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

Salomon C, Yee S, Scholz-Romero K, Kobayashi M, Vaswani K, Kvaskoff D, Illanes SE, Mitchell MD, Rice GE - Front Pharmacol (2014)

Bottom Line: HTR-8/SVneo cells were significantly more (~30%) invasive than JEG-3 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.

View Article: PubMed Central - PubMed

Affiliation: Centre for Clinical Diagnostics, Royal Brisbane and Women's Hospital, University of Queensland Centre for Clinical Research Brisbane, QLD, Australia ; Department of Obstetric and Gynaecology, Faculty of Medicine, Universidad de los Andes Santiago, Chile.

ABSTRACT

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 × 10(8) ± 2.5 × 10(8) particles/10(6) cells) compared to JEG-3 (2.86 × 10(8) ± 0.78 × 10(8) particles/10(6) 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.

No MeSH data available.


Related in: MedlinePlus

EVT-derived exosomes effects on hVSMC migration. hVSMC were grown to confluence in 231 media and a wound was made using 96 well WoundMaker (see Materials and Methods). hVSMC Migration was measured in absence or presence of 100 ug/ml of exosomes from JEG-3 and HTR-8/SVneo cells and mitomycin C (100 ng/ml) for 48 h. Exosome particles were subjected to sonication (+sonication) or heat inactivation (+heat) before exposure to hVSMC cells (A,B) hVSMC proliferation and migration in the presence of Mitomycin C, respectively. (C) Time course of wound closure for hVSMC expressed as relative wound density (%). (D) Area under curves from data in (C). (E) Time-dependent uptake of exosomes using a real time imaging system analysis. Top: images after 24 h; Bottom: Graphical representation of exosome uptake. (F) Fluorescent microscopy analysis of exosome uptake (40X). Data represent an n = 12 well each point with 3 different cells culture. Values are mean ± SD. In (D)***p < 0.001 vs. all condition except exo-HTR-8/SVneo + heat; and †p < 0.05 vs. control. In (E)*p < 0.001 vs. corresponding values at 0 h; ‡p < 0.05 vs. corresponding values control at 4 h; †p < 0.05 vs. corresponding values control at 8 and 4 h.
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Figure 4: EVT-derived exosomes effects on hVSMC migration. hVSMC were grown to confluence in 231 media and a wound was made using 96 well WoundMaker (see Materials and Methods). hVSMC Migration was measured in absence or presence of 100 ug/ml of exosomes from JEG-3 and HTR-8/SVneo cells and mitomycin C (100 ng/ml) for 48 h. Exosome particles were subjected to sonication (+sonication) or heat inactivation (+heat) before exposure to hVSMC cells (A,B) hVSMC proliferation and migration in the presence of Mitomycin C, respectively. (C) Time course of wound closure for hVSMC expressed as relative wound density (%). (D) Area under curves from data in (C). (E) Time-dependent uptake of exosomes using a real time imaging system analysis. Top: images after 24 h; Bottom: Graphical representation of exosome uptake. (F) Fluorescent microscopy analysis of exosome uptake (40X). Data represent an n = 12 well each point with 3 different cells culture. Values are mean ± SD. In (D)***p < 0.001 vs. all condition except exo-HTR-8/SVneo + heat; and †p < 0.05 vs. control. In (E)*p < 0.001 vs. corresponding values at 0 h; ‡p < 0.05 vs. corresponding values control at 4 h; †p < 0.05 vs. corresponding values control at 8 and 4 h.

Mentions: A VSMC was used to establish the effects of EVT-derived exosomes on cell migration. An anti-proliferative drug Mitomycin C (50 and 100 ng/ml) reduces significantly (p < 0.01) VSMC proliferation (Figure 4A). A dose of 100 ng/ml decreased VSMC migration compared to 50 ng/ml of Mitomycin C and control (Figure 4B). The effects of exosomes (100 μg protein/ml) isolated from JEG-3 and HTR-8/SVneo cultured under 8% O2 on VSMC migration under 8% O2 are presented in Figures 4C,D. All experiments were done in the presence of Mitomycin C (100 ng/ml). The rate of wound closure was significantly increased in the presence of HTR-8/SVneo-derived exosome compared to control (-exosomes) as measured by ST50 (15.57 ± 0.32 vs. 25.09 ± 0.58, p < 0.01) (Table 1). Area under curves analysis showed that HTR-8/SVneo-derived exosome increased ~35 ± 0.2% VSMC migration compared to control. Similarly, exosomes from JEG-3 cells increased VSMC migration ~12 ± 0.1% compared to values in the absence of exosomes (control), however, the effect was smaller compared to exosomes from HTR-8/SVneo. Exosomes were exposed to heat inactivation before incubation on VSMC; however, heat inactivation did not affect the effect of exosomes on VSMC migration. In contrast, sonication completely abolished the HTR-8/SVneo and JEG-3-derived exosomes effect on VSMC migration.


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

Salomon C, Yee S, Scholz-Romero K, Kobayashi M, Vaswani K, Kvaskoff D, Illanes SE, Mitchell MD, Rice GE - Front Pharmacol (2014)

EVT-derived exosomes effects on hVSMC migration. hVSMC were grown to confluence in 231 media and a wound was made using 96 well WoundMaker (see Materials and Methods). hVSMC Migration was measured in absence or presence of 100 ug/ml of exosomes from JEG-3 and HTR-8/SVneo cells and mitomycin C (100 ng/ml) for 48 h. Exosome particles were subjected to sonication (+sonication) or heat inactivation (+heat) before exposure to hVSMC cells (A,B) hVSMC proliferation and migration in the presence of Mitomycin C, respectively. (C) Time course of wound closure for hVSMC expressed as relative wound density (%). (D) Area under curves from data in (C). (E) Time-dependent uptake of exosomes using a real time imaging system analysis. Top: images after 24 h; Bottom: Graphical representation of exosome uptake. (F) Fluorescent microscopy analysis of exosome uptake (40X). Data represent an n = 12 well each point with 3 different cells culture. Values are mean ± SD. In (D)***p < 0.001 vs. all condition except exo-HTR-8/SVneo + heat; and †p < 0.05 vs. control. In (E)*p < 0.001 vs. corresponding values at 0 h; ‡p < 0.05 vs. corresponding values control at 4 h; †p < 0.05 vs. corresponding values control at 8 and 4 h.
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Figure 4: EVT-derived exosomes effects on hVSMC migration. hVSMC were grown to confluence in 231 media and a wound was made using 96 well WoundMaker (see Materials and Methods). hVSMC Migration was measured in absence or presence of 100 ug/ml of exosomes from JEG-3 and HTR-8/SVneo cells and mitomycin C (100 ng/ml) for 48 h. Exosome particles were subjected to sonication (+sonication) or heat inactivation (+heat) before exposure to hVSMC cells (A,B) hVSMC proliferation and migration in the presence of Mitomycin C, respectively. (C) Time course of wound closure for hVSMC expressed as relative wound density (%). (D) Area under curves from data in (C). (E) Time-dependent uptake of exosomes using a real time imaging system analysis. Top: images after 24 h; Bottom: Graphical representation of exosome uptake. (F) Fluorescent microscopy analysis of exosome uptake (40X). Data represent an n = 12 well each point with 3 different cells culture. Values are mean ± SD. In (D)***p < 0.001 vs. all condition except exo-HTR-8/SVneo + heat; and †p < 0.05 vs. control. In (E)*p < 0.001 vs. corresponding values at 0 h; ‡p < 0.05 vs. corresponding values control at 4 h; †p < 0.05 vs. corresponding values control at 8 and 4 h.
Mentions: A VSMC was used to establish the effects of EVT-derived exosomes on cell migration. An anti-proliferative drug Mitomycin C (50 and 100 ng/ml) reduces significantly (p < 0.01) VSMC proliferation (Figure 4A). A dose of 100 ng/ml decreased VSMC migration compared to 50 ng/ml of Mitomycin C and control (Figure 4B). The effects of exosomes (100 μg protein/ml) isolated from JEG-3 and HTR-8/SVneo cultured under 8% O2 on VSMC migration under 8% O2 are presented in Figures 4C,D. All experiments were done in the presence of Mitomycin C (100 ng/ml). The rate of wound closure was significantly increased in the presence of HTR-8/SVneo-derived exosome compared to control (-exosomes) as measured by ST50 (15.57 ± 0.32 vs. 25.09 ± 0.58, p < 0.01) (Table 1). Area under curves analysis showed that HTR-8/SVneo-derived exosome increased ~35 ± 0.2% VSMC migration compared to control. Similarly, exosomes from JEG-3 cells increased VSMC migration ~12 ± 0.1% compared to values in the absence of exosomes (control), however, the effect was smaller compared to exosomes from HTR-8/SVneo. Exosomes were exposed to heat inactivation before incubation on VSMC; however, heat inactivation did not affect the effect of exosomes on VSMC migration. In contrast, sonication completely abolished the HTR-8/SVneo and JEG-3-derived exosomes effect on VSMC migration.

Bottom Line: HTR-8/SVneo cells were significantly more (~30%) invasive than JEG-3 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.

View Article: PubMed Central - PubMed

Affiliation: Centre for Clinical Diagnostics, Royal Brisbane and Women's Hospital, University of Queensland Centre for Clinical Research Brisbane, QLD, Australia ; Department of Obstetric and Gynaecology, Faculty of Medicine, Universidad de los Andes Santiago, Chile.

ABSTRACT

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 × 10(8) ± 2.5 × 10(8) particles/10(6) cells) compared to JEG-3 (2.86 × 10(8) ± 0.78 × 10(8) particles/10(6) 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.

No MeSH data available.


Related in: MedlinePlus