Endothelial Nitric Oxide Synthase Polymorphism Is Associated with Delayed Cerebral Ischemia Following Aneurysmal Subarachnoid Hemorrhage

doi:10.1016/j.wneu.2017.02.062

World Neurosurgery

Volume 101, May 2017, Pages 514-519

https://doi.org/10.1016/j.wneu.2017.02.062 Get rights and content

Background and Purpose

Nitric oxide is critical in the regulation of cerebral blood flow and smooth muscle proliferation. It is synthesized by 3 nitric oxide synthase (NOS) isoforms: neuronal, inducible, and endothelial NOS (eNOS). Aneurysmal subarachnoid hemorrhage (aSAH) causes endothelial dysfunction that, in turn, contributes to pathophysiologic processes surrounding aSAH. Previous studies reported an association of an eNOS single nucleotide polymorphism (SNP) with the clinical sequelae of aSAH. Here, we further elucidate the impact of this eNOS SNP on the clinical course after aSAH.

Methods

The Cerebral Aneurysm Renin Angiotensin System study prospectively enrolled aSAH patients at 2 academic institutions in the United States from 2012–2015. Blood samples from all patients enrolled in the study were used for genetic evaluation using 5′exonuclease (Taqman) genotyping assays. Associations between the eNOS SNP rs2070744 (786 T->C) and clinical course after aSAH were analyzed.

Results

Samples from 149 aSAH patients were available for analysis. The C allele of the eNOS SNP independently predicted an increased risk for delayed cerebral ischemia (OR = 2.936, 95% CI 1.048–8.226, P = 0.040). The eNOS SNP rs2070744 was not associated with functional outcome or size of aneurysm at the time of rupture.

Conclusions

The present study is the first to demonstrate that the C allele of the eNOS SNP 786 T->C rs2070744 is independently associated with an increased risk for delayed cerebral ischemia following aSAH.

Introduction

Delayed cerebral ischemia (DCI) is associated with poor outcome and mortality following aneurysmal subarachnoid hemorrhage (aSAH).¹ Genetic, molecular, and a number of clinical factors have been linked to DCI, emphasizing its multifactorial pathophysiologic mechanism involving both macrovasculature and microvasculature.2, 3, 4, 5 Cortical spreading ischemia results in microvascular spasms.⁶ Endothelial damage, proinflammatory processes, and impairment of the fibrinolytic cascade after aSAH contribute to cerebral microthrombembolism.⁷ Large-vessel cerebral vasospasm reduces cerebral blood flow and increases the risk for cerebral infarction.⁸ However, DCI may occur in the absence of radiographic vasospasm.9, 10, 11 Conversely, even severe radiographic vasospasm does not inevitably result in DCI.¹²

Nitric oxide (NO) serves as a key molecule for maintenance of cerebral blood flow and regulation of platelet aggregation, leukocyte adhesion and migration, and smooth muscle proliferation.13, 14 The gaseous molecule is synthesized from L-arginine by 1 of the 3 nitric oxide synthase (NOS) isoforms: neuronal (nNOS), inducible (iNOS), and endothelial NOS (eNOS). Following aSAH, endothelial dysfunction and relative NO depletion have been observed and may contribute to the development of DCI. Genetic polymorphisms of the gene encoding eNOS may alter NO synthesis, thus having an impact on the clinical course after aSAH.13, 14

Section snippets

Methods

The Cerebral Aneurysm Renin Angiotensin System study prospectively enrolled aSAH patients at 2 academic institutions in the United States from 2012–2015.3, 4, 15, 16, 17, 18 Institutional review board approval was obtained at both institutions, and written informed consent was obtained from participants or their proxy.

Blood samples were collected within 72 hours of admission and used for genetic evaluation. The eNOS 786 T->C single nucleotide polymorphism (SNP) rs2070744 (Table 1) was detected

Patient Characteristics

Blood samples from 149 aSAH patients were analyzed. Patient characteristics are shown in Table 2. The majority of ruptured aneurysms was less than 7 mm in maximum diameter (62.4%) and located in the anterior circulation (80.5%). Hunt & Hess 1–3 and modified Fisher CT 1–2 made up 77.2% and 49.2%, respectively. Clinical vasospasm and DCI occurred in 22.8% and 21.2%, respectively. Of 31 patients with DCI, 80.6% also experienced clinical vasospasm. A total of 34 patients had clinical vasospasm, and

Discussion

There were 3 key findings of the present study: (1) the C allele of the eNOS SNP 786 T->C rs2070744 was independently associated with an increased risk for DCI; (2) size of the ruptured aneurysm size did not correlate with eNOS SNP rs2070744; (3) functional outcome at discharge and last follow-up were not linked to the eNOS SNP rs2070744 genotype.

Conclusion

The C allele of the eNOS SNP 786 T->C rs2070744 was independently associated with an increased risk for DCI in the present study. Pathophysiologically, the C allele is thought to result in decreased eNOS activity and lower NO levels in the cerebral circulation. There was no correlation with ruptured aneurysm size or functional outcome.

Acknowledgments

We would like to thank the participants in this study and the efforts of the neurosurgical research coordinators at Inova Health System for their work and contribution to the Cerebral Aneurysm Renin Angiotensin System Study.

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Enhancing S-nitrosoglutathione reductase decreases S-nitrosylation of Drp1 and reduces neuronal apoptosis in experimental subarachnoid hemorrhage both in vivo and in vitro
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Our research confirms that 12 h after SAH occurs, the NO level in the brain tissue is significantly increased. The high NO level at this stage is related to the increased expression of NOS, especially iNOS and nNOS (Lan et al., 2020; Hendrix et al., 2017). Excessive NO is converted into GSNO to S-nitrosylate intracellular proteins, affecting protein localization, stability, and protein-protein interactions (Kolár and Nohejlová, 2014; Pirie et al., 2021).
Subarachnoid hemorrhage (SAH) is a hemorrhagic stroke with a high mortality and disability rate. Nitric oxide (NO) can promote blood supply through vasodilation, leading to protein S-nitrosylation. However, the function of S-nitrosylation in neurons after SAH remains unclear. Excessive NO in the pathological state is converted into S-nitrosoglutathione (GSNO) and stored in cells, which leads to high S-nitrosylation of intracellular proteins and causes nitrosative stress. S-nitrosoglutathione reductase (GSNOR) promotes GSNO degradation and protects cells from excessive S-nitrosylation. We conducted an in vivo rat carotid puncture model and an in vitro neuron hemoglobin intervention. The results showed that SAH induction increased NO, GSNO, neuron protein S-nitrosylation, and neuronal apoptosis, while decreasing the level and activity of GSNOR. GSNOR overexpression by lentivirus decreased GSNO but had little effect on NO. GSNOR overexpression also improved short- and long-term neurobehavioral outcomes in rats and alleviated nitrosative stress. Furthermore, GSNOR reduced neuronal apoptosis and played a neuroprotective role by alleviating Drp1 S-nitrosylation, reducing mitochondrial division. Thus, the regulation of GSNOR in early brain injury and neuronal denitrosylation may play an important role in neuroprotection.
A Gene Map of Brain Injury Disorders
2021, The Molecular Immunology of Neurological Diseases
Traumatic brain injury (TBI) is a serious threat worldwide affecting the normal lives of people of every age, including children. It causes high rates of death and morbidity and affects males more than females. Majority of the less severe brain injuries are left undiagnosed due to complex multifactorial pathophysiology. It is now clear that various genetic factors influence the outcome of posttraumatic brain damage. The identification of the genes responsible for pathological outcomes post TBI can help in devising a therapeutic strategy for the management of brain injury disorders. These differentially expressed genes (DEGs) also have the potential as molecular markers having higher reliability in the diagnosis. However, we have limited knowledge of the genes that influence the outcome of brain injury. Coordinated efforts by various research groups across the globe with the help of genome-wide association studies (GWASs) have made possible the identification of genes and associated single nucleotide polymorphisms (SNPs) underlying the outcome of TBI. Nevertheless, it has to be taken into consideration that most of the studies related to brain injury genetics are “candidate gene association studies,” which are prone to fail in replicating the results during independent cohorts. Besides, various environmental factors in association with the genetic factors have to be taken into account to assess the progression and outcome of post brain injury diseases.
Nomogram for predicting delayed cerebral ischemia after aneurysmal subarachnoid hemorrhage in the Chinese population
2020, Journal of Stroke and Cerebrovascular Diseases
Citation Excerpt :
In a prospective clinical study of 504 patients with aSAH, researchers11 determined that female sex, history of diabetes mellitus, and poor clinical grade are independent, early risk factors for DCI. Although some gene signatures have been associated with increased risk of DCI, their clinical applicability in the preoperative risk estimation of DCI remains uncertain.12,13 Accurate preoperative estimation of DCI can help surgeons to select the optimal surgical procedures, enable timely imaging examinations, and administer appropriate drug treatment for patients based on a risk-benefit assessment.
Delayed cerebral ischemia is a serious complication of aneurysmal subarachnoid hemorrhage with debilitating and fatal consequences. Lack of well-established risk factors impedes early identification of high-risk patients with delayed cerebral ischemia. A nomogram provides personalized, evidence-based, and accurate risk estimation. To offset the lack of a predictive tool, we developed a nomogram to predict delayed cerebral ischemia before performing surgical interventions for aneurysmal subarachnoid hemorrhage to aid surgical decision-making.
We retrospectively collected data from 887 consecutive eligible Chinese patients who underwent surgical clipping or endovascular coiling for aneurysmal subarachnoid hemorrhage. Patients who previously underwent surgery formed the training cohort (n = 621) for nomogram development; those who underwent surgery later formed the validation cohort (n = 266) to confirm the performance of the model. A multivariate logistic regression analysis identified the independent risk factors associated with delayed cerebral ischemia, which were then incorporated into the nomogram.
Delayed cerebral ischemia was identified in 158/621 patients (25.4%) in the training cohort and in 66/266 patients (24.8%) in the validation cohort. Preoperative factors associated with delayed cerebral ischemia were age > 65 years, modified Fisher grade of 3–4, ruptured aneurysm in the anterior circulation, Hunt–Hess grade of 4–5, high blood pressure on admission, and plasma homocysteine level ≥ 10 μmol/L. Incorporating these six factors in the nomogram achieved efficient concordance indices of 0.73 (95% confidence interval, 0.68–0.77) and 0.65 (95% confidence interval, 0.57–0.72) in predicting delayed cerebral ischemia in the training and validation cohorts, respectively.
Our model can help determine an individual's risk of developing delayed cerebral ischemia in the Chinese population, and thereby, facilitate reasonable treatment-related decision-making.
Correlation Between Altered DNA Methylation of Intergenic Regions of ITPR3 and Development of Delayed Cerebral Ischemia in Patients with Subarachnoid Hemorrhage
2019, World Neurosurgery
Citation Excerpt :
In a meta-analysis of 18 studies, Hu et al.2 found an elevated risk of DCI in patients with SAH with the apolipoprotein E genotype. Hendrix et al.31 showed that the C allele of the endothelial nitric oxide synthase SNP was significantly associated with the occurrence of DCI (OR, 2.936; 95% CI, 1.048–8.226). Using genome-wide association studies, Kim et al.4 reported that rs999662 reached genome-wide significance (P = 3.39 × 10–8) for cerebral vasospasm as per the TCD velocity.
Delayed cerebral ischemia (DCI) is related to the major causes of morbidity and mortality in patients following subarachnoid hemorrhage (SAH); however, little is known about the role of epigenetics in the pathogenesis of DCI. We investigated the specific DNA methylation profile that may affect the expression of inositol 1-,4-,5-trisphosphate receptor (ITPR3) responsible for cerebral vasospasm following SAH.
We prospectively studied patients with SAH between March 2015 and October 2018. The degree of methylation in the distal intergenic region (IGR) located on ITPR3 and gene expression were measured using methylation-specific polymerase chain reaction (MSP) and quantitative real-time polymerase chain reaction (qPCR). To investigate the regulatory mechanims of DNA hypermethylation, we further analyzed the mRNA expression of DNA methyltransferase (DNMT1) and ten-eleven translocation enzymes (TET1, TET2, and TET3).
A total of 42 patients were included in our analysis. Patients with SAH and DCI had significantly higher levels of methylation intensity of distal IGR upstream of ITPR3 than those without DCI (median, 0.941 [interquartile range (IQR), 0.857–0.984] versus (0.670 [IQR, 0.543–0.761]; P < 0.001). In addition, patients with DCI showed decreased mRNA expression of ITPR3 compared with patients without DCI (median, 0.039 [IQR, 0.030–0.045] vs. 0.047 [IQR, 0.038–0.064]; P = 0.0328). Patients with DCI had higher DNMT1 expression (P < 0.001) and lower TET1 expression (P = 0.040) than those without DCI; however, differences in TET2 and TET3 levels between the 2 groups were not statistically significant.
Hypermethylation of the distal IGR located upstream of ITPR3 is related to greater DCI development in patients with SAH. Further studies of the precise mechanisms of methylation degree and DCI development using in vitro and in vivo models are needed.
The role of endothelial nitric oxide synthase −786 T/C polymorphism in cardiac instability following aneurysmal subarachnoid hemorrhage
2017, Nitric Oxide - Biology and Chemistry
Citation Excerpt :
In a prior publication, our group has already demonstrated that the C allele of the eNOS SNP -786 T/C was independently associated with an increased risk for cerebral infarctions following aSAH. An association of a specific genotype with an unfavorable outcome at discharge or follow-up has not been observed [39]. The C allele of the eNOS SNP -786 T/C has been associated with decreased NO synthesis subsequently resulting in an increased risk for coronary artery spasms [8–13].
Cardiac abnormalities are observed frequently after aneurysmal subarachnoid hemorrhage (aSAH). A subset of aSAH patients develops neurogenic cardiomyopathy, likely induced by catecholamine excess. Genetic polymorphisms of the endothelial nitric oxide synthase (eNOS) gene have been linked to decreased nitric oxide (NO) levels, coronary artery spasm, and myocardial infarction. The role of the eNOS single nucleotide polymorphism (SNP) −786 T/C in cardiac instability following aSAH has not been previously investigated.
From 2012 to 2015, aSAH patients were prospectively enrolled in the Cerebral Aneurysm Renin Angiotensin System (CARAS) study at two academic institutions. Blood samples were used to assess the eNOS SNP -786 T/C rs2070744 through 5'exonuclease (Taqman) genotyping assays. Associations between this polymorphism and cardiac instability following aSAH were analyzed.
Multivariable analysis demonstrated a dominant effect of the C allele of eNOS SNP -786 T/C on cardiac instability in patients with aSAH. A lower Glasgow Coma Scale score and a history of ischemic vascular disease were also associated with cardiac instability. Furthermore, cardiac instability independently predicted poor functional outcome upon discharge from the hospital.
The C allele of the eNOS SNP -786 T/C was independently associated with an increased risk for cardiac instability following aSAH. Cardiac instability itself was a risk factor for an unfavorable functional outcome upon discharge from the hospital.
Association of Plasminogen Activator Inhibitor 1 (SERPINE1) Polymorphisms and Aneurysmal Subarachnoid Hemorrhage
2017, World Neurosurgery
Citation Excerpt :
Institutional review board approval was obtained at both institutions, and written informed consent was obtained from participants or their proxy. The study design has been reported in detail in prior publications.12-17 In short, blood samples obtained within 72 hours of admission were used to evaluate 6 common SERPINE1 single nucleotide polymorphisms (SNPs) via 5′ exonuclease (Taqman) genotyping assays (Table 1).
Genetic variations of the serine proteinase inhibitor family E member 1 (SERPINE1) gene, which encodes plasminogen activator inhibitor 1, correlate with serum levels of its product and are associated with thrombophilia and coronary atherosclerosis. Various SERPINE1 ;gene polymorphisms have been identified. However, only the functional 5G/4G polymorphism has been assessed in the context of aneurysmal subarachnoid hemorrhage (aSAH). We assessed associations of 6 SERPINE1 polymorphisms with the clinical sequelae of aSAH.
From 2012 to 2015, patients with aSAH were prospectively enrolled into the CARAS (Cerebral Aneurysm Renin Angiotensin System) study at 2 major academic institutions. Blood samples were used to evaluate 6 common SERPINE1 single nucleotide polymorphisms via 5′ exonuclease (Taqman) genotyping assays.
There was an association of the AA genotype of rs2227631 with the 4G/4G genotype and of the GG genotype of rs7242 with the AA genotype of rs2227684. In multivariable analysis, patients with the AA genotype of rs2227631 and 4G/4G genotype had an increased risk for developing delayed cerebral ischemia. Patients with the GG genotype of rs7242 and AA genotype of rs2227684 had a decreased risk for a poor functional outcome.
SERPINE1 gene polymorphisms were associated with delayed cerebral ischemia and functional outcome after aSAH. These associations may arise from alterations of plasminogen activator inhibitor 1 levels.

View all citing articles on Scopus

: Conflict of interest statement: We would like to thank the Department of Anesthesiology at the University of Alabama at Birmingham, the Brain Aneurysm Foundation, and family of Timothy P. Susco for their generous support of the present study.

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Original ArticleEndothelial Nitric Oxide Synthase Polymorphism Is Associated with Delayed Cerebral Ischemia Following Aneurysmal Subarachnoid Hemorrhage

Background and Purpose

Methods

Results

Conclusions

Introduction

Section snippets

Methods

Patient Characteristics

Discussion

Conclusion

Acknowledgments

Exp Neurol

Clin Neurol Neurosurg

World Neurosurg

Neurosci Lett

Am J Cardiol

Am J Cardiol

Prediction of delayed cerebral ischemia, rebleeding, and outcome after aneurysmal subarachnoid hemorrhage

Stroke

Delayed cerebral ischemia after subarachnoid hemorrhage: a systematic review of clinical, laboratory, and radiological predictors

Stroke

Association of nosocomial infections with delayed cerebral ischemia in aneurysmal subarachnoid hemorrhage

J Neurosurg

Associations of renin-angiotensin system genetic polymorphisms and clinical course after aneurysmal subarachnoid hemorrhage

J Neurosurg

Aneurysmal subarachnoid hemorrhage. Complications and outcome in a hospital population

Stroke

Cortical spreading ischaemia is a novel process involved in ischaemic damage in patients with aneurysmal subarachnoid haemorrhage

Brain

Microthrombosis after aneurysmal subarachnoid hemorrhage: an additional explanation for delayed cerebral ischemia

J Cereb Blood Flow Metab

The relationship between delayed infarcts and angiographic vasospasm after aneurysmal subarachnoid hemorrhage

Neurosurgery

Relationship between vasospasm, cerebral perfusion, and delayed cerebral ischemia after aneurysmal subarachnoid hemorrhage

Neuroradiology

Diagnosing delayed cerebral ischemia with different CT modalities in patients with subarachnoid hemorrhage with clinical deterioration

Stroke

Predictors of cerebral infarction in aneurysmal subarachnoid hemorrhage

Stroke

Definition of delayed cerebral ischemia after aneurysmal subarachnoid hemorrhage as an outcome event in clinical trials and observational studies: proposal of a multidisciplinary research group

Stroke

Original Article
Endothelial Nitric Oxide Synthase Polymorphism Is Associated with Delayed Cerebral Ischemia Following Aneurysmal Subarachnoid Hemorrhage