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

Abstract

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.

Figure 1

Impact of L-Trp on arterial rings of the chorionic plate of normal term placentas. The data show the magnitude of L-Trp- versus vehicle control- elicited relaxation of U46619-precontracted arterial rings pre-incubated overnight (b–e) or not (a) with TNFα and IFNγ. (a) Freshly isolated, non-stimulated arteries (P = 0.528). (b) Cytokine-stimulated arterial rings respond differently to L-Trp than stimulated and non-stimulated controls (P < 0.001); the controls did not react differently. (c) Relaxation following the application of 8 mM L-Trp in the presence (1MetDTrp + L-Trp; P ≤ 0.01) or absence (L-Trp; P ≤ 0.001) of 1 mM of the IDO-blocker 1MethylDTrp. (d) Relaxation following the application of 8 mM L-Trp in the presence (INCB + L-Trp; P ≤ 0.001) or absence (L-Trp; P ≤ 0.0001) of 30 μM of the IDO1-blocker INCB024360. (e) Reactivity of endothelium-denuded stimulated arterial rings to L-Trp differs from its (denuded) control group (P < 0.001). There is also a difference between the two control groups (denuded versus non-denuded, P = 0.034). Results are mean ± SD of at least 2 rings for each condition, obtained from at least 3 placentas; **P ≤ 0.01 (linear regression model in a,b and e; Dunnett’s test in c and d).

Figure 2

Impact of L-Trp on the vessel back pressure in placental cotyledons. (a) Representative experiment from a set of 5 independent experiments showing the time course of the vessel back pressure during perfusion with 8 mM L-Trp, after attaining a phase (10 min) of stable pressure (black bar). (b) Representative experiment from a set of 6 independent experiments showing the time course after attaining a phase of stable pressure (black bar) followed by perfusion of the cotyledon with U46619 (10 nM). After attaining stable (10 min) vessel back pressure again, the cotyledons were perfused with L-Trp (8 mM) and U46619 (10 nM). Each pressure point shows the average of 60 measurements. Results are given as mean ± SD. (c) Linear regression model. In both sets of experiments the decrease of the vessel back pressure during perfusion with 8 mM L-Trp is significant. Without preconstriction (dashed line, 5 experiments), the coefficient for the linear relationship between pressure and time is −0.175 (SE 0.020, P < 0.001). Following preconstriction (continuous line, 6 experiments), the coefficient for the linear relationship between pressure and time is −0.651 (SE 0.059, p < 0.001).

Figure 3

IDO1-mRNA and -protein expression, and IDO activity in chorionic tissue of normal and pathological placentas. (a) IDO1 mRNA in the pre-term controls (n = 5), term placentas (n = 10), IUGR (n = 6) and PE (n = 13) was analyzed by RT-qPCR. Results were normalized to the expression of the endothelial marker CD34 and calculated as a fold change relative to the pre-term control. (b) IDO1 Western Blot of chorionic tissue of IUGR and PE. Samples originate from pre-term controls (n = 5), term controls (n = 11), IUGR (n = 6) and PE (n = 13). The gels are grouped in pre-term control (blue line), term control (grey line), IUGR (red line) and PE (orange line). Fibroblasts stimulated with IFNγ and TNFα were used as a positive control (black line). (c) Densitometric analysis of IDO1 protein by Western Blot in the pre-term controls (n = 5), term placentas (n = 11), IUGR (n = 6) and PE (n = 13). The results were normalized to GAPDH protein and calculated as fold change relative to the pre-term control. (d) IDO activity was measured in the pre-term controls (n = 11), term placentas (n = 9), IUGR (n = 7) and PE (n = 13) by LC/MS. IDO1 activity is expressed as kynurenine formed per mg tissue protein per min. Results are given as mean ± SD. *P ≤ 0.05, **P ≤ 0.01 (Dunnett’s test in a, c and d).

Figure 4

Immunohistochemical stainings of serial paraffin sections from pre-term control, IUGR and PE placental tissue. Samples were stained for CD34 II (first row), IDO1 (second row) and the macrophage marker CD68 (third row) and compared to pre-term controls (first column; blue), IUGR (second column; red) and PE (third column; orange). This PE sample contains a focus of chronic villitis and intervillositis (arrows). The scale bars represent 100 μm.

Figure 5

Effect of L-Trp on chorionic plate arteries from PE and IUGR pregnancies. The data show the magnitude of the L-Trp- and vehicle control- elicited relaxation of U46619-precontracted arterial rings pre-incubated overnight (b) or not (a,c) with TNFα and IFNγ. (a) P = 0.216. (b) ***P ≤ 0.001. (c) ***P ≤ 0.001. The data were determined at least in triplicates (using a minimum of 3 rings per placenta) from 3 experiments using different placentas. Results are mean ± SD (linear regression model).