Fig 1.
Effects of progesterone on trophoblast cells.
Twenty-four hours after the treatment with 17β-estradiol (E2) and progesterone (P), (A, E) total cell numbers (n = 6 each), (B, F) cell viability (n = 6, n = 5, respectively), (C, G) total-β2GPI levels in culture supernatants (n = 6, n = 5, respectively), and (D, H) the relative levels of reduced-β2GPI in culture supernatants (n = 4 each) were assessed in (A, B, C, D) HTR-8/SVneo and (E, F, G, H) primary culture human trophoblast (4 samples) cells. All bars represent the mean ± SEM. Dot plots for each measurement value were superimposed onto the bar graph. Data were evaluated by one-way ANOVA (a) or Kruskal-Wallis ANOVA on Ranks (b) with Shapiro–Wilk normality test and Brown–Forsythe test, followed by Student-Newman-Keuls multiple comparison test.
Fig 2.
Effects of progesterone on hepatocyte cells.
Twenty-four hours after the treatment with 17β-estradiol (E2) and progesterone (P), total-β2GPI levels (n = 4) and the relative levels of reduced-β2GPI (n = 4) in culture supernatants (n = 4) were assessed in HepG2 cells. All bars represent the mean ± SEM. Dot plots for each measurement value were superimposed onto the bar graph. Data were evaluated by one-way ANOVA (a) or Kruskal-Wallis ANOVA on Ranks (b) with Shapiro–Wilk normality test and Brown–Forsythe test, followed by Student-Newman-Keuls multiple comparison test.
Fig 3.
To investigate the effects of β2GPI on trophoblast and hepatocyte cells, APOH gene expression plasmid DNA (pCMV3-APOH) or control plasmid DNA (pCMV3-control) was transferred into (A) HTR-8/SVneo or (B) HepG2 cells and thioredoxin-1 (TRX-1) and thioredoxin reductase (TRX-R) were then added to generate free thiols within β2GPI. Total-β2GPI levels in culture supernatants (n = 6) and the relative levels of reduced (free thiol) β2GPI in culture supernatants (n = 6) were assessed. All bars represent the mean ± SEM. Dot plots for each measurement value were superimposed onto the bar graph. Data were evaluated by one-way ANOVA (a) or Kruskal-Wallis ANOVA on Ranks (b) with Shapiro–Wilk normality test and Brown–Forsythe test, followed by Student-Newman-Keuls multiple comparison test, or the Shapiro–Wilk normality test and Student’s t-test (c).
Fig 4.
β2GPI redox status on trophoblast cells. (A) The secretion of placental growth factor (PlGF) (n = 9) and soluble fms-like tyrosine kinase-1 (sFlt-1) (n = 7) were assessed in HTR-8/SVneo trophoblast cells following the transfer of APOH gene expression plasmid DNA (pCMV3-APOH) or control plasmid DNA (pCMV3-control) and subsequent treatment with thioredoxin-1 (TRX-1) and thioredoxin reductase (TRX-R). (B) After incubation with different concentrations (0, 0.4, 4.0, 40, or 400 nM) of native recombinant β2GPI (native rβ2GPI), most of which was in its oxidized form (See S2 Fig), or TRX-1-treated rβ2GPI, which generates free thiols within β2GPI (reduced-β2GPI), for 24 hours, the secretion of PlGF (n = 6) and sFlt-1 (n = 6) in HTR-8/SVneo cells were assessed. (C) A migration assay was performed 24 hours after the addition of native or TRX-1-treated recombinant β2GPI (rβ2GPI) (400 nM). Migratory cells on the bottom of the membrane were stained, and cell numbers were assessed and quantified at OD 540 nm after extraction. (D) The levels of total-β2GPI and reduced-β2GPI in culture supernatants were assessed in HepG2 liver cells 24 hours after the addition of sFlt-1 (0, 1, 10, and 100 ng/ml). All bars represent the mean ± SEM. Dot plots for each measurement value were superimposed onto the bar graph. Data were evaluated by one-way ANOVA (a) or Kruskal-Wallis ANOVA on Ranks (b) with Shapiro–Wilk normality test and Brown–Forsythe test, followed by Student-Newman-Keuls multiple comparison test. Scale bars: 200 μm.
Table 1.
Patients characteristics.
Fig 5.
Circulating levels of total- and reduced- (free thiol)
β2GPI in women with pre-eclampsia (PE). Peripheral blood samples were collected at term before delivery. Total and reduced (free thiol) β2GPI levels in circulating blood samples were assessed in the control (n = 14) and PE (n = 26) groups. All bars represent the mean ± SEM. Dot plots for each measurement value were superimposed onto the bar graph. Data were evaluated by the Shapiro–Wilk normality test and Student’s t-test (*P < 0.05, **P < 0.01).