Dysregulation of Glucocorticoid Receptor Homeostasis and Glucocorticoid-Associated Genes in Umbilical Cord Endothelial Cells of Diet-Induced Obese Pregnant Sheep

Abstract

Maternal obesity (MO) is associated with offspring cardiometabolic diseases that are hypothesized to be partly mediated by glucocorticoids. Therefore, we aimed to study fetal endothelial glucocorticoid sensitivity in an ovine model of MO. Rambouillet/Columbia ewes were fed either 100% (control) or 150% (MO) National Research Council recommendations from 60 d before mating until near-term (135 days gestation). Sheep umbilical vein and artery endothelial cells (ShUVECs and ShUAECs) were used to study glucocorticoid receptor (GR) expression and function in vitro. Dexamethasone dose-response studies of gene expression, activation of a glucocorticoid response element (GRE)-dependent luciferase reporter vector, and cytosolic/nuclear GR translocation were used to assess GR homeostasis. MO significantly increased basal GR protein levels in both ShUVECs and ShUAECs. Increased GR protein levels did not result in increased dexamethasone sensitivity in the regulation of key endothelial gene expression such as endothelial nitric oxide synthase, plasminogen activator inhibitor 1, vascular endothelial growth factor, or intercellular adhesion molecule 1. In ShUVECs, MO increased GRE-dependent transactivation and FKBP prolyl isomerase 5 (FKBP5) expression. ShUAECs showed generalized glucocorticoid resistance in both dietary groups. Finally, we found that ShUVECs were less sensitive to dexamethasone-induced activation of GR than human umbilical vein endothelial cells (HUVECs). These findings suggest that MO-mediated effects in the offspring endothelium could be further mediated by dysregulation of GR homeostasis in humans as compared with sheep.

Keywords: endothelial cell; fetal programming; glucocorticoid; maternal obesity.

Figure 1

Maternal obesity upregulates GR protein expression in fetal umbilical vein and artery endothelial cells. Confluent and quiescent cells were treated with various doses of dexamethasone (0.04, 0.2, and 1 µM) for 24h and then analyzed for GR expression as described in the Section 4. (A) ShUVEC GR mRNA levels, (B) ShUVEC GR protein levels, (C) representative immunoblots for ShUVEC GR and ACTB, (D) ShUAEC GR mRNA levels, (E) ShUAEC GR protein levels, and (F) representative immunoblots for ShUAEC GR and ACTB. Bar graphs represent the mean ± SEM for each group (n = 5–6 lambs/diet group). * p < 0.05 control (CO) vs. obesogenic (MO) diet; ^† p < 0.05 basal vs. dexamethasone.

Figure 2

Maternal obesity alters basal fetal endothelial cell gene expression but does not affect dexamethasone response. Confluent and quiescent ShUVECs and ShUAECs were treated with various doses of dexamethasone (0.04, 0.2, and 1 µM) for 24 h and then analyzed for NOS3 (A), SERPINE1 (B), VEGFA (C), and ICAM1 (D) expression as described in the Section 4. A 2-way ANOVA followed by post hoc analysis was used to determine differences between the four groups (CO-A: control ShUAEC, MO-A: Obese ShUAEC, CO-V: control ShUVEC, and MO-V: obese ShUVEC, n = 5–6/group). * p < 0.05 control vs. obesogenic diet for each type of cell, ^§ p < 0.05 ShUAECs vs. ShUVECs within the same diet group.

Figure 3

Maternal obesity decreases GRE-dependent transcriptional activation in ShUVECs. Cells were transfected with a GRE-dependent luciferase reporter vector and treated with various doses of dexamethasone (0.04, 0.2, and 1 µM) to stimulate luciferase expression. Results are shown for basal and dexamethasone-stimulated GRE-mediated transactivation in ShUVECs (A) and ShUAECs (B). Bar graphs represent the mean ± SEM for each group (n = 4–5 lambs/diet group). * p < 0.05 control (CO) vs. obesogenic (MO) diet; ^† p < 0.05 basal vs. dexamethasone.

Figure 4

Maternal obesity decreases GR nuclear translocation in ShUVECs and ShUAECs. Confluent and quiescent cells were treated with solvent (DMSO) or dexamethasone at low (0.1 µM) and high (1 µM) concentrations for 15 min to study GR nuclear translocation as explained in the Section 4. Nuclear-to-cytosolic GR ratios and representative immunoblots are shown for ShUVECs (A,B) and ShUAECs (C,D). Bar graphs represent the mean ± SEM (n = 4–5/group). * p < 0.05 control (CO) vs. obesogenic (MO) diet; ^† p < 0.05 basal vs. dexamethasone.

Figure 5

Maternal obesity increases dexamethasone sensitivity to FKBP5 upregulation in ShUVECs. Confluent and quiescent ShUVECs and ShUAECs were treated with various doses of dexamethasone (0.04, 0.2, and 1 µM) for 24h and then analyzed for FKBP5 expression as described in the Section 4. (A) ShUVEC FKBP5 mRNA levels, (B) ShUVEC FKBP5 protein levels, (C) representative immunoblots for ShUVEC FKBP5 and ACTB, (D) ShUAEC FKBP5 mRNA levels, (E) ShUAEC FKBP5 protein levels, and (F) representative immunoblots for ShUAEC FKBP5 and ACTB. Bar graphs represent the mean ± SEM for each group (n = 5–6 lambs/diet group). p < 0.05 control (CO) vs. obesogenic (MO) diet, ^† p < 0.05 basal vs. dexamethasone.

Figure 6

ShUVECs show decreased sensitivity to glucocorticoids compared with HUVECs. ShUVECs from control lambs of another sheep breed (Western Mix) were studied next to previously characterized dexamethasone-sensitive HUVECs. (A) GRE-dependent transactivation of a luciferase reporter vector is shown as relative Firefly/Renilla luciferase activity (n = 6 WM-ShUVECs, n = 12 HUVECs). (B) GR and (C) RELA nuclear translocation were studied by immunoblotting using HSP70 and TBP as control markers (D) (n = 4/group). ^† p < 0.05 basal vs. dexamethasone, ^§ p < 0.05 WM-ShUVECs vs. HUVECs.

Figure 7

Maternal obesity increases p65 NF-kB (RELA) levels in ShUVECs and ShUAECs. Confluent and quiescent cells were treated with solvent (DMSO) or dexamethasone (Dex) to examine RELA expression by immunoblotting as explained in the Section 4. (A,B) ShUVEC total basal and dexamethasone-stimulated levels of RELA (A) and representative immunoblots (B). (C,D) ShUAEC total basal and dexamethasone-stimulated RELA expression (C) and representative immunoblots (D). (E,F) Nuclear-to-cytosolic RELA ratios (E) and representative immunoblots (F) are shown for ShUVECs. Bar graphs represent the mean ± SEM (n = 4–5/group). * p < 0.05 control (CO) vs. obesogenic (MO) diet.

Figure 8

Summarized findings of MO effects on GR homeostasis and key endothelial gene expression. The current hypothesis is that MO-originated inflammatory lipid mediators induce epigenetic changes that lead to offspring endothelial dysfunction. In this MO model, fetal endothelial cells show partial to generalized glucocorticoid resistance that could be a species-specific protective mechanism against cortisol-mediated endothelial dysfunction.