Recombinant protein diannexin prevents preeclampsia-like symptoms in a pregnant mouse model via reducing the release of microparticles

doi:10.1007/s11684-021-0918-6

Front. Med.

2022, Vol. 16

Issue (6) : 919-931 https://doi.org/10.1007/s11684-021-0918-6

RESEARCH ARTICLE

Recombinant protein diannexin prevents preeclampsia-like symptoms in a pregnant mouse model via reducing the release of microparticles

Han Guo¹, Yuncong Zhang², Yaxin Chu¹, Shuo Yang¹, Jie Zhang¹, Rui Qiao¹(

)

¹. Laboratory Medicine, Peking University Third Hospital, Beijing 100191, China
². Department of Clinical Laboratory, Peking University International Hospital, Beijing 102206, China

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Abstract

Preeclampsia (PE) is characterized by placenta-mediated pregnancy complication. The only effective treatment for PE is the delivery of the placenta. However, this treatment may cause preterm birth and neonatal death. Therefore, preventing PE is needed. The mechanism of PE involves abnormal placentation, which leads to the release of anti-angiogenic and inflammatory mediators into maternal circulation. These mediators contribute to systemic vascular dysfunction, inflammatory responses, and excessive thrombin generation. Microparticles (MPs) are reportedly involved in PE by promoting the thromboinflammatory response. This study describes a strategy to prevent PE by reducing MP release using the recombinant protein, diannexin. Results showed that the patients with PE had elevated MP number and procoagulant activity and increased NLRP3 inflammasome activation. Additionally, diannexin remarkably reduced the release of MPs from activated cells by binding to phosphatidylserine exposed on the surface of activated cells. Moreover, in vivo results showed that diannexin could prevent PE-like symptoms by decreasing MPs and NLRP3 inflammasome activation in pregnant mice. Furthermore, diannexin effectively inhibited trophoblast cell activation and NLRP3 inflammasome activation in vitro. These findings suggested that diannexin inhibited MP release and might be an effective therapeutic strategy for preventing PE.

Keywords preeclampsia recombinant protein diannexin microparticle NLRP3 inflammasome phosphatidylserin

Corresponding Author(s): Rui Qiao

Just Accepted Date: 06 July 2022 Online First Date: 03 November 2022 Issue Date: 16 January 2023

Cite this article:

Han Guo,Yuncong Zhang,Yaxin Chu, et al. Recombinant protein diannexin prevents preeclampsia-like symptoms in a pregnant mouse model via reducing the release of microparticles[J]. Front. Med., 2022, 16(6): 919-931.

URL:

https://academic.hep.com.cn/fmd/EN/10.1007/s11684-021-0918-6
https://academic.hep.com.cn/fmd/EN/Y2022/V16/I6/919

Fig.1 Elevated circulating MPs and increased NLRP3 inflammasome activation in patients with PE. (A, B) The number and procoagulant activity of MPs in healthy pregnant women (HP) and patients with PE. (C, D) Expression of NLRP3, caspase 1, and IL-1β in the placenta tissues of HP and patients with PE (C, representative immunoblots; D, bar graph summarizing the results). (E, F) Immunohistochemistry analysis of NLRP3, caspase 1, and IL-1β levels in placenta tissues from HP and patients with PE (E, immunohistochemistry images; F, bar graph summarizing the semi-quantification). Bar represents 50 μm. Data are shown as mean ± SEM from 14 healthy pregnant women and 24 patients with PE. ^*P < 0.05, Student’s t-test.

Fig.2 Recombinant protein diannexin inhibited MP release via PS binding. (A) PS microcapsules and PC microcapsules were detected by transmission electron microscopy. (B) PS microcapsules and PC microcapsules treated as described were visualized by confocal microscopy. (C, D) The binding capacity of diannexin to unstimulated or stimulated platelets was visualized by confocal microscopy. (E) Interactions of diannexin with PS-exposing MPs were detected by flow cytometry. (F, G) Flow cytometric detection of PMPs generated by platelets (F) or EMPs generated by EA.Hy-926 cells (G) pretreated with different diannexin doses and stimulators. PS, phosphatidylserine; PC, phosphatidylcholine; MPs, microparticles. Data are shown as mean ± SEM (n = 3). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 for comparisons between the indicated groups. Paired t-test was performed in E–G, and Student’s t test was performed in D.

Fig.3 Diannexin ameliorates the PE-like phenotype induced by MPs in pregnant mice. (A–C) Blood pressure, P/C, and plasma sFlt-1 levels in the control, PE model, and diannexin-treated groups were tested on day 12.5 p.c. (D) Representative images of the uterus, placentae, and embryo. Dark arrows show the absorbed placentae, and yellow arrows show impaired embryo development. The bar represents 1 cm. (E–G) Bar graphs quantifying embryonic survival (E), embryo height (F), and placental diameter (G). SBP, systolic blood pressure; MBP, mean blood pressure; DBP, diastolic blood pressure; P/C, urine protein/creatinine ratio; sFlt-1, soluble fms-like tyrosine kinase 1; p.c., post coitum. The data represent the mean ± SEM of all placentae and embryos from five pregnant females per group. The mean embryo height and placental diameter from one mouse is presented as one sample point. ^*P < 0.05, ^**P < 0.01 for comparisons between control and PE model group; ^#P < 0.05 for comparisons between the PE model and diannexin group. Student’s t-test was performed.

Fig.4 Diannexin lowered the levels of circulating MPs and placental NLRP3 inflammasome activation in PE model mice. (A, B) The number of circulating MPs (A) and endothelial MPs (B) in the plasma from the control, PE model, and diannexin-treated groups. (C–E) IFN-γ (C), IL-1β (D), and IL-6 (E) concentrations in the three groups. (F, G) Western blot images (F) and bar graphs (G) of NLRP3, caspase 1, and IL-1β levels in placental tissues from the three groups. (H–K) Immunohistochemical images (H, I) and bar graphs (J, K) of NLRP3 and caspase 1 levels in placental tissues from the three groups. The bar represents 50 μm. (L) IL-β levels in placental tissue homogenates from the three groups. GTC, trophoblast giant cell; SpTC, spongiotrophoblast cell. Data are shown as the mean ± SEM of at least three placentae from five pregnant females per group. ^*P < 0.05, ^**P < 0.01 for comparisons between the control and PE model groups; ^#P < 0.05, ^##P < 0.01 for comparisons between the PE model and diannexin-treated group. Student’s t-test was performed.

Fig.5 Diannexin reduced the NLRP3 inflammasome activation induced by MPs in BeWo and SWAN-71 cells. (A, E) NLRP3 and caspase 1 expression in BeWo (A) and SWAN-71 (E) cells treated with diannexin and/or MPs according to Western blot. (B, F) Bar graphs of the results for BeWo (B) and SWAN-71 cells (F). (C, G) IL-1β levels in the culture supernatant from BeWo (C) and SWAN-71cell cultures (G). (D, H) Number of TMPs in the supernatants from BeWo (D) and SWAN-71 cell cultures (H). TMPs, trophoblast microparticles. Data are presented as mean ± SEM (n = 3). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 for comparisons of indicated groups. Student’s t-test was performed.

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