Estradiol stimulates vasodilatory and metabolic pathways in cultured human endothelial cells

Abstract

Vascular effects of estradiol are being investigated because there are controversies among clinical and experimental studies. DNA microarrays were used to investigate global gene expression patterns in cultured human umbilical vein endothelial cells (HUVEC) exposed to 1 nmol/L estradiol for 24 hours. When compared to control, 187 genes were identified as differentially expressed with 1.9-fold change threshold. Supervised principal component analysis and hierarchical cluster analysis revealed the differences between control and estradiol-treated samples. Physiological concentrations of estradiol are sufficient to elicit significant changes in HUVEC gene expression. Notch signaling, actin cytoskeleton signaling, pentose phosphate pathway, axonal guidance signaling and integrin signaling were the top-five canonical pathways significantly regulated by estrogen. A total of 26 regulatory networks were identified as estrogen responsive. Microarray data were confirmed by quantitative RT-PCR in cardiovascular meaning genes; cyclooxygenase (COX)1, dimethylarginine dimethylaminohydrolase (DDAH)2, phospholipase A2 group IV (PLA2G4) B, and 7-dehydrocholesterol reductase were up-regulated by estradiol in a dose-dependent and estrogen receptor-dependent way, whereas COX2, DDAH1 and PLA2G4A remained unaltered. Moreover, estradiol-induced COX1 gene expression resulted in increased COX1 protein content and enhanced prostacyclin production. DDAH2 protein content was also increased, which in turn decreased asymmetric dimethylarginine concentration and increased NO release. All stimulated effects of estradiol on gene and protein expression were estrogen receptor-dependent, since were abolished in the presence of the estrogen receptor antagonist ICI 182780. This study identifies new vascular mechanisms of action by which estradiol may contribute to a wide range of biological processes.

Figure 1. Supervised hierarchical cluster of HUVEC gene expression changes in response to estradiol.

263 probe sets of genes significantly regulated by greater than 1.8-fold change were used for 2D hierarchical clustering. Each row represents an individual probe set, and each column represents a pool of cells (C1, C2 and C3: control samples; E1, E2 and E3: estradiol-treated samples). 129 up- (red) or 134 down- (green) were regulated (P value<0.05).

Figure 2. Supervised principal component analysis (PCA).

Microarray hybridizations were performed using total RNA from HUVEC exposed to 1 nmol/L estradiol for 24 h. The gene expression profiles of 3 pools of control cells (blue) and 3 pools of cells treated with estradiol 1 nmol/L (red) were compared using PCA. The three-dimensional (3D) plot view of gene expression data (including all probe sets on U133 Plus 2.0 GeneChip) is shown, with respect to their correlation to the first three principal components.

Figure 3. Top ten signaling and metabolic pathways regulated by estradiol.

For the functional categorization of genes, Fischer's exact test was used to calculate a p value (shown as bars) determining the probability that each biological function assigned to the network is due to chance alone. The ratio (shown as squares) represents the number of differentially expressed genes in a given pathway divided by total number of genes that make up that canonical pathway.

Figure 4. The most significant network regulated by estradiol is centered on TGFB1.

Networks of genes were algorithmically generated with the IPA software based on their connectivity and assigned a score. The intensity of the node color indicates the degree of up- (red) or down- (green) regulation. A continuous line means a direct relationship between the two genes, whereas a discontinuous line indicates an indirect association. The most significant network regulated by estradiol includes 33 genes with an assigned score of 62 and is centered on TGFB1.

Figure 5. QRT-PCR confirms some estradiol up-regulated selected genes from the microarray analysis.

HUVEC were exposed to different estradiol concentrations (0,01–100 nmol/L), and to 1 µmol/L ICI182780 alone or plus 1 nmol/L estradiol, and the relative expression of the genes was quantified: (A) COX1, (B) DDAH2, (C) PLA2G4B, and (D) DHCR7. Data are percentage of control values and are mean ± SEM of 5–19 values (4–6 different experiments). * p<0.05, ** p<0.01 or *** p<0.001 vs. control cells, † p<0.05 vs. 0.01 nmol/L estradiol, and ‡ p<0.05 vs. 1 nmol/L estradiol.

Figure 6. Unregulated genes in microarray analysis were also unchanged by QRT-PCR.

HUVEC were exposed to different estradiol concentrations (0,01–100 nmol/L), and to 1 µmol/L ICI182780 alone or plus 1 nmol/L estradiol, for 24 hours. Total cellular RNA was extracted, and the relative expression of the genes was quantified using specific primers for (A) COX2, (B) DDAH1 and (C) PLA2G4A. The GADPH gene was used as control as described in Materials and Methods. Data are expressed as percentage of control values and are mean ± SEM of 5–17 values corresponding to 5 different experiments.

Figure 7. Estrogen receptor alpha and beta protein expression in HUVEC.

Cells were exposed to 1 nmol/L estradiol with or without 1 µmol/L ICI182780 for 24 hours, and protein expression of (A) ERα and (B) ERβ were measured as stated in Materials and methods. A typical immunoblotting image and relative levels assessed by densitometry of bands of 66-kDa (ERα) or 56-kDa (ERβ) are presented. Data are percentage of control values and are mean ± SEM of 6 values (3 different experiments).

Figure 8. Estradiol up-regulated COX1 protein expression results in increased prostacyclin production through ER.

HUVEC were exposed to 1 nmol/L estradiol with or without 1 µmol/L ICI182780, and protein expression of (A) COX1 and (B) COX2 and prostacyclin production (C) were measured as stated in Materials and methods. Data are percentage of control values and are mean ± SEM of 6–8 values (3–4 different experiments). * p<0.05 or ** p<0.01 vs. control cells, and † p<0.05 vs. estradiol-alone treated cells.

Figure 9. Estradiol up-regulated DDAH2 protein expression results in decreased ADMA production and increased NO release mediated by ER.

HUVEC were exposed to 1 nmol/L estradiol with or without 1 µmol/L ICI182780, and protein expression of (A) DDAH1 and (B) DDAH2, along with (C) ADMA levels and NO production, were measured as stated in Materials and methods. Data are percentage of control values and are mean ± SEM of 9–12 values (4 different experiments). * p<0.05 or ** p<0.01 vs. control cells, and † p<0.05 vs. estradiol-alone treated cells.