RESUMO
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.
Assuntos
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
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.
Assuntos
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
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
Pervasive transcription of the human genome is responsible for the production of a myriad of non-coding RNA molecules (ncRNAs) some of them with regulatory functions. The pivotal role of ncRNAs in cardiovascular biology has been unveiled in the last decade, starting from the characterization of the involvement of micro-RNAs in cardiovascular development and function, and followed by the use of circulating ncRNAs as biomarkers of cardiovascular diseases. The human non-coding secretome is composed by several RNA species that circulate in body fluids and could be used as biomarkers for diagnosis and outcome prediction. In cardiovascular diseases, secreted ncRNAs have been described as biomarkers of several conditions including myocardial infarction, cardiac failure, and atrial fibrillation. Among circulating ncRNAs, micro-RNAs (miRNAs), long noncoding RNAs (lncRNAs) and circular RNAs (circRNAs) have been proposed as biomarkers in different cardiovascular diseases. In comparison with standard biomarkers, the biochemical nature of ncRNAs offers better stability and flexible storage conditions of the samples, and increased sensitivity and specificity. In this review we describe the current trends and future prospects of the use of the ncRNA secretome components as biomarkers of cardiovascular diseases, including the opening questions related with their secretion mechanisms and regulatory actions.
Assuntos
Doenças Cardiovasculares/sangue , MicroRNAs/sangue , RNA Longo não Codificante/sangue , RNA/sangue , Biomarcadores/sangue , Doenças Cardiovasculares/genética , Humanos , MicroRNAs/genética , RNA/genética , RNA Circular , RNA Longo não Codificante/genéticaRESUMO
Background: Detailed and invasive clinical investigations are required to identify the causes of haematuria. Highly unbalanced patient population (predominantly male) and a wide range of potential causes make the ability to correctly classify patients and identify patient-specific biomarkers a major challenge. Studies have shown that it is possible to improve the diagnosis using multi-marker analysis, even in unbalanced datasets, by applying advanced analytical methods. Here, we applied several machine learning algorithms to classify patients from the haematuria patient cohort (HaBio) by analysing multiple biomarkers and to identify the most relevant ones. Materials and methods: We applied several classification and feature selection methods (k-means clustering, decision trees, random forest with LIME explainer and CACTUS algorithm) to stratify patients into two groups: healthy (with no clear cause of haematuria) or sick (with an identified cause of haematuria e.g., bladder cancer, or infection). The classification performance of the models was compared. Biomarkers identified as important by the algorithms were also analysed in relation to their involvement in the pathological processes. Results: Results showed that a high unbalance in the datasets significantly affected the classification by random forest and decision trees, leading to the overestimation of the sick class and low model performance. CACTUS algorithm was more robust to the unbalance in the dataset. CACTUS obtained a balanced accuracy of 0.747 for both genders, 0.718 for females and 0.803 for males. The analysis showed that in the classification process for the whole dataset: microalbumin, male gender, and tPSA emerged as the most informative biomarkers. For males: age, microalbumin, tPSA, cystatin C, BTA, HAD and S100A4 were the most significant biomarkers while for females microalbumin, IL-8, pERK, and CXCL16. Conclusions: CACTUS algorithm demonstrated improved performance compared with other methods such as decision trees and random forest. Additionally, we identified the most relevant biomarkers for the specific patient group, which could be considered in the future as novel biomarkers for diagnosis. Our results have the potential to inform future research and provide new personalised diagnostic approaches tailored directly to the needs of the individuals.
RESUMO
Extracellular vesicles, especially the larger fraction (LEVs - large extracellular vesicles), are believed to be an important means of intercellular communication. Earlier studies on LEVs have shown their healing properties, especially in the vascular cells of diabetic patients. Uptake of LEVs by endothelial cells and internalization of their cargo have also been demonstrated. Endothelial cells change their properties under hyperglycemic conditions (HGC), which reduces their activity and is the cause of endothelial dysfunction. The aim of our study was to investigate how human umbilical vein endothelial cells (HUVECs) change their biological properties: shape, mobility, cell surface stiffness, as well as describe the activation of metabolic pathways after exposure to the harmful effects of HGC and the administration of LEVs released by endothelial cells. We obtained LEVs from HUVEC cultures in HGC and normoglycemia (NGC) using the filtration and ultracentrifugation methods. We assessed the size of LEVs and the presence of biomarkers such as phosphatidylserine, CD63, beta-actin and HSP70. We analyzed the LEVs uptake efficiency by HUVECs, HUVEC shape, actin cytoskeleton remodeling, surface stiffness and finally gene expression by mRNA analysis. Under HGC conditions, HUVECs were larger and had a stiffened surface and a strengthened actin cortex compared to cells under NGC condition. HGC also altered the activation of metabolic pathways, especially those related to intracellular transport, metabolism, and organization of cellular components. The most interesting observation in our study is that LEVs did not restore cell motility disturbed by HGC. Although, LEVs were not able to reverse this deleterious effect of HGC, they activated transcription of genes involved in protein synthesis and vesicle trafficking in HUVECs.
Assuntos
Vesículas Extracelulares , Hiperglicemia , Humanos , Vesículas Extracelulares/metabolismo , Hiperglicemia/metabolismo , Células Endoteliais da Veia Umbilical Humana , Movimento Celular , Comunicação CelularRESUMO
Recent years have brought great focus on the development of drug delivery systems based on extracellular vesicles (EVs). Considering the possible applications of EVs as drug carriers, the isolation process is a crucial step. To solve the problems involved in EV isolation, we developed and validated a new EV isolation method-low-vacuum filtration (LVF)-and compared it with two commonly applied procedures-differential centrifugation (DC) and ultracentrifugation (UC). EVs isolated from endothelial cell culture media were characterized by (a) Transmission Electron Microscopy (TEM), (b) Nanoparticle Tracking Analysis (NTA), (c) Western blot and (d) Attenuated Total Reflection Fourier-Transform Infrared Spectroscopy (ATR-FTIR). Additionally, the membrane surface was imaged with Environmental Scanning Electron Microscopy (ESEM). We found that LVF was a reproducible and efficient method for EV isolation from conditioned media. Additionally, we observed a correlation between ATR-FTIR spectra quality and EV and protein concentration. ESEM imaging confirmed that the actual pore diameter was close to the values calculated theoretically. LVF is an easy, fast and inexpensive EV isolation method that allows for the isolation of both ectosomes and exosomes from high-volume sources with good repeatability. We believe that it could be an efficient alternative to commonly applied methods.
RESUMO
Drug delivery systems are created to achieve the desired therapeutic effect of a specific pharmaceutical compound. Numerous drawbacks and side effects such as unfavorable pharmacokinetics, lack of tissue selectivity, immunogenicity, increased systemic clearance and toxicity, have been observed for currently available drug delivery systems (DDSs). The use of natural and artificial extracellular vesicles (EVs) in drug delivery may help to solve the aforementioned problems faced by different DDSs. Due to their self-origin, small size, flexibility, the presence of multiple adhesive molecules on their surfaces as well as their function as biomolecules carriers, EVs are the perfect candidates for DDSs. Currently, several drug delivery systems based on EVs have been proposed. While the great potential of these particles in targeted drug delivery has been recognized in cancer, hepatitis C, neurodegenerative diseases, inflammatory states etc., this field is still in the early stage of development. Unfortunately, the use of EVs from natural sources (cell cultures, body fluids) results in numerous problems in terms of the heterogeneity of isolated vesicle population as well as the method of isolation thereof, which may influence vesicle composition and properties. Therefore, there is a significant need for the synthesis of artificial EV-based DDSs under strictly controlled laboratory conditions and from well-defined biomolecules (proteins and lipids). Vesicle-mimetic delivery systems, characterized by properties similar to natural EVs, will bring new opportunities to study the mechanisms of DDS internalization and their biological activity after delivering their cargo to a target cell.
Assuntos
Sistemas de Liberação de Medicamentos , Vesículas Extracelulares , Preparações Farmacêuticas/administração & dosagem , HumanosRESUMO
BACKGROUND AND AIMS: Diabetic foot ulcers (DFUs) are linked to amputations and premature deaths. Negative pressure wound therapy (NPWT) has been used for DFUs. The mechanism of NPWT's action may be associated with its influence on circulating molecules. We assessed NPWT's effect on the plasma levels of angiopoietin-2 (Ang2), a key regulator of angiogenesis, and its microvesicular receptors (Tie2) as well as the microvesicles (MVs) themselves in DFU patients. MATERIALS AND METHODS: We included 69 patients with type 2 diabetes mellitus (T2DM) and neuropathic, noninfected DFUs-49 were treated with NPWT and 20 were treated with standard therapy (ST). Assigning patients to the NPWT group was not random but based on DFU characteristics, especially wound area. Ang2 was measured by ELISA in the entire group, while in a subgroup of 19 individuals on NPWT and 10 on ST, flow cytometry was used to measure Tie2+ and the corresponding isotype control (Iso+) and annexin V (AnnV+) as well as total MVs. Measurements were performed at the beginning and after 8 ± 1 days of therapy. RESULTS: Treatment groups were similar for basic characteristics but differed by their median DFU areas (10.3 (4.2-18.9) vs. 1.3 (0.9-3.4) cm2, p = 0.0001). At day 0, no difference was observed in Ang2 levels, total MVs, MV Tie+, and MV AnnV+ between the groups. Ang2 decreased after 8 days in the NPWT group, unlike in the ST group (3.54 (2.40-5.40) vs. 3.32 (2.33-4.61), p = 0.02, and 3.19 ± 1.11 vs. 3.19 ± 1.29 ng/mL, p = 0.98, respectively). No other parameters were identified that may have been influenced by the NPWT treatment. CONCLUSION: NPWT in T2DM patients with neuropathic, noninfected DFU seems to lead to reduction of the Ang2 level. Influencing the level of Ang2 may constitute one of NPWT-related mechanisms to accelerate wound healing.
Assuntos
Angiopoietina-2/sangue , Pé Diabético/terapia , Tratamento de Ferimentos com Pressão Negativa , Cicatrização , Idoso , Biomarcadores/sangue , Pé Diabético/sangue , Pé Diabético/diagnóstico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Tratamento de Ferimentos com Pressão Negativa/efeitos adversos , Neovascularização Fisiológica , Projetos Piloto , Receptor TIE-2/sangue , Fatores de Tempo , Resultado do TratamentoRESUMO
AIMS: Numerous studies confirmed the involvement of extracellular vesicles in cancer development and progression. The present study was designed to investigate the glycan composition of ectosomes derived by human cutaneous melanoma (CM) cell lines with the use of lectins. MAIN METHODS: Ectosomes released by primary (WM115, WM793) and metastatic (WM266-4, WM1205Lu) CM cells were isolated from conditioned media by sequential centrifugation. Proteins from ectosomes, the whole cell extracts and membrane fractions were probed with a panel of lectins using Western Blot and flow cytometry and compared in terms of disease stage and glycosignature. KEY FINDINGS: Ectosomal proteins revealed enrichment (mainly with fucose and complex type N-glycans with bisecting GlcNAc) or depletion of specific glycoepitopes in comparison to the parental cell membranes. Moreover, similar lectin binding patterns were observed between related cell lines. It is the first study to characterize the glycosylation of ectosome proteins released by CM cells. SIGNIFICANCE: Our data indirectly supports the findings that ectosomes derive from particular regions of the cell membrane contain a unique glycan composition, which could serve as a specific sorting signal. If proven correct, the hypothesis that glycan-based protein sorting is a major mechanism for protein incorporation into ectosomes may provide new means to control vesicular content and have possible clinical implications.
Assuntos
Micropartículas Derivadas de Células/química , Epitopos/química , Melanoma/metabolismo , Polissacarídeos/química , Neoplasias Cutâneas/metabolismo , Biomarcadores/química , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Movimento Celular , Progressão da Doença , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica , Glicosilação , Humanos , Lectinas/química , Metástase Linfática , Melanoma Maligno CutâneoRESUMO
OBJECTIVES: Bone infections are treated with antibiotics administered intravenously, antibiotic-releasing bone cements or collagen sponges placed directly in the infected area. These approaches render limited effectiveness due to the lack of site specificity and invasiveness of implanting cements and sponges. To address these limitations, we developed a novel polysaccharide hydrogel-based injectable system that enables controlled delivery of gentamicin (GENT). Its advantages are minimal invasiveness, and localized and finely regulated release of the drug. METHODS: GENT was incorporated both directly within the gellan gum hydrogel and into poly(L-lactide-co-glycolide) nanoparticles embedded into the hydrogel. RESULTS: We confirmed the injectability of the system and measured extrusion force was 15.6 ± 1.0 N, which is suitable for injections. The system set properly after the injection as shown by rheological measurements. Desired burst release of the drug was observed within the first 12 h and the dose reached ~27% of total GENT. Subsequently, GENT was released gradually and sustainably: ~60% of initial dose within 90 days. In vitro studies confirmed antimicrobial activity of the system against Staphylococcus spp. and cytocompatibility with osteoblast-like cells. CONCLUSIONS: Developed injectable system enables minimally invasive, local and sustained delivery of the pharmaceutically relevant doses of GENT to combat bone infections.