Effects of an acute exercise bout in hypoxia on extracellular vesicle release in healthy and prediabetic subjects.

The purpose of this study is to investigate exosome-like vesicle (ELV) plasma concentrations and markers of multivesicular body (MVB) biogenesis in skeletal muscle in response to acute exercise. Seventeen healthy [body mass index (BMI): 23.5 ± 0.5 kg·m-2] and 15 prediabetic (BMI: 27.3 ± 1.2 kg·m-2) men were randomly assigned to two groups performing an acute cycling bout in normoxia or hypoxia ([Formula: see text] 14.0%). Venous blood samples were taken before (T0), during (T30), and after (T60) exercise, and biopsies from m. vastus lateralis were collected before and after exercise. Plasma ELVs were isolated by size exclusion chromatography, counted by nanoparticle tracking analysis (NTA), and characterized according to international standards, followed by expression analyses of canonical ELV markers in skeletal muscle. In the healthy normoxic group, the total number of particles in the plasma increased during exercise from T0 to T30 (+313%) followed by a decrease from T30 to T60 (-53%). In the same group, an increase in TSG101, CD81, and HSP60 protein expression was measured after exercise in plasma ELVs; however, in the prediabetic group, the total number of particles in the plasma was not affected by exercise. The mRNA content of TSG101, ALIX, and CD9 was upregulated in skeletal muscle after exercise in normoxia, whereas CD9 and CD81 were downregulated in hypoxia. ELV plasma abundance increased in response to acute aerobic exercise in healthy subjects in normoxia, but not in prediabetic subjects, nor in hypoxia. Skeletal muscle analyses suggested that this tissue did not likely play a major role of the exercise-induced increase in circulating ELVs.

Prediagnostic circulating inflammation biomarkers and esophageal squamous cell carcinoma: A case-cohort study in Japan.

Esophageal squamous cell carcinoma (ESCC) is the predominant histologic subtype of esophageal cancer worldwide. Measurements of circulating inflammation-related biomarkers may inform etiology or provide noninvasive signatures for early diagnosis. We therefore examined levels of inflammation molecules for associations with ESCC risk. Using a case-cohort study designed within the Japan Public Health Center-based Prospective Study, we measured baseline plasma levels of 92 biomarkers using a multiplex assay in a subcohort of 410 randomly selected participants and 66 participants with incident ESCC (including four cases that occurred in the subcohort). ESCC hazard ratios (HRs) were calculated for 2-4 quantiles of each biomarker by Cox proportional hazards regression models with age as the time metric, adjusted for sex, smoking and alcohol use. Twenty analytes were undetectable in nearly all samples. Of the remaining 72, 12 biomarkers (FGF19, ST1A1, STAMBP, AXIN1, CASP8, NT3, CD6, CDCP1, CD5, SLAMF1, OPG and CSF1) were associated with increased ESCC risk (ptrend < 0.05) with HRs per quantile 1.28-1.65. Seven biomarkers (CXCL6, CCL23, CXCL5, TGFA, CXCL1, OSM and CCL4) were inversely associated with HRs 0.57-0.72. FGF19, CASP8, STAMBP, ST1A1 and CCL-4 met statistical significance with false discovery rate correction. Associations did not differ <5 vs. ≥5 years between blood collection and ESCC diagnosis. CASP8, STAMBP and ST1A1 were strongly correlated (p < 0.05). Our study expands the range of inflammation molecules associated with the development of this highly lethal neoplasia. Correlations among these novel biomarkers suggest a possible shared pathway. These findings need replication and could further delineate ESCCs molecular mechanisms of carcinogenesis.

The ESCRT-III pathway facilitates cardiomyocyte release of cBIN1-containing microparticles.

Microparticles (MPs) are cell-cell communication vesicles derived from the cell surface plasma membrane, although they are not known to originate from cardiac ventricular muscle. In ventricular cardiomyocytes, the membrane deformation protein cardiac bridging integrator 1 (cBIN1 or BIN1+13+17) creates transverse-tubule (t-tubule) membrane microfolds, which facilitate ion channel trafficking and modulate local ionic concentrations. The microfold-generated microdomains continuously reorganize, adapting in response to stress to modulate the calcium signaling apparatus. We explored the possibility that cBIN1-microfolds are externally released from cardiomyocytes. Using electron microscopy imaging with immunogold labeling, we found in mouse plasma that cBIN1 exists in membrane vesicles about 200 nm in size, which is consistent with the size of MPs. In mice with cardiac-specific heterozygous Bin1 deletion, flow cytometry identified 47% less cBIN1-MPs in plasma, supporting cardiac origin. Cardiac release was also evidenced by the detection of cBIN1-MPs in medium bathing a pure population of isolated adult mouse cardiomyocytes. In human plasma, osmotic shock increased cBIN1 detection by enzyme-linked immunosorbent assay (ELISA), and cBIN1 level decreased in humans with heart failure, a condition with reduced cardiac muscle cBIN1, both of which support cBIN1 release in MPs from human hearts. Exploring putative mechanisms of MP release, we found that the membrane fission complex endosomal sorting complexes required for transport (ESCRT)-III subunit charged multivesicular body protein 4B (CHMP4B) colocalizes and coimmunoprecipitates with cBIN1, an interaction enhanced by actin stabilization. In HeLa cells with cBIN1 overexpression, knockdown of CHMP4B reduced the release of cBIN1-MPs. Using truncation mutants, we identified that the N-terminal BAR (N-BAR) domain in cBIN1 is required for CHMP4B binding and MP release. This study links the BAR protein superfamily to the ESCRT pathway for MP biogenesis in mammalian cardiac ventricular cells, identifying elements of a pathway by which cytoplasmic cBIN1 is released into blood.

Exploring Potential Proteomic Biomarkers for Prognosis of Infective Endocarditis through Profiled Autoantibodies by an Immunomics Protein Array Technique.

Though infective endocarditis (IE) is a life-threatening cardiac infection with a high mortality rate, the effective diagnostic and prognostic biomarkers for IE are still lacking. The aim of this study was to explore the potential applicable proteomic biomarkers for IE through the Immunome™ Protein Array system. The system was employed to profile those autoantibodies in IE patients and control subjects. Our results showed that interleukin-1 alpha (IL1A), nucleolar protein 4 (NOL4), tudor and KH domain-containing protein (TDRKH), G antigen 2B/2C (GAGE2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and X antigen family member 2 (XAGE2) are highly differentially-expressed among IE and non-IE control. Furthermore, bactericidal permeability-increasing protein (BPI), drebrin-like protein (DBNL), signal transducing adapter molecule 2 (STAM2), cyclin-dependent kinase 16 (CDK16), BAG family molecular chaperone regulator 4 (BAG4), and nuclear receptor-interacting protein 3 (NRIP3) are differentially-expressed among IE and healthy controls. On the other hand, those previously identified biomarkers for IE, including erythrocyte sedimentation rate, C-reactive protein, rheumatoid factor, procalcitonin, and N-terminal-pro-B-type natriuretic peptide demonstrated only minor significance. With scientific rationalities for those highly differentially-expressed proteins, they could serve as potential candidates for diagnostic biomarkers of IE for further analysis.

Clinical and prognostic importance of XIAP and USP8 in advanced stages of non-small cell lung cancer.

PURPOSE: To investigate the relationship of the apoptosis regulators X-linked inhibitor of apoptosis (XIAP) and ubiquitin specific protease 8 (USP8) with clinical parameters, survival and response to chemotherapy in patients with advanced stages of non-small cell lung cancer (NSCLC). METHODS: The study included 34 NSCLC patients (28 females, 6 males) and 44 healthy individuals (17 males, 27 females) as a control group. XIAP and USP8 levels were determined by ELISA. RESULTS: The median serum XIAP level of the patients and the control group showed no significant difference. USP8 level was higher in patients than in controls (p<0.0001). In univariate analysis, there was no significant relationship between XIAP and USP8 serum levels and age, sex, performance status, weight loss, stage of disease, histopatological type and response to chemotherapy. Response to chemotherapy did not differ between the high and low XIAP and USP8 groups . There was no significant difference in progression- free survival (PFS) (p=0.432 and p=0.50, respectively) and overall survival (OS) (p=0.989 and p=0.90, respectively) between the low and high XIAP and USP8 groups. CONCLUSION: No relationship was found in serum XIAP and USP8 levels with clinical parameters, response to chemotherapy, PFS and OS in patients with advanced stages of NSCLC.

High serum extracellular vesicle miR-10b expression predicts poor prognosis in patients with acute myeloid leukemia.

BACKGROUND: Increasing evidence have demonstrated that serum extracellular vesicle microRNAs (EV-miRNAs) are promising noninvasive biomarkers for various cancer types. OBJECTIVE: In this study, we aimed to investigate and evaluate the potential clinical significance of serum EV-miR-10b for acute myeloid leukemia (AML). METHODS: Blood samples were collected from a cohort of 95 de novo AML patients and 80 healthy individuals. Of all AML patients, 51 patients were considered as cytogenetic normal AML (CN-AML). Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was performed to measure the expression levels of serum EV-miR-10b. RESULTS: The extracellular vesicles we extracted from the serum samples were positive for EV/exosome markers including TSG101, CD63, Flotillin-1 and CD9. The expression levels of serum EV-miR-10b were significantly higher in AML/CN-AML patients than healthy controls. In addition, serum EV-miR-10b expression was strongly correlated with aggressive clinical characteristics. Moreover, receiver operating characteristic analysis showed serum EV-miR-10b yielded an area under the curve of 0.875, with 77.89% specificity and 82.50% sensitivity in identifying AML patients from normal controls. Furthermore, AML patients with higher serum EV-miR-10b expression had significantly shorter survival and serum EV-miR-10b was demonstrated to be an independent prognostic marker for overall survival in AML. CONCLUSIONS: Taken together, serum EV-miR-10b might serve as a promising biomarker for predicting the prognosis of AML.

Exosome enrichment by ultracentrifugation and size exclusion chromatography.

Exosomes are a subset of extracellular vesicles (EVs) that have important roles in intercellular communication. They contain and carry bioactive molecules within their membranes which are delivered to target cells. Reproducible isolation and enrichment of these exosomes will aid in evaluation of cellular communication. We present an approach that involved the pre-processing of plasma, combined with ultracentrifugation (UC) and size exclusion chromatography (SEC) to isolate EVs and subsequently enrich exosomes. Four variations of this approach (denoted methods I to IV) were compared. Coupling an ultracentrifugation method with size exclusion chromatography (Method II) provided the best yield by nanoparticle tracking analyses (NTA), the presence of the exosomal markers CD63, Flotillin-1 and TSG-101 (immunoblotting) and showed exosome morphology using transmission electron microscopy (TEM). This method provides an efficient way to enrich the exosomes from blood (plasma), which could be potentially employed for clinical diagnostic assessment and therapeutic intervention.

Serum proteome mapping of EGF transgenic mice reveal mechanistic biomarkers of lung cancer precursor lesions with clinical significance for human adenocarcinomas.

Atypical adenomatous hyperplasia (AAH) of the lung is a pre-invasive lesion (PL) with high risk of progression to lung cancer (LC). However, the pathways involved are uncertain. We searched for novel mechanistic biomarkers of AAH in an EGF transgenic disease model of lung cancer. Disease regulated proteins were validated by Western immunoblotting and immunohistochemistry (IHC) of control and morphologically altered respiratory epithelium. Translational work involved clinical resection material. Collectively, 68 unique serum proteins were identified by 2DE-MALDI-TOF mass spectrometry and 13 reached statistical significance (p < 0.05). EGF, amphiregulin and the EGFR endosomal sorting protein VPS28 were induced up to 5-fold while IHC confirmed strong induction of these proteins. Furthermore, ApoA1, α-2-macroglobulin, and vitamin-D binding protein were nearly 6- and 2-fold upregulated in AAH; however, ApoA1 was oppositely regulated in LC to evidence disease stage dependent regulation of this tumour suppressor. Conversely, plasminogen and transthyretin were highly significantly repressed by 3- and 20-fold. IHC confirmed induced ApoA1, Fetuin-B and transthyretin expression to influence calcification, inflammation and tumour-infiltrating macrophages. Moreover, serum ApoA4, ApoH and ApoM were 2-, 2- and 6-fold repressed; however tissue ApoM and sphingosine-1-phosphate receptor expression was markedly induced to suggest a critical role of sphingosine-1-phosphate signalling in PL and malignant transformation. Finally, a comparison of three different LC models revealed common and unique serum biomarkers mechanistically linked to EGFR, cMyc and cRaf signalling. Their validation by IHC on clinical resection material established relevance for distinct human lung pathologies. In conclusion, we identified mechanistic biomarker candidates recommended for in-depth clinical evaluation.

Exosomal microRNA Biomarkers: Emerging Frontiers in Colorectal and Other Human Cancers.

Diagnostic strategies, particularly non-invasive blood-based screening approaches, are gaining increased attention for the early detection and attenuation of mortality associated with colorectal cancer (CRC). However, the majority of current screening approaches are inadequate at replacing the conventional CRC diagnostic procedures. Yet, due to technological advances and better understanding of molecular events underlying human cancer, a new category of biomarkers are on the horizon. Recent evidence indicates that cells release a distinct class of small vesicles called 'exosomes', which contain nucleic acids and proteins that reflect and typify host-cell molecular architecture. Intriguingly, exosomes released from cancer cells have a distinct genetic and epigenetic makeup, which allows them to undertake their tumorigenic function. From a clinical standpoint, these unique cancer-specific fingerprints present in exosomes appear to be detectable in a small amount of blood, making them very attractive substrates for developing cancer biomarkers, particularly noninvasive diagnostic approaches.

Muscle Releases Alpha-Sarcoglycan Positive Extracellular Vesicles Carrying miRNAs in the Bloodstream.

In the past few years, skeletal muscle has emerged as an important secretory organ producing soluble factors, called myokines, that exert either autocrine, paracrine or endocrine effects. Moreover, recent studies have shown that muscle releases microRNAs into the bloodstream in response to physical exercise. These microRNAs affect target cells, such as hormones and cytokines. The mechanisms underlying microRNA secretion are poorly characterized at present. Here, we investigated whether muscle tissue releases extracellular vesicles (EVs), which carry microRNAs in the bloodstream under physiological conditions such as physical exercise. Using density gradient separation of plasma from sedentary and physically fit young men we found EVs positive for TSG101 and alpha-sarcoglycan (SGCA), and enriched for miR-206. Cytometric analysis showed that the SGCA+ EVs account for 1-5% of the total and that 60-65% of these EVs were also positive for the exosomal marker CD81. Furthermore, the SGCA-immuno captured sub-population of EVs exhibited higher levels of the miR-206/miR16 ratio compared to total plasma EVs. Finally, a significant positive correlation was found between the aerobic fitness and muscle-specific miRNAs and EV miR-133b and -181a-5p were significantly up-regulated after acute exercise. Thus, our study proposes EVs as a novel means of muscle communication potentially involved in muscle remodeling and homeostasis.

Identification of a new target of miR-16, Vacuolar Protein Sorting 4a.

RATIONALE: The rationale was to utilize a bioinformatics approach to identify miRNA binding sites in genes with single nucleotide mutations (SNPs) to discover pathways in heart failure (HF). OBJECTIVE: The objective was to focus on the genes containing miRNA binding sites with miRNAs that were significantly altered in end-stage HF and in response to a left ventricular assist device (LVAD). METHODS AND RESULTS: BEDTools v2.14.3 was used to discriminate SNPs within predicted 3'UTR miRNA binding sites. A member of the miR-15/107 family, miR-16, was decreased in the circulation of end-stage HF patients and increased in response to a LVAD (p<0.001). MiR-16 decreased Vacuolar Protein Sorting 4a (VPS4a) expression in HEK 293T cells (p<0.01). The SNP rs16958754 was identified in the miR-15/107 family binding site of VPS4a which abolished direct binding of miR-16 to the 3'UTR of VPS4a (p<0.05). VPS4a was increased in the circulation of end-stage HF patients (p<0.001), and led to a decrease in the number of HEK 293T cells in vitro (p<0.001). CONCLUSIONS: We provide evidence that miR-16 decreases in the circulation of end-stage HF patients and increases with a LVAD. Modeling studies suggest that miR-16 binds to and decreases expression of VPS4a. Overexpression of VPS4a decreases cell number. Together, these experiments suggest that miR-16 and VPS4a expression are altered in end-stage HF and in response to unloading with a LVAD. This signaling pathway may lead to reduced circulating cell number in HF.