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A gestational profile of placental exosomes in maternal plasma and their effects on endothelial cell migration.

Salomon C, Torres MJ, Kobayashi M, Scholz-Romero K, Sobrevia L, Dobierzewska A, Illanes SE, Mitchell MD, Rice GE - PLoS ONE (2014)

Bottom Line: 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).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).Pregnancy is associated with a dramatic increase in the number of exosomes present in plasma and maternal plasma exosomes are bioactive.

View Article: PubMed Central - PubMed

Affiliation: University of Queensland Centre for Clinical Research, Centre for Clinical Diagnostics, Royal Brisbane and Women's Hospital, Queensland, Australia.

ABSTRACT
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.

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Characterisation of exosome from maternal circulation.Exosome were isolated from plasma of non-pregnant women and women uncomplicated pregnancies during first, second and third trimester by differential and buoyant density centrifugation (see Methods). (A–D) Representative vesicle size distribution isolated from maternal circulation across the pregnancy (first, second and third trimester) using a NanoSight NS500 instrument. Fractions 1 to 10, represent fractions collected after buoyant density centrifugation. (A) 200,000×g pellet (before sucrose density fractionation); (B) fractions 1–4; (C) fractions 5–8 enriched exosome population; (D) fractions 9–10. (E) percentage of vesicles before (white bar  = starting solution: 100%) and after (blue: fractions 1–4; red: fractions 5–8 and orange: fractions 9–10) of the exosome purification using a continuos sucrose gradient. (F) Representative Western blot for exosome enriched markers: CD63, CD9 and CD81. Exosome density is represented as red square. (G) Representative electron micrograph of 200,000×g supernatant (1) and exosome fractions (pooled enriched exosome population from fractions 5 to 8). In G, Scale bar 100 nm.
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pone-0098667-g001: Characterisation of exosome from maternal circulation.Exosome were isolated from plasma of non-pregnant women and women uncomplicated pregnancies during first, second and third trimester by differential and buoyant density centrifugation (see Methods). (A–D) Representative vesicle size distribution isolated from maternal circulation across the pregnancy (first, second and third trimester) using a NanoSight NS500 instrument. Fractions 1 to 10, represent fractions collected after buoyant density centrifugation. (A) 200,000×g pellet (before sucrose density fractionation); (B) fractions 1–4; (C) fractions 5–8 enriched exosome population; (D) fractions 9–10. (E) percentage of vesicles before (white bar  = starting solution: 100%) and after (blue: fractions 1–4; red: fractions 5–8 and orange: fractions 9–10) of the exosome purification using a continuos sucrose gradient. (F) Representative Western blot for exosome enriched markers: CD63, CD9 and CD81. Exosome density is represented as red square. (G) Representative electron micrograph of 200,000×g supernatant (1) and exosome fractions (pooled enriched exosome population from fractions 5 to 8). In G, Scale bar 100 nm.

Mentions: Exosome were isolated using the gold standard methods and purified by a sucrose continuous gradient. Nanoparticle tracking analysis showed that we obtained particles between 50 and 200 nm (Figure 1 A–D and table 2). In addition, after the sucrose continuous gradient we obtained an exosome enriched fractions corresponding ∼70% of the total particles after the ultracentrifugation (200,000×g) (Figure 1E). Maternal plasma exosomes isolated by differential and sucrose density gradient centrifugation were characterised by a buoyant density of 1.126 to 1.187 g/ml (fractions 5 to 8), the presence of CD63, CD81 and CD9 as assessed by Western blot (Figure 1F) and as ∼100 nm diameter particles when imaged by electron microscopy (Figure 1G).


A gestational profile of placental exosomes in maternal plasma and their effects on endothelial cell migration.

Salomon C, Torres MJ, Kobayashi M, Scholz-Romero K, Sobrevia L, Dobierzewska A, Illanes SE, Mitchell MD, Rice GE - PLoS ONE (2014)

Characterisation of exosome from maternal circulation.Exosome were isolated from plasma of non-pregnant women and women uncomplicated pregnancies during first, second and third trimester by differential and buoyant density centrifugation (see Methods). (A–D) Representative vesicle size distribution isolated from maternal circulation across the pregnancy (first, second and third trimester) using a NanoSight NS500 instrument. Fractions 1 to 10, represent fractions collected after buoyant density centrifugation. (A) 200,000×g pellet (before sucrose density fractionation); (B) fractions 1–4; (C) fractions 5–8 enriched exosome population; (D) fractions 9–10. (E) percentage of vesicles before (white bar  = starting solution: 100%) and after (blue: fractions 1–4; red: fractions 5–8 and orange: fractions 9–10) of the exosome purification using a continuos sucrose gradient. (F) Representative Western blot for exosome enriched markers: CD63, CD9 and CD81. Exosome density is represented as red square. (G) Representative electron micrograph of 200,000×g supernatant (1) and exosome fractions (pooled enriched exosome population from fractions 5 to 8). In G, Scale bar 100 nm.
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Related In: Results  -  Collection

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pone-0098667-g001: Characterisation of exosome from maternal circulation.Exosome were isolated from plasma of non-pregnant women and women uncomplicated pregnancies during first, second and third trimester by differential and buoyant density centrifugation (see Methods). (A–D) Representative vesicle size distribution isolated from maternal circulation across the pregnancy (first, second and third trimester) using a NanoSight NS500 instrument. Fractions 1 to 10, represent fractions collected after buoyant density centrifugation. (A) 200,000×g pellet (before sucrose density fractionation); (B) fractions 1–4; (C) fractions 5–8 enriched exosome population; (D) fractions 9–10. (E) percentage of vesicles before (white bar  = starting solution: 100%) and after (blue: fractions 1–4; red: fractions 5–8 and orange: fractions 9–10) of the exosome purification using a continuos sucrose gradient. (F) Representative Western blot for exosome enriched markers: CD63, CD9 and CD81. Exosome density is represented as red square. (G) Representative electron micrograph of 200,000×g supernatant (1) and exosome fractions (pooled enriched exosome population from fractions 5 to 8). In G, Scale bar 100 nm.
Mentions: Exosome were isolated using the gold standard methods and purified by a sucrose continuous gradient. Nanoparticle tracking analysis showed that we obtained particles between 50 and 200 nm (Figure 1 A–D and table 2). In addition, after the sucrose continuous gradient we obtained an exosome enriched fractions corresponding ∼70% of the total particles after the ultracentrifugation (200,000×g) (Figure 1E). Maternal plasma exosomes isolated by differential and sucrose density gradient centrifugation were characterised by a buoyant density of 1.126 to 1.187 g/ml (fractions 5 to 8), the presence of CD63, CD81 and CD9 as assessed by Western blot (Figure 1F) and as ∼100 nm diameter particles when imaged by electron microscopy (Figure 1G).

Bottom Line: 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).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).Pregnancy is associated with a dramatic increase in the number of exosomes present in plasma and maternal plasma exosomes are bioactive.

View Article: PubMed Central - PubMed

Affiliation: University of Queensland Centre for Clinical Research, Centre for Clinical Diagnostics, Royal Brisbane and Women's Hospital, Queensland, Australia.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus