Endothelial indoleamine 2,3-dioxygenase-1 regulates the placental vascular tone and is deficient in intrauterine growth restriction and pre-eclampsia

Indoleamine 2,3-dioxygenase-1 (IDO1) mediates the degradation of L-tryptophan (L-Trp) and is constitutively expressed in the chorionic vascular endothelium of the human placenta with highest

levels in the microvasculature. Given that endothelial expression of IDO1 has been shown to regulate vascular tone and blood pressure in mice under the condition of systemic inflammation,

we asked whether IDO1 is also involved in the regulation of placental blood flow and if yes, whether this function is potentially impaired in intrauterine growth restriction (IUGR) and

pre-eclampsia (PE). In the large arteries of the chorionic plate L-Trp induced relaxation only after upregulation of IDO1 using interferon gamma and tumor necrosis factor alpha. However, ex

vivo placental perfusion of pre-constricted cotyledonic vasculature with L-Trp decreases the vessel back pressure without prior IDO1 induction. Further to this finding, IDO1 protein

expression and activity is reduced in IUGR and PE when compared to gestational age–matched control tissue. These data suggest that L-Trp catabolism plays a role in the regulation of

placental vascular tone, a finding which is potentially linked to placental and fetal growth. In this context our data suggest that IDO1 deficiency is related to the pathogenesis of IUGR and

PE.

L-Tryptophan (L-Trp) is the least abundant essential amino acid1. It is required for protein synthesis and also as precursor of key biomolecules such as serotonin, melatonin and NAD+ 

1,2,3,4. L-Trp catabolism mainly follows the kynurenine (kyn) pathway (KP), with the first and rate-limiting step being the oxidation of the indole ring of L-Trp. This step is catalyzed by

one of three enzymes capable of converting of L-Trp into N-formylkynurenine5,6, namely tryptophan 2,3-dioxygenase (TDO), indoleamine 2,3-dioxygenase-1 (IDO1) and −2 (IDO2). These cytosolic

enzymes display varying expression patterns2,7,8, a factor which likely dictates their relative ability to contribute to the degradation of L-Trp along the KP.

It is well established that IDO1 is induced during infection where it displays antimicrobial activity9 and immune regulation10,11. Indeed, recent studies have probed the ability of the

enzyme to mediate the immune system in tumour survival12. In the term placenta high levels of IDO1 expression and activity have been reported, where it is implicated in establishing and

maintaining feto-maternal tolerance13,14,15,16, a role mediated via the induction of regulatory T cells17.

Other recent works have shown that during inflammation, IDO1 becomes expressed in vascular endothelial cells and contributes to arterial relaxation18. The enzyme is constitutively expressed

on both the maternal and the chorionic vascular endothelium with a positive gradient towards the feto-maternal interface13. This led us to question whether expression of IDO1 in the fetal

vessels of the chorion is involved in the regulation of placental perfusion and consequently placental and fetal growth. Furthermore, we wondered whether pathologies which display reduced

fetal growth (such as intrauterine growth retardation (IUGR) and preeclampsia (PE))19,20, are associated with a defect in IDO1-mediated regulation of placental vascular tone. In fact,

previous reports present some evidence in favour of this hypothesis. For instance, L-Trp degradation is reduced in placental tissue in IUGR21, and IDO1 expression is decreased in PE22,23. In

addition, disruption of the IDO1 gene induces IUGR and preeclampsia phenotypes in pregnant mice24. Changes in the chorionic vasculature are associated with IUGR, as increased vascular

resistance leads to abnormal Doppler waveforms of the umbilical artery25. Remodeling of the arteries of placental stem villi contributes to IUGR26. However, the role of IDO1 in the

regulation of the placental vascular tone and its potential link with these pregnancy complications has not been investigated so far.

As a basis for subsequent functional studies involving chorionic plate arteries, we studied IDO1 expression at the level of mRNA and protein in freshly isolated primary PLAECs and in PLAECs

stimulated with IFNγ (80 ng/mL) and TNFα (80 ng/mL) for 48 h27 (Supplementary Fig. S1). As revealed by qPCR, IDO1 mRNA expression was markedly augmented following cytokine stimulation

(Supplementary Fig. S1a). IDO1 protein contents varied between the different isolations, and markedly increased upon cytokine stimulation (Supplementary Fig. S1b). The calculated MW of IDO1

is 45 kDa but the observed band was found at a somewhat lower level as previously described9. Immunocytochemical staining of cellular pellets failed to detect IDO1 protein in non-stimulated

cells (Supplementary data, Fig. S1c1). However, strong IDO1-staining was found in PLAECs stimulated with IFNγ and TNFα (Supplementary Fig. S1c2).

Compared to vehicle control, L-Trp had no discernible effects on U46619 pre-constricted arterial rings (P = 0.528) (Fig. 1a). In contrast, L-Trp- elicited relaxation of the arterial rings

preincubated overnight with IFNγ and TNFα was significantly higher compared to vehicle-treated control rings (P