Hypoxia and the anticoagulants dalteparin and acetylsalicylic acid affect human placental amino acid transport.

BACKGROUND: Anticoagulants, e.g. low-molecular weight heparins (LMWHs) and acetylsalicylic acid (ASA) are prescribed to women at risk for pregnancy complications that are associated with impaired placentation and placental hypoxia. Beyond their role as anticoagulants these compounds exhibit direct effects on trophoblast but their impact on placental function is unknown. The amino acid transport systems A and L, which preferably transfer essential amino acids, are well-described models to study placental nutrient transport. We aimed to examine the effect of hypoxia, LMWHs and ASA on the activity of the placental amino acid transport systems A and L and associated signalling mechanisms. METHODS: The uptake of C14-MeAIB (system A) or H3-leucin (system L) was investigated after incubation of primary villous fragments isolated from term placentas. Villous tissue was incubated at 2% O2 (hypoxia), 8% O2 and standard culture conditions (21% O2) or at 2% O2 and 21% O2 with dalteparin or ASA. Activation of the JAK/STAT or mTOR signalling pathways was determined by Western analysis of total and phosphorylated STAT3 or Raptor. RESULTS: Hypoxia decreased system A mediated MeAIB uptake and increased system L mediated leucine uptake compared to standard culture conditions (21% O2). This was accompanied by an impairment of STAT3 and a stimulation of Raptor signalling. System L activity increased at 8% O2. Dalteparin treatment reduced system A and system L activity under normoxic conditions and ASA (1 mM) decreased system A and L transporter activity under normoxic and hypoxic conditions. CONCLUSIONS: Our data underline the dependency of placental function on oxygen supply. LMWHs and ASA are not able to reverse the effects of hypoxia on placental amino acid transport. These findings and the uncovering of the signalling mechanisms in more detail will help to understand the impact of LMWHs and ASA on placental function and fetal growth

LDH release and secretion of hCG into the culture medium under hypoxic conditions (2% O₂) compared to 21% O₂ (A) and after treatment of placental villous explants with dalteparin or ASA at an oxygen level of 21% (B, C).

<p>Data are presented as relative means ± SEM. *p<0.05 compared to untreated control (set to 1).</p

Relative placental system A (A) and L amino acid transporter activities (B) after 2 h of incubation of villous explants at 2% O₂ or 8% O₂ compared to 21% O₂.

<p>Normalized ratio of pSTAT3/STAT3 (C) and pRaptor/Raptor (D) at different oxygen concentrations. Values are relative means ± SEM. *p<0.05 compared to system A and L activities or ratio of pSTAT3/STAT3 and pRaptor/Raptor incubated at 21% O₂ (set to 1).</p

Protein expression of pSTAT3/STAT3 (A, B) and pRaptor/Raptor (C, D) after 2 h of incubation with ASA (0.01 mM, 0.1 mM, 1 mM) at different oxygen levels (21% and 2% O₂).

<p> Representative Western blot of STAT3, pSTAT3 and β-actin (A, C). Representative bars (B, D) show effects on pSTAT3/STAT3 and pRaptor/Raptor ratios compared to untreated control. Data are presented as relative means ± SEM. *p<0.05 compared to untreated control (set to 1).</p

The table summarizes the different effects of dalteparin, ASA and hypoxia on system A and L amino acid transport, pSTAT3/STAT3 and pRaptor/Raptor ratio.

<p>↑ = increase; ↓ = decrease; – = no significant change.</p

System A (A, C) and L (B, D) amino acid transporter activities in placental villous explants after 2 h of incubation with dalteparin (0.025 IU/ml, 0.25 IU/ml, 2.5 IU/ml) or with ASA (0.01 mM, 0.1 mM, 1 mM) at 21% and 2% O₂.

<p>Values are relative means ± SEM. *p<0.05 compared to untreated control (set to 1).</p

Protein expression of pSTAT3/STAT3 (A, B) and pRaptor/Raptor (C, D) in placental villous explants after incubation with dalteparin (0.025 IU/ml, 0.25 IU/ml, 2.5 IU/ml) at 21% and 2% O₂.

<p>Representative Western blot of STAT3, pSTAT3 and β-actin (A) or Raptor, pRaptor and β-actin (C). Representative bar graph showing pSTAT3/STAT3 (B) and pRaptor/Raptor (D) ratios for dalteparin treated villous fragments compared to control. Data are presented as relative means ± SEM. *p<0.05 compared to untreated control (set to 1).</p