Aspirin and heparin effect on basal and antiphospholipid antibody modulation of trophoblast function

Abstract

Objective: Low molecular weight (LMW) heparin, with or without aspirin (acetylsalicylic acid [ASA]), is used to prevent complications in antiphospholipid syndrome in pregnancy. Our objective was to elucidate the actions of low-dose LMW heparin and ASA on basal and antiphospholipid antibody-induced modulation of trophoblast function.

Methods: The human first-trimester trophoblast cell line (HTR-8) was treated with or without antiphospholipid antibody in the presence of no medication, low-dose LMW heparin, low-dose ASA, or combination therapy. Interleukin (IL)-6, IL-8, IL-1β, growth-regulated oncogene-α, vascular endothelial growth factor (VEGF), placental growth factor, soluble FMS-like tyrosine kinase-1, and soluble endoglin were measured in the supernatant. Cell migration was performed using a two-chamber assay.

Results: Low molecular weight heparin improved basal trophoblast migration and induced potent increases in growth-regulated oncogene-α and soluble FMS-like tyrosine kinase-1. Aspirin did not affect basal function. Combined therapy promoted migration but did not reverse the LMW heparin-induced soluble FMS-like tyrosine kinase-1 effect. Antiphospholipid antibody increased IL-8, IL-1β, growth-regulated oncogene-alpha, VEGF, placental growth factor, and soluble endoglin secretion, while decreasing cell migration and IL-6 and soluble FMS-like tyrosine kinase-1 secretion. The antiphospholipid antibody-induced cytokine changes were best reversed with LMW heparin, with partial reversal of IL-8 and IL-1β upregulation. The antiphospholipid antibody-induced angiogenic changes were worsened by LMW heparin, with increased soluble FMS-like tyrosine kinase-1 secretion. The therapies did not reverse antiphospholipid antibody-induced decrease in migration.

Conclusion: In the absence of antiphospholipid antibodies, LMW heparin induces potentially detrimental proinflammatory and antiangiogenic profile in the trophoblast. In the presence of antiphospholipid antibodies, single-agent LMW heparin may be the optimal therapy to counter trophoblast inflammation, but also induces an antiangiogenic response. These findings may explain the inability of current therapies to consistently prevent adverse outcomes.

Figure 1

Effects of low molecular weight (LMW) heparin, acetylsalicylic acid (ASA), and combination therapy on basal trophoblast cytokine/chemokine production. HTR-8 cells were treated for 72 hours with: 1) media-only, no treatment control; 2) LMW heparin (10 µg/mL); 3) ASA (10 µg/mL); or 4) combination LMW heparin and ASA (10 µg/mL each). Supernatants were analyzed for (A) interleukin (IL)-6, (B) IL-8, (C) growth-regulated oncogene-alpha, and (D) IL-1β. (*P<.001; †P<.05; N=3).

Figure 2

Effects of low molecular weight (LMW) heparin, acetylsalicylic acid (ASA), and combination therapy on basal trophoblast angiogenic factor production. HTR-8 cells were treated for 72 hours with: 1) media-only, no treatment control, 2) LMW heparin (10 µg/mL); 3) ASA (10 µg/mL); or 4) combination LMW heparin and ASA (10 µg/mL each). Supernatants were analyzed for (A) vascular endothelial growth factor (VEGF), (B) placental growth factor, (C) soluble FMS-like tyrosine kinase-1 (sFlt-1), and (D) soluble endoglin. *P<.05; N=3.

Figure 3

Effects of low molecular weight (LMW) heparin, acetylsalicylic acid (ASA), and combination therapy on basal trophoblast cell migration. HTR-8 cells were treated for 72 hours with: 1) media-only, no treatment control, 2) LMW heparin (10 µg/mL); 3) ASA (10 µg/mL); or 4) combination LMW heparin and ASA (10 µg/mL each). Cell migration was assessed using a colorimetric assay. Bar charts show changes in the percentage of cell migration compared to the no-treatment control. *P<.05; N=3.

Figure 4

Effects of low molecular weight (LMW) heparin, acetylsalicylic acid (ASA), and combination therapy on antiphospholipid antibody-mediated modulation of trophoblast cytokine/chemokine production. HTR-8 cells were treated with no treatment (NT), antiphospholipid antibodies (ID2, 20 µg/mL), or antiphospholipid antibodies in the presence of: 1) LMW heparin (10 µg/mL); 2) ASA (10 µg/mL); or 3) combination LMW heparin and ASA (10 µg/mL each). Supernatants were analyzed for (A) interleukin (IL)-6, (B) IL-8, (C)) growth-regulated oncogene-alpha, and (D) IL-1β. *P<.05; †P<.05 when compared to antiphospholipid antibodies; N=3.

Figure 5

Effects of low molecular weight (LMW) heparin, acetylsalicylic acid (ASA), and combination therapy on antiphospholipid antibody-mediated modulation of trophoblast angiogenic factor production. HTR-8 cells were treated with no treatment (NT), antiphospholipid antibodies (ID2, 20 µg/mL), or antiphospholipid antibodies in the presence of: 1) LMW heparin (10 µg/mL); 2) ASA (10 µg/mL); or 3) combination LMW heparin and ASA (10 µg/mL each). Supernatants were analyzed for (A) vascular endothelial growth factor (VEGF), (B) placental growth factor, (C) sFlt-1, and (D) sEng by enzyme-linked immunosorbent assay (ELISA). *P<.05; †P<.05 when compared to antiphospholipid antibodies; N=3.

Figure 6

Effects of low molecular weight (LMW) heparin, acetylsalicylic acid (ASA), and combination therapy on antiphospholipid antibody-mediated modulation of trophoblast cell migration. HTR-8 cells were treated with no treatment (NT), antiphospholipid antibodies (ID2, 20 µg/mL), or antiphospholipid antibodies in the presence of: 1) LMWH (10 µg/mL); 2) ASA (10 µg/mL); or 3) combination LMW heparin and ASA (10 µg/mL each). Cell migration was assessed using a colorimetric assay. Bar charts show changes in the percentage of cell migration compared to the no-treatment control. *P<.05; †P<.05 when compared to mouse anti-human β2-GPI monoclonal IgG1; N=3).