Influence of Estradiol and Estrogen Receptors on NOS3 Expression and Nitric Oxide Release: Implications for Intracranial Aneurysm Formation and Rupture

Abstract

Objective

The prevalence of intracranial aneurysms (IAs) and aneurysmal subarachnoid hemorrhage (aSAH) due to IA rupture, is higher in postmenopausal compared with premenopausal women and men in the same age group. This is presumably due to reduced levels of the vascular protective hormone, estradiol. Experimental studies have revealed direct effects of estradiol on the vascular endothelium. Our aim was to determine the influence of estradiol and estrogen receptors on cerebral vasculature and the role of estrogen-mediated nitric oxide (NO) pathway in the formation and rupture of IAs.

Materials and Methods

We measured circulatory estradiol and NO levels and the relative expression of genes involved in estrogen-mediated NO synthesis in 349 aSAH patients and 360 age- and gender-matched controls. We observed significantly reduced serum estradiol and NO levels in aSAH patients compared with controls.

Results

The expression of endothelial NO synthase gene ( NOS3 ), estrogen receptor genes ( ESR1 , ESR2 ), and the kinases ( SRC , AKT1 , PIK3R1 ) involved in NO synthesis was significantly reduced in IA tissues compared with controls. Serum estradiol positively and significantly correlated with NO levels.

Conclusion

Our results indicate that the activity of endothelial NO synthase and the bioavailability of NO may be regulated by estradiol and estrogen receptors to maintain cerebral vascular tone.

Introduction

The majority of nontraumatic subarachnoid hemorrhage (SAH) occurs due to the rupture of focal outpouchings of the intracranial arterial wall called intracranial aneurysms (IAs). ¹ The prevalence of aneurysmal SAH (aSAH) is higher in postmenopausal compared with premenopausal women and men in the same age group. ^{1 2} Since the female preponderance in aSAH incidence surges postmenopause, hormonal factors, especially estrogen, have been implicated in IA formation and rupture. ³ Experimental studies have shown that estradiol, the physiologically active form of estrogen, plays an important role in the regulation and maintenance of vascular tone. ^{4 5} The actions of estradiol are mediated by estrogen receptors (ERs)—ERα and ERβ (encoded by ESR1 and ESR2 genes, respectively). ⁶ In a meta-analysis of microarray databases related to human IAs, researchers analyzed the expression profiles from six microarray data available in the Gene Expression Omnibus database and identified 94 differentially expressed genes in IAs, that were specifically associated with KEGG–ER signaling pathway (hsa04915), including the estradiol-mediated, nitric oxide (NO) signaling pathway. ⁷ Estradiol acts by increasing the activity of endothelial NO synthase (eNOS) and the release of the gaseous signaling molecule, NO. ^{4 5} NO is a vasodilator which maintains endothelial integrity, vascular tone, and cerebral hemodynamics. ⁸ Thus, estradiol decreases membrane permeability and prevents vascular endothelial activation. It regulates vascular tone by inducing vasorelaxation. ⁹

The mechanisms involved in IA formation and rupture in postmenopausal women have not been elucidated. We hypothesized that the estradiol-ER-mediated NO synthesis pathway may be involved in IA etiopathogenesis. Hence, in this study we measured circulatory NO and estradiol levels, expression of ER genes, and genes involved in estrogen-mediated NO synthesis pathway ( NOS3 , SRC , AKT1 , and PIK3R1 ), to evaluate the role of estradiol and NO in IA formation and rupture.

Materials and Methods

Subjects and Sample Collection

This is a prospective, case–control study, conducted at the National Institute of Mental Health and Neuro Sciences (NIMHANS), Bengaluru, Karnataka, India, a tertiary care center for neurological disorders. This study was approved by the Institute Ethics Committee and informed consent was obtained from all subjects willing to participate in the research. The study was performed in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans. Cases consisted of aSAH patients, aged between 18 and 80 years. aSAH was diagnosed on the basis of clinical and neuroimaging findings. Control group consisted of healthy individuals, age-matched, and clinically normal with no history of cerebral vascular disease.

Five milliliters of blood was drawn from each subject, plasma and serum separated, and stored at –20°C until analysis of NO and estradiol levels. Gene expression was studied in tissue samples. Aneurysm wall was collected from patients undergoing surgical clipping and superficial temporal artery (STA) obtained from patients undergoing surgery for nonvascular diseases was used as control tissue.

Biochemical Analyses

Plasma NO concentrations were measured as total NO _x (nitrite and nitrate) by Griess method using a spectrophotometer. ¹⁰ Inter- and intra-assay coefficient of variation was 1.97 and 5.17%, respectively.

Serum estradiol was estimated by enzyme-linked immunosorbent assay using commercially available kits (ES180S, Calbiotech, California, United States). The sensitivity of the assay was 3.94 pg/mL. Inter- and intra-assay coefficient of variation was 1.72 and 3.7%, respectively.

Relative Quantitation of Gene Expression

Total ribonucleic acid (RNA) was isolated from IA and STA tissue samples using TRI reagent (# T9424, Merck Sigma Aldrich, Missouri, United States). The high-capacity complementary deoxyribonucleic acid (cDNA) reverse transcription kit (4374966, ThermoFisher Scientific-Applied Biosystems, California, United States) was used for reverse transcription of total RNA into cDNA. Quantitative real-time polymerase chain reaction (RT-PCR) was performed using the TaqMan universal PCR master mix (4324018, ThermoFisher Scientific-Applied Biosystems) and TaqMan gene expression probes. Amplifications were run using Applied Biosystems 7500 fast RT-PCR system (ThermoFisher Scientific-Applied Biosystems). The expressions of all genes were normalized against the reference gene glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ) expression and the relative gene expression was calculated using the 2 ^−ΔΔCt method.

Statistical Analysis

Statistical tool GraphPad Prism v.6.0.1 (GraphPad Software, La Jolla, California, United States) was used for analysis. For categorical variables, chi-square test was used to assess dissimilarity in demographic characteristics between patients and controls. Mann–Whitney test was performed for continuous parameters and to compare the relative messenger RNA (mRNA) expression levels of various genes. The relative quantification of various gene expressions is expressed as log ₂ fold change. Spearman rank test was used for correlation analysis between various parameters. Two-sided p -value of < 0.05 was considered statistically significant.

Results

Characteristics of the Study Group

A total of 709 subjects were enrolled, including 349 patients with aSAH and 360 healthy controls. Demographic details of the study participants are given in Table 1 . Of the 349 cases, 160 were males and 189 females and 122 women had attained menopause. Mean age of cases was 55.15 ± 9.77 years. Control group consisted of 190 males and 170 females and 86 of them had attained menopause. The average age of the control group was 53.22 ± 10.25 years. Vascular risk factors such as smoking, alcohol consumption, and hypertension were more prevalent in cases compared with controls.

Table 1. Demographic details of the subjects of the study.

Parameters	Cases, N  = 349 (%)	Controls, N  = 360 (%)	p -Value a
Age (years, SD)	55.15 ± 9.77	53.22 ± 10.25
Gender (F/M)	189/160	170/190
Hypertension	110 (31.51)	40 (11.11)	< 0.0001 ***
Diabetes	40 (11.46)	38 (10.55)	0.7201
Smoking	98 (28.08)	30 (8.33)	< 0.0001 ***
Alcohol consumption	65 (18.62)	25 (6.94)	< 0.0001 ***
Serum estradiol level (pmol/L)	Median ± SEM	Median ± SEM	p -Value b
Overall ( n )	27.80 ± 1.370 (343)	30.46 ± 1.894 (352)	0.0480 c
Premenopausal women ( n )	47.81 ± 5.352 (60)	64.46 ± 3.054 (70)	0.0447 c
Postmenopausal women ( n )	14.53 ± 1.490 (125)	16.5 ± 1.714 (95)	0.0361 c
Men ( n )	28.56 ± 1.785 (158)	31.68 ± 1.658 (187)	0.2671
Plasma NOx (µmol/L)	Median ± SEM	Median ± SEM	p -Value b
Overall ( n )	11.06 ± 0.74 (343)	28.07 ± 1.15 (352)	< 0.0001 ***
Premenopausal women ( n )	13.73 ± 2.259 (60)	34.17 ± 2.192 (70)	< 0.0001 ***
Postmenopausal women ( n )	12.44 ± 1.378 (125)	21.39 ± 2.087 (95)	0.0089 **
Men ( n )	10.63 ± 0.7961 (158)	28.20 ± 1.653 (187)	< 0.0001 ***

Abbreviations: F, female; M, male; SD, standard deviation; SEM, standard error of the mean.

^aTwo-sided chi-square test.

^b Mann–Whitney U test.

^cp  < 0.05 is statistically significant.

^**p <0.01 (Highly significant)

^***p  <0.0001 (Very highly significant)

Cerebral Vasculature Expresses ERα and ERβ

The gene expression of ERs was checked in control cerebral vessels (STA) to determine the relative abundance of ER subtypes in cerebral vasculature. Both subtypes of ERs are expressed in cerebral vessels. However, the expression of ERα is significantly higher compared with the expression of ERβ in STA ( Fig. 1 ).

Fig. 1.

Relative expression of estrogen receptor (ER) genes ( ESR1 and ESR2 ) in control tissue samples ( N  = 20). The relative gene expression is presented as mean ± standard error of the mean (SEM). * p -Value < 0.05 is statistically significant as determined by the Mann–Whitney U test.

Relative Expression of Genes in IA Associated with ER-Mediated Signaling in NO Synthesis

The expressions of both ER genes ( ESR1 and ESR2 ) were significantly downregulated in cerebral aneurysmal tissue compared with control tissues ( Fig. 2 ). The mRNA levels of genes in the NO synthetic pathway ( NOS3 , SRC , AKT1 , and PIK3R1 ), were significantly downregulated in IA tissues. Expression of signaling kinases such as SRC was decreased significantly in aneurysm samples compared with controls ( p  = 0.0449). Expression of AKT1 ( p  = 0.0035) and PIK3R1 ( p  = 0.0027) was significantly decreased. NOS3 expression was also reduced in aneurysm tissues compared with controls ( p  = 0.0010).

Fig. 2.

Relative expression of genes associated with vasodilation in aneurysm ( N  = 20) and control ( N  = 20) tissue samples. The messenger ribonucleic acid (mRNA) levels of ( A ) ESR1 , ( B ) ESR2 , ( C ) SRC , ( D ) AKT1 , ( E ) PIK3R1 , and ( F ) NOS3 —the genes in nitric oxide (NO) pathway. The relative gene expression is presented as mean ± standard error of the mean (SEM). * p -Value < 0.05 is statistically significant as determined by the Mann–Whitney U test.

The relationship between expression of vasodilator- NOS3 and ERs was analyzed using Spearman test. Nearly significant positive correlation was observed between ESR1 and NOS3 expression. However, no correlation between ESR2 and NOS3 mRNA levels was observed ( Fig. 3A ).

Fig. 3.

Scatter plot indicating correlation between ( A ) the expression levels of [ΔCt] of estrogen receptor genes and NOS3 gene in intracranial aneurysm (IA) tissue samples and ( B ) the plasma NOx level and estradiol levels. Spearman's rank test was used to calculate correlation ( r ) and p -value < 0.05 is statistically significant.

Status of Plasma Nitric Oxide Metabolites (NOx) in aSAH Cases and Controls

The concentration of plasma total NOx ranged from 2.73 to 109 µmol/L in control samples. Median plasma NOx levels were significantly lower among cases (11.06 µmol/L) compared with controls (28.07 µmol/L) ( p  < 0.0001) ( Table 1 ). Overall correlation analysis revealed that circulatory estradiol levels positively and significantly correlated with NOx levels ( Fig. 3B ).

Discussion

The vascular endothelium is a monolayer of endothelial cells present at the interface between the vessel wall and blood. The endothelium is mainly involved in the maintenance of vascular integrity and its dysfunction can lead to various vascular pathologies like hypertension, atherosclerosis, ischemic stroke, etc. ¹¹ Previous studies have demonstrated direct effects of estradiol on the endothelium. ^{8 9 11} Estradiol acts by increasing the activity of eNOS and bioavailability of NO. Plasma NO levels were found to be increased and the levels of endothelin, a vasoconstrictor, decreased in postmenopausal women who received hormone therapy, when compared with controls who did not receive hormone replacement therapy (HRT). ^{4 5}

In this study, we first confirmed that both subtypes of ERs are expressed in cerebral vasculature, and found that expression ERα was significantly higher compared with the expression of ERβ. We next analyzed expression of genes in the estrogen-mediated NO synthesis pathway in IA tissues. The expression of ER genes and genes in the NO pathway ( NOS3 , SRC , AKT1 , PIK3R1 ) were significantly downregulated in IA tissues compared with controls. Expression of NOS3 was downregulated in IA tissues and its levels positively correlated with ESR1 expression. A positive correlation was observed between circulatory estradiol and NOx levels in healthy subjects. This may indicate that estradiol-ER plays a crucial role in vasodilation mediated by NO. As the estradiol level is reduced in postmenopausal women, reduced NO causes vasoconstriction leading to increased hemodynamic stress.

Sexual dimorphism has been reported in the basal NO release from the endothelium. ^{12 13} In females, eNOS protein levels were significantly higher (9.2-fold) in cerebral microvessels, than in males. ¹⁴ NOS3 knockout mice subjected to unilateral carotid artery ligation were predisposed to the formation of IAs. ¹⁵ Also, single-nucleotide polymorphisms (SNPs) in the NOS3 are associated with the increased risk for aSAH in various populations. ^{16 17}

Estradiol can activate eNOS by both genomic and nongenomic signaling pathways. NOS3 promoter has ERE-like motif, a transcription binding site for ER and is transcriptionally regulated by estradiol-ER in association with other transcription factors such as Sp-1. ¹⁸ ERα but not ERβ, is shown to be involved in the estradiol-mediated NO release in the cardiovascular system. ^{19 20} Chambliss and coworkers ²¹ demonstrated that ERα was localized on plasma membrane, and Figtree et al showed that the plasma membrane-bound, truncated ERα isoform is responsible for nongenomic, rapid release of NO in the endothelial cells. ^{22 25} The membrane-bound ERα activates a series of kinase signaling cascade involving tyrosine kinase Src, serine/threonine kinase, phosphatidylinositol-3-OH kinase (PI3K) activation of protein kinase B (Akt), and mitogen-activated protein kinase (MAPK), finally leading to the activation of eNOS by phosphorylation on serine 1177. ^{8 22 25}

In a study on premenopausal women, Gavin et al found that the ERα protein expression in endothelial cells was reduced by 30% during the early follicular phase, which is characterized by low estrogen compared with the luteal phase with high estrogen. The ERα expression in postmenopausal women was reduced by 33% when compared with expression levels in premenopausal women in the luteal phase of the menstrual cycle. Furthermore, a strong positive correlation was observed between ERα expression and endothelial-dependent dilation as well as ERα abundance and eNOS levels and activity. ²⁶ Furthermore, polymorphisms in genes may affect receptor expression. SNPs in ERs, in combination with reduced estradiol levels in postmenopausal women, may impair normal hormonal compensatory mechanisms and potentially diminish the vascular protective effects of estradiol. ²⁷

The formation and rupture of cerebral aneurysms is complex with multifactorial etiology. Our study suggests that postmenopausal decline in estrogen may play a role in the etiopathogenesis of this disease via NO pathway dysfunction, which contributes to a vulnerable vascular environment that facilitates initiation, promotes growth, and ultimately increases the risk of rupture. However, this is only a correlation study, and the cellular or molecular mechanisms mediated by estradiol have to be elucidated using animal models and cell lines as the action of estradiol in regulating transcription of target genes is tissue- and temporal-specific. Further, the precise molecular and cellular mechanism behind the vascular protective effects of estradiol needs to be established experimentally. Human specimens have great heterogeneity as the genetic backgrounds and the gene–environment interaction leading to disease pathology varies. The limitation of our work was that we were unable to do any protein studies in the tissues as the surgically excised human IA tissue samples obtained were small and limited. However, we had analyzed the expression of numerous other mRNAs and identified several that were either downregulated (antioxidant enzymes genes) or upregulated (genes involved in inflammation, vascular remodeling, and oxidative stress) in IA tissues compared with control tissues. Further studies with imaging of control subjects to rule out unruptured aneurysms would strengthen the conclusions. This specific limitation of our study and its impact on interpretation need to be addressed in future studies.

In conclusion, we speculate that the sudden and sharp decline in estradiol during menopause, which had conferred protection against vascular diseases earlier in the life, may increase the risk of aSAH incidence in women. Hence, estradiol HRT may prove to be an effective strategy for preventing IA formation or rupture and reduce the burden of aSAH in postmenopausal women. However, precise knowledge on timing/dosage/route of administration, duration, and side effects are necessary.

Funding Statement

Funding This work was supported by the Department of Science and Technology (Project No.SR/WOS-A/LS-656/2016), New Delhi, India.