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Extracellular vesicles (EVs) carry a wide range of molecules, such as proteins, RNAs, and DNA. EVs are secreted from a wide range of cells, including placental cells. Interestingly, EVs secreted from placental cells have been identified in maternal circulation as early as 6 weeks of gestation, and their concentration increases with the gestational age. While there is growing interest in elucidating the role of exosomes during normal and complicated pregnancies, progress in the field has been delayed because of the inability to quantify placental EVs from the maternal circulation. Recent reports have demonstrated the presence of placental-type alkaline phosphatase (PLAP) EVs only in the blood of pregnant women, indicating that PLAP is a marker to identify EVs secreted from the placenta. Therefore, here we describe a workflow to quantify placental EVs from maternal circulation using a targeted proteomics approach based on selecting specific peptides identified in the PLAP protein., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Caveolae proteins play diverse roles in cancer development and progression. In prostate cancer, non-caveolar caveolin-1 (CAV1) promotes metastasis, while CAVIN1 attenuates CAV1-induced metastasis. Here, we unveil a novel mechanism linking CAV1 to selective loading of exosomes with metastasis-promoting microRNAs., Results: We identify hnRNPK as a CAV1-regulated microRNA binding protein. In the absence of CAVIN1, non-caveolar CAV1 drives localisation of hnRPNK to multi-vesicular bodies (MVBs), recruiting AsUGnA motif-containing miRNAs and causing their release within exosomes. This process is dependent on the lipid environment of membranes as shown by cholesterol depletion using methyl-β-cyclodextrin or by treatment with n-3 polyunsaturated fatty acids. Consistent with a role in bone metastasis, knockdown of hnRNPK in prostate cancer PC3 cells abolished the ability of PC3 extracellular vesicles (EV) to induce osteoclastogenesis, and biofluid EV hnRNPK is elevated in metastatic prostate and colorectal cancer., Conclusions: Taken together, these results support a novel pan-cancer mechanism for CAV1-driven exosomal release of hnRNPK and associated miRNA in metastasis, which is modulated by the membrane lipid environment., (© 2021 The Authors. Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.)

Background: The "nonclassic" apparent mineralocorticoid excess (NC-AME) has been identified in approximately 7% of general population. This phenotype is characterized by low plasma renin activity (PRA), high serum cortisol (F) to cortisone (E) ratio, low cortisone, high Fractional Excretion of potassium (FEK) and normal-elevated systolic blood pressure (SBP). An early detection and/or identification of novel biomarkers of this phenotype could avoid the progression or future complications leading to arterial hypertension. Isolation of extracellular vesicles, such as exosomes, in specific biofluids support the identification of tissue-specific RNA and miRNA, which may be useful as novel biomarkers. Our aim was to identify miRNAs within urinary exosomes associated to the NC-AME phenotype., Methods: We perform a cross-sectional study in a primary care cohort of 127 Chilean subjects. We measured BP, serum cortisol, cortisone, aldosterone, PRA. According to the previous reported, a subgroup of subjects was classified as NC-AME (n = 10). Urinary exosomes were isolated and miRNA cargo was sequenced by Illumina-NextSeq-500., Results: We found that NC-AME subjects had lower cortisone (p < 0.0001), higher F/E ratio (p < 0.0001), lower serum potassium (p = 0.009) and higher FEK 24 h (p = 0.03) than controls. We found miR-204-5p (fold-change = 0.115; p 0.001) and miR-192-5p (fold-change = 0.246; p 0.03) are both significantly downregulated in NC-AME. miR-192-5p expression was correlated with PRA (r = 0.45; p 0.028) and miR-204-5p expression with SBP (r = - 0.48, p 0.027) and F/E ratio (r = - 0.48; p 0.026)., Conclusions: These findings could support a potential role of these miRNAs as regulators and novel biomarkers of the NC-AME phenotype.

Exosomes are small nanovesicles that carry bioactive molecules which can be delivered to neighbouring cells to modify their biological functions. Studies have showed that exosomes from ovarian cancer (OVCA) cells can alter the cell migration and proliferation of cells within the tumour microenvironment, an effect modulated by the invasiveness capacity of their originating cells. Using an OVCA cell line xenograph mouse model, we showed that exosomes derived from a high invasiveness capacity cell line (exo-SKOV-3) promoted metastasis in vivo compared with exosomes from a low invasiveness capacity cell line (exo-OVCAR-3). Analysis from an in vivo imaging system (IVIS) revealed that exo-SKOV-3 formed metastatic niches, whereas exo-OVCAR-3 formed colonies of clustered cells close to the site of injection. Interestingly, kinetic parameters showed that the half-maximal stimulatory time (ST 50 ) of tumour growth with exo-OVCAR-3 (4.0 ± 0.31 weeks) was significantly lower compared with the ST 50 in mice injected with exo-SKOV-3 (4.5 ± 0.32 weeks). However, the number of metastic nodes in mice injected with exo-SKOV-3 was higher compared with exo-OVCAR-3. Using a quantitative mass spectrometry approach (SWATH MS/MS) followed by bioinformatics analysis using the Ingenuity Pathway Analysis (IPA), we identified a total of 771 proteins. Furthermore, 40 of these proteins were differentially expressed in tumour tissues from mice injected with exo-SKOV-3 compared with exo-OVCAR-3, and associated with Wnt canonical pathway (β-catenin). Finally, we identified a set of proteins which had elevated expression in the circulating exosomes in association with tumour metastasis. These observations suggest that exosomal signalling plays an important role in OVCA metastasis., (© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Context: Molecules produced by adipose tissue (AT) function as an endocrine link between maternal AT and fetal growth by regulating placental function in normal women and women with gestational diabetes mellitus (GDM)., Objective: We hypothesized that AT-derived exosomes (exo-AT) from women with GDM would carry a specific set of proteins that influences glucose metabolism in the placenta., Design: Exosomes were isolated from omental AT-conditioned media from normal glucose tolerant (NGT) pregnant women (n = 65) and pregnant women with GDM (n = 82). Sequential window acquisition of all theoretical fragment ion spectra mass spectrometry was used to construct a small ion library from AT and exosomal proteins, followed by ingenuity pathway analysis to determine the canonical pathways and biofunctions. The effect of exosomes on human placental cells was determined using a Human Glucose Metabolism RT2 Profiler PCR array., Results: The number of exosomes (vesicles/μg of tissue/24 hours) was substantially (1.7-fold) greater in GDM than in NGT, and the number of exosomes correlated positively with the birthweight Z score. Ingenuity pathway analysis of the exosomal proteins revealed differential expression of the proteins targeting the sirtuin signaling pathway, oxidative phosphorylation, and mechanistic target of rapamycin signaling pathway in GDM compared with NGT. GDM exo-AT increased the expression of genes associated with glycolysis and gluconeogenesis in placental cells compared with the effect of NGT exo-AT., Conclusions: Our findings are consistent with the possibility that AT exosomes play an important role in mediating the changes in placental function in GDM and might be responsible for some of the adverse consequences in this pregnancy complication, such as fetal overgrowth., (Copyright © 2019 Endocrine Society.)

Exosomes are membrane-bound nanovesicles that transport molecular signals between cells. This study determined changes in maternal plasma exosome proteomics contents in term and preterm births. Maternal plasma (MP) samples were collected from group 1: term not in labor (TNIL, n = 13); group 2: term in labor (TL, n = 11); group 3: preterm premature rupture of membranes (pPROM, n = 8); and group 4: preterm birth (PTB, n = 13). Exosomes isolated from plasma by differential density centrifugation followed by size exclusion chromatography were characterized by morphology (electron microscopy), quantity and size (nanoparticle tracking analysis), and markers (western blot). A quantitative, information-independent acquisition [sequential windowed acquisition of all theoretical mass spectra (SWATH-MS)] approach was used to determine the protein profile in exosomes. Ingenuity Pathway Analysis determined pathways associated with the protein profile identified in exosomes. MP exosomes were spherical, had a mean diameter of 120 nm, and were positive for exosomal proteins CD63 and TSG101 irrespective of pregnancy status. No distinct changes in exosome quantities were seen in maternal circulation across the groups. SWATH-MS identified 72 statistically significant proteins across the groups studied. Bioinformatics analysis showed the proteins within the exosomes in TNIL, TL, pPROM, and PTB target pathways mainly associated with inflammatory and metabolic signals. Exosomal data suggest that homeostatic imbalances, specifically inflammatory and endocrine signaling, might disrupt pregnancy maintenance resulting in labor-related changes both at term and preterm. Reflection of physiologic changes in exosomes is suggestive of its usefulness as biomarkers and cellular function indicators., (Copyright © 2019 Endocrine Society.)

Despite decades of research in the field of human reproduction, the mechanisms responsible for human parturition still remain elusive. The objective of this study was to describe the changes in the exosomal miRNA concentrations circulating in the maternal plasma between mothers delivering term and preterm neonates, across gestation using a longitudinal study design. This descriptive study identifies the miRNA content in exosomes present in maternal plasma of term and preterm birth (PTB) (n = 20 and n = 10 per each gestational period, respectively) across gestation (i.e., first, second, and third trimesters and at the time of delivery). Changes in exosomal miRNA signature in maternal plasma during term and preterm gestation were determined using the NextSeq 500 high-output 75 cycles sequencing platform. A total of 167 and 153 miRNAs were found to significantly change (P < 0.05) as a function of the gestational age across term and PTB pregnancies, respectively. Interestingly, a comparison analysis between the exosomal miRNA profile between term and PTB reveals a total of 173 miRNAs that significantly change (P < 0.05) across gestation. Specific trends of changes (i.e., increase, decrease, and both) as a function of the gestational age were also identified. The bioinformatics analyses establish that the differences in the miRNA profile are targeting signaling pathways associated with TGF-β signaling, p53, and glucocorticoid receptor signaling, respectively. These data suggest that the miRNA content of circulating exosomes in maternal blood might represent a biomolecular "fingerprint" of the progression of pregnancy.

Several factors including placental hormones (PH) released from the human placenta have been associated with the development of insulin resistance and gestational diabetes mellitus (GDM). However, circulating levels of PH does not correlate well with maternal insulin sensitivity across gestation, suggesting that other, previously unrecognized, mechanisms may be involved. The levels of circulating exosomes are higher in GDM compared to normal. GDM derived exosomes produce greater release of pro-inflammatory cytokines from endothelial cells compared to exosomes from normal, suggesting that their contents may differ compared to normal pregnancies. Using a quantitative, information-independent acquisition (Sequential Windowed Acquisition of All Theoretical Mass Spectra [SWATH]) approach, differentially abundant circulating exosome proteins are identified in women with normal glucose tolerance (NGT) and GDM at the time of GDM diagnosis. A total of 78 statistically significant proteins in the relative expression of exosomal proteins in GDM are compared with NGT. Bioinformatic analysis shows that the exosomal proteins in GDM target pathways are mainly associated with energy production, inflammation, and metabolism. Finally, an independent cohort of patients is used to validate some of the proteins identified by SWATH. The data obtained may be of utility in elucidating the underlying physiological mechanisms associated with insulin resistance in GDM., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Chemoresistance is one of the major obstacles in the treatment of cancer patients. It poses a fundamental challenge to the effectiveness of chemotherapy and is often linked to relapse in patients. Chemoresistant cells can be identified in different types of cancers; however, ovarian cancer has one of the highest rates of chemoresistance-related relapse (50% of patients within 5 years). Resistance in cells can either develop through prolonged cycles of treatment or through intrinsic pathways. Mechanistically, the problem of drug resistance is complex mainly because numerous factors are involved, such as overexpression of drug efflux pumps, drug inactivation, DNA repair mechanisms and alterations to and/or mutations in the drug target. Additionally, there is strong evidence that circulating miRNAs participate in the development of chemoresistance. Recently, miRNAs have been identified in exosomes, where they are encapsulated and hence protected from degradation. These miRNAs within exosomes (exo-miRNAs) can regulate the gene expression of target cells both locally and systemically. Exo-miRNAs play an important role in disease progression and can potentially facilitate chemoresistance in cancer cells. In addition, and from a diagnostic perspective, exo-miRNAs profiles may contribute to the development of predictive models to identify responder and non-responder chemotherapy. Such model may also be used for monitoring treatment response and disease progression. Exo-miRNAs may ultimately serve as both a predictive biomarker for cancer response to therapy and as a prognostic marker for the development of chemotherapy resistance. Therefore, this review examines the potential role of exo-miRNAs in chemotherapy in ovarian cancer.

Ovarian cancer has resulted in over 140 000 deaths reported annually worldwide. This is often attributed to cellular changes in the microenvironment, including increased migration of mesenchymal stem cells (MSCs) and endothelial cells (ECs) to facilitate metastasis. Recently, the ability of exosomes to communicate signals between cells (and promote cancer progression) has been established. In the present study, we explored the effect of exosomes on cells present in the tumour microenvironment. Exosomes were isolated from ovarian cancer cells with different invasive capacity (high = SKOV-3 and low = OVCAR-3) by differential and buoyant density centrifugation and characterised using nanoparticle tracking analysis (NTA), Western blot, and EM. Exosome secretion was positively correlated with invasiveness of releasing cells. Proteomic analyses identified common and unique proteins between exosomes from SKOV-3 and OVCAR-3 with gene ontology analyses revealing that these exosomes are involved in the regulation of cell migration. Since the tumour microenvironment contains multiple cell types, including MSCs and ECs, we examined the effect of these exosomes on MSC and EC migration. Exosomes promoted MSC and EC migration in a time- and concentration-dependent manner. The effect of exosomes isolated from SKOV-3 on cell migration was significantly higher compared with exosomes from OVCAR-3. Thus, we suggest that exosomes from ovarian cancer cells contain a specific set of proteins that are representative of its cell of origin and the invasive capacity., (© 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Our objective was to determine the amniotic fluid-derived exosomal proteomic profile in patients who had spontaneous preterm birth (PTB) or preterm premature rupture of membranes (pPROM) compared with those who delivered at term. A cross-sectional study of a retrospective cohort was used to quantify and determine the protein content of exosomes present in amniotic fluid, in PTB or pPROM, and normal term labor (TL) or term not in labor (TNIL) pregnancies. Exosomes were isolated by differential centrifugation and quantified using nanocrystals (Qdot) coupled to CD63 and placental alkaline phosphatase (PLAP) by fluorescence nanoparticle tracking analysis. The exosomal proteomic profile was identified by liquid chromatography-tandem mass spectrometry, and a small ion library was constructed to quantify the proteomic data by Sequential Window Acquisition of All Theoretical analysis. Ingenuity Pathway Analysis determined canonical pathways and biofunctions associated with dysregulated proteins. Amniotic fluid exosomes have similar shape and quantity regardless of the conditions; however, the PLAP/CD63 ratios for TL, PTB, and pPROM were significantly higher (∼3.8-, ∼4.4-, and ∼3.5-fold, respectively) compared with TNIL. The PLAP/CD63 ratio was also significantly higher (∼1.3-fold) in PTB compared with pPROM. Biological functions primarily indicated nonspecific inflammatory response regardless of condition, but unique profiles were also identified in cases (PTB and pPROM) compared with term. Amniotic fluid exosomes provide information specific to normal and abnormal parturition. Inflammatory marker enrichment and its uniqueness in term and preterm pregnancies support the value of exosomes in determining underlying physiology associated with term and preterm parturition.

Introduction: Placenta-derived exosomes may represent an additional pathway by which the placenta communicates with the maternal system to induce maternal vascular adaptations to pregnancy and it may be affected during Fetal growth restriction (FGR). The objective of this study was to quantify the concentration of total and placenta-derived exosomes in maternal and fetal circulation in small fetuses classified as FGR or small for gestational age (SGA)., Methods: Prospective cohort study in singleton term gestations including 10 normally grown fetuses and 20 small fetuses, sub-classified into SGA and FGR accordingly to birth weight (BW) percentile and fetoplacental Doppler. Exosomes were isolated from maternal and fetal plasma and characterized by morphology, enrichment of exosomal proteins, and size distribution by electron microscopy, western blot, and nanoparticle tracking analysis, respectively. Total and specific placenta-derived exosomes were determined using quantum dots coupled with CD63 +ve and placental-type alkaline phosphatase (PLAP) +ve antibodies, respectively., Results: Maternal concentrations of CD63 +ve and PLAP +ve exosomes were similar between the groups (all p > 0.05). However, there was a significant positive correlation between the ratio of placental-derived to total exosomes (PLAP +ve ratio) and BW percentile, [rho = 0.77 (95% CI: 0.57 to 0.89); p = 0.0001]. The contribution of placental exosomes to the total exosome concentration in maternal and fetal circulation showed a significant decrease among cases, with lower PLAP +ve ratios in FGR compared to controls and SGA cases., Discussion: Quantification of placental exosomes in maternal plasma reflects fetal growth and it may be a useful indicator of placental function., (Copyright © 2018. Published by Elsevier Ltd.)

Exosomes are membrane-bound nanovesicles that transport molecular signals (e.g., proteins) between cells and are released from a wide range of cells, including the human placenta. Interestingly, the levels of exosomes present in maternal circulation are higher in preeclamptic pregnancies and their protein content profile change in response to the microenvironment milieu. Through the discovery of candidate biomarkers, mass spectrometry (MS)-based proteomics may provide a better understanding of the pathophysiology underlying pregnancy-associated disorders. With advances in sample preparation techniques, computational methodologies, and bioinformatics, MS-based proteomics have addressed the challenge of identifying and quantifying thousands of proteins and peptides from a variety of complex biological samples. Despite increasing interest in biomarker diagnostics, the complex nature of biological matrices (e.g., plasma) poses a challenge for candidate biomarker discovery. Here we describe a workflow to prepare exosomes for proteomic analysis.

Exosomes are small (~100 nm) vesicles that carry a wide range of molecules including proteins, RNAs, and DNA. Exosomes are secreted from a wide range of cells including placental cells. Interestingly, exosomes secreted from placental cells have been identified in maternal circulation as early as in 6 weeks of gestation, and their concentration increases with the gestational age. While there is growing interest in elucidating the role of exosomes during normal and complicated pregnancies (such as preeclampsia), progress in the field has been delayed because of the inability to isolate placental exosomes from maternal circulation. Therefore, here we describe a workflow to isolate placental exosomes from maternal circulation.

Our understanding of how cells communicate has undergone a paradigm shift since the recent recognition of the role of exosomes in intercellular signaling. In this study, we investigated whether oxygen tension alters the exosome release and miRNA profile from extravillous trophoblast (EVT) cells, modifying their bioactivity on endothelial cells (EC). Furthermore, we have established the exosomal miRNA profile at early gestation in women who develop pre-eclampsia (PE) and spontaneous preterm birth (SPTB). HTR-8/SVneo cells were used as an EVT model. The effect of oxygen tension (i.e. 8% and 1% oxygen) on exosome release was quantified using nanocrystals (Qdot®) coupled to CD63 by fluorescence NTA. A real-time, live-cell imaging system (Incucyte™) was used to establish the effect of exosomes on EC. Plasma samples were obtained at early gestation (<18 weeks) and classified according to pregnancy outcomes. An Illumina TrueSeq Small RNA kit was used to construct a small RNA library from exosomal RNA obtained from EVT and plasma samples. The number of exosomes was significantly higher in EVT cultured under 1% compared to 8% oxygen. In total, 741 miRNA were identified in exosomes from EVT. Bioinformatic analysis revealed that these miRNA were associated with cell migration and cytokine production. Interestingly, exosomes isolated from EVT cultured at 8% oxygen increased EC migration, whilst exosomes cultured at 1% oxygen decreased EC migration. These changes were inversely proportional to TNF-α released from EC. Finally, we have identified a set of unique miRNAs in exosomes from EVT cultured at 1% oxygen and exosomes isolated from the circulation of mothers at early gestation, who later developed PE and SPTB. We suggest that aberrant exosomal signalling by placental cells is a common aetiological factor in pregnancy complications characterised by incomplete SpA remodeling and is therefore a clinically relevant biomarker of pregnancy complications.

Recent studies report that 35% of women are either overweight or obese at reproductive age. The placenta continuously releases exosomes across gestation and their concentration is higher in pregnancy complications. While there is considerable interest in elucidating the role of exosomes during gestation, important questions remain to be answered: i) Does maternal BMI affect the exosomal profile across gestation? and ii) What is the contribution of placenta-derived exosomes to the total number of exosomes present in maternal plasma across gestation? Plasma samples were classified according to the maternal BMI into three groups (n = 15 per group): Lean, overweight, and obese. Total exosomes and specific placenta-derived exosomes were determined by Nanoparticle Tracking Analysis (NanoSight™) using quantum dots coupled with CD63 or PLAP antibodies. The effect of exosomes on cytokine (IL-6, IL-8, IL-10 and TNF-α) release from endothelial cells was established by cytokine array analysis (Bioplex-200). The total number of exosomes present in maternal circulation was strongly correlated with maternal BMI. Between ∼12% and ∼25% of circulating exosomes in maternal blood are of placental origin during gestation, and the contribution of placental exosomes to the total exosomal population decreases with higher maternal BMI across gestation. Exosomes increase IL-6, IL-8 and TNF-α release from endothelial cells, an effect even higher when exosomes were isolated from obese women compared to lean and overweight. This study established that maternal BMI is a factor that explains a significant component of the variation in the exosomes data. Exosomes may contribute to the maternal systemic inflammation during pregnancy., (Copyright © 2016. Published by Elsevier Ltd.)