contribute to NAFLD susceptibility, first in individuals with contrasting phenotypes (with early-onset NAFLD vs. very low aminotransferases) and then in a large validation cohort. Rare PNPLA3 variants were not detected by sequencing coding regions and intron-exon boundaries either in 142 patients with early-onset NAFLD nor in 100 healthy individuals with alanine aminotransferase <22/20 IU/mL. Besides rs738409 I148M, the rs2294918 G>A polymorphism (E434K sequence variant) was over-represented in NAFLD (adjusted P = 0.01). In 1,447 subjects with and without NAFLD, the 148M-434E (P < 0.0001), but not the 148M-434K, haplotype (P > 0.9), was associated with histological NAFLD and steatohepatitis. Both the I148M (P = 0.0002) and E434K variants (P = 0.044) were associated with serum ALT levels, by interacting with each other, in that the 434K hampered the association with liver damage of the 148M allele (P = 0.006). The E434K variant did not affect PNPLA3 enzymatic activity, but carriers of the rs2294918 A allele (434K) displayed lower hepatic PNPLA3 messenger RNA and protein levels (P < 0.05). CONCLUSIONS: Rare loss-of-function PNPLA3 variants were not detected in early-onset NAFLD. However, PNPLA3 rs2294918 E434K decreased PNPLA3 expression, lessening the effect of the I148M variant on the predisposition to steatosis and liver damage. This suggests that the PNPLA3 I148M variant has a codominant negative effect on triglycerides mobilization from lipid droplets, mediated by inhibition of other lipases.