Impact of High-Mobility Group Box 1 Polymorphism on Delayed Cerebral Ischemia After Aneurysmal Subarachnoid Hemorrhage

Accepted Manuscript Impact of high-mobility group box-1 polymorphism on delayed cerebral ischemia following aneurysmal subarachnoid hemorrhage Philipp Hendrix, MD, Paul M. Foreman, MD, Mark R. Harrigan, MD, Winfield S. rd Fisher, 3 , MD, Nilesh A. Vyas, MD, Robert H. Lipsky, PhD, Minkuan Lin, PhD, Beverly C. Walters, MD, MSc, R. Shane Tubbs, PhD, Mohammadali M. Shoja, MD, Jean-Francois Pittet, MD, Mali Mathru, MD, Christoph J. Griessenauer, MD PII:

S1878-8750(17)30147-X

DOI:

10.1016/j.wneu.2017.01.121

Reference:

WNEU 5218

To appear in:

World Neurosurgery

Received Date: 23 December 2016 Revised Date:

29 January 2017

Accepted Date: 30 January 2017

Please cite this article as: Hendrix P, Foreman PM, Harrigan MR, Fisher 3rd WS, Vyas NA, Lipsky RH, Lin M, Walters BC, Tubbs RS, Shoja MM, Pittet J-F, Mathru M, Griessenauer CJ, Impact of highmobility group box-1 polymorphism on delayed cerebral ischemia following aneurysmal subarachnoid hemorrhage, World Neurosurgery (2017), doi: 10.1016/j.wneu.2017.01.121. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Impact of high-mobility group box-1 polymorphism on delayed cerebral ischemia following aneurysmal subarachnoid hemorrhage Philipp Hendrix MD1, Paul M. Foreman MD2, Mark R. Harrigan MD2, Winfield S.

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Fisher 3rd MD2, Nilesh A. Vyas MD3, Robert H. Lipsky PhD3,4, Minkuan Lin PhD4,

Beverly C. Walters MD, MSc2,3,4, R. Shane Tubbs, PhD5, Mohammadali M. Shoja MD6, Jean-Francois Pittet MD7, Mali Mathru MD7, Christoph J. Griessenauer MD8,9

Department of Neurosurgery, Saarland University Medical Center and Saarland

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University Faculty of Medicine, Homburg/Saar, Germany Department of Neurosurgery, University of Alabama at Birmingham, AL 3

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Department of Neurosciences, Inova Health System, Falls Church, VA

Department of Molecular Neuroscience, George Mason University, Fairfax, VA 5

Seattle Science Foundation, Seattle, WA

Neuroscience Research Center, Tabriz University of Medical Sciences, Tabriz, Iran 7

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Department of Anesthesiology, University of Alabama at Birmingham, AL

Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA Department of Neurosurgery, Geisinger Health System, Danville, PA

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Running head: High-mobility group box-1 polymorphism and subarachnoid hemorrhage Key words: high-mobility group box-1, polymorphism, aneurysm, subarachnoid hemorrhage, outcome, delayed cerebral ischemia

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Corresponding author: Philipp Hendrix, MD Klinik für Neurochirurgie

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Universität des Saarlandes Kirrberger Straße 100, Gebäude 90.5 66421 Homburg/ Saar, Germany

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Phone: +49-6841-1624400

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[email protected]

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Fax: +49-6841-1624480

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Abstract

Background and Purpose

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The high-mobility group box-1 protein (HMGB1) is an eukaryotic, ubiquitously expressed protein that serves as a biomarker for various diseases and is involved in the promotion of a proinflammatory response to cell injury. In aneurysmal subarachnoid

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hemorrhage (aSAH), elevated HMGB1 levels have been linked to poor outcome and an increased risk for cerebral vasospasm. The role of HMGB1 polymorphisms in aSAH has

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not been previously investigated.

Methods

Aneurysmal subarachnoid hemorrhage patients and controls enrolled in the prospective, two-center CARAS (Cerebral Aneurysm Renin Angiotensin System) study were

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evaluated. The 3814 C/G HMGB1 single nucleotide polymorphism (SNP) rs2249825 was detected using 5’exonuclease (Taqman) genotyping assays from blood samples from

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aSAH patients and controls. Associations between aSAH and its clinical sequelae with

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the HMGB1 SNP were assessed.

Results

Samples from 149 aSAH patients and 50 controls were available for analysis. No increased risk for aSAH associated with the SNP was found compared to the control group. Delayed cerebral ischemia (DCI) was defined as a cerebral infarction at the time of discharge from the ICU and identified in 21.2% of aSAH patients. In multivariable

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logistic regression analysis, the G allele of rs2249825 was independently associated with DCI (OR = 5.695, 95% CI 1.804 – 17.975, p = 0.003).

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Conclusions

The minor allele G of rs2249825 was associated with an increased risk for DCI, or cerebral infarction, following aSAH. This may be attributable to an increased HMGB1

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protein expression in these patients.

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Introduction

High-mobility group box-1 protein (HMGB1) is expressed in eukaryotic cells and was

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originally described as a non-histone protein interacting with deoxyribonucleic acid (DNA). It stabilizes nucleosome formation and is involved in regulation of gene expression 1,2. Severe stress or cell death lead to passive release of HMGB1 by eukaryotic

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cells 3. During apoptosis, however, HMGB1 undergoes modification preventing leakage of the naïve protein into the extracellular space 4. Proinflammatory cells like monocytes,

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macrophages, and dendritic cells actively secret HMGB1 in the extracellular space 5. There, HMGB1 recruits inflammatory cells to the site of injury and promotes proinflammatory cytokine and chemokine production

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. The HMGB1 has been

identified as a biomarker for various diseases including autoimmune disorders, cancer, 3,5,6

. In the central nervous system, HMGB1 induces astrocyte

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trauma, and infection

activation to a proinflammatory status that releases chemokines attracting monocytes 7. The HMGB1 has been shown to be elevated in cerebrospinal fluid (CSF) and plasma of

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patients with aSAH and release of HMGB1 in the acute phase of aSAH may be related to its clinical sequelae including cerebral vasospasm and delayed cerebral ischemia 8–10. The

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role of the 3814 C/G HMGB1 single nucleotide polymorphism (SNP) rs2249825 on aSAH and its clinical sequelae has not been previously investigated.

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Methods

Aneurysmal subarachnoid hemorrhage patients and controls from the CARAS

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(Cerebral Aneurysm Renin Angiotensin System) study were included. Briefly, this was a prospective, two-center study of genetic polymorphisms that enrolled patients in the United States from 2012 to 2015. Institutional review board approval was obtained at

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both participating centers and included subjects and controls were required to give informed written consent. Blood samples from the CARAS study

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were used to

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detect the 3814 C/G HMGB1 single nucleotide polymorphism (SNP) rs2249825 using an 5’exonuclease (Taqman) genotyping assay according to the vendor (Thermo Fisher Scientific Inc., Cambridge, MA) (Table 1). Approximately 10% of the DNA samples were randomly selected to test reproducibility of Taqman assays. All of the replication

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samples produced concordant genotypes.

Patients suffering from aneurysmal subarachnoid hemorrhage were treated in accordance 15.

The control group contained trauma

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with guidelines for the management of aSAH

patients, age ≥ 19 years, with unremarkable CTAs of head and neck (no cerebral

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aneurysm or other vascular malformation) and without known genetic risk factors for cerebral aneurysm formation. Eligibility for CARAS study required enrollment of both aSAH patients and controls within 72 hours of admission.

Definition of clinical vasospasm and delayed cerebral ischemia

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Clinical vasospasm was defined as a new focal or global neurological deficit, or deterioration of at least 2 points on the Glasgow Coma Scale not owing to another clinical process covering hydrocephalus, aneurysm rebleeding, electrolyte disturbance,

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seizure, infection, fever, metabolic disturbance, cerebral edema, or surgical complication. The diagnosis of clinical vasospasm was adjudicated by consensus of the study team after consideration of corroborating evidence from neurovascular imaging and treated with 16.

Hyperdynamic therapy aimed for a goal systolic

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hyperdynamic therapy as first line

blood pressure > 160 mmHg, accomplished with either permissive hypertension or

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vasopressor therapy. Medical refractory clinical vasospasm was treated in the endovascular suite at the discretion of the treating neurosurgeon. Delayed cerebral ischemia (DCI) was delineated as low-density areas on computed tomography (CT) that corresponded to a vascular distribution or a magnetic resonance

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imaging (MRI) demonstrating a hyperintense area on diffusion weighted image sequence with a corresponding hypointense apparent diffusion coefficient sequence correlate that corresponds with a vascular territory. Computed tomography scans or MRI were

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performed in all patients upon transfer from the ICU to the ward. Infarctions or contusion seen on postoperative day 1 imaging were considered procedurally related and were not

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considered DCI.

Functional outcome assessment

Using the modified Rankin scale (mRS), functional outcome was recorded at the time of discharge from the acute hospital setting, and at last follow-up. Scores 3 – 6 were

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designated unfavorable outcome. All outcome data were obtained blinded to the results of the genetic analysis. Functional outcome was assessed either in clinic or via telephone

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interview with the patient or with a surrogate if the patient was unable to participate 17.

Statistical analysis

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Statistical analysis was performed using the software package SPSS (SPSS version 23; SPSS, Inc., Chicago, IL). Continuous variables are presented as mean ± standard

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deviation and categorical variables are presented as frequency and percent. Wilcoxon rank sum, Student’s t-test, Chi-square, and Fisher’s exact tests were performed as appropriate. Characteristics from all patients and the HMGB1 SNP polymorphism were tested in univariable analysis to determine predictors of aSAH. In patients suffering from

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aSAH, aneurysm characteristics and HMGB1 polymorphism were tested in univariable analysis to determine predictors of the following dependent variables: clinical vasospasm, DCI, unfavorable functional outcome (mRS 3-6) at discharge and upon follow-up.

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Factors predictive in univariable analysis (p < 0.15) were entered into a multivariable

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logistic regression analysis. P-values of ≤ 0.05 were considered statistically significant.

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Results

Patient and control characteristics

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Mean age of aSAH patients and controls was 54.9±12.5 and 50.6±18.6 years, respectively (p = 0.07). There was a higher proportion of African-Americans in the aSAH group as compared to the control group (p = 0.003). History of hypertension (p = 0.013)

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and family history of cerebral aneurysms (p = 0.025) were more frequent in the aSAH group. Sex and rate of current or former smokers was evenly distributed between the two

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groups. Following aSAH, clinical vasospasm and DCI were diagnosed in 22.8% and 21.2% of patients, respectively. A favorable functional outcome (mRS 0-2) at discharge from hospital and last follow-up was accomplished in 46.7% and 61.7% of patients,

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respectively (Table 2).

Association of the HMGB1 polymorphism and aneurysmal subarachnoid hemorrhage

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The 3814 C/G HMGB1 SNP rs2249825 was in Hardy-Weinberg equilibrium in aSAH patients and controls. Multivariable analysis did not reveal an association between aSAH

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and rs2249825 (p = 0.129) (Table 3).

Associations of the HMGB1 polymorphism and the clinical course after aneurysmal subarachnoid hemorrhage

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In multivariable logistic regression analysis, there was a dominant effect of the G allele of rs2249825 on DCI (OR = 5.695, 95% CI 1.804 – 17.975, p = 0.003) when controlling for Hunt and Hess grade (OR = 1.805, 95% CI 1.132 – 2.880, p = 0.013), rerupture (OR =

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14.933, 95% CI 1.495 – 149.125, p = 0.021), cerebral edema (OR = 6.195, 95% CI 1.995 – 19.233, p = 0.002), and ventriculitis (OR = 38.826, 95% CI 2.931 – 514.387, p = 0.006) (Table 4). There was no association with clinical vasospasm, functional outcome at

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discharge or at follow-up in multivariable logistic regression analysis.

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Discussion

The present study is the first to demonstrate that the G allele of 3814 C/G HMGB1 SNP

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rs2249825 was associated with development of DCI, or cerebral infarction, after aSAH and represents an important step in elucidating the role of HMGB1 in aSAH. The presence of the G allele results in a new binding site for v-myb. V-myb is a retroviral

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oncogene that interacts with the mim-1 promoter and activates the mim-1 enhancer which

Animal models of aSAH and HMGB1

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enhances the expression of HMGB1 18–21.

In a rat model, there was a significant increase in neuronal HMBG1 expression as evidenced by increased mRNA and protein levels within 2 hours of aSAH. The protein

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was translocated from the nucleus to the cytosol. Subsequently both active and passive release of HMGB1 into the extracellular space promoted brain injury 22. The increase of HMGB1 expression appears to be sustained for several days. Murakami et al. showed a

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significant increase of HMGB1 expression in the microglia as late as day 5 following induction of aSAH in a rabbit model

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Zhao et al. also investigated the HMGB1

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expression in a rat aSAH model and found an increased expression of HMGB1 within the walls of the basilar artery, particularly on day 3 and day 5 after aSAH. This correlated with histological thickening of the media of the basilar artery and the reduction its lumen. The authors postulated that HMGB1 may be involved in the development of cerebral vasospasm 24.

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Aneurysmal subarachnoid hemorrhage and HMGB1 in humans Patients suffering from an acute ischemic stroke display elevated HMGB1 plasma levels 25.

Plasma HMGB1 levels have been shown to significantly correlate with functional 10.

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outcome, in hospital mortality, one-year-mortality, and cerebral vasospasm in aSAH

Nakahara et al. found that along with other chemokines, HMGB1 levels were markedly elevated in the CSF of patients suffering from aSAH with unfavorable outcome compared

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to favorable outcome 9. King et al. made similar observations. In their cohort, HMGB1 was exclusively detected in the CSF of patients suffering from aSAH when comparing to

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CSF from a control cohort suffering from normal pressure hydrocephalus. HMGB1 levels correlated with neurological outcome and functional outcome, but not with the extent of subarachnoid blood on CT 8. Release of HMGB1 in the acute phase of aSAH may be

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related to development of its clinical sequelae such as DCI 8.

Genetic and pathopysiologic mechanisms underlying DCI are poorly understood. Proinflammatory processes, microthrombembolism

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cortical spreading depolarization

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microvascular spasm owing to

and large-vessel cerebral vasospasm

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are involved

in the complex multifactorial pathogenesis of DCI. Aneurysmal subarachnoid

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hemorrhage is associated with an inflammatory response that appears to be at least partially mediated by HMGB1. There is evidence that elevated HMGB1 levels are associated with an increased risk for cerebral vasospasm

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Although we did not

observe an increased risk for clinical vasospasm in the present study, we demonstrated that the G allele of rs2249825 was associated with almost 6-times greater odds for DCI following aSAH. The dissociation of clinical vasospasm and DCI may be explained as

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following. Delayed cerebral ischemia was purely a radiographic diagnosis; a cerebral infarction at the time of discharge from the ICU. Clinical vasospasm, however, may not necessarily result in a cerebral infarction if it is detected early and treated aggressively

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with hyperdynamic therapy and endovascular interventions. Therefore, the HMGB1 SNP potentially enhances HMGB1 expression that is involved in the promotion of proinflammatory processes and spasms of the cerebral vasculature ultimately increasing

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Future directions

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the risk for cerebral infarctions.

The receptors for advance glycation end products and toll-like receptors bind HMGB1 and promote downstream signal transduction 2. Inhibition of downstream signaling via HMGB1-antibodies and molecules interacting with receptor-ligand interaction have been

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studied. They effectively neutralize or attenuate experimentally induced deleterious effects like septic shock, inflammation, tumor growth, or autoimmune arthritis 2,3,5. With further elucidation of the clinical significance of HMGB1, mechanisms modulating

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HMGB1 signaling might represent an attractive model to establish new treatments for

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patients suffering from aSAH.

Limitations

The study did not assess whether the HMGB1 polymorphism altered transcription or the biological character of the proteins encoded and HMGB1 plasma or CSF levels were not assessed. Another limitation of the study is the sample size. Larger samples provide more accurate data in association studies. Narrowing of the cerebral vasculature is frequently

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seen on angiographic imaging within the first days following aSAH. The study did not systematically screen for radiographic vasospasm, but focused on clinical vasospasm that subsequently was confirmed by radiological imaging. We did not observe an association

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of clinical vasospasm with HMGB1 SNP. However, we cannot exclude that clinicallysilent radiographic vasospasm was more frequent than clinical vasospasm. Therefore, we

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cannot rule out HMGB1 SNP to be associated with radiographic vasospasm.

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Conclusions

Presence of the minor allele G of rs2249825 is an independent predictor for DCI

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following aSAH. This may be attributable to an increased HMGB1 protein expression in

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these patients.

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Disclosures

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None.

Acknowledgements

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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

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CARAS Study.

Sources of Funding

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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

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Figure legend

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their generous support of the present study.

Figure 1. Proposed mechanism of HMGB1 in aneurysmal subarachnoid hemorrhage. The minor allele G of rs2249825 may result in increased HMGB1 expression predisposing to delayed cerebral ischemia (*).

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Table 1. HMG polymorphism Alleles

Minor allele frequency rs2249825 (3814C->G) C/G MAF 0.15 (G)* * http://useast.ensembl.org/Homo_sapiens/Info/Index?redirect=no

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HMG polymorphism

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Table 2. Patient and control characteristics Controls N=50 50.6±18.6

P value

85 (57.0%) 60 (40.3%) 4 (2.7%)

41 (82%) 7 (14%) 2 (4%)

0.003

35 (23.5%) 114 (76.5%) 13 (8.6%) 90 (60.4%)

18 (36%) 32 (64%) 8 (16%) 20 (40%)

132 (88.6%) 14 (9.4%) 3 (2.0%)

39 (78.0%) 11 (22.0%) 0

0.044

58 (38.9%) 18 (12.1%) 73 (49%) 14 (9.4%) 0.9 ± 2.7

21 (42%) 9 (18%) 20 (40%) 0 (0%)

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0.07

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Age Race Caucasian African American Other Gender Male Female Ischemic vascular disease Hypertension Antithrombotics None Antiplatelets Anticoagulation Smoking Never Former Current Family history Days for symptom onset to admission (days, mean ± SD) Aneurysm size <7 mm 7–12 mm 13–24 mm ≥25 mm Location Anterior Posterior Hunt & Hess grade 1-3 4-5 Fisher CT grade 1-2 3-4 Modified Fisher CT grade1 1-2 3-4 Hijdra scale1 0 1

aSAH N=149 54.9±12.5

120 (80.5%) 29 (19.5%)

115 (77.2%) 34 (22.8%) 23 (15.4%) 126 (84.6%) 59 (49.2%) 61 (50.8%) 29 (24.2%) 10 (8.3%)

0.086

0.153 0.013

0.025

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2 24 (20.0%) 3 20 (16.7%) 4 7 (5.8%) 5 4 (3.3%) 6 11 (9.2%) 7 5 (4.2%) 8 4 (3.3%) 9 0 10 5 (4.2%) 11 1 (0.8%) Treatment2 Microsurgical clipping 76 (52.4%) Endovascular treatment 69 (47.6%) Rerupture 7 (4.7%) Clinical vasospasm 34 (22.8%) DCI3 31 (21.2%) Hyponatremia 56 (37.6%) Infection (blood stream, 61 (40.9%) pneumonia, urinary tract) Ventriculitis 4 (2.7%) Antiepileptic drug 62 (41.6%) administration ICU stay (days, mean ± 11.9 ± 7.9 SD) Hospital stay (days, mean ± 15.9 ± 11.3 SD) Discharge mRS 0-2 71 (47.7%) 3-6 78 (52.3%) mRS at last follow-up 0-2 92 (61.7%) 3-6 57 (38.3%) Length of follow-up (days, 249.7 ± 187.8 mean ± SD) 1 Data was not available for 29 patients. 2 Data was not available for 4 aneurysms. 3 Data was not available for 3 patients.

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Table 3. Genotype frequencies of HMG polymorphism P value 0.129

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Controls 29 18 3 0.926

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Polymorphism Genotype aSAH rs2249825 CC 109 (3814C->G) CG 35 GG 5 HWE 0.306 HWE = Hardy-Weinberg equilibrium

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Table 4. Predictors of outcome measures in aSAH in multivariable logistic regression analysis

5.695 (1.804 – 17.975)

0.013 0.021 0.002 0.006

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Delayed cerebral ischemia 1.805 (1.132 – 2.880) 14.933 (1.495 – 149.125) 6.195 (1.995 – 19.233) 38.826 (2.931 – 514.387)

0.003

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Higher Hunt and Hess grade Rerupture Cerebral edema Ventriculitis SNP rs2249825 (CG and GG vs CC genotype)

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ACCEPTED MANUSCRIPT * The high-mobility group box-1 protein (HMGB1) is a biomarker for cell death and in aneurysmal subarachnoid hemorrhage (aSAH), elevated HMGB1 levels have been linked to poor outcome and an increased risk for cerebral vasospasm.

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* Delayed cerebral ischemia (DCI) was defined as a cerebral infarction at the time of discharge from the ICU and identified in 21.2% of aSAH patients.

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* In multivariable logistic regression analysis, the G allele of the 3814 C/G HMGB1 single nucleotide polymorphism (SNP) rs2249825 was independently associated with DCI (OR =

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protein expression in these patients.

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5.695, 95% CI 1.804 – 17.975, p = 0.003). This may be attributed to an increased HMGB1

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aneurysmal subarachnoid hemorrhage (aSAH)

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Cerebral Aneurysm Renin Angiotensin System (CARAS) cerebrospinal fluid (CSF) Computed tomography (CT)

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CT angiography (CTA) deoxyribonucleic acid (DNA)

digital subtraction angiography (DSA) modified Rankin scale (mRS)

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Delayed cerebral ischemia (DCI)

high-mobility group box-1 protein (HMGB1)

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single nucleotide polymorphisms (SNPs)

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December 23rd, 2016

All authors have no conflicts of interest to report.

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Sincerely,

Philipp Hendrix MD

Mark R. Harrigan MD Winfield S. Fisher 3rd MD Nilesh A. Vyas MD Robert H. Lipsky PhD Minkuan Lin PhD

Shane Tubbs, PhD

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Beverly C. Walters MD, MSc

Mohammadali M. Shoja MD

Mali Mathru MD

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Jean-Francois Pittet MD

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Paul M. Foreman MD

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Christoph J. Griessenauer MD