Miharu Kobayashi

Exosomal signaling during hypoxia mediates microvascular endothelial cell migration and vasculogenesis.

Vasculogenesis and angiogenesis are critical processes in fetal circulation and placental vasculature development. Placental mesenchymal stem cells (pMSC) are known to release paracrine factors (some of which are contained within exosomes) that promote angiogenesis and cell migration. The aims of this study were: to determine the effects of oxygen tension on the release of exosomes from pMSC; and to establish the effects of pMSC-derived exosomes on the migration and angiogenic tube formation of placental microvascular endothelial cells (hPMEC). pMSC were isolated from placental villi (8–12 weeks of gestation, n = 6) and cultured under an atmosphere of 1%, 3% or 8% O2. Cell-conditioned media were collected and exosomes (exo-pMSC) isolated by differential and buoyant density centrifugation. The dose effect (5–20 µg exosomal protein/ml) of pMSC-derived exosomes on hPMEC migration and tube formation were established using a real-time, live-cell imaging system (Incucyte™). The exosome pellet was resuspended in PBS and protein content was established by mass spectrometry (MS). Protein function and canonical pathways were identified using the PANTHER program and Ingenuity Pathway Analysis, respectively. Exo-pMSC were identified, by electron microscopy, as spherical vesicles, with a typical cup-shape and diameters around of 100 nm and positive for exosome markers: CD63, CD9 and CD81. Under hypoxic conditions (1% and 3% O2) exo-pMSC released increased by 3.3 and 6.7 folds, respectively, when compared to the controls (8% O2; p<0.01). Exo-pMSC increased hPMEC migration by 1.6 fold compared to the control (p<0.05) and increased hPMEC tube formation by 7.2 fold (p<0.05). MS analysis identified 390 different proteins involved in cytoskeleton organization, development, immunomodulatory, and cell-to-cell communication. The data obtained support the hypothesis that pMSC-derived exosomes may contribute to placental vascular adaptation to low oxygen tension under both physiological and pathological conditions.

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.

Hypoxia-induced changes in the bioactivity of cytotrophoblast-derived exosomes.

Migration of extravillous trophoblasts (EVT) into decidua and myometrium is a critical process in the conversion of maternal spiral arterioles and establishing placenta perfusion. EVT migration is affected by cell-to-cell communication and oxygen tension. While the release of exosomes from placental cells has been identified as a significant pathway in materno-fetal communication, the role of placental-derived exosomes in placentation has yet to be established. The aim of this study was to establish the effect of oxygen tension on the release and bioactivity of cytotrophoblast (CT)-derived exosomes on EVT invasion and proliferation. CT were isolated from first trimester fetal tissue (n = 12) using a trypsin-deoxyribonuclease-dispase/Percoll method. CT were cultured under 8%, 3% or 1% O2 for 48 h. Exosomes from CT-conditioned media were isolated by differential and buoyant density centrifugation. The effect of oxygen tension on exosome release (µg exosomal protein/106cells/48 h) and bioactivity were established. HTR-8/SVneo (EVT) were used as target cells to establish the effect (bioactivity) of exosomes on invasion and proliferation as assessed by real-time, live-cell imaging (Incucyte™). The release and bioactivity of CT-derived exosomes were inversely correlated with oxygen tension (p<0.001). Under low oxygen tensions (i.e. 1% O2), CT-derived exosomes promoted EVT invasion and proliferation. Proteomic analysis of exosomes identified oxygen-dependent changes in protein content. We propose that in response to changes in oxygen tension, CTs modify the bioactivity of exosomes, thereby, regulating EVT phenotype. Exosomal induction of EVT migration may represent a normal process of placentation and/or an adaptive response to placental hypoxia.

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.

Hypoxia regulates the response of trophoblast-derived exosomes to hyperglycemia and displays a difference placental exosome profile in plasma from patients with gestational diabetes mellitus

Figures will be available only online at UNIV OF PITTSBURGH on June 19, 2015 rsx.sagepub.com Downloaded from at UNIV OF PITTSBURGH on June 19, 2015 rsx.sagepub.com Downloaded from Thrsday O als Scientific Abstracts Reproductive Sciences Vol. 22, Supplement 1, March 2015 57AINTRODUCTION: Statin use inadvertently during pregnancy and proposed use of statins for the treatment of preeclampsia, led us to question the evidence behind their current contraindicated status. Several studies have evaluated the relationship between statin use in pregnancy with fetal outcome but their results have not been quantitatively assessed by meta-analysis. Our objective was to undertake a systematic review of all published clinical evidence to assess the effects of statin use in pregnancy on subsequent fetal wellbeing. METHODS: A comprehensive search strategy was performed of all electronic databases and the Merck reporting database for studies published from 1966 to 2014. Two reviewers independently screened citations and undertook study quality assessment and data extraction. We obtained summary estimates of adverse fetal events that were classified as potentially fatal, clinically significant morbidity or minor adverse event. We identified 602 titles and reviewed 30 articles for inclusion and exclusion criteria. Meta-analysis was performed on seven studies (3 cohort, 3 case-series and 1 case-control). RESULTS: Of the 922 cases of statin exposure in pregnancy, 27 exposures were associated with lethal or clinically significant fetal morbidity and 10 with minor adverse events. Statin exposure was limited to the first trimester in all but two cases. The pooled rate of lethal or clinically significant fetal abnormalities in pregnant women exposed to statins was 0.01 (95% CI 0.00-0.04), less than the European rate of 0.026 (95% CI 2.54- 2.57)EUROCAT. The rate of fetal abnormality for simvastatin was 0.03 (95% CI 0.00-0.08), atorvostatin 0.11 (95% CI 0.00-0.52), pravastatin 0.01 (95% CI 0.00-0.2) and lovastatin use 0.04 (95% CI 0.00-0.28). Systems based anomalies were also calculated, congenital heart disease was 0.8 (95% CI 0.02-0.12) compared with the background rate of 0.79 (95% CI 0.78- 0.80). CONCLUSIONS: The published data suggests that statins may not be teratogenic when given inadvertently during pregnancy and prospective studies such as The StAmP Trial may provide more dataIntroduction. Being born small increases cardio-renal disease risk, with males exhibiting more se vere phenotypes than females. These disease risks are not limited to the first generation (F1) but may be transmitted to subsequent generations (F2 and F3). The F3 maternal line represents the first generation that is not directly exposed to the initial insults. There is limited evidence of paternal line transmission. W e characterized nephron number and cardio-renal phenotype of F3 of fspring born to normally grown and growth restricted (F1) mothers or fathers. Methods. Late gestation rat uteroplacental insuf ficiency was induced (Restricted) or sham (Control) surgery in F0. Rats were anaesthetized with 4% isoflurane and 650ml.min -1 oxygen flow (reduced to 3.2% isoflurane and 250ml.min -1 oxygen flow when suturing). T o generate F3 paternal line offspring, F1 Control and Restricted males were mated with normal females and the F2 Control and Restricted males were then mated with normal females. F3 maternal line of fspring were similarily generated. F3 body weights were measured from birth to 12 months. Nephron number w as quantified using unbiased sterology at postnatal day 35. 24h renal excretions, creatinine clearance and tail cuf f blood pressure were measured at 6 (maternal and paternal lines) and 12 (maternal line only) months of age. All data were analysed by t-test within a gender and line. Results. Although F1 offspring were born small, F2 and F3 offspring (both maternal and paternal lines) had normal birth weights. F3 body weight was not dif ferent from birth to 12 months of age with no dif ferences in kidney, heart or adipose weights at 6 months between groups or lines. F3 male and female nephron endowment and blood pressure w as not different between groups for paternal and maternal lines. F3 maternal line renal function was normal at 6 months of age. Ho wever, at 12 months, although creatinine clearance (eGFR) was normal, renal dysfunction emerged in F3 maternal line males (proteinuria) and females (increased urinary creatinine excretion). F3 offspring from fathers born small had e vidence of impaired eGFR (reduced creatinine clearance). Conclusions. F3 offspring, born to F1 growth restricted mothers or f athers are not programmed to be born of low birth weight but de veloped renal dysfunction in the absence of obesity . The proteinuria that emerged with aging in the F3 maternal line male of fspring in the absence of nephron deficits, hypertension and reduced eGFR, suggests tubulointerstitial injury mediated either through podocyte dysfunction/depletion or solely via proteinuria. In contrast, F3 paternal line of fspring had glomerular dysfunction (reduced eGFR), indicati ve of glomerular filtration deficits. Our paternal line results highlight sustained transgenerational inheritance of renal dysfunction. Since nephron number was preserved our results propose the progression of renal dysfunction via the “fibrosis hypothesis” rather than the “Brenner h ypothesis”. Our findings provide no vel evidence of transgenerational transmission of renal dysfunction to F3 offspring from both maternal and paternal lines.INTRODUCTION: Preeclampsia is a vascular disorder in pregnancyand is biochemical characterization by high soluble Flt-1 and lowplacenta growth factor as well as an imbalance in redox homeostasis.During conditions of high oxidative stress, cysteine residues on keyproteins are reversibly altered by S-glutathionylation, modifying theirfunction. Glutaredoxin-1 (Glrx) enzymatically catalyzes the removal of S-glutathione adducts, conferring reversible signaling dynamics toproteins with redox-sensitive cysteines. The role of Glrx in preeclampsiais unknown.METHODS: Immunohistochemistry and Western blot analysis for Glrx orglutathione were conducted on human placenta samples collected pre-termfrom early onset preeclamptic patients (n=10) or non-preeclamptic induceddeliveries (n=9). Human endothelial cells were infected with adenovirusencoding Glrx or LacZ prior to the cells being exposed to hypoxia (0.1%O2, 24h) to measure changes in soluble Flt-1 (sFlt-1). Quantitative PCRand ELISA were used to measure sFlt-1 at mRNA and protein level.RESULTS: Immunohistochemical staining for GSH revealed lowerS-glutathionylation adducts in preeclampsia placenta in comparison tocontrols. Glrx expression, which catalyses de-glutathionylation wasenhanced in early onset preeclampsia compared to pre-term controlsamples. In contrast, no change was observed in preeclamptic and IUGRplacentas at full term. In endothelial cells overexpressing Glrx, sFlt-1expression was dramatically enhanced at mRNA (3-fold P 0.01, n=4) after hypoxia andoverexpressing Glrxin mice enhanced levels of circulating sFlt-1 during in vivo ischemia.CONCLUSIONS: Enhanced Glrx expression in preeclamptic placentain line with an apparent decrease in S-glutathionylation may leavekey proteins susceptible to irreversible oxidation in conditions of highoxidative stress.

Cross‐Talk Between Hypoxia and the Tumour via Exosomes

Cancer is one of the leading causes of death worldwide, and this is often attributed to the nonspecific symptoms. Additionally, delayed diagnosis and a lack of treatment options negatively impact prognosis. Recently, the role of extracellular vesicles in cancer progression, specifically, in metastasis and in the capacity of several tumours to invade and colonise specific organs has been established. Reduced oxygen tension due to imbalanced oxygen supply and consumption is termed hypoxia and is one of the most commonly observed features in solid tumours. This is often correlated with poor cancer prognosis. Several reports have established that low oxygen tension (i.e. hypoxia) is a common feature of the tumour microenvironment often enhancing the process of epithelial‐to‐mesenchymal transition (EMT) in cancer cells, thus promoting tumouri‐ genesis and metastasis. Furthermore, hypoxia increases the number of extracellular vesicles released from cancer cells and also modifies their bioactivity and function. The aim of this chapter is to review the association between the tumour microenvironment and extracellular vesicles (EVs), focusing on a specific subpopulation of EVs of endocytic origin, termed exosomes.

Abstract 4379: Exosomes isolated from ovarian cancer cells transfer oncogenic features to the target cells promoting epithelial to mesenchymal transition

Introduction: Recent studies establish the role of exosomes in cell-cell communication in cancer. As adaptation to hypoxia is a critical step in tumor progression, the aim of this study was to test the hypotheses that hypoxia promotes epithelial to mesenchymal transition (EMT) and that exosomes isolated from hypoxic cancer cells transfer oncogenic properties to target cells. Methods: CaOV-3 cell line was used as model of ovarian cancer. Cells were cultured under 8% O2 (normoxia) and 1% O2 (hypoxia) for 48 hours. Exosomes were isolated from cell-conditioned media by differential and buoyant density centrifugation. Exosomes were characterised by the presence of TSG101 using Western Blot, size distribution (Nanosight™) and morphology by electron microscopy. Exosomal protein and miRNA content were determined using liquid chromatography mass spectrometry (5600 Triple TOF, AB Sciex,) and an Illumina NextSeq 500 Platform, respectively. The effects of exosomes released from CaOV-3 incubated under 1% O2 on EMT induction in CaOV-3 cells cultured under 8% O2 were assessed by the measuring the ratio of E-cadherin (epithelial marker) to N-cadherin (mesenchymal marker) by Wester Blot and the expression of 84 key genes involve in the EMT (RT 2 Profiler™ PCR Array, QIAGEN). Results: Exosomes were identified as spherical vesicles with a typical cup-shape, diameters ranging from 50 to 100 nm with the expression of TSG101. Exosome release from ovarian cancer cells was ∼4-fold higher under hypoxic than normoxic conditions (p Conclusion: The data obtained is consistent with the hypothesis that exosomes released from cancer cells modify the phenotype of target cells by transferring pro-oncogenic molecules inducing cancerous phenotype of receipt cells, contributing to tumour growth and metastasis. Citation Format: Miharu Kobayashi, Laura Cohelo, Dominic Guanzon, Katherin Scholz-Romero, Melissa Brown, Richard Kline, Li Li, Gregory Rice, Carlos Salomon. Exosomes isolated from ovarian cancer cells transfer oncogenic features to the target cells promoting epithelial to mesenchymal transition. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4379.

Hypoxia increases the exosome release from ovarian cancer cells and promotes the epithelial-mesenchymal transition of target cells

Figures will be available only online at UNIV OF PITTSBURGH on June 19, 2015 rsx.sagepub.com Downloaded from at UNIV OF PITTSBURGH on June 19, 2015 rsx.sagepub.com Downloaded from Thrsday O als Scientific Abstracts Reproductive Sciences Vol. 22, Supplement 1, March 2015 57AINTRODUCTION: Statin use inadvertently during pregnancy and proposed use of statins for the treatment of preeclampsia, led us to question the evidence behind their current contraindicated status. Several studies have evaluated the relationship between statin use in pregnancy with fetal outcome but their results have not been quantitatively assessed by meta-analysis. Our objective was to undertake a systematic review of all published clinical evidence to assess the effects of statin use in pregnancy on subsequent fetal wellbeing. METHODS: A comprehensive search strategy was performed of all electronic databases and the Merck reporting database for studies published from 1966 to 2014. Two reviewers independently screened citations and undertook study quality assessment and data extraction. We obtained summary estimates of adverse fetal events that were classified as potentially fatal, clinically significant morbidity or minor adverse event. We identified 602 titles and reviewed 30 articles for inclusion and exclusion criteria. Meta-analysis was performed on seven studies (3 cohort, 3 case-series and 1 case-control). RESULTS: Of the 922 cases of statin exposure in pregnancy, 27 exposures were associated with lethal or clinically significant fetal morbidity and 10 with minor adverse events. Statin exposure was limited to the first trimester in all but two cases. The pooled rate of lethal or clinically significant fetal abnormalities in pregnant women exposed to statins was 0.01 (95% CI 0.00-0.04), less than the European rate of 0.026 (95% CI 2.54- 2.57)EUROCAT. The rate of fetal abnormality for simvastatin was 0.03 (95% CI 0.00-0.08), atorvostatin 0.11 (95% CI 0.00-0.52), pravastatin 0.01 (95% CI 0.00-0.2) and lovastatin use 0.04 (95% CI 0.00-0.28). Systems based anomalies were also calculated, congenital heart disease was 0.8 (95% CI 0.02-0.12) compared with the background rate of 0.79 (95% CI 0.78- 0.80). CONCLUSIONS: The published data suggests that statins may not be teratogenic when given inadvertently during pregnancy and prospective studies such as The StAmP Trial may provide more dataIntroduction. Being born small increases cardio-renal disease risk, with males exhibiting more se vere phenotypes than females. These disease risks are not limited to the first generation (F1) but may be transmitted to subsequent generations (F2 and F3). The F3 maternal line represents the first generation that is not directly exposed to the initial insults. There is limited evidence of paternal line transmission. W e characterized nephron number and cardio-renal phenotype of F3 of fspring born to normally grown and growth restricted (F1) mothers or fathers. Methods. Late gestation rat uteroplacental insuf ficiency was induced (Restricted) or sham (Control) surgery in F0. Rats were anaesthetized with 4% isoflurane and 650ml.min -1 oxygen flow (reduced to 3.2% isoflurane and 250ml.min -1 oxygen flow when suturing). T o generate F3 paternal line offspring, F1 Control and Restricted males were mated with normal females and the F2 Control and Restricted males were then mated with normal females. F3 maternal line of fspring were similarily generated. F3 body weights were measured from birth to 12 months. Nephron number w as quantified using unbiased sterology at postnatal day 35. 24h renal excretions, creatinine clearance and tail cuf f blood pressure were measured at 6 (maternal and paternal lines) and 12 (maternal line only) months of age. All data were analysed by t-test within a gender and line. Results. Although F1 offspring were born small, F2 and F3 offspring (both maternal and paternal lines) had normal birth weights. F3 body weight was not dif ferent from birth to 12 months of age with no dif ferences in kidney, heart or adipose weights at 6 months between groups or lines. F3 male and female nephron endowment and blood pressure w as not different between groups for paternal and maternal lines. F3 maternal line renal function was normal at 6 months of age. Ho wever, at 12 months, although creatinine clearance (eGFR) was normal, renal dysfunction emerged in F3 maternal line males (proteinuria) and females (increased urinary creatinine excretion). F3 offspring from fathers born small had e vidence of impaired eGFR (reduced creatinine clearance). Conclusions. F3 offspring, born to F1 growth restricted mothers or f athers are not programmed to be born of low birth weight but de veloped renal dysfunction in the absence of obesity . The proteinuria that emerged with aging in the F3 maternal line male of fspring in the absence of nephron deficits, hypertension and reduced eGFR, suggests tubulointerstitial injury mediated either through podocyte dysfunction/depletion or solely via proteinuria. In contrast, F3 paternal line of fspring had glomerular dysfunction (reduced eGFR), indicati ve of glomerular filtration deficits. Our paternal line results highlight sustained transgenerational inheritance of renal dysfunction. Since nephron number was preserved our results propose the progression of renal dysfunction via the “fibrosis hypothesis” rather than the “Brenner h ypothesis”. Our findings provide no vel evidence of transgenerational transmission of renal dysfunction to F3 offspring from both maternal and paternal lines.INTRODUCTION: Preeclampsia is a vascular disorder in pregnancyand is biochemical characterization by high soluble Flt-1 and lowplacenta growth factor as well as an imbalance in redox homeostasis.During conditions of high oxidative stress, cysteine residues on keyproteins are reversibly altered by S-glutathionylation, modifying theirfunction. Glutaredoxin-1 (Glrx) enzymatically catalyzes the removal of S-glutathione adducts, conferring reversible signaling dynamics toproteins with redox-sensitive cysteines. The role of Glrx in preeclampsiais unknown.METHODS: Immunohistochemistry and Western blot analysis for Glrx orglutathione were conducted on human placenta samples collected pre-termfrom early onset preeclamptic patients (n=10) or non-preeclamptic induceddeliveries (n=9). Human endothelial cells were infected with adenovirusencoding Glrx or LacZ prior to the cells being exposed to hypoxia (0.1%O2, 24h) to measure changes in soluble Flt-1 (sFlt-1). Quantitative PCRand ELISA were used to measure sFlt-1 at mRNA and protein level.RESULTS: Immunohistochemical staining for GSH revealed lowerS-glutathionylation adducts in preeclampsia placenta in comparison tocontrols. Glrx expression, which catalyses de-glutathionylation wasenhanced in early onset preeclampsia compared to pre-term controlsamples. In contrast, no change was observed in preeclamptic and IUGRplacentas at full term. In endothelial cells overexpressing Glrx, sFlt-1expression was dramatically enhanced at mRNA (3-fold P 0.01, n=4) after hypoxia andoverexpressing Glrxin mice enhanced levels of circulating sFlt-1 during in vivo ischemia.CONCLUSIONS: Enhanced Glrx expression in preeclamptic placentain line with an apparent decrease in S-glutathionylation may leavekey proteins susceptible to irreversible oxidation in conditions of highoxidative stress.

Network and canonical pathway analysis of placental mesenchymal stem cells-derived exosomes

Jelmer R Prins, Leigh R Guerin, Bihong Zhang, John E Schjenken, Simon C Barry, Sarah A Robertson