Figure 2
Figure 2. Maternal platelets mediate EV-induced PE-like phenotype in mice. (A) Double immunofluorescence staining showing activated platelets (CD62P, P-selectin; red) in the placenta (left), but not in the kidney (right), after injections of mouse endothelial cell–derived EVs. CD62P (red) colocalizes (yellow) with GP1bα (green) indicating presence of activated platelets within the placenta (Placenta, top left panel). Colocalization (yellow) of activated platelets (CD62P; red) and Gcm-1+ syncytiotrophoblast (arrows, green) indicates direct contact of activated platelets with syncytiotrophoblasts, which line maternal blood spaces within the placenta (Placenta, bottom left panel); conventional immunofluorescence analyses; arrowheads, autofluorescence of erythrocytes. (B-C) Depletion of maternal platelets using anti-GP1bα antibody, inhibition of maternal platelets using aspirin, genetic platelet deficiency (p45 NF-E2−/− mice), or genetically superimposed diminished platelet activation (Gαq deficiency) protects pregnant female mice from EV-induced impaired pregnancy outcome. Pregnancy outcome at day 12.5 p.c. after EV injection (mouse endothelial cell–derived EVs) at days 10.5 p.c. and 11.5 p.c. into pregnant females mated to WT males. (B) Representative images of uterus (top), placentae (middle), and embryos (bottom) along with (C) bar graphs quantifying embryonic survival. (D-E) Plasma sFlt-1 levels (D) and proteinuria (E, bar graph summarizing data of P/C ratio) are normal despite EV treatment in platelet-depleted, aspirin-treated, NFE-2−/−, or Gαq−/− pregnant female mice. Scale bar represents (A) 80 µm for placenta and 10 µm for kidney, and (B) 1 mm for embryo and placenta. Data represent mean ± SEM of at least 8 placentae or embryos analyzed from at least 3 different litters of each group. Control mice (C) were injected with the supernatant obtained after the last PBS wash during EV isolation. *P < .05 (relative to control, C); #P < .05 (relative to EV). (C-E) ANOVA. ASA, acetylsalicylic acid; DAPI, 4′,6-diamidino-2-phenylindole.

Maternal platelets mediate EV-induced PE-like phenotype in mice. (A) Double immunofluorescence staining showing activated platelets (CD62P, P-selectin; red) in the placenta (left), but not in the kidney (right), after injections of mouse endothelial cell–derived EVs. CD62P (red) colocalizes (yellow) with GP1bα (green) indicating presence of activated platelets within the placenta (Placenta, top left panel). Colocalization (yellow) of activated platelets (CD62P; red) and Gcm-1+ syncytiotrophoblast (arrows, green) indicates direct contact of activated platelets with syncytiotrophoblasts, which line maternal blood spaces within the placenta (Placenta, bottom left panel); conventional immunofluorescence analyses; arrowheads, autofluorescence of erythrocytes. (B-C) Depletion of maternal platelets using anti-GP1bα antibody, inhibition of maternal platelets using aspirin, genetic platelet deficiency (p45 NF-E2−/− mice), or genetically superimposed diminished platelet activation (Gαq deficiency) protects pregnant female mice from EV-induced impaired pregnancy outcome. Pregnancy outcome at day 12.5 p.c. after EV injection (mouse endothelial cell–derived EVs) at days 10.5 p.c. and 11.5 p.c. into pregnant females mated to WT males. (B) Representative images of uterus (top), placentae (middle), and embryos (bottom) along with (C) bar graphs quantifying embryonic survival. (D-E) Plasma sFlt-1 levels (D) and proteinuria (E, bar graph summarizing data of P/C ratio) are normal despite EV treatment in platelet-depleted, aspirin-treated, NFE-2−/−, or Gαq−/− pregnant female mice. Scale bar represents (A) 80 µm for placenta and 10 µm for kidney, and (B) 1 mm for embryo and placenta. Data represent mean ± SEM of at least 8 placentae or embryos analyzed from at least 3 different litters of each group. Control mice (C) were injected with the supernatant obtained after the last PBS wash during EV isolation. *P < .05 (relative to control, C); #P < .05 (relative to EV). (C-E) ANOVA. ASA, acetylsalicylic acid; DAPI, 4′,6-diamidino-2-phenylindole.

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