Retos y oportunidades en el estudio de vesículas extracelulares: contexto institucional a nivel mundial y situación actual en Colombia / Challenges and opportunities in the study of extracellular vesicles: Global institutional context and national state of the art

Resumen En la última década se ha incrementado el número de estudios y publicaciones sobre las vesículas extracelulares y los exosomas. En Colombia, ha habido interés y avances en su estudio, lo que se evidencia en el aumento de publicaciones y proyectos de investigación. Sin embargo, este es un campo de investigación aún en desarrollo, con desafíos analíticos y limitaciones técnicas, por lo cual, en el planteamiento de los proyectos de investigación y desarrollo, es necesario considerar cuál es el estado del campo científico a nivel mundial en cuanto a la nomenclatura y la clasificación de las vesículas extracelulares, las técnicas, recursos, requisitos y especificaciones de calidad y las instituciones que regulan el campo. La respuesta a esta pregunta permitirá desarrollar estudios que cumplan con los estándares internacionales, y las exigencias y recomendaciones institucionales. Sin embargo, la información científica disponible se encuentra dispersa y no todos los aspectos son tratados a cabalidad. En este actualización se condensa la información disponible y se presentan los términos oficiales para denominar las vesículas extracelulares y la nomenclatura aceptada actualmente, así como la evolución del campo, la homogenización de los parámetros experimentales, el establecimiento de autoridades científicas, instituciones y recursos, y las recomendaciones que se han generado a nivel mundial para el desarrollo de investigaciones en vesículas extracelulares, incluidos su aislamiento, caracterización y estudio funcional. Por último, se analiza el contexto nacional de una forma crítica, teniendo en cuenta las fortalezas institucionales, los errores usualmente cometidos, y las técnicas y tecnologías analíticas disponibles.

Abstract In the last decade, the number of studies and publications on extracellular vesicles (EV) and exosomes has boomed. Colombia has displayed interest and progress in their study as shown in the increase of research project publications and products. However, this research field is still developing and has its own analytical challenges and technical limitations. For planning research projects and developing EV studies it is necessary to consider what is the state of the scientific field worldwide concerning EV nomenclature and classification, available techniques, resources, requirements and quality specifications, and the institutions that regulate the field. Answering this question will elicit EV studies that comply with international standards and respond to institutional demands and recommendations. However, the scientific information available is scattered and not all the aspects are considered in full. In this update, the available information is condensed and the official terms and currently defined nomenclature is presented, as well as the evolution of the field, the homogenization of the experimental parameters, the establishment of scientific authorities, institutions, and resources, and the recommendations generated worldwide for their development and research including their isolation, characterization, and functional studies. Finally, I analyzed the national context in a critical way, considering institutional strengths, common mistakes, and available analytical techniques and technologies.

Level and clinical significance of vitreous microparticles in proliferative diabetic retinopathy / 中华眼底病杂志

Objective:To observe the level of microparticles in the vitreous of patients with proliferative diabetic retinopathy (PDR), and preliminarily explore the role of microparticles in the pathogenesis of PDR.Methods:A case control study. From January to December 2018, 54 cases of 54 eyes of PDR patients (PDR group) and 20 cases of non-diabetic retinopathy patients (control group), who were diagnosed and treated with vitrectomy (PPV) in the Department of Ophthalmology, Tianjin Medical University General Hospital vitreous samples were included in the study. Among 54 eyes in the PDR group, there were 42, 21, and 17 eyes with vitreous hemorrhage (VH), traction retinal detachment (TRD), and previous intravitreal injection of drugs, respectively. Among the 20 eyes of the control group, idiopathic macular hole, idiopathic anterior macular membrane, vitreous macular traction syndrome, and complete lens dislocation were 6, 6, 2, and 6 eyes, respectively. The PDR group was divided into uncombined TRD group and combined TRD group according to PDR stage and whether TRD occurred, with 33 and 21 eyes, respectively. According to the presence or absence of VH, they were divided into groups with VH and without VH, with 42 eyes and 12 eyes, respectively. According to whether anti-vascular endothelial growth factor (VEGF) drugs were injected into the intravitreal cavity 3 days before PPV, they were divided into anti-VEGF drug group and no anti-VEGF drug group, with 17 eyes and 37 eyes respectively. The levels of retinal photoreceptor cells (RMP), platelets (PMP), endothelial cells (EMP) and phosphatidylserine (PS-MP) expressing on the membrane surface in the sample were detected by flow cytometry. The comparison between the two groups of samples was performed by t test, and the comparison between multiple groups of samples was performed by one-way analysis of variance or Mann-Whitney test. Results:Compared with the control group, the vitreous RMP level of the PDR group was significantly decreased, and the EMP and PMP levels were significantly increased. The differences were statistically significant ( t=-2.361, 5.064, 3.531; P=0.018, <0.001, 0.001). There was no statistically significant difference in PS-MP levels between the two groups ( t=-1.617, P=0.110). Compared with the TRD group, the levels of RMP and PMP in the vitreous of the TRD group were significantly increased, and the difference was statistically significant ( t=-2.221, -2.098; P=0.031, 0.041). The level of EMP in the vitreous body of the anti-VEGF drug group was significantly lower than that of the non-anti-VEGF drug group, however, it was still higher than the control group. The difference was statistically significant ( Z=-2.430, -2.499; P=0.015, 0.012). The level of PMP in the vitreous body of the eye without VH was significantly higher than that in the group with VH, and the difference was statistically significant ( t=-3.097, P=0.003). Conclusions:The elevated levels of EMP and PMP in the vitreous of PDR patients may be related to the damage of retinal capillaries; intravitreal injection of anti-VEGF drugs before surgery can reduce the level of EMP. VH may be related to the procoagulant effect of PMP.

Circulating microsome proteins in the pathogenesis of preeclampsia / 中华检验医学杂志

Objective In this study, we aimed to detect the level of total circulating microparticles (MPs) in pregnant women with preeclampsia (PE) and analyze the proteome of MPs to explore their roles in the pathogenesis and progression of PE. Methods 98 pregnant women with PE, 54 healthy pregnant women, and 51 healthy non-pregnant women were enrolled from December 2016 to June 2018, whose MP levels were detected by flow cytometry and compared. Proteins extracted from the MPs were analyzed by high performance liquid chromatography mass spectrometry.Results The total MP level of the healthy pregnant group was significantly higher than thatof the non-pregnant group [159.87 (113.25, 218.18)/μl vs 94.10 (53.35, 140.23)/μl, P=0.004], but was not significantly different from that of the PE group. By proteomic profiling, 30 differential proteins were obtained between healthy pregnant women and healthy non-pregnant women, which were closely related to biological processes such as complements, coagulation cascades, angiogenesis and so on; 14 differential proteins were found between PE patients and healthy pregnant women, which were closely related to biological processes such as coagulation cascades, complements and inflammatory reactions, angiogenesis and so forth. Conclusions The level of circulating MPs may reflect the hypercoagulability of preeclampsia. In addition, circulating MPs may be involved in the pathogenesis of PE through various pathways by carrying different proteins, which indicates their potential value in the intervention of PE.

Current progress on the study of microparticles in ocular fundus diseases / 中华眼底病杂志

Microparticles are small vesicles that are released by budding of the plasma membrane during cellular activation and apoptotic cell breakdown.A spectrum of cell types can release microparticles including endothelial cells,platelets,macrophages,lymphocytes and tumor cells.Biological effects of microparticles mainly include procoagulant activity,inhibition of inflammation and cancer progression.The present study shows that vitreous microparticles isolated from proliferative diabetic retinopathy (PDR) stimulated endothelial cell proliferation and increased new vessel formation,promoting the pathological neovascularization in PDR patients.Oxidative stress induces the formation of retina pigment epithelium-derived microparticles carrying membrane complement regulatory proteins,which is associated with drusen formation and age related macular degeneration.Microparticles from lymphocyte (LMP) play an important role in anti-angiogenesis by altering the gene expression pattern of angiogenesis-related factors in macrophages.Besides,LMP are important proapoptotic regulators for retinoblastoma cells through reduction of spleen tyrosine kinase expression and upregulation of the p53-p21 pathway which ultimately activates caspase-3.However,how to apply the microparticles in the prevention and treatment of retinal diseases is a major challenge,because the study of the microparticles in the fundus diseases is still limited.Further studies conducted would certainly enhance the application of microparticles in the fundus diseases.

Cell Derived Microparticles in Gingival Crevicular Fluid from Periodontitis Patients with Type 2 Diabetes

Abstract Cell-derived microparticles (MPs) have been described as vital contributors to the inflammatory process. However, its role in the periodontal disease pathogenesis remains unclear. Therefore, we aimed to detect the presence neutrophil (CD66b+) and platelet (CD41b+) derived microparticles in gingival crevicular fluid from individuals having periodontitis aggravated by type 2 diabetes. Twelve patients (56.2 ±7.2 yrs) with severe form of chronic periodontitis aggravated by type 2 diabetes were included. Clinical and metabolic data were gathered. Gingival crevicular fluid was collected using filter strips from deep and shallow sites. MPs were detected by flow cytometry according to their size (< 1 µm) and the expression of surface markers (CD66b for neutrophil-derived MPs and CD41b for platelet-derived MPs). All samples were positive for the antibodies. Median levels of CD66b+ MPs and CD41b+ MPs were, respectively, 3,677.0 (2,553.2 - 9,059.8) MP/µL and 520.7 (432.9 - 766.1) MP/µL in deep sites. In shallow sites, the corresponding values were 2,644.9 (1,451.5 - 3,858.9) MP/µL and 371.2 (287.2 - 692.7) MP/µL. There was no significant difference between deep and shallow sites (p>0.05). In conclusion, this study reported the presence of neutrophil and platelet derived microparticles in gingival crevicular fluid from individuals having severe periodontitis and type 2 diabetes.

Resumo As micropartículas derivadas de células (MPs) têm sido descritas como contribuintes vitais para o processo inflamatório. No entanto, seu papel na patogênese da doença periodontal permanece obscuro. Por isso, nosso objetivo foi detectar a presença de micropartículas derivadas de neutrófilos (CD66b +) e plaquetas (CD41b +) no fluido gengival de indivíduos com periodontite e diabetes tipo 2. Doze pacientes (56,2 ± 7,2 anos) com periodontite crônica severa e diabetes tipo 2 foram incluídos no estudo. Foram coletados dados clínicos e metabólicos. O fluido gengival foi coletado usando tiras de filtro de papel em sítios rasos e profundos. As MPs foram detectadas por citometria de fluxo de acordo com o seu tamanho (<1 μm) e pela expressão de marcadores de superfície (CD66b para MPs derivadas de neutrófilos e CD41b para MPs derivadas de plaquetas). Todas as amostras foram positivas para os anticorpos. Os níveis médios de CD66b + MPs e CD41b + MPs foram, respectivamente, 3.677.0 (2,553.2 - 9,059.8) MP/μL e 520.7 (432.9 - 766.1) MP/μL nos sítios profundos. Nos sítios rasos, os valores correspondentes foram 2,644.9 (1,451.5 - 3,858.9) MP/μL e 371.2 (287.2 - 692.7) MP/μL. Não houve diferença significativa entre os sítios rasos e profundos (p>0.05). Concluindo, o presente estudo reportou a presença de micropartículas derivadas de neutrófilos e plaquetas no fluido gengival de pacientes com periodontite e com diabetes tipo 2 .

A Meta-Analysis of Circulating Microvesicles in Patients with Myocardial Infarction / Uma Meta-Análise de Microvesículas Circulantes em Pacientes com Infarto do Miocárdio

Abstract Background: Cell-derived microvesicles (MVs) are vesicles released from activated or apoptotic cells. However, the levels of MVs in myocardial infarction have been found inconsistent in researches. Objective: To assess the association between MVs and myocardial infarction by conducting a meta-analysis. Methods: A systematic literature search on PubMed, Embase, Cochran, Google Scholar electronic database was conducted. Comparison of the MVs levels between myocardial infarction patients and healthy persons were included in our study. Standard Mean Difference (SMD) and 95% confidence interval (CI) in groups were calculated and meta-analyzed. Results: 11 studies with a total of 436 participants were included. Compared with the health persons, AMVs [SMD = 3.65, 95% CI (1.03, 6.27)], PMVs [SMD = 2.88, 95% CI (1.82, 3.93),] and EMVs [SMD = 2.73, 95% CI (1.13, 4.34)], levels were higher in patients with myocardial infarction. However, LMVs levels [SMD = 0.73, 95% CI (-0.57, 2.03)] were not changed significantly in patients with myocardial infarction. Conclusions: AMVs, PMVs and EMVs might be potential biomarkers for myocardial infarction.

Resumo Fundamentos: As microvesículas derivadas de células (MVs) são vesículas liberadas de células ativadas ou apoptóticas. No entanto, os níveis de MVs no infarto do miocárdio foram encontrados inconsistentes nas pesquisas. Objetivo: Avaliar a associação entre MV e infarto do miocárdio por meio de uma meta-análise. Métodos: Foi realizada uma pesquisa sistemática na literatura em PubMed, Embase, Cochran e no banco de dados eletrônico do Google Scholar. Uma comparação dos níveis de MV entre pacientes com infarto do miocárdio e pessoas saudáveis foi incluída no nosso estudo. A Diferença Média Padrão (DMP) e o intervalo de confiança (IC) de 95% nos grupos foram calculadas e meta-analisadas. Resultados: Foram incluídos 11 estudos com um total de 436 participantes. Em comparação com as pessoas saudáveis, as MVA [DMP = 3,65, IC 95% (1,03, 6,27)], MVPs [DMP = 2,88, IC 95% (1,82, 3,93)] e MVEs [DMP = 2,73, IC 95% (1,13, 4.34)], foram maiores em pacientes com infarto do miocárdio. No entanto, os níveis de MVL [DMP = 0,73, IC 95% (-0,57, 2,03)] não foram alterados significativamente em pacientes com infarto do miocárdio. Conclusões: MVAs, MVPs e MVEs podem ser biomarcadores potenciais para o infarto do miocárdio.

The effects of indoxyl sulfate-induced endothelial microparticles on neointimal hyperplasia formation in an ex vivo model

PURPOSE: Neointimal hyperplasia (NH) is considered to be one of the main causes of vascular access occlusion in patients receiving hemodialysis. Endothelial injury and TGF-β-mediated proliferation of vascular smooth muscle cells (VSMCs) induce NH. Endothelial microparticles (EMPs) are also increased by endothelial injury. We aimed to investigate the effects of EMPs and TGF-β expression on VSMC proliferation and their contributions to NH formation in an ex vivo model. METHODS: EMPs were collected from the culture media of human umbilical vein endothelial cells treated with indoxyl sulfate (IS, 250 µg/mL) after ultracentrifugation at 100,000 × g. Porcine internal jugular veins were isolated and treated with EMPs (2 × 10⁶ /mL) or left untreated for 12 days and subsequently compared with TGF-β (10 ng/mL)-treated venous tissue. Intima-media thickness and NH area were assessed using a digital program. Masson's trichrome staining and immunohistochemistry (IHC) analysis for α-smooth muscle actin, phosphorylated Akt, ERK1/2, p38 mitogen-activated protein kinase (MAPK), and Smad3 were performed on each vein sample. RESULTS: NH and VSMC proliferation developed to a significantly greater degree in EMP-treated veins compared to controls, with similar patterns seen in TGF-β-stimulated samples. IHC analysis demonstrated that EMPs markedly increased phosphorylation of Akt, ERK1/2, p38 MAPK, and Smad3 in areas of venous NH formation. CONCLUSION: Our results showed that IS-induced EMPs provoked massive VSMC proliferation and NH formation via activation of the TGF-β signaling pathways. Further investigation is needed to elucidate the precise mechanism of EMP activity on vascular access stenosis in vivo.

Effect of methotrexate packaged by tumor derived microparticles combined with radiotherapy on proliferation of cancer stem cells in lung adenocarcinoma in vitro / 肿瘤研究与临床

Objective To explore the effect of methotrexate packaged by tumor derived microparticles (T-MP MTX) combined with radiotherapy on lung cancer stem cell (CSC) in vitro. Methods T-MP MTX was prepared from non-small cell lung cancer A549 cells. Proliferative changes of A549 cells, bronchial epithelial cells H460 and 16HBE cells treated by T-MP MTX were assayed by MTT method. Cell cycles of A549 cells in blank group and T-MP MYX group were examined by fluorescence activated cell sorting (FACS). The effect of T-MP MTX combined with radiotherapy on CSCs was assessed by tumor sphere formation experiment and animal experiment. The expressions of stemness relative genes (such as β-catenin, Nanog, SOX-2 and KLF4) were measured by Western blot. Results T-MP MTX dose-dependently inhibited the cell growth in A549 cells, but didn't in H460 cells and 16HBE cells. The S cycle ratio of A549 cells in blank group and T-MP MYX group measured by FACS were (15.83±3.14)%and (47.47±6.69)%, respectively. S cycle ratio of T-MP MYX group was notably higher compared with that of blank group (t=7.411, P=0.002). Further study revealed that the number of tumor sphere in blank group, control group, 2 Gy group, 4 Gy group and 6 Gy group was (268.9±22.4), (172.4±18.7), (48.3±5.1), (16.3±3.5) and (5.1±3.1), respectively. The number of tumor sphere in other groups was decreased compared with that in blank group (F=228.291, P=0.000). The numbers of tumor sphere in 2 Gy group, 4 Gy group and 6 Gy groups was also reduced compared with that in control group. Importantly, the number of tumor sphere in these groups were decreased dramatically as the dose of radiotherapy increased (F=95.142, P=0.000). The results of tumor sphere volume were similar with the number of tumor sphere. Western blot experiment showed that T-MP MTX treatment in A549 cells decreased the expression of stemness relative genes (β-catenin, Nanog, SOX-2 and KLF4), and its role was reinforced when radiotherapy was combined. Animal experiment implied that activity of luciferase in T-MP MTX group was decreased compared with that in blank group (P=0.000), and the activity of luciferase in T-MP MTX plus 2 Gy group was reduced significantly (t=6.887, P=0.002). Conclusions T-MP MTX has a potential to sensibilize radiotherapy, and it will synergistically inhibit the proliferation of CSCs when combined with radiotherapy. Moreover, its mechanism may be related with T-MP MTX activating CSCs from hypometabolism state and blocking process of cell cycle.

Novos Horizontes para abordagem do infarto agudo do miocárdio com supradesnivelamento do segmento ST / New horizons for approach of ST-Segment elevation mycardial infarction

Nas últimas décadas, houve redução da mortalidade por infarto agudo do miocárdio com supradesnivelamento do segmento ST (IAMCSST) associada a um conjunto de ações que une avanços tecnológicos e políticas públicas. Entretanto, sua característica de doença tempo-dependente ainda é responsável por elevado número de casos de morte súbita e as consequências da reperfusão tardia ou ineficiente estão relacionadas à ocorrência de insuficiência cardíaca e maior morbimortalidade. Sob esse ponto de vista, foram revisados três diferentes aspectos: o impacto das arritmias ventriculares no atendimento pré-hospitalar; a influência do fator de Von Willebrand e o papel das micropartículas no diagnóstico da doença.

In the past decades, there was a reduction in mortality from ST segment elevation acute myocardial infarction (STEMI) associated with a set of actions combining technological advances and public policies. However, its characteristic of a time-dependent disease is still responsible for a high number of cases of sudden death and the consequences of late or inefficient reperfusion are related to heart failure and increased morbidity and mortality. From this point of view, three different aspects were reviewed: the impact of ventricular arrhythmias in prehospital care; the influence of Von Willebrand factor and the role of microparticles in the diagnosis of the disease.

Microparticles as Potential Biomarkers of Cardiovascular Disease / Micropartículas como Possíveis Biomarcadores da Doença Cardiovascular

Primary prevention of cardiovascular disease is a choice of great relevance because of its impact on health. Some biomarkers, such as microparticles derived from different cell populations, have been considered useful in the assessment of cardiovascular disease. Microparticles are released by the membrane structures of different cell types upon activation or apoptosis, and are present in the plasma of healthy individuals (in levels considered physiological) and in patients with different pathologies. Many studies have suggested an association between microparticles and different pathological conditions, mainly the relationship with the development of cardiovascular diseases. Moreover, the effects of different lipid-lowering therapies have been described in regard to measurement of microparticles. The studies are still controversial regarding the levels of microparticles that can be considered pathological. In addition, the methodologies used still vary, suggesting the need for standardization of the different protocols applied, aiming at using microparticles as biomarkers in clinical practice.

A prevenção primária da doença cardiovascular constitui uma opção de grande relevância pelos seus impactos na saúde. Alguns biomarcadores têm sido considerados úteis na avaliação da doença cardiovascular, dentre eles micropartículas originadas de diferentes populações de células. Micropartículas são estruturas liberadas pela membrana de diferentes tipos celulares após ativação ou apoptose, presentes tanto no plasma de indivíduos saudáveis (níveis considerados fisiológicos) quanto em portadores de diferentes doenças. Muitos estudos têm sugerido uma associação entre micropartículas e diferentes condições patológicas, destacando-se a relação com o desenvolvimento das doenças cardiovasculares. Além disso, têm sido descritos os efeitos de diferentes terapias hipolipemiantes na mensuração de micropartículas. Os estudos ainda são controversos quanto aos níveis de micropartículas que possam ser considerados patológicos, e os métodos utilizados ainda são variados, o que sugere a necessidade da padronização dos diferentes protocolos utilizados, visando à utilização de micropartículas como biomarcadores úteis na prática clínica.

Effect of Abciximab on the Levels of Circulating Microparticles in Patients with Acute Myocardial Infarction Treated by Primary Angioplasty

BACKGROUND AND OBJECTIVES: We investigated the effect of the additional use of abciximab during percutaneous coronary intervention (PCI) on the level of procoagulant microparticles (MPs) in patients with ST-segment elevation myocardial infarction (STEMI) who had undergone primary PCI. SUBJECTS AND METHODS: In this study, we studied 86 patients with STEMI (72 men, age 58+/-13) who had undergone primary PCI. The decision to administer abciximab immediately prior to PCI was left to the discretion of the operator. Blood samples for analysis of MPs were obtained from the femoral artery before and after PCI. MPs with procoagulant potential were measured using a commercial kit. The cellular origins of MPs were determined by antigenic capture with specific antibodies. RESULTS: Procoagulant MPs captured onto annexin V were not changed significantly after PCI {13.4+/-13.2 nM vs. 13.2+/-16.1 nM phosphatidylserine equivalent (PS eq), p=0.479}. Abciximab was used in 30 of 86 patients (35%) immediately prior to PCI. In patients who had undergone PCI without abciximab, no significant change in the level of MPs was observed after PCI. However, in the abciximab group, the level of circulating MPs was significantly decreased after PCI (12.0+/-10.7 nM vs. 7.8+/-11.7 nM PS eq, p=0.018). Levels of endothelial- and platelet-derived MPs also showed a significant reduction after PCI in the abciximab group. CONCLUSION: Primary PCI with additional abciximab significantly reduced the level of procoagulant MPs regardless of their cellular origins in patients with STEMI.