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The complexity of cardiovascular diseases (CVDs), which remains the leading cause of death worldwide, makes the current clinical pathway for cardiovascular risk assessment unsatisfactory, as there remains a substantial unexplained residual risk. Simultaneous assessment of a large number of plasma proteins may be a promising tool to further refine risk assessment, and lipoprotein-associated proteins have the potential to fill this gap. Technical advances now allow for high-throughput proteomic analysis in a reproducible and cost-effective manner. Proteomics has great potential to identify and quantify hundreds of candidate marker proteins in a sample and allows the translation from isolated lipoproteins to whole plasma, thus providing an individual multiplexed proteomic fingerprint. This narrative review describes the pathophysiological roles of atherogenic apoB-containing lipoproteins and the recent advances in their mass spectrometry-based proteomic characterization and quantitation for better refinement of CVD risk assessment.
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Apolipoproteínas B , Doenças Cardiovasculares , Humanos , Proteômica , Lipoproteínas , Espectrometria de MassasRESUMO
The Human Plasma Proteome has always been the most investigated compartment in proteomics-based biomarker discovery, and is considered the largest and deepest version of the human proteome, reflecting the state of the body in health and disease. Even if efforts have been always dedicated to the refinement of proteomic approaches to investigate more deeply the plasma proteome, it should not be forgotten that also highly abundant plasma proteins, like human serum albumin (HSA), often neglected in these studies, might provide fundamental physiological functions in plasma, and should be better considered. This review summarizes the important roles of HSA in the context of cardiovascular diseases (CVD), and in particular in heart failure. Notwithstanding much attention has been historically directed toward the association of HSA levels and CVD risk, the advances in the field of mass spectrometry research allow also a better characterization of the effects of oxidative modifications that could alter not only the structure but also the function of HSA.
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Albuminas , Doenças Cardiovasculares , Insuficiência Cardíaca , Humanos , Proteoma/metabolismo , ProteômicaRESUMO
Adipose tissue is classically considered the primary site of lipid storage, but in recent years has garnered appreciation for its broad role as an endocrine organ, capable of remotely signaling to other tissues to alter their metabolic program. The adipose tissue is now recognized as a crucial regulator of cardiovascular health, mediated by the secretion of several bioactive products, with a wide range of endocrine and paracrine effects on the cardiovascular system. Thanks to the development and improvement of high-throughput mass spectrometry, the size and components of the human secretome have been characterized. In this review, we summarized the recent advances in mass spectrometry-based studies of the cell and tissue secretome for the understanding of adipose tissue biology, which may help to decipher the complex molecular mechanisms controlling the crosstalk between the adipose tissue and the cardiovascular system, and their possible clinical translation.
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Lipid-lowering therapies are widely used to prevent the development of atherosclerotic cardiovascular disease (ASCVD) and related mortality worldwide. "Omics" technologies have been successfully applied in recent decades to investigate the mechanisms of action of these drugs, their pleiotropic effects, and their side effects, aiming to identify novel targets for future personalized medicine with an improvement of the efficacy and safety associated with the treatment. Pharmacometabolomics is a branch of metabolomics that is focused on the study of drug effects on metabolic pathways that are implicated in the variation of response to the treatment considering also the influences from a specific disease, environment, and concomitant pharmacological therapies. In this review, we summarized the most significant metabolomic studies on the effects of lipid-lowering therapies, including the most commonly used statins and fibrates to novel drugs or nutraceutical approaches. The integration of pharmacometabolomics data with the information obtained from the other "omics" approaches could help in the comprehension of the biological mechanisms underlying the use of lipid-lowering drugs in view of defining a precision medicine to improve the efficacy and reduce the side effects associated with the treatment.
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Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Inibidores de Hidroximetilglutaril-CoA Redutases , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Hipolipemiantes , Medicina de Precisão , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/tratamento farmacológico , LipídeosRESUMO
In Antarctica, ice-free areas can be found along the coast, on mountain peaks, and in the McMurdo Dry Valleys, where microorganisms well-adapted to harsh conditions can survive and reproduce. Metabolic analyses can shed light on the survival mechanisms of Antarctic soil communities from both coastal sites, under different plant coverage stages, and inner sites where slow-growing or dormant microorganisms, low water availability, salt accumulation, and a limited number of primary producers make metabolomic profiling difficult. Here, we report, for the first time, an efficient protocol for the extraction and the metabolic profiling of Antarctic soils based on the combination of NMR spectroscopy and mass spectrometry (MS). This approach was set up on samples harvested along different localities of Victoria Land, in continental Antarctica, devoid of or covered by differently developed biological crusts. NMR allowed for the identification of thirty metabolites (mainly sugars, amino acids, and organic acids) and the quantification of just over twenty of them. UPLC-MS analysis identified more than twenty other metabolites, in particular flavonoids, medium- and long-chain fatty acids, benzoic acid derivatives, anthracenes, and quinones. Our results highlighted the complementarity of the two analytical techniques. Moreover, we demonstrated that their combined use represents the "gold standard" for the qualitative and quantitative analysis of little-explored samples, such as those collected from Antarctic soils.
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Solo , Espectrometria de Massas em Tandem , Solo/química , Regiões Antárticas , Projetos Piloto , Cromatografia Líquida , Metabolômica/métodosRESUMO
The human long pentraxin PTX3 has complex regulatory roles at the crossroad of innate immunity, inflammation, and tissue repair. PTX3 can be produced by various cell types, including vascular endothelial cells (ECs), in response to pro-inflammatory cytokines or bacterial molecules. PTX3 has also been involved in the regulation of cardiovascular biology, even if ambiguous results have been so far provided in both preclinical and clinical research. In this study, we compared the proteomic profiles of human ECs (human umbilical vein ECs, HUVECs), focusing on differentially expressed proteins between the control and PTX3-silenced ECs. We identified 19 proteins that were more abundant in the proteome of control ECs and 23 proteins that were more expressed in PTX3-silenced cells. Among the latter, proteins with multifunctional roles in angiogenesis, oxidative stress, and inflammation were found, and were further validated by assessing their mRNAs with RT-qPCR. Nevertheless, the knock down of PTX3 did not affect in vitro angiogenesis. On the contrary, the lack of the protein induced an increase in pro-inflammatory markers and a shift to the more oxidative profile of PTX3-deficient ECs. Altogether, our results support the idea of a protective function for PTX3 in the control of endothelial homeostasis, and more generally, in cardiovascular biology.
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Proteoma , Componente Amiloide P Sérico , Humanos , Componente Amiloide P Sérico/metabolismo , Proteína C-Reativa/metabolismo , Proteômica , Células Endoteliais da Veia Umbilical Humana/metabolismo , Neovascularização Patológica , Inflamação/genética , Inflamação/metabolismoRESUMO
Prenylcysteine Oxidase 1 (PCYOX1) is an enzyme involved in the degradation of prenylated proteins. It is expressed in different tissues including vascular and blood cells. We recently showed that the secretome from Pcyox1-silenced cells reduced platelet adhesion both to fibrinogen and endothelial cells, suggesting a potential contribution of PCYOX1 into thrombus formation. Here, we show that in vivo thrombus formation after FeCl3 injury of the carotid artery was delayed in Pcyox1-/- mice, which were also protected from collagen/epinephrine induced thromboembolism. The Pcyox1-/- mice displayed normal blood cells count, vascular procoagulant activity and plasma fibrinogen levels. Deletion of Pcyox1 reduced the platelet/leukocyte aggregates in whole blood, as well as the platelet aggregation, the alpha granules release, and the αIIbß3 integrin activation in platelet-rich plasma, in response to adenosine diphosphate (ADP) or thrombin receptor agonist peptide (TRAP). Washed platelets from the Pcyox1-/- and WT animals showed similar phosphorylation pathway activation, adhesion ability and aggregation. The presence of Pcyox1-/- plasma impaired agonist-induced WT platelet aggregation. Our findings show that the absence of PCYOX1 results in platelet hypo-reactivity and impaired arterial thrombosis, and indicates that PCYOX1 could be a novel target for antithrombotic drugs.
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Liases de Carbono-Enxofre/metabolismo , Células Endoteliais , Trombose , Animais , Plaquetas/metabolismo , Cisteína/análogos & derivados , Células Endoteliais/metabolismo , Fibrinogênio/metabolismo , Camundongos , Oxirredutases/metabolismo , Ativação Plaquetária , Agregação Plaquetária , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Trombose/metabolismoRESUMO
Heart failure (HF) is a complex disease due to the intricate interplay of several mechanisms, which therefore implies the need for a multimarker strategy to better personalize the care of patients with HF. In this study, we developed a targeted mass spectrometry approach based on multiple reaction monitoring (MRM) to measure multiple circulating protein biomarkers, involved in cardiovascular disease, to address their relevance in the human HF, intending to assess the feasibility of the workflow in the disease monitoring and risk stratification. In this study, we analyzed a total of 60 plasma proteins in 30 plasma samples from eight control subjects and 22 age- and gender- matched HF patients. We identified a panel of four plasma proteins, namely Neuropilin-2, Beta 2 microglobulin, alpha-1-antichymotrypsin, and complement component C9, that were more abundant in HF patients in relation to disease severity and pulmonary dysfunction. Moreover, we showed the ability of the combination of these candidate proteins to discriminate, with sufficient accuracy, HF patients from healthy subjects. In conclusion, we demonstrated the feasibility and potential of a proteomic workflow based on MRM mass spectrometry for the evaluation of multiple proteins in human plasma and the identification of a panel of biomarkers of HF severity.
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Biomarcadores/análise , Insuficiência Cardíaca/sangue , Proteômica/métodos , Adulto , Idoso , Estudos de Casos e Controles , Complemento C9/análise , Feminino , Humanos , Modelos Lineares , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Neuropilina-2/análise , Consumo de Oxigênio , Proteoma , Risco , alfa 1-Antitripsina/análise , Microglobulina beta-2/análiseRESUMO
Despite major progress in treating skeletal muscle disease associated with dystrophinopathies, cardiomyopathy is emerging as a major cause of death in people carrying dystrophin gene mutations that remain without a targeted cure even with new treatment directions and advances in modelling abilities. The reasons for the stunted progress in ameliorating dystrophin-associated cardiomyopathy (DAC) can be explained by the difficulties in detecting pathophysiological mechanisms which can also be efficiently targeted within the heart in the widest patient population. New perspectives are clearly required to effectively address the unanswered questions concerning the identification of authentic and effectual readouts of DAC occurrence and severity. A potential way forward to achieve further therapy breakthroughs lies in combining multiomic analysis with advanced preclinical precision models. This review presents the fundamental discoveries made using relevant models of DAC and how omics approaches have been incorporated to date.
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Cardiomiopatias/patologia , Biologia Computacional/métodos , Distrofina/deficiência , Genoma , Proteoma/análise , Transcriptoma , Animais , Cardiomiopatias/etiologia , Cardiomiopatias/metabolismo , HumanosRESUMO
Platelets are a heterogeneous small anucleate blood cell population with a central role both in physiological haemostasis and in pathological states, spanning from thrombosis to inflammation, and cancer. Recent advances in proteomic studies provided additional important information concerning the platelet biology and the response of platelets to several pathophysiological pathways. Platelets circulate systemically and can be easily isolated from human samples, making proteomic application very interesting for characterizing the complexity of platelet functions in health and disease as well as for identifying and quantifying potential platelet proteins as biomarkers and novel antiplatelet therapeutic targets. To date, the highly dynamic protein content of platelets has been studied in resting and activated platelets, and several subproteomes have been characterized including platelet-derived microparticles, platelet granules, platelet releasates, platelet membrane proteins, and specific platelet post-translational modifications. In this review, a critical overview is provided on principal platelet proteomic studies focused on platelet biology from signaling to granules content, platelet proteome changes in several diseases, and the impact of drugs on platelet functions. Moreover, recent advances in quantitative platelet proteomics are discussed, emphasizing the importance of targeted quantification methods for more precise, robust and accurate quantification of selected proteins, which might be used as biomarkers for disease diagnosis, prognosis and therapy, and their strong clinical impact in the near future.
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Plaquetas/metabolismo , Plaquetas/fisiologia , Biomarcadores/metabolismo , Humanos , Ativação Plaquetária , Inibidores da Agregação Plaquetária/metabolismo , Inibidores da Agregação Plaquetária/farmacologia , Processamento de Proteína Pós-Traducional , Proteoma/metabolismo , Proteômica/métodos , Transdução de SinaisRESUMO
INTRODUCTION: Since the completion of genome sequencing, gene silencing technologies have emerged as powerful tools to study gene functions in various biological processes, both in vivo and in vitro. Moreover, they have also been proposed as therapeutic agents to inhibit selected genes in a variety of pathological conditions, such as cancer, neurodegenerative, and cardiovascular diseases. Area covered: This review summarizes the mechanisms of action and applications of genome editing tools, from RNA interference to clustered regularly interspaced short palindromic repeats-based systems, in research and in clinics. We describe their essential role in high-throughput genetic screens and, in particular, in functional proteomics studies, to identify diagnostic markers and therapeutic targets. Indeed, gene silencing and proteomics have been extensively integrated to study global proteome changes, posttranslational modifications, and protein-protein interactions. Expert commentary: Functional proteomics approaches that leverage gene silencing tools have been successfully applied to examine the role of several genes in various contexts, leading to a deeper knowledge of biological pathways and disease mechanisms. Recent developments of gene silencing tools have improved their performance, also in terms of off-targets effects reduction, paving the way for a wider therapeutic application of these systems.
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Inativação Gênica , Proteômica , Pesquisa Translacional Biomédica , Animais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Ensaios de Triagem em Larga Escala , HumanosRESUMO
AIMS: Epidemiological studies strongly suggest a link between stress, depression, and cardiovascular diseases (CVDs); the mechanistic correlation, however, is poorly understood. A single-nucleotide polymorphism in the BDNF gene (BDNFVal66Met), associated with depression and anxiety, has been proposed as a genetic risk factor for CVD. Using a knock-in mouse carrying the BDNFVal66Met human polymorphism, which phenocopies psychiatric-related symptoms found in humans, we investigated the impact of this SNP on thrombosis. METHODS AND RESULTS: BDNFMet/Met mice displayed a depressive-like phenotype concomitantly with hypercoagulable state and platelet hyperreactivity. Proteomic analysis of aorta secretome from BDNFMet/Met and wild-type (WT) mice showed differential expression of proteins involved in the coagulation and inflammatory cascades. The BDNF Met allele predisposed to carotid artery thrombosis FeCl3-induced and to death after collagen/epinephrine injection. Interestingly, transfection with BDNFMet construct induced a prothrombotic/proinflammatory phenotype in WT cells. SIRT1 activation, using resveratrol and/or CAY10591, prevented thrombus formation and restored the physiological levels of coagulation and of platelet markers in BDNFMet/Met mice and/or cells transfected with the Met allele. Conversely, inhibition of SIRT1 by sirtinol and/or by specific siRNA induced the prothrombotic/proinflammatory phenotype in WT mice and cells. Finally, we found that BDNF Met homozygosity is associated with increased risk of acute myocardial infarction (AMI) in humans. CONCLUSION: Activation of platelets, alteration in coagulation pathways, and changes in vessel wall protein expression in BDNFMet/Met mice recapitulate well the features occurring in the anxiety/depression condition. Furthermore, our data suggest that the BDNFVal66Met polymorphism contribute to the individual propensity for arterial thrombosis related to AMI.
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Fator Neurotrófico Derivado do Encéfalo/genética , Transtorno Depressivo/genética , Polimorfismo de Nucleotídeo Único/genética , Trombose/genética , Animais , Transtornos de Ansiedade/genética , Aorta/fisiologia , Coagulação Sanguínea/genética , Artérias Carótidas/fisiologia , Trombose das Artérias Carótidas/genética , Modelos Animais de Doenças , Feminino , Heterozigoto , Homozigoto , Humanos , Masculino , Camundongos Transgênicos , Pessoa de Meia-Idade , Isquemia Miocárdica/genética , Proteínas do Tecido Nervoso/metabolismo , Ativação Plaquetária/genética , Inibidores da Agregação Plaquetária/farmacologia , Receptores de Superfície Celular/metabolismo , Resveratrol , Transdução de Sinais/fisiologia , Sirtuína 1/antagonistas & inibidores , Estilbenos/farmacologiaRESUMO
The Mitochondrial Human Proteome Project aims at understanding the function of the mitochondrial proteome and its crosstalk with the proteome of other organelles. Being able to choose a suitable and validated enrichment protocol of functional mitochondria, based on the specific needs of the downstream proteomics analysis, would greatly help the researchers in the field. Mitochondrial fractions from ten model cell lines were prepared using three enrichment protocols and analyzed on seven different LC-MS/MS platforms. All data were processed using neXtProt as reference database. The data are available for the Human Proteome Project purposes through the ProteomeXchange Consortium with the identifier PXD007053. The processed data sets were analyzed using a suite of R routines to perform a statistical analysis and to retrieve subcellular and submitochondrial localizations. Although the overall number of identified total and mitochondrial proteins was not significantly dependent on the enrichment protocol, specific line to line differences were observed. Moreover, the protein lists were mapped to a network representing the functional mitochondrial proteome, encompassing mitochondrial proteins and their first interactors. More than 80% of the identified proteins resulted in nodes of this network but with a different ability in coisolating mitochondria-associated structures for each enrichment protocol/cell line pair.
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Mitocôndrias/química , Proteoma/fisiologia , Proteômica/normas , Linhagem Celular , Cromatografia Líquida , Humanos , Itália , Proteínas Mitocondriais/análise , Mapas de Interação de Proteínas/fisiologia , Espectrometria de Massas em TandemRESUMO
INTRODUCTION: Protein prenylation is a ubiquitous covalent post-translational modification characterized by the addition of farnesyl or geranylgeranyl isoprenoid groups to a cysteine residue located near the carboxyl terminal of a protein. It is essential for the proper localization and cellular activity of numerous proteins, including Ras family GTPases and G-proteins. In addition to its roles in cellular physiology, the prenylation process has important implications in human diseases and in the recent years, it has become attractive target of inhibitors with therapeutic potential. Areas covered: This review attempts to summarize the basic aspects of prenylation integrating them with biological functions in diseases and giving an account of the current status of prenylation inhibitors as potential therapeutics. We also summarize the methodologies for the characterization of this modification. Expert commentary: The growing body of evidence suggesting an important role of prenylation in diseases and the subsequent development of inhibitors of the enzymes responsible for this modification lead to the urgent need to identify the full spectrum of prenylated proteins that are altered in the disease or affected by drugs. Proteomic tools to analyze prenylated proteins are recently emerging, thanks to the advancement in the field of mass spectrometry coupled to enrichment strategies.
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Prenilação de Proteína/genética , Processamento de Proteína Pós-Traducional/genética , Proteínas/genética , Proteômica , Cisteína/genética , HumanosRESUMO
The mitral valve is a highly complex structure which regulates blood flow from the left atrium to the left ventricle (LV) avoiding a significant forward gradient during diastole or regurgitation during systole. The integrity of the mitral valve is also essential for the maintenance of normal LV size, geometry, and function. Significant advances in the comprehension of the biological, functional, and mechanical behavior of the mitral valve have recently been made. However, current knowledge of protein components in the normal human mitral valve is still limited and complicated by the low cellularity of this tissue and the presence of high abundant proteins from the extracellular matrix. We employed here an integrated proteomic approach to analyse the protein composition of the normal human mitral valve and reported confident identification of 422 proteins, some of which have not been previously described in this tissue. In particular, we described the ability of pre-MS separation technique based on liquid-phase IEF and SDS-PAGE to identify the largest number of proteins. We also demonstrated that some of these proteins, e.g. αB-Crystallin, septin-11, four-and-a-half LIM domains protein 1, and dermatopontin, are synthesised by interstitial cells isolated from human mitral valves. These initial results provide a valuable basis for future studies aimed at analysing in depth the mitral valve protein composition and at investigating potential pathogenetic molecular mechanisms. Data are available via ProteomeXchange with identifier PXD004397.
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Eletroforese em Gel Bidimensional/métodos , Valva Mitral/química , Proteoma/análise , Proteômica/métodos , Adulto , Feminino , Humanos , Imuno-Histoquímica/métodos , Masculino , Pessoa de Meia-Idade , Proteoma/químicaRESUMO
Gestational diabetes (GDM) is the most common complication of pregnancy and it is associated with maternal and fetal short- and long-term consequences. GDM modifies placental structure and function, but many of the underlying mechanisms are still unclear. The aim of this study is to develop and compare two different methods, based respectively on gel-based and gel-free proteomics, in order to investigate the placental proteome in the absence or in the presence of GDM and to identify, through a comparative approach, possible changes in protein expression due to the GDM condition. Placenta homogenates obtained by pooling six control samples and six samples from GDM pregnant women were analyzed by two-dimensional (2D) electrophoresis coupled with mass spectrometry [nano-liquid chromatography (nano-LC) tandem mass spectrometry (MS/MS) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS)] and by a label-free mass spectrometry method based on LC-MS(E). The gel-based approach highlights 13 over-expressed proteins and 16 under-expressed proteins, while the label-free method shows the over- expression of 10 proteins and the under-expression of nine proteins. As regards 2D gel electrophoresis, a comparison between two different protein identification methods, based respectively on nLC-electrospray ionization-MS/MS and MALDI-MS/MS, was performed taking into consideration the sequence coverage, the MASCOT score and the exponentially modified protein abundance index. The analysis of the complex proteome through an integrated strategy revealed that the quantitative gel-free and label-free MS approach might be suitable to identify candidate markers of GDM.
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Diabetes Gestacional , Eletroforese em Gel Bidimensional , Placenta , Proteômica , Feminino , Humanos , Gravidez , Proteoma , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em TandemRESUMO
This paper describes a microproteomic workflow that is useful for simultaneously identifying and quantifying proteins from a minimal number of morphotypically heterogeneous cultured adherent cells. The analytical strategy makes use of laser capture microdissection, an effective means of harvesting pure cell populations, and label-free mass spectrometry. We optimised the workflow with particular reference to cell fixation which is crucial for successful laser-based microdissection and also downstream molecular studies. In addition, we defined the minimum number of cells to be isolated and analysed for satisfactory proteome coverage. To set up this workflow, we choose human monocyte-derived macrophages spontaneously differentiated in vitro. These cells, under our culture conditions, show distinct morphotypes, reminiscent of the heterogeneity observed in tissues in various homeostatic and pathological states, e.g. atherosclerosis. This optimised workflow may provide new insights into biology and pathology of heterogeneous cell in culture, particularly when other cell selection approaches are not suitable.
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Microdissecção e Captura a Laser/métodos , Macrófagos/química , Espectrometria de Massas/métodos , Proteômica/métodos , Células Cultivadas , Humanos , Macrófagos/metabolismo , Proteínas/química , Proteínas/metabolismo , Fluxo de TrabalhoRESUMO
The process of adipogenesis involves the differentiation of preadipocytes into mature adipocytes. Excessive adipogenesis promotes obesity, a condition that increasingly threatens global health and contributes to the rapid rise of obesity-related diseases. We have recently shown that prenylcysteine oxidase 1 (PCYOX1) is a regulator of atherosclerosis-disease mechanisms, which acts through mechanisms not exclusively related to its pro-oxidant activity. To address the role of PCYOX1 in the adipogenic process, we extended our previous observations confirming that Pcyox1-/-/Apoe-/- mice fed a high-fat diet for 8 or 12 weeks showed significantly lower body weight, when compared to Pcyox1+/+/Apoe-/- mice, due to an evident reduction in visceral adipose content. We herein assessed the role of PCYOX1 in adipogenesis. Here, we found that PCYOX1 is expressed in adipose tissue, and, independently from its pro-oxidant enzymatic activity, is critical for adipogenesis. Pcyox1 gene silencing completely prevented the differentiation of 3T3-L1 preadipocytes, by acting as an upstream regulator of several key players, such as FABP4, PPARγ, C/EBPα. Proteomic analysis, performed by quantitative label-free mass spectrometry, further strengthened the role of PCYOX1 in adipogenesis by expanding the list of its downstream targets. Finally, the absence of Pcyox1 reduces the inflammatory markers in adipose tissue. These findings render PCYOX1 a novel adipogenic factor with possible pathophysiological or therapeutic potential.
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Sacubitril/Valsartan, used for the treatment of heart failure (HF), is a combination of two drugs, an angiotensin receptor inhibitor, and a neprilysin inhibitor, which activates vasoactive peptides. Even though its beneficial effects on cardiac functions have been demonstrated, the mechanisms underpinning these effects remain poorly understood. To achieve more mechanistic insights, we analyzed the profiles of circulating miRNAs in plasma from patients with stable HF with reduced ejection function (HFrEF) and treated with Sacubitril/Valsartan for six months. miRNAs are short (22-24 nt) non-coding RNAs, which are not only emerging as sensitive and stable biomarkers for various diseases but also participate in the regulation of several biological processes. We found that in patients with high levels of miRNAs, specifically miR-29b-3p, miR-221-3p, and miR-503-5p, Sacubitril/Valsartan significantly reduced their levels at follow-up. We also found a significant negative correlation of miR-29b-3p, miR-221-3p, and miR-503-5p with VO2 at peak exercise, whose levels decrease with HF severity. Furthermore, from a functional point of view, miR-29b-3p, miR-221-3p, and miR-503-5p all target Phosphoinositide-3-Kinase Regulatory Subunit 1, which encodes regulatory subunit 1 of phosphoinositide-3-kinase. Our findings support that an additional mechanism through which Sacubitril/Valsartan exerts its functions is the modulation of miRNAs with potentially relevant roles in HFrEF pathophysiology.
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Human serum albumin (HSA) has an important antioxidant activity due to the presence of the reduced cysteine at position 34, which represents the most abundant free thiol in the plasma. In oxidative-based diseases, HSA undergoes S-thiolation (THIO-HSA) with changes in the antioxidant function of albumin that could contribute to the progression of the disease. The aim of this study was to verify, for the first time, the different burdens of THIO-HSA, glycated HSA (GLY-HSA), and advanced glycation end products (AGE) accumulation both in type 2 diabetes mellitus (T2DM) patients and in non-diabetic patients, with or without coronary heart disease (CHD). In this study, we assessed the presence of modified forms of HSA, THIO-HSA, and GLY-HSA by means of mass spectrometry in 33 patients with both T2DM and CHD, in 31 patients with T2DM and without CHD, in 30 patients without diabetes with a history of CHD, and 27 subjects without diabetes and CHD. All the patients' anthropometric and clinical data were recorded including age, sex, duration of diabetes, body mass index (BMI), blood pressure, and history of CHD defined with anamnestic data. Metabolic parameters, such as fasting plasma glucose (FPG), glycated hemoglobin (HbA1c), lipids, pentosidine, AGE, receptor for advanced glycation end-products (RAGE) and its soluble form (sRAGE), were measured. AGE and pentosidine are significantly higher in T2DM patients with and without CHD with respect to non-diabetic patients with CHD and control subjects. RAGE levels are significantly higher in T2DM patients with respect to non-diabetic patients, and among T2DM patients, the group with CHD showed significantly higher RAGE levels than those without CHD (217 ± 171 pg/mL and 140 ± 61 pg/mL, respectively). Albumin isoforms discriminate between non-diabetic patients with CHD and T2DM patients with and without CHD and control subjects, with GLY-HSA levels higher in T2DM with and without CHD, and THIO-HSA higher in CHD patients without T2DM. Finally, we demonstrated that the oxidized forms of HSA can increase the expression of the inflammatory cytokine Tumor Necrosis Factor-alpha (TNFα) in monocytic cells. In patients with CHD, GLY-HSA and THIO-HSA have a different prevalent distribution, the first one prevailing in patients with T2DM and the second one in patients without T2DM. These findings suggest that albumin quality and homeostasis balance between glyco-oxidation and thiolation might have an impact on the antioxidant defense system in cardiovascular diseases.