Figure 2.
Restoring TM prevents EV-induced placental inflammasome activation and pregnancy failure. (A-E) Treatment of EV-injected pregnant C57BL/6 mice with solulin (1 mg/kg body weight) improves pregnancy outcomes. Bar graphs showing placental TM expression (A, bottom: representative immunoblots; GAPDH: loading control), fetal death (B), embryonic height (C), placental inflammasome activation, as reflected by IL-1β and NLRP3 (D, bar graph summarizing the results of immunoblot analyses), and placental cell proliferation (E, assessed by Ki-67 immunostaining, bar graph summarizing the results); n = 8 placentae from 3 different mothers; *P < .05 (relative to control, C), #P < .05 (relative to EV); ANOVA (A-E). (F) Aspirin treatment prevents the loss of placental TM expression (F, bar graph summarizing the results of immunoblot analyses; bottom, representative immunoblots; GAPDH, loading control). n = 8 placentae from 3 different mothers; *P < .05 (relative to control, C); #P < .05 (relative to EV); ANOVA. (G-L) Restoring placental TM expression prevents EV-induced pregnancy failure. Bar graphs showing fetal death (G) or embryonic height (H) and representative images showing the gross morphology of placentae (I, top) and embryos (I, bottom, n = 5 different mothers). Immunoblot images of IL-1β and NLRP3 (J, representative immunoblot; K, bar graph summarizing the results; n = 8 placentae from 3 different mothers). The arrow and arrowhead indicate the cleaved and pro forms of IL-1β, respectively. Expression of the cleaved form was quantified. Ki-67 immunostaining (L, top, representative images taken at ×40 magnification; bottom, bar graph summarizing results; n = 8 placentae from 3 different mothers). *P < .05; Student t test (G-H,K-L). (M) Proposed model summarizing results. EVs and platelet-induced inflammasome activation in trophoblast cells reduce trophoblast TM expression, reducing trophoblast proliferation and causing placental insufficiency. Note that loss of trophoblast TM expression is “downstream” of placental inflammasome activation and hence inflammasome inhibition does not rescue genetic TM deficiency. This model hence describes a unidirectional mechanism through which increased maternal platelet activation, as observed in PE, results in placental insufficiency and embryonic demise. Inhibiting this thromboinflammatory mechanism by solulin or anakinra can restore placental TM levels. ASA, acetylsalicylic acid (aspirin); ATP, adenosine triphosphate.

Restoring TM prevents EV-induced placental inflammasome activation and pregnancy failure. (A-E) Treatment of EV-injected pregnant C57BL/6 mice with solulin (1 mg/kg body weight) improves pregnancy outcomes. Bar graphs showing placental TM expression (A, bottom: representative immunoblots; GAPDH: loading control), fetal death (B), embryonic height (C), placental inflammasome activation, as reflected by IL-1β and NLRP3 (D, bar graph summarizing the results of immunoblot analyses), and placental cell proliferation (E, assessed by Ki-67 immunostaining, bar graph summarizing the results); n = 8 placentae from 3 different mothers; *P < .05 (relative to control, C), #P < .05 (relative to EV); ANOVA (A-E). (F) Aspirin treatment prevents the loss of placental TM expression (F, bar graph summarizing the results of immunoblot analyses; bottom, representative immunoblots; GAPDH, loading control). n = 8 placentae from 3 different mothers; *P < .05 (relative to control, C); #P < .05 (relative to EV); ANOVA. (G-L) Restoring placental TM expression prevents EV-induced pregnancy failure. Bar graphs showing fetal death (G) or embryonic height (H) and representative images showing the gross morphology of placentae (I, top) and embryos (I, bottom, n = 5 different mothers). Immunoblot images of IL-1β and NLRP3 (J, representative immunoblot; K, bar graph summarizing the results; n = 8 placentae from 3 different mothers). The arrow and arrowhead indicate the cleaved and pro forms of IL-1β, respectively. Expression of the cleaved form was quantified. Ki-67 immunostaining (L, top, representative images taken at ×40 magnification; bottom, bar graph summarizing results; n = 8 placentae from 3 different mothers). *P < .05; Student t test (G-H,K-L). (M) Proposed model summarizing results. EVs and platelet-induced inflammasome activation in trophoblast cells reduce trophoblast TM expression, reducing trophoblast proliferation and causing placental insufficiency. Note that loss of trophoblast TM expression is “downstream” of placental inflammasome activation and hence inflammasome inhibition does not rescue genetic TM deficiency. This model hence describes a unidirectional mechanism through which increased maternal platelet activation, as observed in PE, results in placental insufficiency and embryonic demise. Inhibiting this thromboinflammatory mechanism by solulin or anakinra can restore placental TM levels. ASA, acetylsalicylic acid (aspirin); ATP, adenosine triphosphate.

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