Aspirin Inhibits Fibronectin Expression and Reverses Fibronectin-Mediated Cell Invasiveness by Activating Akt Signaling in Preeclampsia

Abstract

Preeclampsia is a severe gestational hypertensive disorder that may lead to maternal multiple organ dysfunction and adverse fetal outcomes. Aspirin provides a protective effect by reducing the risk of preeclampsia; however, its mechanism of action is unclear. Fibronectin (FN) is a key factor in cell motility and is associated with preeclampsia. Here, we demonstrated that cellular FN expression was elevated in the placenta of preeclamptic patients. The functional roles of plasma and cellular FN in trophoblasts were investigated by treating HTR-8/SVneo cells with exogenous recombinant human FN protein (rhFN) and siRNA, respectively. Trophoblast migration and invasion were inhibited by rhFN and facilitated by FN knockdown. Moreover, rhFN activated ERK and Akt signaling in trophoblasts, and FN-suppressed cell motility was rescued by ERK and/or Akt inhibitors. In this study, aspirin suppressed trophoblast cellular FN expression and reversed FN-mediated cell functions, including cell migration, invasion, and ERK/Akt signal changes. Taken together, the results of this study revealed the effects of FN on trophoblast motility and signaling; aspirin inhibits FN expression and reverses FN-mediated trophoblast biology. These results provide a drug mechanism for disease prevention and a target for preeclampsia intervention.

Keywords: Akt; aspirin; fibronectin; preeclampsia; trophoblast invasion.

Figure 1

Placental fibronectin (FN) expression is elevated in patients with preeclampsia. The protein concentration of FN in the placenta was measured in preeclamptic patients (P1-3 and PE) and women with normal pregnancies (C1, C1, C3, and control) using (A) Western blotting (n = 3 in both groups) and (B) ELISA (n = 20 in both groups). (C) The plasma FN concentration was measured in preeclamptic patients (n = 35) and normal controls (n = 45) using ELISA. Data are presented as the means ± SEMs. N.S. denotes no significance, * p < 0.05 compared with the corresponding control.

Figure 2

Fibronectin (FN) inhibits trophoblast migration and invasion without affecting cell viability. HTR-8/SVneo cells were treated with fibronectin (0, 2, and 5 μg/mL, rhFN) in serum-free medium for 24 h and then processed for (A) cell viability measurements after 24 h of culture and (B,C) cell migration and invasion assays for the next 24 h using a transwell system. HTR-8/SVneo cells were transfected with scrambled siRNA (si-CTL) or si-FN for 24 h, and cells were subjected to (D) cellular FN expression in the cell lysate using Western blotting and quantification of Western blotting, (E) cell viability, (F) cell migration, and (G) cell invasion assays. After FN knockdown in HTR-8/SVneo cells with si-FN for 24 h, cells were treated with rhFN (5 μg/mL) for another 24 h before (H) migration and (I) invasion assays. Data are presented as the mean ± SEMs. N.S. denotes no significance, * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001 compared with the corresponding control. ^### p < 0.005, ^#### p < 0.001 compared with samples at the same rhFN concentration. Scale bar: 100 μM.

Figure 3

Fibronectin (FN) regulates trophoblast invasiveness and migration by activating ERK and Akt signaling. Phospho-ERK, ERK, phospho-Akt, and Akt were analyzed at different timepoints (0, 0.25, 0.5, 1, 2, 4, 8, and 24 h) after cells were treated with rhFN (5 μg/mL) using Western blotting and quantification of Western blotting in (A) HTR-8/SV neo and (B) FN-knockdown cells. (C,D) HTR-8/SVnel cells were pretreated with rhFN (5 μg/mL) for 24 h and then U0126 (10 μM) or Akt inhibitor (1 μM) for another 4 h before (C) cell migration and (D) cell invasion assays. Data are presented as the means ± SEMs. N.S. denotes no significance, * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001 compared with the control treated with the same inhibitor. ^# p < 0.05, ^## p < 0.01, ^### p < 0.005, ^#### p < 0.001 compared with samples at the same rhFN concentration without ERK treatment or Akt inhibitors. Scale bar: 100 μM.

Figure 4

Aspirin inhibits fibronectin (FN) expression and reverses FN-mediated trophoblast migration and invasion in HTR-8/SVneo cells. (A) Different doses of aspirin (0, 0.1, 0.5, 1, and 2 mM) were used to treat HTR-8/SVneo cells for 24 h, and then cellular FN expression in the cell lysate was analyzed and quantified using Western blotting. (B–D) HTR-8/SVneo cells were treated with rhFN (5 μg/mL) for 24 h and then aspirin (1 mM) for another 24 h, and the cells were then subjected to (B) cell viability, (C) cell migration, and (D) cell invasion assays. After FN knockdown of HTR-8/SVneo cells with si-FN (100 nM) for 24 h, cells were treated with aspirin (1 mM) for another 24 h before determining (E) cellular FN expression using Western blotting and its quantification, (F) cell viability, (G) migration, and (H) invasion assays. Data are presented as the means ± SEMs. N.S. denotes no significance, * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001 compared with the control treated with the same reagents. ^# p < 0.05, ^## p < 0.01, ^### p < 0.005, ^#### p < 0.001 compared with the samples at the same rhFN or si-FN concentration without aspirin treatment. Scale bar: 100 μM.