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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.

SUBMITTER: Sobrino A

PROVIDER: S-EPMC2785884 | biostudies-literature | 2009 Dec

REPOSITORIES: biostudies-literature

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Estradiol stimulates vasodilatory and metabolic pathways in cultured human endothelial cells.

Sobrino Agua A Mata Manuel M Laguna-Fernandez Andrés A Novella Susana S Oviedo Pilar J PJ García-Pérez Miguel Angel MA Tarín Juan J JJ Cano Antonio A Hermenegildo Carlos C

PloS one 20091214 12

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 ...[more]

PMID: 20011585

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Project description:Estrogen has been reported to have beneficial effects on vascular biology through direct actions on endothelium. Together with transcription factors, miRNAs are the major drivers of gene expression and signaling networks. The objective of this study was to identify a comprehensive regulatory network (miRNA-transcription factor-downstream genes) that controls the transcriptomic changes observed in endothelial cells exposed to estradiol. miRNA/mRNA interactions were assembled using our previous microarray data of human umbilical vein endothelial cells (HUVEC) treated with 17β-estradiol (E2) (1 nmol/L, 24 h). miRNA-mRNA pairings and their associated canonical pathways were determined using Ingenuity Pathway Analysis software. Transcription factors were identified among the miRNA-regulated genes. Transcription factor downstream target genes were predicted by consensus transcription factor binding sites in the promoter region of E2-regulated genes by using JASPAR and TRANSFAC tools in Enrichr software. miRNA-target pairings were filtered by using differentially expressed miRNAs and mRNAs characterized by a regulatory relationship according to miRNA target prediction databases. The analysis identified 588 miRNA-target interactions between 102 miRNAs and 588 targets. Specifically, 63 upregulated miRNAs interacted with 295 downregulated targets, while 39 downregulated miRNAs were paired with 293 upregulated mRNA targets. Functional characterization of miRNA/mRNA association analysis highlighted hypoxia signaling, integrin, ephrin receptor signaling and regulation of actin-based motility by Rho among the canonical pathways regulated by E2 in HUVEC. Transcription factors and downstream genes analysis revealed eight networks, including those mediated by JUN and REPIN1, which are associated with cadherin binding and cell adhesion molecule binding pathways. This study identifies regulatory networks obtained by integrative microarray analysis and provides additional insights into the way estradiol could regulate endothelial function in human endothelial cells.

Project description:Endothelial progenitor cells (EPCs) repair damaged endothelium and promote capillary formation, processes involving receptor tyrosine kinases (RTKs) and heme oxygenase 1 (HO-1). Because estradiol augments vascular repair, we hypothesize that estradiol increases EPC proliferation and capillary formation via RTK activation and induction of HO-1. Physiological concentrations of estradiol (10 nmol/L) increased EPC-induced capillary sprout and lumen formation in matrigel/fibrin/collagen systems. Propyl-pyrazole-triol (PPT; 100 nmol/L; estrogen receptor [ER]-alpha agonist), but not diarylpropionitrile (ER-beta agonist), mimicked the stimulatory effects of estradiol on capillary formation, and methyl-piperidino-pyrazole (ER-alpha antagonist) abolished the effects of estradiol and PPT. Three different RTK activators (vascular endothelial growth factor, hepatocyte growth factor, and stromal derived growth factor 1) mimicked the capillary-stimulating effects of estradiol and PPT. SU5416 (RTK inhibitor) blocked the stimulatory effects of estradiol and PPT on capillary formation. Estradiol increased HO-1 expression by 2- to 3-fold, an effect blocked by SU5416, and PPT mimicked the effects of estradiol on HO-1. The ability of estradiol to enhance capillary formation, increase expression of HO-1, and augment phosphorylation of extracellular signal-regulated kinase 1/2, Akt, and vascular endothelial growth factor receptor 2 was mimicked by its cell-impermeable analog BSA estradiol. Actinomycin (transcription inhibitor) did not alter the effects of estradiol on RTK activity or vascular endothelial growth factor secretion. We conclude that estradiol via ER-alpha promotes EPC-mediated capillary formation by a mechanism that involves nongenomic activation of RTKs and HO-1 activation. Estradiol in particular and ER-alpha agonists in general may promote healing of injured vascular beds by promoting EPC activity leading to more rapid endothelial recovery and capillary formation after injury.

Project description:Tetrahydrobiopterin (BH4) is a key redox-active cofactor in endothelial isoform of NO synthase (eNOS) catalysis and is an important determinant of NO-dependent signaling pathways. BH4 oxidation is observed in vascular cells in the setting of the oxidative stress associated with diabetes. However, the relative roles of de novo BH4 synthesis and BH4 redox recycling in the regulation of eNOS bioactivity remain incompletely defined. We used small interference RNA (siRNA)-mediated "knockdown" GTP cyclohydrolase-1 (GTPCH1), the rate-limiting enzyme in BH4 biosynthesis, and dihydrofolate reductase (DHFR), an enzyme-recycling oxidized BH4 (7,8-dihydrobiopterin (BH2)), and studied the effects on eNOS regulation and biopterin metabolism in cultured aortic endothelial cells. Knockdown of either DHFR or GTPCH1 attenuated vascular endothelial growth factor (VEGF)-induced eNOS activity and NO production; these effects were recovered by supplementation with BH4. In contrast, supplementation with BH2 abolished VEGF-induced NO production. DHFR but not GTPCH1 knockdown increased reactive oxygen species (ROS) production. The increase in ROS production seen with siRNA-mediated DHFR knockdown was abolished either by simultaneous siRNA-mediated knockdown of eNOS or by supplementing with BH4. In contrast, addition of BH2 increased ROS production; this effect of BH2 was blocked by BH4 supplementation. DHFR but not GTPCH1 knockdown inhibited VEGF-induced dephosphorylation of eNOS at the inhibitory site serine 116; these effects were recovered by supplementation with BH4. These studies demonstrate a striking contrast in the pattern of eNOS regulation seen by the selective modulation of BH4 salvage/reduction versus de novo BH4 synthetic pathways. Our findings suggest that the depletion of BH4 is not sufficient to perturb NO signaling, but rather that concentration of intracellular BH2, as well as the relative concentrations of BH4 and BH2, together play a determining role in the redox regulation of eNOS-modulated endothelial responses.

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Estradiol stimulates vasodilatory and metabolic pathways in cultured human endothelial cells.

Publications

Estradiol stimulates vasodilatory and metabolic pathways in cultured human endothelial cells.

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