Extracellular vesicles (EVs) have recently emerged as a relevant way of cell to cell communication, and its analysis has become an indirect approach to assess the cell/tissue of origin status. ...However, the knowledge about their nature and role on metabolic diseases is still very scarce. We have established an insulin resistant (IR) and two lipid (palmitic/oleic) hypertrophied adipocyte cell models to isolate EVs to perform a protein cargo qualitative and quantitative Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH) analysis by mass spectrometry. Our results show a high proportion of obesity and IR-related proteins in pathological EVs; thus, we propose a panel of potential obese adipose tissue EV-biomarkers. Among those, lipid hypertrophied vesicles are characterized by ceruloplasmin, mimecan, and perilipin 1 adipokines, and those from the IR by the striking presence of the adiposity and IR related transforming growth factor-beta-induced protein ig-h3 (TFGBI). Interestingly, functional assays show that IR and hypertrophied adipocytes induce differentiation/hypertrophy and IR in healthy adipocytes through secreted EVs. Finally, we demonstrate that lipid atrophied adipocytes shed EVs promote macrophage inflammation by stimulating IL-6 and TNFα expression. Thus, we conclude that pathological adipocytes release vesicles containing representative protein cargo of the cell of origin that are able to induce metabolic alterations on healthy cells probably exacerbating the disease once established.
Background
Uveal melanoma (UM) has a high tendency to cause liver metastases. Metastatic disease is fatal, with a low survival rate. There are two large groups of UMs that, according to their risk of ...metastatic disease, can be divided into risk subgroups based on histopathological, cytogenetic and molecular characteristics. The presence of somatic mutations in certain genes may explain the origin and prognosis of these tumours.
Methods
Forty‐six UM samples previously classified as high or low metastatic risk according to chromosome 3 copy number status were tested for somatic mutations. A multi‐gene targeting strategy was adopted, and sequencing was performed using AmpliSeq technology.
Results
Mutations were found in all major UM‐related genes. BAP1 mutations confer an increased risk of metastases in high‐risk tumours; thus, this gene acts as a strong prognostic predictor in UM. The presence of somatic mutations in LZTS1 did not show significant differences in the risk of metastases.
Conclusions
This result supports the idea that exploring mutations and copy number variations in UM provides insights into patient outcomes. Genetic tests allow the determination of accurate personalized molecular profiles with a fundamental prognostic purpose.
The detection of metastases in patients with a diagnosis of uveal melanoma (UM) is a controversial issue. While only 1% of the patients have detectable metastases at the time of diagnosis, up to 30% ...of them will develop liver metastases within 5 years of treatment. UM spreads hematogenously, therefore, blood biomarkers may be helpful for prognosis and monitoring the disease progression. Despite the great progress achieved thanks to the genetic analysis of UM biopsies, this is an invasive technique and is limited by the heterogeneity of the tumor. The present review considers the current understanding in the field regarding biomarkers for the diagnosis and prognosis of UM and its metastasis, primarily to the liver. General covered topics include non-conventional markers such as proteins previously identified in cutaneous melanoma and UM cell lines, circulating tumor cells, microRNAs (miRNA), and circulating DNA, and how each may be critical in the development of novel blood biomarkers for UM.
The extracellular vesicles (EVs) have emerged as key players in metabolic disorders rising as an alternative way of paracrine/endocrine communication. In particular, in relation to adipose tissue ...(AT) secreted EVs, the current knowledge about its composition and function is still very limited. Nevertheless, those vesicles have been lately suggested as key players in AT communication at local level, and also with other metabolic peripheral and central organs participating in physiological homoeostasis, and also contributing to the metabolic deregulation related to obesity, diabetes, and associated comorbidities. The aim of this review is to summarize the most relevant data around the EVs secreted by adipose tissue, and especially in the context of obesity, focusing in its protein cargo. The description of the most frequent proteins identified in EVs shed by AT and its components, including their changes under pathological status, will give the reader a whole picture about the membrane/antigens, and intracellular proteins known so far, in an attempt to elucidate functional roles, and also suggesting biomarkers and new paths of therapeutic action.
Brown adipose tissue (BAT) is a key target for the development of new therapies against obesity due to its role in promoting energy expenditure; BAT secretory capacity is emerging as an important ...contributor to systemic effects, in which BAT extracellular vesicles (EVs) (i.e., batosomes) might be protagonists. EVs have emerged as a relevant cellular communication system and carriers of disease biomarkers. Therefore, characterization of the protein cargo of batosomes might reveal their potential as biomarkers of the metabolic activity of BAT. In this study, we are the first to isolate batosomes from lean and obese Sprague–Dawley rats, and to establish reference proteome maps. An LC-SWATH/MS analysis was also performed for comparisons with EVs secreted by white adipose tissue (subcutaneous and visceral WAT), and it showed that 60% of proteins were exclusive to BAT EVs. Precisely, batosomes of lean animals contain proteins associated with mitochondria, lipid metabolism, the electron transport chain, and the beta-oxidation pathway, and their protein cargo profile is dramatically affected by high fat diet (HFD) intervention. Thus, in obesity, batosomes are enriched with proteins involved in signal transduction, cell communication, the immune response, inflammation, thermogenesis, and potential obesity biomarkers including UCP1, Glut1, MIF, and ceruloplasmin. In conclusion, the protein cargo of BAT EVs is affected by the metabolic status and contains potential biomarkers of thermogenesis activity.
Extracellular vesicles (EVs) have lately arisen as new metabolic players in energy homeostasis participating in intercellular communication at the local and distant levels. These nanosized lipid ...bilayer spheres, carrying bioactive molecular cargo, have somehow changed the paradigm of biomedical research not only as a non-classic cell secretion mechanism, but as a rich source of biomarkers and as useful drug-delivery vehicles. Although the research about the role of EVs on metabolism and its deregulation on obesity and associated pathologies lagged slightly behind other diseases, the knowledge about their function under normal and pathological homeostasis is rapidly increasing. In this review, we are focusing on the current research regarding adipose tissue shed extracellular vesicles including their characterization, size profile, and molecular cargo content comprising miRNAs and membrane and intra-vesicular proteins. Finally, we will focus on the functional aspects attributed to vesicles secreted not only by adipocytes, but also by other cells comprising adipose tissue, describing the evidence to date on the deleterious effects of extracellular vesicles released by obese adipose tissue both locally and at the distant level by interacting with other peripheral organs and even at the central level.
More than 50% of patients with uveal melanoma end up developing metastases. Currently, there is no standard first-line treatment that facilitates proper management of the metastatic disease.
A ...systematic review of the last 40 years in PubMed with an exhaustive and strict selection of studies was conducted, in which the unit of measurement was overall survival (OS) expressed in Kaplan-Meier curves or numerically.
After the selection process, 110 articles were included. Regional therapies, such as intra-arterial liver chemotherapy (OS: 2, 9-22 months), isolated liver perfusion (OS: 9, 6-27, 4 months), or selective internal radiation therapy (OS: 18 months in monotherapy and 26 months in combination with other therapies) showed some superiority when compared to systemic therapies, such as chemotherapy (OS: 4, 6-17 months), immunotherapy (OS: 5-19, 1 month), immunosuppression (OS: 11 months), or targeted therapy (OS: 6-12 months), without being significant.
The results of this review suggest that there are no important differences in OS when comparing the different current treatment modalities. Most of the differences found seem to be explained by the heterogenicity of the different studies and the presence of biases in their design, rather than actual extensions of patient survival.
Las vesículas extracelulares (VEs) reflejan el estado fisiopatológico de sus células de origen y constituyen un reservorio de información renal accesible en la orina. Cuando la biopsia no es una ...opción, las VEs se presentan como un centinela de funcionalidad y daño, constituyendo una aproximación no invasiva. Sin embargo, el análisis de las VEs de la orina requiere de un aislamiento previo que ralentiza y dificulta su traslación a la práctica clínica. El objetivo de este trabajo es mostrar la aplicabilidad de la tecnología «sensor de imágenes de reflectancia interferométrica de una sola partícula» (SP-IRIS) mediante la plataforma ExoView® para el análisis directo de las VEs de la orina y de proteínas implicadas en funcionalidad renal.
La tecnología ExoView® permite la cuantificación y el fenotipado de las VEs presentes en la orina y la cuantificación de sus proteínas, de membrana e internas. En este trabajo se aplica esta tecnología a la cuantificación de las VEs de la orina y de sus proteínas con expresión renal tubular, amnionless (AMN) y secreted frizzled-related protein 1 (SFRP1), empleando únicamente 5μl de orina. La expresión tubular se confirmó por inmunohistoquímica.
El tamaño medio de las VEs analizadas fue de 59±16nm para las capturadas por la tetraspanina CD63, 61±16nm para las capturadas por la tetraspanina CD81 y 59±10 nm para la tetraspanina CD9, siendo CD63 la subpoblación de VEs mayoritaria en la orina (48,92%). La distribución de AMN y SFRP1 en las 3 tetraspaninas de captura resultó ser similar para ambas proteínas, expresándose mayoritariamente en CD63 (48,23% para AMN y 52,1% para SFRP1).
Este trabajo evidencia la aplicabilidad y las ventajas de la técnica ExoView® para el análisis directo de las VEs de la orina, y su cargo proteico en relación con el túbulo renal. El empleo de volúmenes mínimos, 5μl, y el tiempo total de análisis no superior a 3h facilita la traslación a la práctica clínica diaria de las VEs como fuente de información diagnóstica.
Extracellular vesicles (EVs) reflect the pathophysiological state of their cells of origin and are a reservoir of renal information accessible in urine. When biopsy is not an option, EVs present themselves as sentinels of function and damage, providing a non-invasive approach. However, the analysis of EVs in urine requires prior isolation, which slows down and hinders translation to clinical practice. The aim of this study is to show the applicability of the “single particle interferometric reflectance imaging sensor” (SP-IRIS) technology through the ExoView® platform for the direct analysis of urine EVs and proteins involved in renal function.
The ExoView® technology enables the quantification and phenotyping of EVs present in urine and the quantification of their membrane and internal proteins. We have applied this technology to the quantification of urinary EVs and their proteins with renal tubular expression, amnionless (AMN) and secreted frizzled-related protein 1 (SFRP1), using only 5μl of urine. Tubular expression was confirmed by immunohistochemistry.
The mean size of the EVs analysed was 59±16nm for those captured by tetraspanin CD63, 61±16nm for those captured by tetraspanin CD81, and 59±10 nm for tetraspanin CD9, with CD63 being the majority EVs subpopulation in urine (48.92%). The distribution of AMN and SFRP1 in the three capture tetraspanins turned out to be similar for both proteins, being expressed mainly in CD63 (48.23% for AMN and 52.1% for SFRP1).
This work demonstrates the applicability and advantages of the ExoView® technique for the direct analysis of urine EVs and their protein content in relation to the renal tubule. The use of minimum volumes, 5μl, and the total analysis time not exceeding three hours facilitate the translation of EVs to daily clinical practice as source of diagnostic information.
Uveal melanoma (UM) is the most common primary intraocular tumor in adulthood. Approximately 50% of patients develop metastatic disease, which typically affects the liver and is usually fatal within ...one year. This type of cancer is heterogeneous in nature and is divided into two broad groups of tumors according to their susceptibility to develop metastasis. In the last decade, chromosomal abnormalities and the aberrant expression of several signaling pathways and oncogenes in uveal melanomas have been described. Recently, importance has been given to the association of the mentioned deregulation with the expression of non-coding RNAs (ncRNAs). Here, we review the different classes of ncRNAs—such as long non-coding RNAs (lncRNAs) and microRNAs (miRNAs)—and their contribution to the development of UM. Special attention is given to miRNAs and their regulatory role in physiopathology and their potential as biomarkers. As important agents in gene regulation, ncRNAs have a huge potential for opening up therapeutic pathways, predicting response to treatment, and anticipating patient outcome for UM.
Several studies showed an association between metabolic syndrome (MetS) and Parkinson's disease (PD). The linking mechanisms remain unclear. MetS promotes low-grade peripheral oxidative stress and ...inflammation and dysregulation of the adipose renin-angiotensin system (RAS). Interestingly, brain RAS dysregulation is involved in the progression of dopaminergic degeneration and PD. Circulating extracellular vesicles (EVs) from MetS fat tissue can cross the brain-blood barrier and may act as linking signals. We isolated and characterized EVs from MetS and control rats and analyzed their mRNA and protein cargo using RT-PCR and the ExoView R200 platform, respectively. Furthermore, cultures of the N27 dopaminergic cell line and the C6 astrocytic cell line were treated with EVs from MetS rats. EVs were highly increased in MetS rat serum, which was inhibited by treatment of the rats with the angiotensin type-1-receptor blocker candesartan. Furthermore, EVs from MetS rats showed increased pro-oxidative/pro-inflammatory and decreased anti-oxidative/anti-inflammatory RAS components, which were inhibited in candesartan-treated MetS rats. In cultures, EVs from MetS rats increased N27 cell death and modulated C6 cell function, upregulating markers of neuroinflammation and oxidative stress, which were inhibited by the pre-treatment of cultures with candesartan. The results from rat models suggest EVs and their RAS cargo as a mechanism linking Mets and PD.
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