RESUMO
The subject of the paper is a review of multidimensional data analysis methods, which is the canonical analysis with its various variants and its use in omics data research. The dynamic development of high-throughput methods, and with them the availability of large and constantly growing data resources, forces the development of new analytical approaches that allow the review of the analyzed processes, taking into account data from various levels of the organization of living organisms. The multidimensional perspective allows for the assessment of the analyzed phenomenon in a more realistic way, as it generally takes into account much more data (including OMICs data). Without omitting the complexity of an organism, the method simplifies the multidimensional view, finally giving the result so that the researcher can draw practical conclusions. This is particularly important in medical sciences, where the study of pathological processes is usually aimed at developing treatment regimens. One of the primary methods for studying biomedical processes in a multidimensional approach is the canonical correlation analysis (CCA) with various variants. The use of CCA unique methodologies for simultaneous analysis of multiset biomolecular data opens up new avenues for studying previously undiscovered processes and interdependencies such as e.g. in the tumor microenvironment (TME) connected to intercellular communication. Because of the huge and still untapped potential of canonical correlation, in this review available implementations of CCA techniques are presented. In particular, the possibility of using the technique of canonical correlation analysis for OMICs data is emphasized.
Assuntos
Análise de Correlação CanônicaRESUMO
BACKGROUND: The aim of this study was to investigate the role of urine-derived extracellular vesicles (uEVs) in diabetic kidney disease (DKD) in patients diagnosed with type 2 diabetes mellitus (T2DM). METHODS: UEVs were characterized by size distribution and microRNA content by next-generation small RNA sequencing and quantitative reverse transcription PCR. RESULTS: A subset of sixteen miRNAs enriched in T2DM patients with DKD, including hsa-miR-514a-5p, hsa-miR451a, hsa-miR-126-3p, hsa-miR-214, or hsa-miR503 was identified. Eight miRNAs as hsa-miR-21-3p, hsa-miR-4792, hsa-miR375, hsa-miR-1268a, hsa-miR-501-5p, or hsa-miR-582 were downregulated. Prediction of potential target genes and pathway enrichment analysis of the Kyoto Encyclopedia of Genes and Genomes (KEGG) confirmed possible functions related to cellular processes such as apoptosis, inflammation, and tissue remodeling, that promote diabetic complications, such as DKD. Among them, hsa-miR-375, hsa-miR-503, and hsa-miR-451a make important contribution. Additionally, downregulated hsa-miR-582-5p has not been reported so far in any diabetes-related pathways. CONCLUSIONS: This study revealed the most significant miRNAs in uEVs of patients with T2DM. However, as this is a bioinformatic prediction that we performed based on the putative targets of the identified miRNAs. Thus, further in vitro functional studies are needed to confirm our findings. Knowing the fact that EVs are crucial in transferring miRNAs, there is a great need toto discover their involvement in the pathomechanism of T2DM-related kidney disease.
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Diabetes Mellitus Tipo 2 , Nefropatias Diabéticas , Vesículas Extracelulares , MicroRNAs , Insuficiência Renal Crônica , Humanos , MicroRNAs/genética , Vesículas Extracelulares/metabolismoRESUMO
Time-of-flight secondary ion mass spectrometry (TOF-SIMS) with the Bi3+ liquid metal ion gun was used to investigate the content of lipids and amino acids (AAs) in extracellular vesicles (EVs). We induced metabolic changes in human pancreatic ß-cells by stimulation with high glucose concentrations (35 mM) and tested the hypothesis of hyperglycemia (HG) has a detrimental effect on lipids and AAs in released EV subpopulations: ectosomes and exosomes. As a result of HG treatment, selected fatty acids (FAs) such as arachidonic, myristic and palmitic acids, changed their abundance in ectosomes and exosomes. Also, intensities of the characteristic peaks for cholesterol (m/z 95.09; 147.07; 161.11; 369.45) along with the molecular ion m/z 386.37 [C27H46O+] under HG conditions, both for ectosomes and exosomes, have changed significantly. Comparative analysis of HG EVs and normoglycemic (NG) ones showed statistically significant differences in the signal intensities of four AAs: valine (m/z 72.08 and 83.05), isoleucine (m/z 86.10), phenylalanine (m/z 120.08 and 132.05) and tyrosine (m/z 107.05 and 136.09). We confirmed that ToF-SIMS is a useful technique to study selected AAs and lipid profiles in various EV subpopulations. Our study is the first demonstration of changes in FAs and AAs in exosomes and ectosomes derived from ß-cells under the influence of HG.
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Micropartículas Derivadas de Células , Vesículas Extracelulares , Hiperglicemia , Aminoácidos , Ácidos Graxos , Humanos , Espectrometria de Massa de Íon Secundário/métodosRESUMO
In this study, we verified the hypothesis that Raman signature of urinary extracellular vesicles (UEVs) can be used to stratify patients with diabetes at various stages of chronic kidney disease (CKD). Patients with type 2 diabetes diagnosed with different stages of CKD and healthy subjects were enrolled in the study. UEVs were isolated using low-vacuum filtration followed by ultracentrifugation. Correlation analysis, multiple linear regression and principal component analysis were used to find differences between spectral fingerprints of UEVs derived from both groups of patients. Electron microscopy and nanoparticle tracking analysis were applied to characterize the size and morphology of UEVs. We observed significant correlations between selected Raman bands measured for UEVs and clinical parameters. We found significant differences in the area under the specific bands originating mainly from proteins and lipids between the study groups. Based on the tryptophan and amide III bands, we were able to predict the estimated glomerular filtration rate (eGFR). Principal component analysis, partial least squares regression (PLSR) and correlation analysis of the UEV Raman spectra supported the results obtained from the direct analysis of Raman spectra. Our analysis revealed that PLSR and a regression model including tryptophan and amide III bands allows to estimate the value of eGFR.
Assuntos
Diabetes Mellitus Tipo 2 , Vesículas Extracelulares , Insuficiência Renal Crônica , Biomarcadores/metabolismo , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/metabolismo , Vesículas Extracelulares/metabolismo , Humanos , Insuficiência Renal Crônica/diagnóstico , Insuficiência Renal Crônica/metabolismo , Análise Espectral Raman , UltracentrifugaçãoRESUMO
Diabetes mellitus type 2 (DM2) has recently become one of the most important health problems in the world. Patients with DM2 with long-term glycaemia are more likely to become infected than the healthy population. Matrix metalloproteinases (MMPs) play a key role in tissue remodeling during various physiological processes. However, it has been reported that certain MMPs are overexpressed during the development of various human diseases. In this study, we analyzed the levels of MMP-3 and MMP-9 in the serum of DM2 patients with and without Epstein-Barr virus (EBV) infection. The study included 115 patients with DM2 hospitalized in the Internal Ward of the Masovian Specialist Hospital in Radom, Poland, who were divided into two groups: EBV-positive and EBV-negative. The levels of MMP-3 and MMP-9 were tested in the serum of patients using the ELISA method, while the presence of EBV in saliva was tested by polymerase chain reaction (PCR). The presented studies showed a significant difference in the concentration of both MMPs in diabetic patients additionally infected with EBV compared to the group of non-infected individuals. It seems that MMPs may be useful biomarkers in the diagnosis, prognosis, and monitoring of diabetes associated with EBV infection.
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Diabetes Mellitus Tipo 2 , Infecções por Vírus Epstein-Barr , Humanos , Herpesvirus Humano 4 , Infecções por Vírus Epstein-Barr/complicações , Metaloproteinase 9 da Matriz , Metaloproteinase 3 da Matriz , Diabetes Mellitus Tipo 2/complicaçõesRESUMO
Protein content of extracellular vesicles (EVs) can modulate different processes during carcinogenesis. Novel proteomic strategies have been applied several times to profile proteins present in exosomes released by urothelial bladder cancer (UBC) cells. However, similar studies have not been conducted so far on another population of EVs, i.e., ectosomes. In the present study we used a shotgun nanoLC-MS/MS proteomic approach to investigate the protein content of ectosomes released in vitro by T-24 UBC cells and HCV-29 normal ureter epithelial cells. In addition, cancer-promoting effects exerted by UBC-derived ectosomes on non-invasive cells in terms of cell proliferation and migratory properties were assessed. In total, 1158 proteins were identified in T-24-derived ectosomes, while HCV-29-derived ectosomes contained a lower number of 259 identified proteins. Qualitative analysis revealed 938 proteins present uniquely in T-24-derived ectosomes, suggesting their potential applications in bladder cancer management as diagnostic and prognostic biomarkers. In addition, T-24-derived ectosomes increased proliferation and motility of recipient cells, likely due to the ectosomal transfer of the identified cancer-promoting molecules. The present study provided a focused identification of biologically relevant proteins in UBC-derived ectosomes, confirming their role in UBC development and progression, and their applicability for further biomarker-oriented studies in preclinical or clinical settings.
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Exossomos/metabolismo , Proteoma , Proteômica , Neoplasias da Bexiga Urinária/metabolismo , Biomarcadores Tumorais , Carcinoma de Células de Transição/metabolismo , Estudos de Casos e Controles , Linhagem Celular Tumoral , Micropartículas Derivadas de Células/metabolismo , Cromatografia Líquida , Biologia Computacional/métodos , Progressão da Doença , Vesículas Extracelulares/metabolismo , Humanos , Proteômica/métodos , Espectrometria de Massas em TandemRESUMO
For this study, we tested and optimized silicon surface functionalization procedures for capturing urinary extracellular vesicles (uEVs). The influence of the silane type (APTES or GOPS) and protein concentration on the efficiency of uEVs binding was investigated. Human lactadherin protein (LACT) was used to capture uEVs. We applied surface characterization techniques, including ellipsometry, atomic force microscopy, and time-of-flight secondary ion mass spectrometry, to observe changes in the biosensor surface after each functionalization step. uEVs were purified by a low-vacuum filtration method and concentrated by ultracentrifugation. The physical parameters of uEVs after the isolation procedure, such as morphology and size distribution, were determined using transmission electron microscopy and tunable resistive pulse sensing methods. We observed a gradual growth of the molecular layer after subsequent stages of modification of the silicon surface. The ToF-SIMS results showed no changes in the mean intensities for the characteristic peaks of amino acids and lipids in positive and negative polarization, in terms of the surface-modifying silane (APTES or GOPS) used. The most optimal concentration of LACT for the tested system was 25 µg/mL.
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Técnicas Biossensoriais/métodos , Desenho de Fármacos , Vesículas Extracelulares/metabolismo , Humanos , Silanos/química , Silício/química , Propriedades de SuperfícieRESUMO
Microvesicles (MVs) are found in several types of body fluids and are promising disease biomarkers and therapeutic targets. This study aimed to develop a novel biofunctionalized surface for binding plasma microvesicles (PMVs) based on a lab-on-a-chip (LOC) approach. A new lactadherin (LACT)-functionalized surface was prepared and examined for monitoring PMVs. Moreover, two different strategies of LACT immobilization on a silicon surface were applied to compare different LACT orientations. A higher PMV to LACT binding efficiency was observed for LACT bonded to an αvß3 integrin-functionalized surface compared with that for LACT directly bonded to a glutaraldehyde-modified surface. Effective binding of PMVs and its components for both LACT immobilization strategies was confirmed using spectral ellipsometry and time-of-flight secondary ion mass spectrometry methods. The proposed PMV capturing system can be used as a foundation to design novel point-of-care (POC) diagnostic devices to detect and characterize PMVs in clinical samples. Graphical Abstract.
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Micropartículas Derivadas de Células/química , Sistemas Automatizados de Assistência Junto ao Leito , Silício/química , Humanos , Dispositivos Lab-On-A-Chip , Microscopia de Força Atômica , Plasma/química , Espectrometria de Massa de Íon Secundário , Propriedades de SuperfícieRESUMO
Cutaneous melanoma (CM) is an aggressive type of skin cancer for which effective biomarkers are still needed. Recently, the protein content of extracellular vesicles (ectosomes and exosomes) became increasingly investigated in terms of its functional role in CM and as a source of novel biomarkers; however, the data concerning the proteome of CM-derived ectosomes is very limited. We used the shotgun nanoLC-MS/MS approach to the profile protein content of ectosomes from primary (WM115, WM793) and metastatic (WM266-4, WM1205Lu) CM cell lines. Additionally, the effect exerted by CM ectosomes on recipient cells was assessed in terms of cell proliferation (Alamar Blue assay) and migratory properties (wound healing assay). All cell lines secreted heterogeneous populations of ectosomes enriched in the common set of proteins. A total of 1507 unique proteins were identified, with many of them involved in cancer cell proliferation, migration, escape from apoptosis, epithelial-mesenchymal transition and angiogenesis. Isolated ectosomes increased proliferation and motility of recipient cells, likely due to the ectosomal transfer of different cancer-promoting molecules. Taken together, these results confirm the significant role of ectosomes in several biological processes leading to CM development and progression, and might be used as a starting point for further studies exploring their diagnostic and prognostic potential.
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Micropartículas Derivadas de Células/metabolismo , Exossomos/metabolismo , Melanoma/metabolismo , Proteômica , Neoplasias Cutâneas/metabolismo , Espectrometria de Massas em Tandem , Biomarcadores , Linhagem Celular Tumoral , Cromatografia Líquida , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Ontologia Genética , Humanos , Melanoma/genética , Neoplasias Cutâneas/genética , Melanoma Maligno CutâneoRESUMO
INTRODUCTION: Diabetes mellitus (DM) is the most common metabolic disease. A WHO report from 2016 indicates that 422 million people worldwide suffer from DM or hyperglycaemia because of impaired glucose metabolism. Chronic hyperglycaemia leads to micro- and macrovessel damage, which may result in life-threatening complications. The Wnt pathway regulates cell proliferation and survival by modulating the expression of genes that control cell differentiation. Three linked Wnt pathways have been discovered thus far: a ß-catenin-dependent pathway and two pathways independent of ß-catenin - the planar cell polarity pathway and calcium-dependent pathway. The Wnt pathway regulates genes associated with inflammation, cell cycle, angiogenesis, fibrinolysis and other molecular processes. AREAS COVERED: This review presents the current state of knowledge regarding the contribution of the Wnt pathway to endothelial ageing under hyperglycaemic conditions and provides new insights into the molecular basis of diabetic endothelial dysfunction. CONCLUSION: The ß-catenin-dependent pathway is a potential target in the prophylaxis and treatment of early-stage diabetes-related vascular complications. However, the underlying molecular mechanisms remain largely undetermined and require further investigation.
Assuntos
Endotélio Vascular/metabolismo , Hiperglicemia/metabolismo , Via de Sinalização Wnt , Animais , Proliferação de Células , Endotélio Vascular/patologia , Humanos , Hiperglicemia/patologiaRESUMO
Background: Platelet-derived microvesicles (PMVs), shed from platelet surface membranes, constitute the majority of circulating microvesicles and have been implicated in procoagulant, pro-inflammatory and pro-atherosclerotic effects. Our aim was to compare plasma PMVs numbers in relation to platelet reactivity during dual antiplatelet therapy (DAPT) with various P2Y12 adenosine diphosphate (ADP) receptor antagonists. Methods: In pre-discharge men treated with DAPT for an acute coronary syndrome, plasma PMVs were quantified by flow cytometry on the basis of CD62P (P-selectin) and CD42 (glycoprotein Ib) positivity, putative indices of PMVs release from activated and all platelets, respectively. ADP-induced platelet aggregation was measured by multiple-electrode aggregometry. Results: Clinical characteristics were similar in patients on clopidogrel (n=16), prasugrel (n=10) and ticagrelor (n=12). Platelet reactivity was comparably reduced on ticagrelor or prasugrel versus clopidogrel (p<0.01). Compared to clopidogrel-treated patients, CD42+/CD62P+ PMVs counts were 3-4-fold lower in subjects receiving ticagrelor (p=0.001) or prasugrel (p<0.05), while CD42+ PMVs were significantly reduced on ticagrelor (by about 6-fold, p<0.001), but not prasugrel (p=0.3). CD42+/CD62P+ PMVs numbers correlated positively to the ADP-induced aggregation on clopidogrel (p<0.01) or prasugrel (p<0.05), which was absent in ticagrelor users (p=0.8). CD42+ PMVs counts were unrelated to platelet reactivity (p>0.5). Conclusions: Higher antiplatelet potency of prasugrel and ticagrelor versus clopidogrel is associated with decreased plasma CD42+/CD62P+ PMVs numbers. However, in contrast to thienopyridines, the association of reduced CD42+/CD62P+ PMVs counts with ticagrelor use appears independent of its anti-aggregatory effect. Despite similar platelet-inhibitory activity of ticagrelor and prasugrel, only the treatment with ticagrelor seems associated with lower total PMVs release. Our preliminary findings may suggest a novel pleiotropic effect of ticagrelor extending beyond pure anti-aggregatory properties of the drug.
Assuntos
Síndrome Coronariana Aguda/tratamento farmacológico , Plaquetas/efeitos dos fármacos , Micropartículas Derivadas de Células/efeitos dos fármacos , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Piridinas/farmacologia , Idoso , Aspirina/uso terapêutico , Plaquetas/metabolismo , Micropartículas Derivadas de Células/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Antagonistas do Receptor Purinérgico P2Y/uso terapêutico , Piridinas/uso terapêutico , Ticagrelor/farmacologia , Ticagrelor/uso terapêuticoRESUMO
Raman spectroscopy was applied to the measurement of urinary and in vitro endothelium-derived extracellular vesicles (EVs) isolated by hydrostatic filtration dialysis (HFD) method. Raman spectra obtained for urinary EVs (UEVs) showed distinct differences in the fingerprint region. In contrast, average Raman spectra of endothelium-derived EVs samples were almost identical. Cluster Analysis of UEVs significantly discriminated diabetic samples from control, moreover endothelium-derived EVs revealed stronger similarity between long hyperglycemia and normoglycemia samples compared to short hyperglycemia. Results obtained from Partial Least Squares analysis corresponded well with integral intensities of selected bands. Our proof-of-concept approach demonstrates the potential for Raman spectroscopy to be used both for identification of EVs molecular signatures in urine samples from patients with type 2 diabetes mellitus and good glycemic control and unsatisfactory glycemic control as well as for in vitro hyperglycemic model. This noninvasive technique may be useful in identifying new biomarkers of diabetes and renal complications.
Assuntos
Diabetes Mellitus Tipo 2/diagnóstico , Células Endoteliais/patologia , Vesículas Extracelulares/patologia , Hiperglicemia/diagnóstico , Diabetes Mellitus Tipo 2/urina , Células Endoteliais/química , Vesículas Extracelulares/química , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Hiperglicemia/urina , Masculino , Análise Espectral Raman/métodos , Urinálise/métodos , Urina/químicaRESUMO
Cancer cells are known to release extracellular vesicles that often promote disease development and progression. The present study investigated the protein content and glycosylation pattern of ectosomes released in vitro by a human primary uveal melanoma Mel202 cell line. Ectosomes released by Mel202 cells were isolated from conditioned media using sequential centrifugation, and a nano-LC-MS/MS approach was used to determine their protein content. Subsequently, proteins from ectosomes, the whole cell extracts, and the membrane fractions were probed with a panel of lectins using Western blotting and flow cytometry to reveal characteristic glycan structures. As many as 2527 unique proteins were identified, and many of them are known to be involved in cancer cell proliferation and altered metabolism, tumor invasion, metastasis, or drug resistance. Lectin-based studies revealed a distinct glycosylation pattern between Mel202-derived ectosomes and the parental cell membranes. Selective enrichment of ectosomal proteins with bisected complex type N-glycans and α2,6-linked sialic acids may be significant for ectosome formation and sequestration. Differences in the surface glycosylation of Mel202 cells and ectosomes supports recent findings that the budding of ectosomes occurs within strictly determined fragments of the plasma membrane, and thus ectosomes contain a unique protein and glycan composition.
Assuntos
Micropartículas Derivadas de Células/metabolismo , Melanoma/metabolismo , Proteoma/metabolismo , Neoplasias Uveais/metabolismo , Biomarcadores/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Membrana Celular/patologia , Micropartículas Derivadas de Células/patologia , Glicosilação , Humanos , Melanoma/patologia , Neoplasias Uveais/patologiaRESUMO
INTRODUCTION: RANTES regulates leukocyte recruitment to areas affected by the inflammatory process. Microvesicles (MVs) belong to a subpopulation of extracellular vesicles and show proangiogenic potential by transferring bioactive molecules to target cells. OBJECTIVES: the aim of this study was to determine the relationship between circulating proangiogenic factors (MVs and RANTES) and diabetes complications in patients with different severities of diabetic retinopathy (DR). CCR5 (CD195) receptors transported by annexin V-labeled MVs were also investigated. PATIENTS AND METHODS: Diabetic patients (n = 61), among whom 35 had confirmed DR classified according to guidelines, and controls (n = 25) were included. MVs were isolated by centrifugation and analyzed using flow cytometry, RANTES was assessed by ELISA. RESULTS: the study group differed from the control group with respect to BMI, age, heart rate and systolic blood pressure. Additionally, glucose and creatinine concentrations were significantly increased: 5.30 [5.09-5.62] vs. 9.38 [7.48-11.55] (p <0.0001) mmol/l and 74.59 [64-84] vs. 89.00 [77.11-105.44] µmol/l (p = 0.0005), respectively. RANTES concentrations were significantly increased in diabetic patients compared to those of controls (15.5 (9.7-18.1) vs. 8.9 (0.9-14.6) µg/ml (p = 0.011)), and RANTES concentration significantly increased with respect to nonproliferative DR progression. Moreover, the number of CCR5-positive MVs was significantly increased in patients with heavy nonproliferative diabetic retinopathy (HNPDR) compared to those with so nonproliferative DR (SNPDR): 1178 [836-2254] vs. 394 [275-799] counts/µl. CONCLUSIONS: Correlation of RANTES concentrations with the stage of nonproliferative DR and the statistically significant dependence of CCR5-positive MVs with disease progression suggest that MVs and RANTES can be considered new biomarkers.
Assuntos
Biomarcadores/sangue , Quimiocina CCL5/sangue , Complicações do Diabetes , Diabetes Mellitus/fisiopatologia , Retinopatia Diabética/etiologia , Receptores CCR5/sangue , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Índice de Gravidade de DoençaRESUMO
Background: Adverse effects of numerous environmental factors, including improperly balanced diets, may accelerate the onset of ailments related to the climacteric period. Objective: The aim of the study was to examine the relationships between diets and the quality of life of working women aged 50-64 years. Material and methods: The study included 274 working women aged 55.4±4.0 years living in Biala Podlaska and the surrounding area. These were women working in various positions (teaching, administrative, economic department) at the State School of Higher Education in Biala Podlaska, Poland and patients of the Health and Rehabilitation Centre in Biala Podlaska. The study was conducted by means of a popular tool used to diagnose quality of life i.e. SF-36 questionnaire (Short Form Health Survey) and the Questionnaire of Eating Behaviour (QEB). Results: In all categories of quality of life (SF-36), apart from pain and general health, there were statistically significant differences between the results of the respondents and the norm for Polish women aged 50 to 60 years. Fruit, vegetables and wholemeal bread were the most frequently consumed products in the healthy diet group, while legumes, fish and curd cheese were the least frequently consumed by the respondents. Of the unhealthy products, the women most often chose sweets (at least once a week), cheese and fried food. Analysis of the effect of a healthy diet on the quality of life showed that a statistically significant correlations were observed in the case of mental health, functioning in society, emotionality, vitality, and well-being. Conclusions: A positive correlation with the application of a healthy diet was observed in all the categories of quality of life. This means that the respondents with healthy diets had a higher quality of life.
Assuntos
Dieta/estatística & dados numéricos , Comportamentos Relacionados com a Saúde , Estado Nutricional , Qualidade de Vida/psicologia , Atitude Frente a Saúde , Dieta/psicologia , Carboidratos da Dieta/administração & dosagem , Gorduras na Dieta/administração & dosagem , Fibras na Dieta/administração & dosagem , Proteínas Alimentares/administração & dosagem , Feminino , Preferências Alimentares/psicologia , Humanos , Pessoa de Meia-Idade , PolôniaRESUMO
BACKGROUND: Extracellular vesicles are small vesicles that contain cytoplasmic and membrane components from their paternal cells. They enter target cells through uptake to transfer their biological cargo. In this study, we investigated the process of endothelial EV internalization and created a 3D visualization of their intracellular distribution. METHODS AND RESULTS: Two immortalized endothelial cell lines that express h-TERT (human telomerase) were used for EV release: microvascular TIME and macrovascular HUVEC. EVs were isolated from the cell culture medium via differential centrifugation and used for the uptake experiments. The size distribution of the EVs was measured using TRPS technology on a qNano instrument. Internalization of EVs was observed using a Zeiss LSM 710 confocal laser microscope after staining of the EVs with PKH26. EVs were observed intracellularly and distributed in the perinuclear region of the target cells. The distribution patterns were similar in both cell lines. CONCLUSION: The perinuclear localization of the internalized EVs shows their biological stability after their uptake to the endothelial cells. The 3D visualization allows the determination of a more accurate location of EVs relative to the donor cell nucleus.
Assuntos
Vesículas Extracelulares/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Imageamento Tridimensional , Endocitose , Células Endoteliais da Veia Umbilical Humana/citologia , HumanosRESUMO
Among the various biomarkers that are used to diagnose or monitor disease, extracellular vesicles (EVs) represent one of the most promising targets in the development of new therapeutic strategies and the application of new diagnostic methods. The detection of circulating platelet-derived microvesicles (PMVs) is a considerable challenge for laboratory diagnostics, especially in the preliminary phase of a disease. In this study, we present a multistep approach to immobilizing and detecting PMVs in biological samples (microvesicles generated from activated platelets and human platelet-poor plasma) on functionalized silicon substrate. We describe the application of time-of-flight secondary ion mass spectrometry (TOF-SIMS) and spectroscopic ellipsometry methods to the detection of immobilized PMVs in the context of a novel imaging flow cytometry (ISX) technique and atomic force microscopy (AFM). This novel approach allowed us to confirm the presence of the abundant microvesicle phospholipids phosphatidylserine (PS) and phosphatidylethanolamine (PE) on a surface with immobilized PMVs. Phosphatidylcholine groups (C5H12N+; C5H15PNO4+) were also detected. Moreover, we were able to show that ellipsometry permitted the immobilization of PMVs on a functionalized surface to be evaluated. The sensitivity of the ISX technique depends on the size and refractive index of the analyzed microvesicles. Graphical abstract Human platelets activated with thrombin (in concentration 1IU/mL) generate population of PMVs (platelet derived microvesicles), which can be detected and enumerated with fluorescent-label method (imaging cytometry). Alternatively, PMVs can be immobilized on the modified silicon substrate which is functionalized with a specific IgM murine monoclonal antibody against human glycoprotein IIb/IIIa complex (PAC-1). Immobilized PMVs can be subjected to label-free analyses by means ellipsometry, atomic force microscopy (AFM) and time-of-flight secondary ion mass spectrometry (TOF-SIMS).
Assuntos
Plaquetas , Vesículas Extracelulares/química , Silício/química , Análise Espectral/métodos , Citometria de Fluxo , Humanos , Lipossomos , Microscopia de Força Atômica , Propriedades de SuperfícieRESUMO
The recognition of the importance of diabetes in vascular disease has greatly increased lately. Common risk factors for diabetes-related vascular disease include hyperglycemia, insulin resistance, dyslipidemia, inflammation, hypercoagulability, hypertension, and atherosclerosis. All of these factors contribute to the endothelial dysfunction which generates the diabetic complications, both macro and microvascular. Knowledge of diabetes-related vascular complications and of associated mechanisms it is becoming increasingly important for therapists. The discovery of microparticles (MPs) and their associated microRNAs (miRNAs) have opened new perspectives capturing the attention of basic and clinical scientists for their potential to become new therapeutic targets and clinical biomarkers. MPs known as submicron vesicles generated from membranes of apoptotic or activated cells into circulation have the ability to act as autocrine and paracrine effectors in cell-to-cell communication. They operate as biological vectors modulating the endothelial dysfunction, inflammation, coagulation, angiogenesis, thrombosis, subsequently contributing to the progression of macro and microvascular complications in diabetes. More recently, miRNAs have started to be actively investigated, leading to first exciting reports, which suggest their significant role in vascular physiology and disease. The contribution of MPs and also of their associated miRNAs to the development of vascular complications in diabetes was largely unexplored and undiscussed. In essence, with this review we bring light upon the understanding of impact diabetes has on vascular biology, and the significant role of MPs and MPs associated miRNAs as novel mediators, potential biomarkers and therapeutic targets in vascular complications in diabetes.
Assuntos
Micropartículas Derivadas de Células/fisiologia , Angiopatias Diabéticas/etiologia , MicroRNAs/metabolismo , Animais , Micropartículas Derivadas de Células/genética , Angiopatias Diabéticas/genética , Angiopatias Diabéticas/fisiopatologia , Dislipidemias/complicações , Endotélio Vascular/fisiopatologia , Humanos , Hiperglicemia/complicações , Inflamação/complicações , Resistência à Insulina , MicroRNAs/genética , Trombofilia/complicaçõesRESUMO
BACKGROUND: There is growing evidence that exposure to titanium dioxide nanoparticles (TiO2 NPs) could be harmful. Previously, we have shown that TiO2 NPs induces endothelial cell dysfunction and damage in glial cells. Considering that inhaled particles can induce systemic effects and the evidence that nanoparticles may translocate out of the lungs, we evaluated whether different types of TiO2 NPs can induce the expression of receptors for adhesion molecules on monocytes (U937 cell line). We evaluated the role of reactive oxygen spices (ROS) on these effects. METHODS: The expression of receptors for early (sLe(x) and PSGL-1) and late (LFA-1, VLA-4 and αVß3) adhesion molecules was evaluated in U937 cells on a time course (3-24 h) using a wide range of concentrations (0.001-100 µg/mL) of three types of TiO2 NPs (<25 nm anatase, 50 nm anatase-rutile or < 100 nm anatase). Cells exposed to TNFα were considered positive controls, and unexposed cells, negative controls. In some experiments we added 10 µmolar of N-acetylcysteine (NAC) to evaluate the role of ROS. RESULTS: All tested particles, starting at a concentration of 0.03 µg/mL, induced the expression of receptors for early and late adhesion molecules. The largest increases were induced by the different molecules after 3 h of exposure for sLe(x) and PSGL-1 (up to 3-fold of the positive controls) and after 18 h of exposure for LFA-1, VLA-4 and αVß3 (up to 2.5-fold of the positive controls). Oxidative stress was observed as early as 10 min after exposure, but the maximum peak was found after 4 h of exposure. Adhesion of exposed or unexposed monocytes to unexposed or exposed endothelial cells was tested, and we observed that monocytes cells adhere in similar amounts to endothelial cells if one of the two cell types, or both were exposed. When NAC was added, the expression of the receptors was inhibited. CONCLUSIONS: These results show that small concentrations of particles may activate monocytes that attach to endothelial cells. These results suggest that distal effects can be induced by small amounts of particles that may translocate from the lungs. ROS play a central role in the induction of the expression of these receptors.
Assuntos
Moléculas de Adesão Celular/metabolismo , Nanopartículas Metálicas/toxicidade , Monócitos/metabolismo , Titânio/química , Humanos , Nanopartículas Metálicas/química , Monócitos/citologia , Células U937RESUMO
Within the first week of the disease, acute kidney injury (AKI) is among the most common causes of mortality in acute pancreatitis (AP). Recently, serum angiopoietin-2 (Ang-2) has been associated with hyperdynamic state of the systemic circulation. The aim of this study was to examine the associations between Ang-2 and the clinical AP severity during the first 72 hours of the disease, and organ disfunction, including AKI. Methods. Study included patients admitted to the surgery ward, diagnosed with AP. AKI was diagnosed according to KDIGO guidelines and renal failure according to modified Marshall scoring system. Ang-2 was determined in serum with ELISA. Results. AP was classified as mild (MAP) in 71% of patients, moderately severe (MSAP) in 22%, and severe (SAP) in 8%. During the first 72 hours of AP, 11 patients developed AKI and 6 developed renal failure. Ang-2 at 24, 48, and 72 hours following the onset of AP symptoms significantly predicted SAP and MSAP, as well as AKI and renal failure. Also, Ang-2 significantly correlated with acute phase proteins as well as with the indicators of renal disfunction. Conclusions. Serum Ang-2 may be a relevant predictor of AP severity, in particular of the development of AP-renal syndrome.