Review Article

Biomarker Discovery in Cerebral Vasospasm after Aneurysmal Subarachnoid Hemorrhage Magdalena M. Przybycien-Szymanska, PhD, and William W. Ashley, Jr, MD, PhD, MBA

Background: Aneurysmal subarachnoid hemorrhage (aSAH) is a devastating problem. Overall, the mortality rate associated with aSAH is 32% to 67%, which makes it the most lethal type of hemorrhagic stroke. Once the aneurysm has been treated, cerebral vasospasm is the leading cause of morbidity and mortality associated with aSAH. Thus, ability to effectively prevent or treat cerebral vasospasm could result in significantly improved survival and quality of life for aSAH patients. Unfortunately, partly because of poor understanding of the mechanisms of vasospasm, current diagnosis and treatment can be inconsistent and/or ineffective. Current treatment methods include primarily medical therapy and endovascular methods. Alone, or in combination, these measures can be of benefit in some patients. However, they are not uniformly efficacious and, on an individual basis, they can present significant risks. These risks include stroke, cardiovascular compromise, and death. More effective diagnosis and treatment strategies could significantly improve patient outcomes after aSAH. Unfortunately, clinically reliable biomarker for cerebral vasospasm has yet to be identified. Biomarker discovery may facilitate earlier diagnosis of vasospasm and improved monitoring of the response to treatment. It may help in stratifying patients into categories of risk to develop vasospasm, which could subsequently guide therapy. Indeed, biomarker research may suggest ‘‘vasospasm phenotypes’’ that can be used to guide the most effective type of therapy for that particular patient. The purpose of this manuscript is to review the current cerebral vasospasm biomarker literature. Methods: An extensive PubMed literature search was performed. We identified over 100 English language articles with key words cerebral vasospasm and biomarkers. Some of these articles and related references were used as the basis of this review. We focused on related human studies performed within the past 10 years. Results: In this review, we focus on recent work identifying molecular markers of cerebral vasospasm following aSAH and the current understanding of the utility of these markers. We highlight novel approaches such as the use of cellular microparticles for the evaluation of cerebral vasospasm. Conclusions: Although multiple molecules have been proposed, no single molecule has been shown to be a clinically reliable biomarker for cerebral vasospasm. This is not surprising based on the complex pathogenesis of cerebral vasospasm. Indeed, it is unlikely that a single biomarker will be clinically effective and reliable for predicting cerebral vasospasm. Instead, cerebral vasospasm may be

From the Department of Neurological Surgery, Loyola University Stritch School of Medicine, Maywood, IL. Received October 21, 2014; revision received January 6, 2015; accepted March 8, 2015.

Address correspondence to William W. Ashley Jr., MD, PhD, MBA, Department of Neurological Surgery, Blg 105/Room 1900, 2160 S First Ave, Maywood, IL 60153. E-mail: [email protected]. 1052-3057/$ - see front matter Ó 2015 by National Stroke Association http://dx.doi.org/10.1016/j.jstrokecerebrovasdis.2015.03.047

Journal of Stroke and Cerebrovascular Diseases, Vol. -, No. - (---), 2015: pp 1-12

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M.M. PRZYBYCIEN-SZYMANSKA AND W.W. ASHLEY JR.

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best predicted by a panel of markers and the temporal progression of their relative levels after aSAH. Many such candidate molecules are reviewed herein and can be categorized as markers of cell damage, inflammation, changes in metabolism and vascular tone as well as microparticle-derived biomarkers. Among these, microparticle-derived biomarkers seem to be promising and lend themselves to further study. Biomarker discovery may facilitate earlier diagnosis of vasospasm and improved monitoring of the response to treatment. Ultimately, it may guide in the development of safer and more effective therapies for the most dreaded of aSAH complications. Key Words: Cerebral aneurysm—microparticles—cerebral vasospasm—biomarkers—subarachnoid hemorrhage. Ó 2015 by National Stroke Association

Aneurysmal subarachnoid hemorrhage (aSAH) represents about 5% of all strokes and affects as many as 30,000 Americans each year. It has associated initial mortality of 10%-15%. Of patients who are able to reach medical care, 25% die within the first 2 weeks, and 20%-30% of the survivors are severely and permanently disabled. Overall, the mortality rate associated with aSAH is 32%-67%, which makes it the most lethal type of hemorrhagic stroke.1 Once the aneurysm has been treated, cerebral vasospasm (CV) is the leading cause of morbidity and mortality associated with aSAH. And 30%-70% of aSAH patients will experience vasospasm, and nearly 15% will die or have devastating neurologic outcomes as a result. According to Bederson et al,2 vasospasm may account for as much as 50% of deaths in patients surviving to treatment after aSAH. The ability to effectively predict, prevent, and treat CV would result in significantly improved survival and quality of life for aSAH patients. Unfortunately, partly because of poor understanding of the mechanism of CV, current diagnosis and treatment can be inconsistent and/or ineffective. CV is a heterogeneous process that has both radiographic and clinical subtypes. Radiographic CV (RCV) can be defined as a complex process involving transient, self-narrowing of intradural subarachnoid arteries as seen on cerebrovascular imaging, such as digital subtraction angiography, magnetic resonance angiography and computed tomography angiography (CTA). For the purposes of this review, radiographic vasospasm also includes vasospasm as evaluated by transcranial Doppler ultrasonography(TCD). Clinical CV (CCV) is a syndrome that usually occurs several days after aSAH and is associated with neurologic deterioration related to initial aSAH and not related to other structural or metabolic causes. It is also known as symptomatic vasospasm and can be related to delayed ischemic neurologic deficit or delayed cerebral infarction. For the purposes of this review, it will be collectively referred to as CCV. Interestingly, sometimes clinical manifestations of CV may not be a direct result of the vessel narrowing seen with RCV. Instead,

both RCV and CCV may be a part of a larger, cell and molecular process that is affecting the entire neurovascular unit. This idea is supported by the fact that there is often a disassociation between radiographic findings and clinical outcomes. Indeed, drugs such as nimodipine (calcium channel blocker) improve clinical outcomes but do not significantly affect vessel caliber. Others, such as clazosentan (endothelin receptor antagonist), significantly improve vessel caliber without an associated improvement in clinical outcome. Vasospasm patients exhibit pathological evidence of endothelial injury, increased contraction of vascular smooth muscle cells, formation of microscopic blood clots (thrombi) within distal vessels, and inflammatory changes. Fisher showed that the thickness of subarachnoid blood clot correlates with the development of CV, suggesting that the process is initiated by a release of blood and blood breakdown products into subarachnoid space. Indeed, the Fisher scale is the tool that is most widely used to assess the extent of aSAH and predict the likelihood of vasospasm.3 Owing to multifactorial nature of CV development, however, our ability to consistently predict the onset and severity of CV is poor. Review of the literature suggests that this process may be mediated by endothelial cell interactions, inflammation, oxidative processes, spreading depolarization, and/or alterations in calcium signaling. Standard therapies for CV include preventive pharmacologic treatments with the oral calcium channel blocker nimodipine, which has been shown to improve poor patient outcomes conventionally associated with CCV.2 Noninvasive medical therapy also includes hypertensive therapy and avoidance of hypovolemia which may help to maintain adequate perfusion2 and avoidance of anemia to help maintain optimal oxygen-carrying capacity. Cerebrospinal fluid (CSF) drainage to treat intracranial hypertension may also help maintain cerebral perfusion pressure. Invasive endovascular methods, in general, include the infusion of vasodilators and balloon angioplasty.2 Alone, or in combination, these measures can be beneficial in some patients; however, they are not uniformly efficacious and can present significant risks that

CEREBRAL VASOSPASM BIOMARKERS

include stroke, cardiovascular compromise, and death. More effective diagnosis and treatment strategies would significantly improve patient outcomes after aSAH. The discovery of reliable biomarkers will guide physicians in stratifying patients into categories of risk to develop CV. The research thus far has yet to fully elucidate the pathogenesis of brain injury following aSAH and clearly define the extent to which biomarkers might reliably indicate the impeding injury. As mentioned, there are multiple theories associated with the development of vasospasm and each represents a possible source of reliable biomarkers. However, it is unlikely that these pathways function independently in the pathogenicity of vasospasm and that a single biomarker will be a realistic prospect. This review focuses on recent work identifying molecular markers of aSAH-related CV and current understanding of the utility of these biomarkers. This review highlights novel neuroproteomics approaches, such as the use of CSF-derived microparticles for the evaluation of CV after aSAH.

An Overview of Human Studies Genetic Markers for aSAH and CV It has been determined that genetic factors play a role in aneurysm development but only a few studies have explored this in CV.1,4-7 Studies performed by Wu et al,6,7 for example, emphasize genotypic variations of apolipoprotein E (ApoE) gene as a risk factor for developing RCV and CCV after spontaneous aSAH, collectively referenced in the study as CV. This association is very interesting as there has been an increasing evidence for a role of variations in ApoE gene (polymorphisms) and risks associated with neurological disorders.8 There are 3 ApoE gene polymorphisms known in humans (ε2, ε3, and ε4). ApoE ε4 allele, for example, has been associated with an increased risk of developing Alzheimer’s disease,8,9 and poor long-term outcome after traumatic brain injury.10 ApoE ε2 and ε4 alleles have been linked to increased risk of developing ischemic stroke and to increased risk of stroke in patients with Alzheimer’s disease.8,9,11 Interestingly, in a case– control study of ApoE gene variations and associated risks for the development of CV after aSAH, investigators showed that the ApoE ε4 allele was associated with an increased risk of CV after spontaneous aSAH.7 Furthermore, an association of ApoE gene promoter polymorphism with the incidence of CV after spontaneous aSAH (as evaluated by TCD combined with determination of patient’s clinical condition) has also been discovered. In this study, investigators found that 2219G/T polymorphism in the ApoE gene promoter was associated with an increased risk of CV.6 These, and other studies, suggest that the ApoE molecule and ApoE gene have the potential to be useful as biomarkers for CV after aSAH.

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Other studies found that a variation in haptoglobin (Hp) gene can be associated with poor patient outcome after aSAH.4,12 Haptoglobin is a hemoglobin-binding molecule that binds hemoglobin released by erythrocytes with high affinity and therefore prevents its oxidative processes.13 The gene has 3 polymorphisms (Hp 1-1, 2-1, and 2-2). A study by Kantor et al12 revealed that in patients with aSAH, Hp 2-2 was correlated with poor prognosis and poor outcome after aSAH. Ohnishi et al4 found that a polymorphism in the Hp gene can predict RCV after aSAH (referred to as angiographic in the study). They reported that Hp 2-2 phenotype was linked with a higher risk of developing RCV after aSAH.4 These, together with other studies, indicated that some genes and their polymorphisms may be associated with a higher risk of developing RCV in aSAH patients (Table 1).

Cell Damage Markers as Biomarkers for CV It is well established that neuronal and astrocytic damage occurs after the initial egress of blood into subarachnoid space.14-16 Mass spectroscopy has advanced protein isolation and identification to yield discoveries in proteins that act as markers for brain injury.14 Proteins that have been studied range in their function but the following mainly are involved in apoptotic cell death. In a prospective study by Lewis et al, calpain- and caspase-mediated spectrin breakdown products were significantly elevated in aSAH patients up to 12 hours before the onset of CCV. Breakdown products of alphaII spectrin undergo cleavage by necrosis and apoptosis, and these studies suggest that necrotic proteolysis may be prevalent after aSAH.17 Another protein involved in cell death cascade, caspase 3, was found to be increased in patients up to 7 days after aSAH.18 This study did not determine, however, correlation between the observed levels of caspase 3 and CCV or RCV after aSAH as the patient population that was included in the study developed hydrocephalus after aSAH and RCV was not evaluated.18 In a study performed by Siman et al, there were 7 neurodegeneration biomarkers found to be upregulated in the CSF of patients who experienced aSAH. These included calpain-derived alpha-spectrin N- and C-terminal fragments (CCSntf and CCSctf, respectively), 14-3-3b and Ϊ, ubiquitin C-terminal hydrolase L1 (UCHL1), neuron-specific enolase (NSE), and S100B.15 Of these, 6 correlated with severity of CV (as evaluated by ultrasonography, angiography, and magnetic resonance imaging, in conjunction with clinical examination), and poor long-term outcome after initial aSAH. More specifically, CCSctf, 14-3-3b, UCHL1, and NSE were significantly upregulated on day 1 after aSAH; all of them except for UCHL1 were upregulated on day 3; 14-3-3b and Ϊ, CCSntf, and NSE were increased on day 5; and 14-3-3b and Ϊ were increased between days 7 and 10 after bleed in patients

M.M. PRZYBYCIEN-SZYMANSKA AND W.W. ASHLEY JR.

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Table 1. Genetic markers associated with CV and worse outcome after aSAH

Study type

No. of patients Imaging

Biomarker

Endogenous role

Authors

Year

Ohnishi et al

2013 Case–control

95

CTA

Haptoglobin 2-2 gene Hemoglobinpolymorphism binding molecule

Wu et al 2011 Case–control

185

TCD

ApoEε4 gene polymorphism

Wu et al 2010 Case–control

101

TCD

Source

Marker Risk presence of CV

Venous blood

+

Cholesterol Venous blood metabolism

+

219T ApoE promoter Cholesterol Venous blood polymorphism metabolism

+

Abbreviations: aSAH, aneurysmal subarachnoid hemorrhage; CV, cerebral vasospasm; CTA, computed tomography angiography; TCD, transcranial Doppler ultrasonography. Table summarizing recent human studies describing genetic variations and their link with a risk of developing CV. A solid black plus (1) in the ‘‘marker presence’’ column indicates that the putative biomarker is present after aSAH. A solid black up arrow ([) in the ‘‘risk of CV’’ column indicates an increased risk of developing CV associated with the presence of the putative marker after aSAH.

who exhibited mild to severe RCV as compared to patients with no RCV. All of these 6 markers were correlated with poor long-term prognosis in aSAH patients regardless of RCV development.15 Neuron-specific enolase, a glycolytic enzyme released from neurons, has been a marker for severity of neuronal damage in conditions such as stroke, encephalitis, and neurodegenerative diseases.18-24 Evidence from a prospective study of Kacira et al verified that NSE elevation is indicative of brain damage in aSAH and showed that levels of NSE in the CSF continue to increase on the seventh day after aSAH. This suggests that neuron damage increases a week after aSAH, although no correlation with RCV was evaluated in this study.18 A study by Siman et al 15 showed that high levels of NSE, among other markers, were correlated with severity of vasospasm and poor patient outcome after aSAH. Based on these and other studies, it is likely that this cell death marker could be useful as a marker for the prognosis of vasospasm; however, more research is needed as some studies were not able to document a meaningful correlation between levels of NSE and CV.25,26 In a study performed by Sanchez-Pena et al, investigators have measured S100B, a protein released from astrocytes and neurons. They demonstrated that mean 15-day levels of S100B were elevated in correlation with poor clinical outcome after aSAH; however, the presence of RCV was not evaluated in this study.27 Although in Siman et al’s15 study, this biomarker was also found to be upregulated after aSAH, there was no significant correlation

with the severity of RCV. Other studies also found no correlation with S100B levels and a time course or severity of different forms of vasospasm25,28,29; therefore, although S100B may serve as a good biomarker for poor patient outcome after aSAH, its value as a marker for RCV remains controversial and requires further research. (UCHL1) is another protein released from neurons and neuroendocrine cells after damage and, as a marker of neuronal loss after aSAH, has been suggested to be a predictor of poor clinical outcome after aSAH. Levels of UCHL1 were elevated in aSAH patients 2 weeks after aSAH and were correlated with poor outcome. In this study, the development of CV was not evaluated.30 A study by Siman et al15 showed that UCHL1 was upregulated on day 3 after aSAH, and high levels of UCHL1 were correlated with the development of RCV and poor patient long-term outcome after aSAH. The value of UCHL1 as a biomarker for the development of RCV needs to be further investigated. Damage to specific cell components is linked to astrocytic and neuronal death. When looking at neurodegeneration, axonal damage is its significant component. Serological protein markers specific for this damage have been detected in patients with aSAH.14,31 One study demonstrated that phosphorylated neurofilament subunit H (pNF-H), a phosphorylated subunit that is resistant to proteases, is elevated in CSF and serum of aSAH patients with poor clinical outcome. In this study, CCV was also evaluated and defined as the acute onset of a focal neurologic deficit or a change in the Glasgow

CEREBRAL VASOSPASM BIOMARKERS

Coma Scale score of 2 or more points. It was confirmed using CTA. Levels of pNF-H were found to be higher in CCV patients as compared to no-CCV patients.31 This study indicated that patients with aSAH have axonal degeneration which adversely affects their clinical prognosis and that pNF-H may serve as a reliable biomarker for the development of CCV; however, its value in the prognosis of the development of RCV needs to be further evaluated. Together, these studies demonstrate that brain damage is directly related to the morbidity and mortality after aSAH and that cell damage markers could potentially be used as biomarkers for CCV and/or RCV (Table 2).

Inflammation Markers as Biomarkers for CV Many studies have demonstrated that there is inflammation caused by the accumulation of blood in subarachnoid space and that inflammation plays a major role in the process of vasospasm development. A vast array of molecules associated with inflammatory processes have been investigated in relation to aSAH and its complications, and those include cytokines (eg, interleukin [IL] 2 and its receptor, IL-6, and IL-8), E-selectins, components of the complement cascade, cellular adhesion molecules (vascular cell adhesion molecule [VCAM] 1, intracellular cell adhesion molecule [ICAM] 1), and tumor necrosis factor alpha (TNFa) among others.14,32,33 Levels of some of these molecules, however, are very transient and tend to decrease within the first few days after aneurysmal rupture.14 Recently, however, there was some development in the field that indicated that specific molecules associated with inflammation may be good biomarkers for development of RCV. TNFa is a proinflammatory cytidine that is associated with oxidative stress, cell death, and recruitment of inflammatory mediators. In their prospective study, Chou et al showed that elevated patient serum TNFa levels correlated with poor patient outcome 3 months after aSAH. In fact, researchers showed that an increase in serum TNFa levels 2-3 days after aSAH was correlated with poor patient outcome both 3 and 6 months after aSAH and the development of RCV.33 These data indicate that TNFa may play a role in the development of RCV after aSAH; however, more research needs to be performed to use it as a predictor for the development of this complication. It has been discovered that acute phase proteins, such as high-sensitivity inflammation marker (C-reactive protein [CRP]), could be used as markers for aSAH. C-reactive protein is produced by cells in response to proinflammatory cytokine, IL-6, and has been used as a very sensitive marker for inflammation in inflammatory diseases.34 In a study by Kacira et al, CRP levels were elevated 3-7 days after aSAH, indicating that inflammation is occurring in aSAH patients.18 Supporting these

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findings, Fountas et al found elevated CRP levels in serum and CSF of patients who experienced RCV after aSAH. More specifically, the levels of CRP were elevated in aSAH patients with the poorest clinical outcomes after aSAH and were correlated with the development of RCV. Patients who developed RCV had increased serum and CSF levels of CRP even on day 0 and 1 after aSAH.35 Together, these studies indicated that CRP could be a reliable marker for the inflammatory processes associated with RCV and could be used as a marker for the prognosis of RCV. Another inflammatory factor to consider in CV disease is endothelial activation of neutrophils. Concentrations of soluble ICAM-1, soluble P-selectin, soluble E-selectin, and ED1-fibronectin can be used as markers of endothelial activation.14,36 A prospective human study by Kim et al showed significant elevation in ICAM-1 and VCAM-1 on days 3 and 7 after aSAH. There was, however, no correlation between changes in levels of these 2 molecules and the development of CV after aSAH. In the study, CV was evaluated via daily TCD and confirmed via CTA.37 Together, these results indicate that cell adhesion molecules, in addition to other inflammatory markers and cell types being sampled in CSF and/or serum of patients with aSAH and RCV, could be potential biomarkers for the development of RCV38; however, more research is warranted to elucidate their specific role (Table 3).

Altered Metabolism Markers as Biomarkers for CV In most patients with aSAH, increased intracerebral pressure reduces cerebral blood flow and thus decreases energy metabolism.39 Most (80%) of the brain’s metabolism is through the cycle of glutamate (GLT) and glutamine (GLN). In astrocytes, GLT is converted to GLN and GLN is released and taken up by neurons where GLN is reconverted to GLT. In a Samuelsson et al39 prospective study, patients who had higher GLT levels had lower intracerebral pressure and better clinical outcome after aSAH, indicating that energy metabolism plays an important role in patients affected by spontaneous aSAH. Jung et al reported that CSF levels of GLT, GLN, histidine, and glycine were correlated with the incidence of RCV on the day that the angiography was performed. In addition, GLU was correlated with the size of ischemia on the day that the measurement was taken.26 In addition, as a result of failure in metabolism described in these and other studies, CSF lactate tends to accumulate and this can act as a predictor of anaerobic glycolysis, glycogenolysis, and poor neurologic status.40,41 These studies suggest that aSAH patients can experience decreased cerebral energy metabolism, and thus a panel of energy metabolism markers may be useful as a potential diagnostic tool for RCV.

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Table 2. Cell damage markers associated with CV and worse outcome after aSAH

Authors

Year

Study type

No. of patients

Siman et al

2011

Prospective

14

Imaging

Biomarker

DSA, MRA, TCD

Neuron-specific enolase

Glycolytic enzyme found in neuronal and neuroendocrine cells

UCHL1

Proteolytically stable protein released from neuronal cells, indicates damage of dendrites and prikarya Cell death markers

CCSctf, 14-3-3b and Ϊ, CCSntf

Endogenous role

Source

Marker level

Risk of CV

CSF

2008

Prospective

30

CTA, DSA

pNF-H

Phospho- form of NF-H normally restricted to axons and resistant to proteases

Venous blood and CSF

Lewis et al

2007

Prospective

20

CTA

SBDP

Products resulting from a cleavage by necrotic/apoptotic proteases of a 280-kD cytoskeletal axonal protein alpha-II spectrin

CSF

Abbreviations: aSAH, aneurysmal subarachnoid hemorrhage; CCSntf and CCSctf, calpain-derived alpha-spectrin N- and C-terminal fragments; CV, cerebral vasospasm; CSF, cerebrospinal fluid; CTA, computed tomography angiography; DSA, digital subtraction angiography; MRA, magnetic resonance angiography; pNF-H, phosphorylated neurofilament subunit H; SBDP, spectrin breakdown products; TCD, transcranial Doppler ultrasonography; UCHL1, ubiquitin-C-terminal hydrolase 1. Table summarizing recent human studies describing the role of various astrocytic and neuronal (including axonal) damage markers in developing CV. A solid black up arrow ([) in the ‘‘marker level’’ column indicates an increase in the levels of the putative biomarker after aSAH. A solid black down arrow (Y) in the ‘‘marker level’’ column indicates a decrease in the levels of the putative biomarker after aSAH. A solid black up arrow ([) in the ‘‘risk of CV’’ column indicates an increased risk of developing CV associated with the change in levels of the putative marker; a solid black dash (-) in the ‘‘risk of CV’’ column indicates the lack of association of the putative biomarker with the development of CV after aSAH; and a solid black down arrow (Y) in the ‘‘risk of CV’’ column indicates a decreased risk of developing CV associated with the change in levels of the putative marker.

M.M. PRZYBYCIEN-SZYMANSKA AND W.W. ASHLEY JR.

Lewis et al

CEREBRAL VASOSPASM BIOMARKERS

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Table 3. Inflammation markers associated with CV and worse outcome after aSAH

Authors

No. of Year Study type patients

Kim et al

2013 Prospective

121

Chou et al

2012 Prospective

52

Fountas et al 2009 Prospective

41

Imaging

Biomarker

Endogenous role

TCD, CTA, ICAM-1 and Cell adhesion molecules, VCAM-1 act as mediators in leucocyte–endothelial cell interactions Cerebral TNFa Proinflammatory cytokine angiography

CTA, DSA

CRP

Source

Marker Risk level of CV

Serum and CSF

Serum and CSF

An acute-phase protein Serum activated by IL-6, very and CSF sensitive to inflammation

Abbreviations: aSAH, aneurysmal subarachnoid hemorrhage; CV, cerebral vasospasm; CRP, C-reactive protein; CSF, cerebrospinal fluid; CTA, computed tomography angiography; DSA, digital subtraction angiography; ICAM-1, intracellular cell adhesion molecule; IL-6, interleukin-6; TCD, transcranial Doppler ultrasonography; TNFa, tumor necrosis factor alpha; VCAM-1, vascular cell adhesion molecule. Table summarizing recent human studies describing the role of various inflammation markers in developing CV. A solid black up arrow ([) in the ‘‘marker level’’ column indicates an increase in the levels of the putative biomarker after aSAH. A solid black up arrow ([) in the ‘‘risk of CV’’ column indicates an increased risk of developing CV associated with the change in levels of the putative marker after aSAH.

ApoE, a protein that acts on lipid clearance and metabolism, when deficient, affects the relaxation of vascular endothelium and decreases functional performance.42 In addition to studies by Wu et al6,7 showing associations between ApoE gene polymorphisms and a risk of developing RCV after spontaneous aSAH described previously, it was hypothesized by Alexander et al42 that ApoE protein may affect RCV (evaluated in the study via cerebral angiogram and/or TCD in combination with clinical neurologic determination) after aSAH. In their prospective study, they showed that ApoE levels were higher in individuals with RCV but not CCV, indicating increased demand after neurologic insult.42 Although Wu et al studies showed a link between ApoE gene polymorphisms and a risk of developing nonspecific (RCV plus CCV) vasospasm after aSAH, studies by Alexander et al suggested that ApoE may be interacting after aSAH but does not necessarily influence the development of CCV although it may influence the development of RCV. The role of ApoE as a predictive marker for RCV and CCV needs to be further evaluated as, because of its role as a marker for many neurodegenerative diseases, it may be found to be a reliable biomarker associated with increased risks for the development of CV after aSAH as well (Table 4).

Markers for Altered Vascular Tone as Biomarkers for CV Vasoconstriction is an abnormal prolonged contraction of smooth muscles of blood vessels causing sustained narrowing of arteries. One of the leading theories in the field is that alterations in calcium metabolism are responsible for the development of RCV.14,40,42 The contraction of

the vasculature involves the release of cytosolic calcium, which leads to the constriction of smooth muscle. The breakdown of blood in subarachnoid space after aSAH releases oxyhemoglobin, which inhibits ATP-dependent calcium pump. This knowledge led Alexander et al to perform a study that measured calcium levels in CSF of aSAH patients. Calcium levels were significantly lower in patients with symptomatic vasospasm (CCV) as compared to those who did not experience CCV.42 As stated previously, the breakdown of red blood cells and hemoglobin in subarachnoid space induces CV. Deoxyhemoglobin is the most prevalent irritant causing vasospasm whereas oxyhemoglobin is found to be elevated in patients with different forms of CV.14,43 Much like calcium, endothelial relaxing factors like nitric oxide–containing compounds affect vasoconstriction that occurs in vasospasm.14 In Lin et al’s44 study, levels of nitrite/nitrate were higher than in control cases but no correlation was found between this elevation and the severity of clinical outcome after aSAH. The same study demonstrated that levels of F2-isoprostanes, products of arachidonic acid oxidation that induce vasoconstriction and platelet aggregation, correlated well with poor clinical outcome after aSAH,44 suggesting that products of oxidative stress could play an important role in the severity of complications and an outcome after aSAH. Recent human studies showed that endothelin-1 (Et-1), a potent vasoconstrictor, may be a reliable marker for the prognosis of CV after aSAH.29,45 Products of hemoglobin degradation together with oxygen radicals may trigger elevation of Et-1 which then acts as a potent vasoconstrictor and its effects last even after its levels return back to baseline.46-48 Bellapart et al described Et-1 as a

M.M. PRZYBYCIEN-SZYMANSKA AND W.W. ASHLEY JR.

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Table 4. Energy metabolism markers associated with CV and worse outcome after aSAH

Authors Jung et al

Year

Study type

2013 Retrospective

Alexander 2008 Prospective et al

No. of patients

Imaging

Biomarker

18

Cerebral arteriography and angiography

50

CTA, TCD

Glutamate, glutamine, histidine, and glycine ApoE

Endogenous role

Source

Energy metabolism

CSF

Catabolism of triglyceride-rich lipoproteins

CSF

Marker Risk level of CV

Abbreviations: aSAH, aneurysmal subarachnoid hemorrhage; ApoE, apolipoprotein E; CV, cerebral vasospasm; CSF, cerebrospinal fluid; CTA, computed tomography angiography; TCD, transcranial Doppler ultrasonography. Table summarizing recent human studies describing the role of various energy metabolism markers in developing CV. A solid black up arrow ([) in the ‘‘marker level’’ column indicates an increase in the levels of the putative biomarker after aSAH. A solid black up arrow ([) in the ‘‘risk of CV’’ column indicates an increased risk of developing CV associated with the change in levels of the putative marker after aSAH.

potent screening marker for RCV in patients with aSAH. The researchers confirmed that CSF and plasma Et-1 levels were markedly increased in patients with aSAH as compared to controls and that these levels were correlated with the incidence of RCV as evaluated via daily TCD and digital subtraction angiography in addition to clinical evaluation. The peak in Et-1 level correlated with the peak in RCV on day 5 after SAH. In addition, plasma levels of Et-1 were higher when compared with CSF levels,29 indicating that measuring blood levels of Et-1 may be reliably used and Et-1 may be potentially valuable as a biomarker for the prognosis of RCV after

aSAH. Thus, products of hemoglobin breakdown and oxidation may play a role in the development of RCV after aSAH, and further investigation is warranted for use of these products as RCV biomarkers (Table 5).

Microparticles as Biomarkers for CV Microparticles are very small (.1-100 mm) membranebound vesicles released by platelets, white blood cells, red blood cells, and, importantly, endothelial cells. In the past, they were believed to be waste products of apoptosis with procoagulant properties but recent studies

Table 5. Markers that play a role in vascular tone modulation

Authors Bellapart et al

Year

Study type

2014 Case–control

No. of patients

Imaging

Biomarker

Endogenous role

20

TCD, DSA Endothelin-1 Potent vasoconstrictor

Alexander 2008 Prospective et al

50

CTA, TCD Calcium

Lin et al

26

CTA, TCD Nitrite/nitrate Nitric oxide (potent vasodilator) production

2006 Case–control

Vasodynamics

Source

Marker Risk level of CV

Venous blood and CSF CSF

CSF

Abbreviations: aSAH, aneurysmal subarachnoid hemorrhage; CV, cerebral vasospasm; CSF, cerebrospinal fluid; CTA, computed tomography angiography; DSA, digital subtraction angiography; TCD, transcranial Doppler ultrasonography. Table summarizing recent human studies describing the role of various biomarkers that play a role in modulation of vascular tone that could play a role in developing CV. A solid black up arrow ([) in the ‘‘marker level’’ column indicates an increase in the levels of the putative biomarker after aSAH. A solid black down arrow (Y) in the ‘‘marker level’’ column indicates a decrease in the levels of the putative biomarker after aSAH. A solid black up arrow ([) in the ‘‘risk of CV’’ column indicates an increased risk of developing CV associated with the change in levels of the putative marker after aSAH; a solid black dash (-) in the ‘‘risk of CV’’ column indicates the lack of association of the putative biomarker with the development of CV after aSAH.

CEREBRAL VASOSPASM BIOMARKERS

Venous blood Cell stimulation or tissue degeneration

Venous blood Cell stimulation, tissue degeneration, coagulation and/or inflammation processes

2010 Lackner et al

Prospective

20

TCD

CD142 (tissue factor), CD41a (platelets), CD235a (erythrocytes), CD146 (endothelial cells), CD66b (neutrophils), and vWF (labeled MP) CD105-labeled endothelial MP TCD, cerebral angiography 22 Prospective 2012 Sanborn et al

Abbreviations: aSAH, aneurysmal subarachnoid hemorrhage; CV, cerebral vasospasm; CTA, computed tomography angiography; MP, microparticles; TCD, transcranial Doppler ultrasonography; vWF, von Willebrand factor. Table summarizing recent human studies describing the role of microparticles in developing CV. A solid black up arrow ([) in the ‘‘marker level’’ column indicates an increase in the levels of the putative biomarker after aSAH. A solid black up arrow ([) in the ‘‘risk of CV’’ column indicates an increased risk of developing CV associated with the change in levels of the putative marker after aSAH.

Risk of CV Marker level Source Endogenous role Biomarker Imaging No. of patients Study type Year Authors

Table 6. Microparticle markers associated with CV and worse outcome after aSAH

suggested their paracrine bioactive and regulatory roles.25,49-53 They can be found in serum, CSF, tears, saliva, and mucus and are released in response to biochemical processes and mechanical stress.52-55 Thus, in addition to being markers of pathophysiologic states, these tiny subcellular particles may play a direct role in the regulation of local and systemic biologic processes such as vasospasm or related ischemia. Relative concentrations of microparticles vary based on pathophysiology and may be specific to injured tissue. Microparticles may be the thread that helps to unify the disparate theories regarding the process of vasospasm. Indeed, recent studies have suggested a role for microparticles in inflammation, endothelial injury, oxidative stress, apoptosis, and spreading depolarization,25,49-52,54-56 all of which have been linked with the development of CV. Studies suggest some relationship to stroke and ischemic brain injury.51,52,55-57 Differential flow cytometry and mass spectroscopy techniques can segregate populations of microparticles, and it has been shown that using these techniques, various disease states, including intracranial and extracranial stenosis, could be differentiated.51-53,55,56,58,59 Based on these studies, investigation of the role of microparticles in the pathogenesis of aSAH and the development of CV was warranted. Recently, one small study showed increased microparticle levels in basal ganglia hemorrhage and an association with poor patient outcome.57 Another study showed that endothelial microparticles are upregulated in the blood of aSAH patients.52 Blood and CSF-derived microparticles represent a way to perform targeted proteomic analysis on cellular components that are released in response to endothelial injury and potentially may be local triggers for the process of vasospasm itself. Only a few studies up to date used microparticle analysis in association with RCV after aSAH. In Lackner et al’s55 recent prospective study, researchers demonstrated that elevated levels of cellular endothelial microparticles (CD105 positive [1]) correlated with RCV as measured via TCD. Sanborn et al52 performed a study in which they analyzed temporal expression of microparticles in plasma following aSAH and their correlation with RCV as evaluated by TCD and angiography. They found that all microparticle subtypes analyzed, including microparticles derived from platelets, endothelial cells, erythrocytes, and neutrophils, were correlated with temporal dynamics of RCV. More specifically, microparticles that were closely associated with thrombosis and endothelial dysfunction had the strongest effect. They had the most durable response and their elevation lasted well into RCV window. Endothelial cell–derived and tissue factor-expressing microparticles were reversely correlated with infarction levels, and their levels predicted infarction as early as on day 1. Microparticles expressing von Willebrand factor or those derived from CD2351 (erythrocyte) cells had sharp initial increase but their

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10 52

levels declined to baseline afterwards. Microparticle analysis may be a useful strategy for further CV biomarker research. More studies, however, need to be performed to elucidate the role of CSF-derived microparticles as biomarkers for CV and possibly their mechanistic role in its development (Table 6).

Summary and Final Remarks The development of CV after aSAH is multifactorial and may involve several distinct but interconnected pathological processes. Based on these processes, multiple biomarkers have been proposed to be used as predictors of CV after aSAH. This review highlights the complexity of CV development and supports the idea that it is very unlikely that a single protein biomarker will be clinically effective and reliable for predicting CV. Instead, CV may be best predicted by a panel of targeted biomarkers and their temporal progression and relative levels after aSAH. Finding such a panel may lead to the development of reliable indicators for the risk of CV development. Because changes in microparticle populations can occur rapidly after a stimulus and can be associated with a wide variety of pathophysiologic states, further investigation of microparticle-derived proteins as an avenue for biomarker discovery is warranted. Acknowledgments: The authors would like to thank Drs. Ramy El Khoury and Elizabeth McKinnon for their aid in literature review as well as funding from Brain Aneurysm Foundation (2011-2012) that supported our preliminary data collection.

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