An endogenous aryl hydrocarbon receptor ligand induces preeclampsia-like phenotypes in rats

Abstract

Preeclampsia (PE) is a hypertensive disorder during human pregnancy. Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor. Exogenous and endogenous AhR ligands can induce hypertension in male rats and mice. Herein, using rats as a model, we tested the hypothesis that over-regulation of endogenous AhR ligands during pregnancy impairs vascular functions by disrupting the transcriptome in the placenta, contributing to the development of PE. Pregnant rats were injected daily with an endogenous AhR ligand, 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE), from gestational day (GD) 10 to 19. Maternal mean blood pressure was measured on GD16-20. Proteinuria and uteroplacental blood flow were monitored on GD20. Placentas collected on GD20 were used to determine changes in vascular density and transcriptome. Compared with the vehicle control, ITE elevated maternal mean blood pressure by 22% and 16% on GD16 and 17, respectively. ITE increased proteinuria by 50% and decreased uteroplacental blood flow by 26%. ITE reduced the placental vascular density by 18%. RNA sequencing analysis revealed that ITE induced 1316 and 2020 differentially expressed genes (DEGs) in female and male placentas, respectively. These DEGs were enriched in pathways relevant to heart diseases, vascular functions and inflammation. Bioinformatics analysis also predicted that ITE altered immune cell infiltration in placentas depending on fetal sex. These data suggest that over-regulation of endogenous AhR ligands may lead to PE with impaired vascular functions and disrupted fetal sex-specific transcriptomes and immune cell infiltration in placentas. These AhR ligand-induced DEGs and pathways may represent promising therapeutic targets for PE-induced cardiovascular dysfunctions. KEY POINTS: An endogenous AhR ligand (ITE) elevated maternal blood pressure and proteinuria in pregnant rats, and decreased uteroplacental blood flow and fetal and placental growth, all of which are hallmarks of preeclampsia. ITE reduced vascular density and altered immune cell distribution in rat placentas. ITE dysregulated transcriptomes in rat placentas in a fetal sex-specific manner. These ITE-dysregulated genes and pathways are highly relevant to diseases of heart, vascular functions and inflammatory responses.

Keywords: AhR ligand; placentas; preeclampsia; sexual dimorphism; transcriptomics; vasculature.

Figure 1. ITE dysregulates tissue growth and cardiovascular functions in pregnant rats

Pregnant rats were treated with ITE (5 mg/kg body weight/day) or DMSO from GD10 to GD19. A and B, maternal body (A) and organ (B) weights were recorded. Each maternal organ weight was adjusted to the body weight of corresponding dam in B. n = 9 and 10 for the ITE and DMSO groups, respectively. C and D, maternal blood pressure (C; n = 6/group for the DMSO and ITE groups, respectively) and uteroplacental blood flow (D; n = 3/group) were measured using the tail‐cuff method and Vevo 2100 ultrasound system, respectively. E, maternal proteinuria was measured (n = 3/group). F‒I, placentas and fetuses were collected (n = 9 and 10 dams for the ITE and DMSO groups, respectively) and weighed. The rulers are in inches. The Mann‒Whitney rank‐sum test or Student's t test was performed to compare differences between the ITE and DMSO groups. FGR: fetal growth restriction. *P < 0.05 versus DMSO (B, D, E, H, I) or DMSO at each corresponding time point (C). [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 2. ITE decreases vascular density and induces apoptosis in rat placentas

Pregnant rats were treated with ITE or DMSO from GD10 to GD19. Placentas were collected on GD20. The placental tissue sections were subjected to CD31 immunostaining followed by haematoxylin counterstaining. A and B, representative images of rat placentas from the ITE and DMSO groups, respectively. The reddish colour indicates CD31‐positive staining. Blue: haematoxylin counterstaining; D: decidua basalis. L: labyrinth zone. B: basal zone. Bar = 1 mm. C and D, the relative thicknesses and areas of the L and B zones were quantified. Ee‒Gg, vascular density and CD31 staining intensity in the L zone. E and F, representative images of CD31 immunostaining in the L zone from the ITE (E) and DMSO (F) groups. The insets in E and F are adjacent tissue sections treated with normal goat IgG as a control. Small arrows: vascular endothelial cells. Large arrow: an area with a density of dead cells and immune cell infiltration. s: maternal sinusoids; es: oedematous stoma. G, vascular area density and CD31 staining intensity were quantified. H‒K, cell apoptosis. The tissue sections were subjected to a TUNEL assay. H and Ii, representative images of two adjacent placental sections from the DMSO group. The sections were pretreated with DNase I (a positive control) (H) or without DNase I (a negative control) (I) followed by slight counterstaining with haematoxylin. J and K, representative images of two adjacent placental sections from the ITE with (J) or without (K; a negative control) recombinant terminal deoxynucleotidyl transferase. Arrows: apoptotic cells. *: blood vessels. Brownish nuclear 3,3′‐diaminobenzidine staining was used to stain apoptotic cells; blue: haematoxylin counter‐staining. *P < 0.05 versus DMSO. Bar = 100 μm. n = 6 placentas from different dams/group. L, Western blotting analysis for CD31 and cleaved caspase‐3. Data normalized to GAPDH are expressed as the means ± SD fold of the control. HUVECs at the caspase‐3 band were treated with staurosporine (200 nM; a positive control for cleaved caspase‐3). The Mann‒Whitney rank‐sum test or Student's t test was performed to compare differences between DMSO and ITE. *P < 0.05 versus DMSO. n = 7 placentas from different dams/group. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 3. ITE dysregulates the transcriptome in rat placentas

A, circos plot illustrating the chromosomal position of DEGs in F (red dots) and M (blue dots) placentas. Each dot represents one gene. The numbers and letters in the outer ring indicate the chromosomal location. For each scatter plot track, dots outside and inside the centreline are up‐ and downregulated genes, respectively. B, volcano plots showing DEGs in F and M placentas. Grey dots: no significant difference; red and green dots: >2‐fold up‐ and downregulation, respectively (FDR‐adjusted P < 0.05) in the ITE group vs. the DMSO group. n = 13, 16, 8, and 13 for the DMSO‐F, DMSO‐M, ITE‐F, and ITE‐M groups, respectively. C, RT‐qPCR validation of ITE‐dysregulated genes in F and M placentas. *Means differ (FDR‐adjusted P < 0.05) from DMSO; †means differ (0.1 > FDR‐adjusted P > 0.05) from DMSO. n = 4/fetal sex/group. F, female; M, male. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 4. ITE dysregulates pathways in rat placentas

A, biological functions. B, disease‐associated biological functions. C, canonical pathway‐associated genes. D, gene networks. Significant enrichment was determined using IPA software (P < 0.05, Fisher's exact test). Dotted line: P = 0.05. F, female; M, male. [Colour figure can be viewed at wileyonlinelibrary.com]