Pregnancies affected by antiphospholipid syndrome are managed with heparin, either alone or in combination with acetylsalicylic acid (ASA), with prevention of maternal and fetal adverse outcomes as the goal.1,2 Although low molecular weight (LMW) heparin is standard with a history of thromboembolism, studies regarding its effectiveness for the prevention of antiphospholipid syndrome-associated adverse pregnancy outcomes have been fraught with contradiction.3 – 8 Furthermore, late pregnancy complications remain prevalent despite heparin therapy.3,4
Similarly, some studies of ASA in antiphospholipid syndrome found an increased live birth rate,7 whereas others did not.9,10 Nonetheless, ASA has gained favor as prophylaxis for recurrent intrauterine growth restriction (IUGR) or pre-eclampsia in nonantiphospholipid syndrome pregnancies. A recent meta-analysis reported that ASA initiated before 16 weeks of gestation decreases risk of recurrent pre-eclampsia (relative risk 0.47, 95% confidence interval [CI] 0.34–0.65), severe pre-eclampsia (relative risk 0.09, 95% CI 0.02–0.37), and IUGR (relative risk 0.44, 95% CI 0.30–0.65), whereas ASA started after 16 weeks of gestation yielded no benefit.11
The effects of these medications on third trimester trophoblasts previously had been reported;12 however, dysregulation of first-trimester trophoblast function is a stronger mediator in the development of adverse pregnancy outcomes.13,14 Previous studies by our group have demonstrated that antiphospholipid antibodies adversely affect human first-trimester trophoblast function by reducing cell migration and inducing a potentially unfavorable proinflammatory cytokine and altered angiogenic factor milieu.15 – 17 Therefore, given the controversies, we sought to elucidate the action of these compounds on human first-trimester trophoblast function in the absence and presence of antiphospholipid antibodies.
MATERIALS AND METHODS
Sterile LMW heparin, enoxaparin sodium injection, 100 mg/mL, was purchased from Aventis Pharmaceuticals. Acetylsalicylic acid was obtained from Sigma-Aldrich and was reconstituted in diluted ethanol and filter-sterilized before use. No significant differences in measured outcomes were seen with ethanol controls.
A mouse antihuman beta-2-glycoprotein-1 (β2-GPI) monoclonal IgG1, designated ID2, was chosen to mimic antiphospholipid syndrome in pregnancy in vitro, because of its well-characterized properties.12,13,18 Similar to human polyclonal antiphospholipid antibodies, ID2 binds β2-GPI when immobilized on a suitable negatively charged surface,19 binds to human first-trimester trophoblast,15,20 and alters first-trimester trophoblast function.15 – 17
The human first trimester extravillous cytotrophoblast cell line, HTR-8, was used,21 and was a gift from Dr. Charles Graham (Queens University, Kingston, Ontario, Canada). Cells were cultured in RPMI 1640 media (Gibco), supplemented with 10% fetal bovine serum (Hyclone), 10 mm Hepes, 0.1 mm minimal essential medium nonessential amino acids, 1 mm sodium pyruvate, and 100 nm penicillin-streptomycin (Gibco). Cells were maintained at 37°C with 5% CO2. These cells respond to antiphospholipid antibodies similarly to primary first-trimester trophoblast cultures.15,17
HTR-8 cells were treated with or without ID2 (20 micrograms/mL) in the presence and absence of: 1) media-only, no treatment control; 2) low-dose LMW heparin (10 micrograms/mL); 3) low-dose ASA (10 micrograms/mL); or 4) combination low-dose LMW heparin plus ASA (10 micrograms/mL each) in OptiMEM (Invitrogen). As control, cells were also treated with ethanol, which had no effect (data not shown). Cell-free supernatants were collected after 72-hour culture, centrifuged at 1,500g for 10 minutes, and stored at −80°C. The concentrations of ASA and LMW heparin used in this study were based on a previous report and equivalent to low-dose medications used in the clinical setting.12,22
Cytokines and angiogenic factor concentrations in the supernatant were evaluated by enzyme-linked immunosorbent assay (Assay Designs) and multiplex assay (Luminex, Austin, TX). Proinflammatory cytokines assayed were interleukin (IL)-6, IL-8, IL-1β, and growth-regulated oncogene-α (a monocyte chemoattractant). Proangiogenic factors assayed were vascular endothelial growth factor (VEGF) and placental growth factor, and antiangiogenic factors assayed were soluble FMS-like tyrosine kinase-1 and soluble endoglin. We have previously demonstrated that antiphospholipid antibodies modulates first-trimester trophoblast secretion of all of these factors.15 – 17
A two-chamber assay was used for the migration studies to measure the spontaneous migratory property of the trophoblast that occurs during normal implantation, which has been previously described by our group.15 The lower chamber for this assay consisted of 24-well tissue culture plates (BD Falcon), which contained 800 microliters of treatment media. Trophoblast cells (1×105 cells in 200 microliters of respective treatment media) were then seeded into a cell culture well insert with 8-micrometer pore size membrane (BD Biosciences), which served as the upper chamber. Cells were treated with the same combinations of ID2, ASA, and LMW heparin as described. After 48-hour incubation, trophoblast migration across the membranes was determined using the QCM 24-Well Colorimetric Cell Migration Assay (Chemicon International). The resulting colored mixture was read in triplicate at 560 nm using a BioRad plate reader and compared with a 100% cell control to determine relative percent migration.
Experiments were performed three times and assayed in duplicate. Data were then pooled and expressed as mean±standard deviation. Statistical significance (P<.05) was determined using analysis of variance.
Low molecular weight heparin and ASA are both known to have anti-inflammatory properties.23 – 25 The first objective of this study therefore was to determine the effects of these therapies on basal cytokine/chemokine production by first-trimester trophoblast. As shown in Figure 1, LMW heparin significantly increased basal growth-regulated oncogene-α secretion by 269%±18.5% (P=.006), without affecting secretion of IL-6, IL-8, and IL-1β. Acetylsalicylic acid alone did not have any significant effect on the basal levels of secreted IL-8, IL-6, growth-regulated oncogene-α, or IL-1β. Treatment of trophoblast with both LMW heparin and ASA fully reversed the upregulation of growth-regulated oncogene-α induced by single-agent LMW heparin therapy.
The next objective of this study was to determine the effect of the therapies on basal trophoblast secretion of proangiogenic (VEGF and placental growth factor) and antiangiogenic factors (soluble FMS-like tyrosine kinase-1 and soluble endoglin). Although a previous report by our group showed that high-dose LMW heparin (100 micrograms/mL) upregulated HTR-8 placental growth factor and soluble FMS-like tyrosine kinase-1 secretion,16 the effects of a low-dose LMW heparin (10 micrograms/mL) on these factors had not yet been evaluated. As shown in Figure 2, treatment with low-dose LMW heparin induced an antiangiogenic state, with a 262.5%±43.6% increase in soluble FMS-like tyrosine kinase-1 (P<.001) and 34.0%±6.3% reduction in VEGF (P<.001). The proangiogenic placental growth factor also increased by 230.2%±85.4% (P<.001). Acetylsalicylic acid had no effects on the production of these angiogenic factors by trophoblasts. Combination treatment resulted in the same effects as single-therapy LMW heparin.
The next objective of this study was to determine the effects of the therapies on basal trophoblast migration. We previously reported that high-dose LMW heparin augmented basal trophoblast migration.17 As shown in Figure 3, single-agent low-dose LMW heparin also induced this promigratory phenomenon, with a 133.6%±9.9% increase in migration (P<.001). Because ASA is also used routinely for prophylaxis against recurrent pre-eclampsia and IUGR,11 we speculated that ASA would have an overall promigratory effect on the first-trimester trophoblast. Treatment with low-dose ASA, however, did not induce any changes in the migratory capacity of HTR-8 trophoblasts. Combination therapy, however, modestly augmented the LMW heparin-dependent increase in migratory capacity by an additional 4.6%±2.1% (P<.001).
Having determined the effects of the treatment regimens on basal trophoblast function, we next sought to determine the effects of LMW heparin and ASA on antiphospholipid antibodies-mediated alterations in the trophoblast function, beginning with cytokine/chemokine production. Consistent with our previous reports,17 treatment of the HTR-8 cell line with the anti-β2GPI monoclonal antibody, ID2 (20 micrograms/mL), significantly decreased IL-6 secretion by 40.7%±11.7% (P<.001) and increased the secretion of IL-8 by 641.3%±376.3% (P<.001), growth-regulated oncogene-α by 541.1%±52.8% (P<.001), and IL-1β by 508.8%±10.5% (P<.001; Fig. 4).
Treatment with LMW heparin did not reverse the antiphospholipid antibodies-induced downregulation of IL-6, but it attenuated the antiphospholipid antibodies-induced elevations of IL-8 and IL-1β secretion (Fig. 4). The augmentation of growth-regulated oncogene-α levels with LMW heparin treatment (Fig. 1C) was maintained in the presence of antiphospholipid antibodies, although the response was more pronounced (Fig. 4C). Single-agent ASA therapy did not affect any of the antiphospholipid antibodies-induced cytokines or chemokine responses (Figs. 4A–D). Combination LMW heparin and ASA treatment did minimally reverse the growth-regulated oncogene-α effect (Fig. 4C), but it did not offer any other additional anti-inflammatory effect.
We next turned our attention to the effects of the therapies on antiphospholipid antibodies-mediated modulation of angiogenic factor production. As previously reported,26 antiphospholipid antibodies significantly increased HTR-8 secretion of VEGF by 145.5%± 18.1% (P<.001), placental growth factor by 224.5%± 45.4% (P<.001), and soluble endoglin by 278.7%± 192.5% (P<.001), while decreasing soluble FMS-like tyrosine kinase-1 by 28.7%±12.4% (P=.012; Fig. 5).
None of the treatment regimens altered the antiphospholipid antibodies-induced changes in levels of VEGF or soluble endoglin (Fig. 5A, D). Treatment with single-agent LMW heparin further augmented the antiphospholipid antibodies-induced placental growth factor secretion by 159.7%±19.5% (P<.001), whereas ASA had no significant effect. The addition of ASA to LMW heparin in combination therapy yielded a similar effect on placental growth factor as treatment with LMW heparin alone (Fig. 5B). As with the basal state, LMW heparin caused a significant elevation of soluble FMS-like tyrosine kinase-1 accumulation in supernatant, even though the antiphospholipid antibodies alone reduced soluble FMS-like tyrosine kinase-1 levels, and combination therapy with ASA was unable to reverse this profound response (Fig. 5C).
The final objective was to determine the effects of the therapies on antiphospholipid antibodies-mediated modulation of trophoblast migration. As seen in Figure 6, and in our previous report,17 antiphospholipid antibodies significantly reduced trophoblast migration by 59.2%±2.4% (P<.001). None of the therapies was able to reverse the effect (Fig. 6).
Despite conflicting data on the efficacy of heparin and ASA therapies in preventing adverse pregnancy outcomes, pregnant women with antiphospholipid syndrome are routinely treated with LMW heparin, either alone or in combination with ASA.9 – 16,18,19,21 Whereas several studies have reported on the anti-inflammatory properties of LMW heparin and ASA,3,27 – 29 there is a paucity of literature on the effects of these therapies on first-trimester trophoblast, a major player in the pathogenesis of a spectrum of obstetrical disorders.13 Given the heterogeneity in clinical outcomes with heparin and ASA therapy, it is increasingly apparent that the action of these therapies, as single agents or in combination, need to be better-elucidated to individualize and optimize therapies.
We focused our current study on human first-trimester trophoblast given their unique susceptibility to antiphospholipid antibodies with β2-GPI activity and their role in the development of pregnancy complications.13,14 Trophoblasts synthesize their own β2-GPI,12 and whereas most cells will only bind β2-GPI on their cell surface under pathologic conditions when there is exteriorization of negatively charge phospholipids on the outer surface of the plasma membrane, the rapid proliferation and differentiation of trophoblasts render them able to express endogenous and bind exogenous β2-GPI on the cell surface under normal physiological conditions.13 Thus, anti-β2-GPI antibodies target the trophoblast surface, establishing the pathogenic placental environment in antiphospholipid syndrome.13
The present study demonstrates the multiplicity and complexity of the effects of LMW heparin and ASA on trophoblast function in the presence and absence of antiphospholipid antibodies. Contrary to previous studies demonstrating anti-inflammatory effects of LMW heparin,3,27 – 29 using our trophoblast in vitro model, single-agent LMW heparin was actually shown to have both proinflammatory and anti-inflammatory effects. Our finding that low molecular weight heparin increased basal and antiphospholipid antibody-induced trophoblast secretion of growth-regulated oncogene-α, a potent monocyte chemoattractant, is novel, although the potential clinical significance and mechanism are currently unknown. Whereas basal IL-6, IL-8, and IL-1β were not affected by either LMW heparin or ASA, the antiphospholipid antibodies-mediated elevations in IL-8 and IL-1β were partially reversed by single-agent LMW heparin. Interestingly, although ASA alone had no effect on basal or antiphospholipid antibodies-induced cytokine production, in combination with LMW heparin, growth-regulated oncogene-α levels in the absence of antiphospholipid antibodies were brought back to baseline, suggesting that ASA can counteract the proinflammatory actions of LMW heparin.
These combined cytokine/chemokine findings may explain the inability of LMW heparin and ASA to consistently reverse the inflammation-induced endpoints seen in antiphospholipid syndrome. None of the therapies proved beneficial to the trophoblast in the absence of antiphospholipid antibodies, although combination therapy is the least detrimental by minimizing the adverse increase in growth-regulated oncogene-α. Although the in vitro model is an isolated system and cannot replicate the complex in vivo milieu in antiphospholipid syndrome patients, these findings underscore the need to better understand the effects of our therapies on the inflammatory milieu and to identify better-targeted therapy in the treatment of pregnancies complicated by antiphospholipid syndrome.
As previously described by our group,16 some derangements in angiogenic factor production in antiphospholipid syndrome mimic those seen in pre-eclampsia, such as elevated soluble endoglin. In pre-eclampsia, circulating levels of VEGF and placental growth factor are markedly reduced, whereas placental-derived soluble FMS-like tyrosine kinase-1 and soluble endoglin are elevated.27,30,31 Alterations in the angiogenic factor balance may be associated with the dysfunctional low-capacitance, high-resistance placenta seen with pre-eclampsia. A similar effect is paradoxically induced with exposure to single-agent LMW heparin, with profound increases in soluble FMS-like tyrosine kinase-1 in the basal and antiphospholipid antibodies-induced states. A recent mechanistic study implicates placental heparanase in the regulation of soluble FMS-like tyrosine kinase-1 release.28 LMW heparin also decreased VEGF, further inducing an overall antiangiogenic milieu. The potentially beneficial placental growth factor response may be counteracted by the profound antiangiogenic soluble FMS-like tyrosine kinase-1 response.
These changes in the angiogenic milieu may explain the inability of LMW heparin and ASA to prevent adverse outcomes in late gestation, such as pre-eclampsia or IUGR. Low molecular weight heparin may prevent early loss by altering the inflammatory milieu in antiphospholipid syndrome patients,13 while setting the foundation for impaired placentation by worsening angiogenesis. In the basal in vitro model, mimicking nonantiphospholipid syndrome patients at high-risk for recurrent adverse outcomes, ASA, the current accepted standard of care, did not affect the angiogenic milieu. More importantly, the paradoxical development of an antiangiogenic milieu mirroring that seen in pre-eclampsia speaks to a need for judicious use of LMW heparin in conditions that are not absolute indications for anticoagulation.
Last, trophoblast migration and invasion into spiral arteries is necessary to establish an adequate placental vascular system. We previously reported that antiphospholipid antibodies limit trophoblast migration by reducing IL-6 production.17 Despite unchanged IL-6 levels with LMW heparin therapy, LMW heparin increased basal trophoblast migration, with further improvement when combined with ASA, albeit marginally. This contradictory upregulation of migration indicates that trophoblast migration is likely regulated by a complex network of factors. Possible factors that merit further investigation include the hypoxia-inducible microRNA-21029 or Nodal,32 a member of the transforming growth factor-β superfamily. However, none of the treatments provided protection against antiphospholipid antibodies-induced downregulation of migration.
Although this in vitro model does not fully mimic the complex milieu in vivo, the strength of our system is that it allows for an uncomplicated way to test the actions of the current treatment regimen on the trophoblast, a crucial player in placental health. The use of a monoclonal antihuman β2-GPI antibody, instead of patient-derived polyclonal antiphospholipid antibodies, and the first-trimester trophoblast cell line instead of primary trophoblast cells, because of the large scale of the experiments, should also be noted. However, we believe these are valid alternatives because our previous studies have shown the monoclonal to behave in a similar manner to patient-derived antibodies and the trophoblast cell line to respond to antiphospholipid antibodies similarly to primary first trimester cultures.15,16 Nonetheless, further evaluation of the effects of LMW heparin and ASA on primary trophoblasts from different gestational ages may further shed light on the effects of these medications. Last, LMW heparin was chosen for this model, instead of unfractionated heparin, because LMW heparin is the standard of care in many clinical settings because of ease of administration and improved patient adherence. However, we have found that unfractionated heparin and LMW heparin have similar effects on the trophoblast.15,17
In summary, LMW heparin, ASA, and combination therapy each impart a distinct set of action on the function of human first-trimester trophoblasts, with both beneficial and potentially detrimental cellular effects. Their mixed effect on the antiphospholipid antibodies-induced pathological state may explain the inability of current therapies to fully reverse the detrimental effects of antiphospholipid syndrome and the conflicting data seen in vivo. Our findings highlight the need for additional investigation into the use of heparin and ASA in the treatment of antiphospholipid syndrome-induced pregnancy complications and the need for new targeted therapeutics.
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