Katherin Scholz-Romero

A Gestational Profile of Placental Exosomes in Maternal Plasma and Their Effects on Endothelial Cell Migration

Studies completed to date provide persuasive evidence that placental cell-derived exosomes play a significant role in intercellular communication pathways that potentially contribute to placentation and development of materno-fetal vascular circulation. The aim of this study was to establish the gestational-age release profile and bioactivity of placental cell-derived exosome in maternal plasma. Plasma samples (n = 20 per pregnant group) were obtained from non-pregnant and pregnant women in the first (FT, 6–12 weeks), second (ST, 22–24 weeks) and third (TT, 32–38 weeks) trimester. The number of exosomes and placental exosome contribution were determined by quantifying immunoreactive exosomal CD63 and placenta-specific marker (PLAP), respectively. The effect of exosomes isolated from FT, ST and TT on endothelial cell migration were established using a real-time, live-cell imaging system (Incucyte). Exosome plasma concentration was more than 50-fold greater in pregnant women than in non-pregnant women (p<0.001). During normal healthy pregnancy, the number of exosomes present in maternal plasma increased significantly with gestational age by more that two-fold (p<0.001). Exosomes isolated from FT, ST and TT increased endothelial cell migration by 1.9±0.1, 1.6±0.2 and 1.3±0.1-fold, respectively compared to the control. Pregnancy is associated with a dramatic increase in the number of exosomes present in plasma and maternal plasma exosomes are bioactive. While the role of placental cell-derived exosome in regulating maternal and/or fetal vascular responses remains to be elucidated, changes in exosome profile may be of clinical utility in the diagnosis of placental dysfunction.

The Effect of Glucose on the Release and Bioactivity of Exosomes From First Trimester Trophoblast Cells

CONTEXT Hyperglycemia and hypoxia are risk factors of metabolic complication during pregnancy. The interactions between oxygen and glucose-sensing pathways that regulate exosome bioactivity from placental cells, however, have not been established. OBJECTIVE The aim of this study was to test the hypothesis that exosomal signaling by placental cells (defined as the number of exosomes released per unit time and their bioactivity) is responsive to extracellular glucose concentration. METHODS First-trimester primary trophoblast cells were incubated with D-glucose (5 mM or 25 mM) under 1%, 3%, or 8% O2 for 48 hours. Exosomes were isolated from cell-conditioned media by differential and buoyant density centrifugation. The total number of exosome vesicles was determined by quantifying immunoreactive exosomal CD63. The effect of exosomes on cytokine (granulocyte macrophage colony-stimulating factor, IL-2, IL-4, IL-6. IL-8, IL-10, interferon-γ, and TNF-α) release from endothelial cells was established by a protein solution array analysis. RESULTS Glucose (25 mM) significantly increased the release of exosomes from trophoblast cells at all oxygen tensions tested (by approximately 2-fold when compared with controls, P < .001). Exosomes (100 μg/mL exosomal protein) released from trophoblast cells significantly increased (P < .05) the release of all cytokines from human umbilical vein endothelial cells when compared with the control (ie, cells without exosomes), with the exception of IL-2 and IL-10 (P > .05). CONCLUSIONS The effects of high glucose on exosomes bioactivity may be recapitulated in vivo and is of clinical relevance in association with maternal insulin resistance (resulting in hyperglycemia) and preeclampsia (associated with placental insufficiency and hypoxia).

Extravillous trophoblast cells-derived exosomes promote vascular smooth muscle cell migration

Background: Vascular smooth muscle cells (VSMCs) migration is a critical process during human uterine spiral artery (SpA) remodeling and a successful pregnancy. Extravillous trophoblast cells (EVT) interact with VSMC and enhance their migration, however, the mechanisms by which EVT remodel SpA remain to be fully elucidated. We hypothesize that exosomes released from EVT promote VSMC migration. Methods: JEG-3 and HTR-8/SVneo cell lines were used as models for EVT. Cells were cultured at 37°C and humidified under an atmosphere of 5% CO2-balanced N2 to obtain 8% O2. Cell-conditioned media were collected, and exosomes (exo-JEG-3 and exo- HTR-8/SVneo) isolated by differential and buoyant density centrifugation. The effects of exo-EVT on VSMC migration were established using a real-time, live-cell imaging system (Incucyte™). Exosomal proteins where identified by mass spectrometry and submitted to bioinformatic pathway analysis (Ingenuity software). Results: HTR-8/SVneo cells were significantly more (~30%) invasive than JEG-3 cells. HTR-8/SVneo cells released 2.6-fold more exosomes (6.39 × 108 ± 2.5 × 108 particles/106 cells) compared to JEG-3 (2.86 × 108 ± 0.78 × 108 particles/106 cells). VSMC migration was significantly increased in the presence of exo-JEG-3 and exo-HTR-8/SVneo compared to control (−exosomes) (21.83 ± 0.49 h and 15.57 ± 0.32, respectively, vs. control 25.09 ± 0.58 h, p < 0.05). Sonication completely abolished the effect of exosomes on VSMC migration. Finally, mass spectrometry analysis identified unique exosomal proteins for each EVT cell line-derived exosomes. Conclusion: The data obtained in this study are consistent with the hypothesis that the release, content, and bioactivity of exosomes derived from EVT-like cell lines is cell origin-dependent and differentially regulates VSMC migration. Thus, an EVT exosomal signaling pathway may contribute to SpA remodeling by promoting the migration of VSMC out of the vessel walls.

Placental exosomes as early biomarker of preeclampsia - Potential role of exosomal microRNAs across gestation.

Context: There is a need to develop strategies for early prediction of patients who will develop preeclampsia (PE) to establish preventive strategies to reduce the prevalence and severity of the disease and their associated complications. Objective: The objective of this study was to investigate whether exosomes and their microRNA cargo present in maternal circulation can be used as early biomarker for PE. Design, Setting, Patients, and Interventions: A retrospective stratified study design was used to quantify total exosomes and placenta‐derived exosomes present in maternal plasma of normal (n = 32 per time point) and PE (n = 15 per time point) pregnancies. Exosomes present in maternal circulation were determined by nanoparticle tracking analysis. An Illumina TruSeq® Small RNA Library Prep Kit was used to construct a small RNA library from exosomal RNA obtained from plasma samples. Results: In presymptomatic women, who subsequently developed PE, the concentration of total exosomes and placenta‐derived exosomes in maternal plasma was significantly greater than those observed in controls, throughout pregnancy. The area under the receiver operating characteristic curves for total exosome and placenta‐derived exosome concentrations were 0.745 ± 0.094 and 0.829 ± 0.077, respectively. In total, over 300 microRNAs were identified in exosomes across gestation, where hsa‐miR‐486‐1‐5p and hsa‐miR‐486‐2‐5p were identified as the candidate microRNAs. Conclusions: Although the role of exosomes during PE remains to be fully elucidated, we suggest that the concentration and content of exosomes may be of diagnostic utility for women at risk for developing PE.

Oxygen tension regulates the miRNA profile and bioactivity of exosomes released from extravillous trophoblast cells-liquid biopsies for monitoring complications of pregnancy

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.

Plasma exosome profiles from dairy cows with divergent fertility phenotypes.

Cell-to-cell communication in physiological and pathological conditions may be influenced by neighboring cells, distant tissues, or local environmental factors. Exosomes are specific subsets of extracellular vesicles that internalize and deliver their content to near and distant sites. Exosomes may play a role in the maternal-embryo crosstalk vital for the recognition and maintenance of a pregnancy; however, their role in dairy cow reproduction has not been established. This study aimed to characterize the exosome profile in the plasma of 2 strains of dairy cow with divergent fertility phenotypes. Plasma was obtained and characterized on the basis of genetic ancestry as fertile (FERT; <23% North American genetics, New Zealand Holstein-Friesian strain, n=8) or subfertile (SUBFERT; >92% North American genetics, North American Holstein-Friesian strain, n=8). Exosomes were isolated by differential and buoyant density centrifugation and characterized by size distribution (nanoparticle tracking analysis, NanoSight NS500, NanoSight Ltd., Amesbury, UK), the presence of CD63 (Western blot), and their morphology (electron microscopy). The total number of exosomes was determined by quantifying the immunoreactive CD63 (ExoELISA kit, System Biosciences), and the protein content established by mass spectrometry. Enriched exosome fractions were identified as cup-shape vesicles with diameters around 100 nm and positive for the CD63 marker. The concentration of exosomes was 50% greater in FERT cows. Mass spectrometry identified 104 and 117 proteins in FERT and SUBFERT cows, of which 23 and 36 were unique, respectively. Gene ontology analysis revealed enrichment for proteins involved in immunomodulatory processes and cell-to-cell communication. Although the role of exosomes in dairy cow reproduction remains to be elucidated, their quantification and content in models with divergent fertility phenotypes could provide novel information to support both physiological and genetic approaches to improving dairy cow fertility.

Placental fibroblast growth factor 21 is not altered in late-onset preeclampsia

BackgroundPreeclampsia (PE) is associated with alterations of placental function. The incidence of PE is higher in insulin resistant states. Women with PE have high circulating levels of the metabolic regulator fibroblast growth factor 21 (FGF21). FGF21 is synthesized in the placenta. The aim of this study was to compare the expression of FGF21, its receptors, downstream targets and transcriptional regulators in placental tissue from pregnancies with and without late-onset PE. Circulating FGF21 in maternal and cord blood was also studied.MethodsmRNA expression was determined by semi-quantitative real-time PCR and normalized for cellular composition in 17 women with and 20 without PE. Protein expression was quantified by Western Blot. FGF21 levels were measured by ELISA in maternal and cord serum of ten mother-baby dyads per condition.ResultsPlacental FGF21 mRNA and protein expression were similar in PE compared with control. Placental mRNA expression of the FGF receptors (1–4) and the co-receptor beta-Klotho was not different between the groups. There was no difference in the expression of the glucose transporters GLUT1, 3 or 4. PPAR-alpha but not PPAR-gamma expression was decreased in PE. Maternal FGF21 serum levels were not significantly different in PE. FGF21 was detected in cord blood of 6 infants (4 PE, 2 controls) but was undetectable in 14 infants.ConclusionsLate-onset PE is not associated with major changes to the expression of FGF21, its receptors or metabolic targets.

Methods to Enrich Exosomes from Conditioned Media and Biological Fluids

Exosomes are nano-vesicles which can transport a range of molecules including but not limited to proteins and miRNA. This ability of exosomes renders them useful in cellular communication often resulting in biological changes. They have several functions in facilitating normal biological processes such as immune responses and an involvement in pregnancy. However, they have also been linked to pathological conditions including cancer and pregnancy complications such as preeclampsia. An understanding for the role of exosomes in preeclampsia is based on the ability to purify and characterize exosomes. There have been several techniques proposed for the enrichment of exosomes such as ultracentrifugation, density gradient separation, and ultrafiltration although there is no widely accepted optimized technique. Here we describe a workflow for isolating exosomes from cell-conditioned media and biological fluids using a combination of centrifugation, buoyant density, and ultrafiltration approaches.

Placental exosomes profile in maternal and fetal circulation in intrauterine growth restriction - Liquid biopsies to monitoring fetal growth

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.

Exosomal Content in the Plasma of Patients with Ovarian Cancer Reflect Tumor State and Induce the Epithelial to Mesenchymal Transition in Target Cells

Figures will be available only online Underline represents presenting author; Asterisk represents senior author; Dagger represents an in-training author.