Complement 5a-mediated trophoblasts dysfunction is involved in the development of pre-eclampsia

Abstract

Pre-eclampsia (PE) is a life-threatening multisystem disorder leading to maternal and neonatal mortality and morbidity. Emerging evidence showed that activation of the complement system is implicated in the pathological processes of PE. However, little is known about the detailed cellular and molecular mechanism of complement activation in the development of PE. In this study, we reported that complement 5a (C5a) plays a pivotal role in aberrant placentation, which is essential for the onset of PE. We detected an elevated C5a deposition in macrophages and C5a receptor (C5aR) expression in trophoblasts of pre-eclamptic placentas. Further study showed that C5a stimulated trophoblasts towards an anti-angiogenic phenotype by mediating the imbalance of angiogenic factors such as soluble fms-like tyrosine kinase 1 (sFlt1) and placental growth factor (PIGF). Additionally, C5a inhibited the migration and tube formation of trophoblasts, while, C5aR knockdown with siRNA rescued migration and tube formation abilities. We also found that maternal C5a serum level was increased in women with PE and was positively correlated with maternal blood pressure and arterial stiffness. These results demonstrated that the placental C5a/C5aR pathway contributed to the development of PE by regulating placental trophoblasts dysfunctions, suggesting that C5a may be a novel therapeutic possibility for the disease.

Keywords: C5a receptor; angiogenesis; arterial stiffness; complement 5a; placenta; pre-eclampsia; trophoblasts.

Figure 1

Histopathologic abnormalities in placentas of PE group (A) HE staining of placental sections from women with PE and compared to normal pregnant women shows an increased level of fibrinoid necrosis (thick arrow) and syncytial knot (thin arrow). (B) Immunofluorescence staining showed decreased expression of CD31 (red, endothelial cell marker) in placentas of women with PE. Scale bar: 100 μm. (C) Statistical data of CD31 staining. N = 6 in each group. Data are shown as mean ± S.E.M.; **P < 0.01.

Figure 2

Expression of complement in maternal circulation and placentas. (A) C5 mRNA levels in human placentas of normal pregnant women and women with PE. Data are shown as mean ± S.E.M., *P < 0.05. (B) Western blot and quantification of C5a protein expression in placentas of two groups. Data are shown as mean ± S.E.M., **P < 0.01. (C) Immunofluorescence analysis of CD11b⁺ macrophages (green) and C5a (red) expression in human placenta. Scale bar: 100 μm. (D) Colocalization of C5aR (red) with Cy7 (green, trophoblast cells marker) in human placentas. Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm.

Figure 3

Expression of angiogenesis‐related factors in human placenta. (A) Relative mRNA levels of anti‐angiogenic factors (IL‐1β, TNF‐α, IL‐6, MCP‐1 and sFlt1) and pro‐angiogenic (PIGF, IL‐10) in normal and pre‐eclamptic placentas. Data are represented as mean ± S.E.M. N = 4–6 in each group. *P < 0.05, **P < 0.01, ***P < 0.001. (B) Immunofluorescence analysis of representative angiogenesis‐related factors (red) and Cy7 (green) in normal and PE placentas. Nuclei were counterstained with DAPI (blue). Scale bar: 60 μm.

Figure 4

Trophoblasts stimulated with C5a display an anti‐angiogenic phenotype. (A) HTR‐8/SVneo cells treated with C5a showed a polarization towards an anti‐angiogenic phenotype with significantly increased mRNA levels of IL‐1β, TNF‐α, IL‐6, MCP‐1, sFlt1 and decreased mRNA level of PIGF and IL‐10. The respective mRNA was normalized to β‐actin housekeeping gene. Data are represented as mean ± S.E.M. N = 3 in each group *P < 0.05, **P < 0.01. (B) Immunofluorescence staining for sFlt1 and PIGF expression in HTR‐8/SVneo cells in the presence of C5a or PBS (CON). Scale bar: 60 μm.

Figure 5

C5a/C5aR axis inhibited the migration and tube formation of trophoblast cells in HTR‐8/SVneo cells. HTR‐8/Svneo cells were transfected with C5aR SiRNA (SiC5aR) or Control SiRNA (CON SiRNA) and treated with C5a or PBS. (A) Transwell assay was used to evaluate the migration capacity of HTR‐8/SVneo cells. Scale bar = 100 μm. Bar graphs showed quantification of migrated cell numbers. **P < 0.01, ***P < 0.001. Data are represented as mean ± S.E.M. (B) Tube formation assay was conducted to analyse the ability of HTR‐8/SVneo cells to form capillary‐like structures. Representative photomicrographs were taken, and bar graphs showed quantification of tubule lengths. Data are presented as mean ± S.E.M.; **P < 0.01; ***P < 0.001. (C and D), HTR‐8/SVneo cells were co‐cultured with macrophages (Thp‐1 cells). Migration and tube formation abilities of HTR‐8/SVneo cells were significantly inhibited. Bar graphs showed quantification of migrated cell numbers. Data are presented as mean ± S.E.M.; ***P < 0.001.

Figure 6

Correlation between maternal serum level of C5a and measures of arterial stiffness. The relationship between maternal serum levels of C5a and systolic blood pressure (A), diastolic blood pressure (B), ba‐PWV (C), cf‐PWV (D) and AIx (E). The linear regression line is shown in case of a significant correlation. (F) Summary of the role of C5a in the pathogenesis of PE.