- Email: [email protected]
Downregulation of Apolipoprotein-E and Apolipoprotein-J in Moyamoya Disease—A Proteome Analysis of Cerebrospinal Fluid
Downregulation of Apolipoprotein-E and Apolipoprotein-J in Moyamoya Disease—A Proteome Analysis of Cerebrospinal Fluid Daina Kashiwazaki,
MD, PhD,
Haruto Uchino,
MD,
and Satoshi Kuroda,
MD, PhD
Background and Purpose: Genetic factors are closely involved in the etiology of moyamoya disease (MMD). However, its postgenomic mechanisms are still unknown. This study was aimed to identify specific biomarkers in the cerebrospinal fluid (CSF) of patients with MMD, using quantitative proteome technique. Methods: This study included 10 patients with MMD and 4 controls. The CSF was collected without blood contamination during surgery. A comparative 2-dimensional gel electrophoresis study (2D-PAGE) was performed. Protein spots that showed significant differences between moyamoya patients and controls were selected for further analysis by mass spectrometry. Results: On 2D-PAGE, 2 proteins were significantly upregulated, and 2 other proteins were downregulated in the CSF of MMD. Further mass spectrometry analysis revealed that haptoglobin and α-1-Bglycoprotein (A1BG) were upregulated. On the other hand, apolipoprotein-E (apoE), apoE precursor, and apolipoprotein-J (apoJ) were significantly downregulated in the CSF of MMD. The observed probability-based MOWSE score was 72 for haptoglobin (P < .05), 521 for A1BG (P < .05), 62 for apoE (P < .05), 72 for apoE precursor (P < .05), and 112 for apoJ (P < .05). Conclusion: Although the role of A1BG in the central nervous system is still unknown, the overexpressed haptoglobin may indicate the inflammation and/or angiogenesis in MMD. The downregulation of apoE and apoJ strongly suggests a critical role of lipid metabolism in the development and progression of MMD. These proteins may be novel biomarkers in shedding light on the pathogenesis of MMD, although further studies would be warranted. Key Words: Apolipoprotein—cerebrospinal fluid—moyamoya disease—proteome analysis. © 2017 National Stroke Association. Published by Elsevier Inc. All rights reserved.
From the Department of Neurosurgery, Graduate School of Medicine and Pharmaceutical Science, University of Toyama University, Toyama, Japan. Received June 22, 2017; revision received July 18, 2017; accepted July 25, 2017. Grant support: This study was supported by a grant from the Research Committee on Moyamoya Disease, sponsored by the Ministry of Health, Labour and Welfare, Japan. Address correspondence to Daina Kashiwazaki, MD, Department of Neurosurgery, Graduate School of Medicine and Pharmaceutical Science, University of Toyama University, 2630 Sugitani, Toyama 930-0194, Japan. E-mail: [email protected]. 1052-3057/$ - see front matter © 2017 National Stroke Association. Published by Elsevier Inc. All rights reserved. https://doi.org/10.1016/j.jstrokecerebrovasdis.2017.07.028
Moyamoya disease (MMD) is an uncommon cerebrovascular disease characterized by a progressive occlusion of the terminal portion of the internal carotid artery and its main branches, resulting in the development of a fine vascular network (moyamoya vessels) at the base of the brain.1,2 Recent studies have disclosed the important role of genetic factors in the pathogenesis of MMD.3,4 In spite of extensive research, however, the precise mechanism of MMD is still unknown. On the other hand, the diagnosis of MMD largely depends on radiological examinations; thus, it is obscure to identify definite MMD. Therefore, it is essential to identify the novel biomarkers that can be used to improve the diagnosis, predict the disease progression, improve our understanding of the pathology, or serve as therapeutic targets for MMD.
Journal of Stroke and Cerebrovascular Diseases, Vol. 26, No. 12 (December), 2017: pp 2981–2987
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Cerebrospinal fluid (CSF) is regarded as an excellent source in identifying the biomarkers for neurological diseases affecting the central nervous system (CNS). Previous studies have shown that the CSF in MMD has the increased concentrations of basic fibroblast growth factor, hepatocyte growth factor, transforming growth factorβ1, soluble vascular cell adhesion molecule type 1, intercellular adhesion molecule-1, E-selectin, and cellular retinoic acid–binding protein.5-9 These proteins are known to be involved in angiogenesis and/or inflammation. However, unknown pathognomonic proteins may still exist because a majority of previous studies are performed using the ELISA technique. Based on these observations, this study was aimed to identify unknown proteins that may be upregulated or downregulated in the CSF of patients with MMD. For this purpose, the quantitative proteomics technique was applied to analyze the whole proteins expressed in the CSF of patients with MMD, using 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry. 2D-PAGE is a powerful technique for analyzing complex protein mixtures by separating numerous numbers of proteins on a single gel according to their relative molecular mass (Mr) and isoelectric point (pI). The technology enables us to differentiate and identify complex protein mixtures extracted from cells, tissues, and other biological samples.
Materials and Methods Sample Collection In this study, the CSF sample was obtained from 10 patients with MMD and 4 controls. All of them met the guideline for the diagnosis set by the Research Committee on Moyamoya Disease of the Ministry of Health, Labor and Welfare of Japan.10 There were 3 males and 7 females. Their mean age was 26.1 ± 17.9 years, ranging from 6 to 48 years. There were 5 children (<20 years) and 5 adults. All 10 patients had a history of transient ischemic attack. None of them had cerebral infarction on magnetic resonance imaging. All of them underwent superficial temporal artery to middle cerebral artery anastomosis and encephalo-duro-myo-arterio-pericranial synangiosis on the involved hemispheres. The surgical procedures for MMD were performed at least after 3 weeks from the last ischemic event. Among 4 control patients, there were 2 males and 2 females. Their mean age was 53.2 ± 10.5 years ranging from 44 to 61 years. CSF was obtained just after the opening of the dura mater. Under a surgical microscope, the small incision was made on the arachnoid membrane over the Sylvian fissure. A 2.5-mL syringe was employed to collect the CSF from the Sylvian cistern without blood contamination. Control specimen was collected from 3 patients with unruptured aneurysms and 1 patient with arteriosclerosis using the same technique during clipping surgery
and superficial temporal artery to middle cerebral artery anastomosis.
2D-PAGE Samples were mixed with lysis buffer (5 M urea, 2 M thiourea, 2% CHAPS, 2% SB3-10, and 1% dithiothreitol) and were subjected individually to ultrafiltration (5 KDa cutoff) for desalting and concentrating proteins. Protein concentrations of these samples were measured by the Protein Assay system (Bio-Rad Laboratories, USA). Aliquots containing 90 micrograms of proteins were applied overnight to Immobiline Drystrip (GE Healthcare BioScience, Japan) by in-gel rehydration.11,12 The rehydrated gels were then gently dried with a filter paper to remove excess fluid, and isoelectric focusing was performed in a Pharmacia Hoefer Multiphor II electrophoresis chamber (GE Healthcare Bio-Science) according to the manufacturer’s instruction. Two-dimensional SDS-PAGE was performed in 9%-18% of acrylamide gradient gels using an IsoDalt electrophoresis chamber (Thermo Fisher Scientific, Japan). The 2-dimensional gels were stained with SYPRO Ruby (Invitrogen, USA) under the manufacturer’s protocols.13 The background subtraction was performed to measure the density of each spot. The SYPRO Rubystained proteins were detected using the Molecular Imager FX (Bio-Rad Laboratories) and were subjected to in-gel digestion. Image analysis and database management were done using the Image Master Platinum image analysis software (GE Healthcare Bio-Science). Differentially regulated proteins were identified below 67 of percent volume from control as downregulated and above 133% of control as upregulated. These proteins were chosen for subsequent analysis to identify the specific proteins.
In-Gel Digestion and Mass Spectrometry Precise procedures were essentially described elsewhere.14 Briefly, protein spots were excised from the dried silver stained 2-dimensional gels and rehydrated for 20 minutes in 100-mM NH4HCO3. The gel spots were then destained for 20 minutes in a solution of 15-mM potassium ferricyanide and 50-mM thiosulfate, rinsed twice in Milli-Q water, and finally dehydrated in 100% acetonitrile until they turned opaque white.15 The spots were then dried in a vacuum centrifuge and subsequently rehydrated in a digestion solution consisting of 50-mM NH4HCO3, 5-mM CaCl2, and .1-µg/µL modified sequence-grade trypsin (Promega, Japan). After overnight incubation at 37°C, the digestion was terminated in 5% trifluoroacetic acid for 20 minutes. Peptides were extracted 3 times (20 minutes each) with 5% trifluoroacetic acid in 50% acetonitrile, and the extracted peptides were pooled and dried in a vacuum centrifuge. The peptides were purified with ZipTip (Millipore, USA) under the manufacturer’s protocol and analyzed by Ultraflex tof/tof (Bruker Daltonics) MALDI mass spectrometer and MASCOT database software (Matrix
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Figure 1. Representative 2-dimensional polyacrylamide gel electrophoretograms of moyamoya patients and control show 5 differentially displayed proteins: haptoglobin (1), α-1-B-glicoprotein (2), apolipoprotein-E (3), apolipoprotein-E precursor (4), and apolipoprotein-J (5). Abbreviation: MMD, moyamoya disease.
Science). The proteins were identified on the basis of m/z values obtained for each protein spot. Database: NCBInr 20101104 (12171356 sequences; 4157546526 residues; taxonomy: Homo sapiens [human] [232072 sequences]) was used to identify the aforementioned proteins. Probability-based MOWSE scores were estimated as ion scores −10*Log(P), where P is the probability that the observed match is a random event. Individual ion scores > 39 indicate identity or extensive homology (P < .05). Protein scores are derived from ions scores as a nonprobabilistic basis for ranking protein hits.
Results The 2D-PAGE pattern of CSF showed the differential expression of 5 proteins between moyamoya patients and controls. The display of 2 proteins (1 and 2) was found to be significantly high in the CSF of MMD. The other
Figure 2. A portion of 2-dimensional gels showing 5 differentially displayed proteins: haptoglobin (1), α-1B-glicoprotein (2), apolipoprotein-E (3), apolipoprotein-E precursor (4), and apolipoprotein-J (5). Abbreviation: MMD, moyamoya disease.
3 proteins (3, 4, and 5) were significantly less in the CSF of moyamoya patients in comparison with controls (Figs 1, 2). The Mr and pI of each spot are shown in Table 1. Comparative electrophoretograms showed that the percent volume of spot 1 was .079 ± .024 (mean ± standard deviation) in moyamoya patients, being significantly higher than .053 ± .013. Likewise, the percent volume of spot 2 showed a significantly increased level (moyamoya patients versus controls: .084 ± .070 versus .053 ± .020). Similarly, spot 3 (moyamoya patients versus controls: .158 ± .063 versus .384 ± .29), spot 4 (moyamoya patients versus controls: .073 ± .025 versus .156 ± .100), and spot 5 (moyamoya patients versus controls: .279 ± .081 versus .420 ± .130) were less represented in moyamoya patients when compared with controls (Fig 3). The protein spots (1-5), which exhibited a significant change in the expression, were identified by MALDI mass spectrometry. As a result, the overdisplayed proteins 1
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Table 1. The proteins significantly changed in the cerebrospinal fluid of patients with moyamoya disease Spot number 1 2 3 4 5
Protein name Haptoglobin Alpha-1-B-glycoprotein ApoE precursor ApoE Apolipoprotein-J
Observed Observed Mr (kDa) pI 18.7 75 32.5 30.1 34.4
6.1 5.4 5.9 5.7 5.4
Abbreviation: apoE, apolipoprotein-E.
and 2 were identified as haptoglobin and α-1-B-glicoprotein (A1BG), respectively. Thus, the less abundant proteins 3, 4, and 5 were identified as apolipoprotein-E (apoE), apoE precursor, and apolipoprotein-J (apoJ). The observed probability-based MOWSE score was 72 for haptoglobin (P < .05), 521 for A1BG (P < .05), 62 for apoE (P < .05), 72 for apoE precursor (P < .05), and 112 for apoJ (P < .05; Fig 4).
Discussion Using the proteome analysis technique, this study identified 5 novel proteins in the CSF, which may be associated with the pathogenesis and/or pathophysiology of MMD. They included haptoglobin, A1BG, apoE, apoE precursor, and apoJ. None of them have been evaluated in previous ELISA studies before. Using the same technique, some investigators have previously tried to detect the proteins specific for MMD. Thus, Hojo et al (1999)
identified an overdisplayed protein with an Mr of 12 kDa and a pI of 5.35 on 2D-PAGE but could not characterize it on SWISS-PROT databases at that time. 16 Subsequently, Kim et al (2003) reported that one similar polypeptide spot (Mr = 13 to 15 kDa, pI = 5 to 5.5) was differentially expressed in the CSF samples of moyamoya patients and identified it as a cellular retinoic acid– binding protein using MALDI mass spectrometry.6 Very recently, Araki et al (2010) employed surface-enhanced laser desorption/ionization time-of-flight mass spectrometry and analyzed the differentially expressed proteins in the CSF. As a result, they found 34 candidates of single biomarker proteins within the range of 1-50 kDa but could not characterize them.17 In this study, 2 proteins are overexpressed in the CSF of patients with MMD. Of these, haptoglobin is a plasma α2-glycoprotein consisting of an alpha and a beta polypeptide chain of 16-20 kDa and 45 kDa. Haptoglobin is produced in the liver. The well-known biological function of haptoglobin is as a carrier of free hemoglobin. Haptoglobin is recognized as an acute-phase protein that acts as an antioxidant by binding free hemoglobin, and one possible mechanism is through its effect on angiogenesis. 18 Haptoglobin is also known as an inflammation-sensitive protein and is induced by proinflammatory cytokines such as IL-6.19 More importantly, haptoglobin acts as an angiogenic factor. Persistent elevation of serum haptoglobin in the setting of inflammation and ischemia is considered to stimulate angiogenesis and tissue repair.20 Therefore, haptoglobin may be involved in the development of collateral circulation through moyamoya vessels and external carotid arteries in MMD.
Figure 3. A bar diagram showing changes in 5 differentially displayed proteins: haptoglobin (1), α-1-Bglicoprotein (2), apolipoprotein-E (3), apolipoprotein-E precursor (4), and apolipoprotein-J (5). The values are expressed as mean ± standard deviation. Abbreviation: MMD, moyamoya disease.
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Figure 4. MASCOT search results and probability plots corresponding to haptoglobin, α-1-B-glicoprotein, apolipoprotein-E (apoE), ApoE precursor, and apolipoprotein-J (apoJ). The m/z-values obtained for each protein are used to identify the protein by searching in the protein database. High probability scores are used to identify the proteins.
A1BG is a 54.3-kDa glycoprotein in human plasma, but its function is still unknown. A1BG has been reported to be overexpressed in pancreatic and hepatocellular cancers.21 There is no information on its role in the CNS, but Rithidech et al (2009) reported that 12 proteins in total, including A1BG, were upregulated in the plasma of pediatric patients with multiple sclerosis.22 Future studies would clarify the significance of A1BG overexpression in the CSF of MMD. On the other hand, this study first demonstrates that apoE, apoE precursor, and ApoJ (clusterin) are downregulated in the CSF of patients with MMD. Interestingly, Koh et al (2010) reported that the apoE precursor is also downregulated in the serum of moyamoya patients.23 Apolipoproteins are proteins that bind lipids to form lipoproteins. They transport the lipids through the lymphatic and circulatory systems. There are 2 major types of apolipoproteins. Apolipoprotein B forms low-density lipoprotein particles, and other apolipoproteins form highdensity lipoprotein particles. Apolipoproteins are known
to play an important role in the transport and metabolism of lipids in the CNS. Specifically, the 3 apolipoprotein genes (APOD, APOE, CLU) are expressed at high levels in the brain, and their expression is dramatically affected by age. Thus, APOD and CLU levels increase 5- to 10-fold from neonatal to adult ages, while APOE levels drop by about 50% over the same period.24 Lipid metabolism is quite important in the CNS because the CNS is the most cholesterol-rich organ in the body. All cholesterol in the CNS is synthetized de novo in the CNS and is efficiently recycled within the CNS, having a long half-life of 1-5 years. Astrocytes are the major source of apoE and apoJ in the CSF.24 ApoE is a 34-kDa glycoprotein that transports cholesterol and other lipids in the plasma and the CNS by binding to cell-surface apoE receptors. The concentration is known to be highest in the liver and the brain. ApoE is the most abundant CSF apolipoprotein.25 ApoE is produced by astrocytes, and its synthesis is stimulated in the event of CNS injury.26 Thus, apoE is an essential
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mediator in maintaining and repairing cell membrane in the CNS and in regulating the synaptic remodeling during or after CNS injury.27 A very recent study has reported that apoE protects the astrocytes against hypoxiainduced apoptosis in a dose-dependent manner.28 Sheng et al (1999) have shown that apoE deficiency accelerates brain damage after global cerebral ischemia.29 ApoE genotype is also a major prognostic factor in patient outcome after aneurysmal subarachnoid hemorrhage.30 Furthermore, apoE is known to represent the most important genetic risk factor for sporadic Alzheimer’s disease.31 Very recently Alata et al reported that the expression of human APOE4 in the mouse was found to lead to a collection of morphological, functional, and molecular changes in cerebral blood vessels, consistent with impaired blood perfusion, and BBB dysfunction.32 These data suggested that apoE may be associated with the pathogenesis of MMD. Our data might be a result of the consumption of apoE. Therefore, the downregulation of apoE and its precursor may provoke cerebrovascular dysfunction and progress occlusive changes in the terminal portion of the internal carotid artery. It may also be involved in neuronal vulnerability against ischemia. In this study, another apolipoprotein, apoJ, is also less abundant in the CSF of moyamoya patients. Human apoJ, also known as clusterin, is a 75- to 80-kDa glycoprotein that serves as both a lipid-transport protein and a molecular chaperone in the cellular stress response.24 ApoJ is ubiquitously produced in the mammalian tissues and is found in all body fluids, including plasma, seminal, milk, urine, and CSF. ApoJ is dramatically upregulated in response to cellular stress, such as cerebral ischemia, and binds to stabilize a diverse range of misfolded proteins. In extracellular space, apoJ binds these proteins and inhibits an inflammatory response.24,33 In fact, apoJ may play a neuroprotective role against cerebral ischemia and contribute to the remodeling of the CNS.34,35 Interestingly, apoJ is induced in the vascular smooth muscle cells during atherosclerosis and neointimal hyperplasia. ApoJ deficiency also augments vascular smooth muscle cell proliferation in vitro and accelerates neointimal hyperplasia in vivo.36 ApoJ prevents endothelial apoptosis by inhibiting caspase-3 activation.37 Thus, apoJ may play a crucial role in keeping a balance between cell proliferation and death. Based on these observations, the downregulation of apoJ may activate caspase-3-involved apoptosis pathway of endothelial cells in MMD. In fact, Takagi et al (2006) found the caspase-3 activation and apoptosis in the arterial specimen of moyamoya patients.38 Endothelial dysfunction and injury may be closely related to the initiation and progression of MMD. There are some limitations in this study. Although the result may indicate the relation between MMD and alteration in the expression level of 5 factors, it is not clear which is the cause or the result. The sample size of this
study is small, so a study with a larger number of samples is warranted.
Conclusion Using the proteome analysis technique, this study demonstrates that the CSF of MMD includes 2 upregulated proteins, haptoglobin and A1BG, and 3 downregulated proteins, apoE, apoE precursor, and apoJ. Although the role of A1BG in the CNS is still unknown, the overexpressed haptoglobin may indicate the inflammation and/or angiogenesis in MMD. Downregulated apoE may be involved in NVU dysfunction and neuronal vulnerability against cerebral ischemia. The decreased content of apoJ may induce the apoptosis of endothelial cells. Altogether, apolipoproteins may be novel biomarkers for shedding light on the pathogenesis of MMD to develop new therapeutic strategies from the viewpoints of lipid metabolism in the intracranial arteries and the brain.
References 1. Kuroda S, Houkin K. Moyamoya disease: current concepts and future perspectives. Lancet Neurol 2008;7:1056-1066. 2. Suzuki J, Takaku A. Cerebrovascular “moyamoya” disease. Disease showing abnormal net-like vessels in base of brain. Arch Neurol 1969;20:288-299. 3. Kamada F, Aoki Y, Narisawa A, et al. A genome-wide association study identifies RNF213 as the first moyamoya disease gene. J Hum Genet 2011;56:34-40. 4. Liu W, Morito D, Takashima S, et al. Identification of RNF213 as a susceptibility gene for moyamoya disease and its possible role in vascular development. PLoS ONE 2011;6:e22542. 5. Hojo M, Hoshimaru M, Miyamoto S, et al. Role of transforming growth factor-beta1 in the pathogenesis of moyamoya disease. J Neurosurg 1998;89:623-629. 6. Kim SK, Yoo JI, Cho BK, et al. Elevation of CRABP-I in the cerebrospinal fluid of patients with moyamoya disease. Stroke 2003;34:2835-2841. 7. Nanba R, Kuroda S, Ishikawa T, et al. Increased expression of hepatocyte growth factor in cerebrospinal fluid and intracranial artery in moyamoya disease. Stroke 2004;35:2837-2842. 8. Soriano SG, Cowan DB, Proctor MR, et al. Levels of soluble adhesion molecules are elevated in the cerebrospinal fluid of children with moyamoya syndrome. Neurosurgery 2002;50:544-549. 9. Takahashi A, Sawamura Y, Houkin K, et al. The cerebrospinal fluid in patients with moyamoya disease (spontaneous occlusion of the circle of Willis) contains high level of basic fibroblast growth factor. Neurosci Lett 1993;160:214-216. 10. Research Committee on the Pathology and Treatment of Spontaneous Occlusion of the Circle of Willis. Guidelines for diagnosis and treatment of moyamoya disease (spontaneous occlusion of the circle of Willis). Neurol Med Chir (Tokyo) 2012;52:245-266. 11. Sanchez JC, Rouge V, Pisteur M, et al. Improved and simplified in-gel sample application using reswelling of dry immobilized pH gradients. Electrophoresis 1997;18:324-327.
PROTEOME ANALYSIS IN MMD 12. Rabilloud T, Valette C, Lawrence JJ. Sample application by in-gel rehydration improves the resolution of twodimensional electrophoresis with immobilized pH gradients in the first dimension. Electrophoresis 1994; 15:1552-1558. 13. Lopez MF, Berggren K, Chernokalskaya E, et al. A comparison of silver stain and SYPRO Ruby Protein Gel Stain with respect to protein detection in two-dimensional gels and identification by peptide mass profiling. Electrophoresis 2000;21:3673-3683. 14. Kristensen DB, Imamura K, Miyamoto Y, et al. Mass spectrometric approaches for the characterization of proteins on a hybrid quadrupole time-of-flight (Q-TOF) mass spectrometer. Electrophoresis 2000;21:430-439. 15. Gharahdaghi F, Weinberg CR, Meagher DA, et al. Mass spectrometric identification of proteins from silver-stained polyacrylamide gel: a method for the removal of silver ions to enhance sensitivity. Electrophoresis 1999;20:601605. 16. Hojo M, Hoshimaru M, Miyamoto S, et al. A cerebrospinal fluid protein associated with moyamoya disease: report of three cases. Neurosurgery 1999;45:170-173, discussion 173-174. 17. Araki Y, Yoshikawa K, Okamoto S, et al. Identification of novel biomarker candidates by proteomic analysis of cerebrospinal fluid from patients with moyamoya disease using SELDI-TOF-MS. BMC Neurol 2010;10:112. 18. Nielsen MJ, Petersen SV, Jacobsen C, et al. A unique loop extension in the serine protease domain of haptoglobin is essential for CD163 recognition of the haptoglobinhemoglobin complex. J Biol Chem 2007;282:1072-1079. 19. Moshage H. Cytokines and the hepatic acute phase response. J Pathol 1997;181:257-266. 20. Cid MC, Grant DS, Hoffman GS, et al. Identification of haptoglobin as an angiogenic factor in sera from patients with systemic vasculitis. J Clin Invest 1993;91:977985. 21. Tian M, Cui YZ, Song GH, et al. Proteomic analysis identifies MMP-9, DJ-1 and A1BG as overexpressed proteins in pancreatic juice from pancreatic ductal adenocarcinoma patients. BMC Cancer 2008;8:241. 22. Rithidech KN, Honikel L, Milazzo M, et al. Protein expression profiles in pediatric multiple sclerosis: potential biomarkers. Mult Scler 2009;15:455-464. 23. Koh EJ, Kim HN, Ma TZ, et al. Comparative analysis of serum proteomes of moyamoya disease and normal controls. J Korean Neurosurg Soc 2010;48:8-13. 24. Elliott DA, Weickert CS, Garner B. Apolipoproteins in the brain: implications for neurological and psychiatric disorders. Clin Lipidol 2010;51:555-573.
2987 25. Montine KS, Bassett CN, Ou JJ, et al. Apolipoprotein E allelic influence on human cerebrospinal fluid apolipoproteins. J Lipid Res 1998;39:2443-2451. 26. Bassett CN, Montine KS, Neely MD, et al. Cerebrospinal fluid lipoproteins in Alzheimer’s disease. Microsc Res Tech 2000;50:282-286. 27. Martinez-Gonzalez NA, Sudlow CL. Effects of apolipoprotein E genotype on outcome after ischaemic stroke, intracerebral haemorrhage and subarachnoid haemorrhage. J Neurol Neurosurg Psychiatry 2006;77: 1329-1335. 28. Zhou S, Wu H, Zeng C, et al. Apolipoprotein e protects astrocytes from hypoxia and glutamate-induced apoptosis. FEBS Lett 2013;587:254-258. 29. Sheng H, Laskowitz DT, Mackensen GB, et al. Apolipoprotein e deficiency worsens outcome from global cerebral ischemia in the mouse. Stroke 1999;30:1118-1124. 30. Guo ZD, Sun XC, Zhang JH. The role of apolipoprotein E in the pathological events following subarachnoid hemorrhage: a review. Acta Neurochir Suppl 2011;110:5-7. 31. Strittmatter WJ, Roses AD. Apolipoprotein E and Alzheimer disease. Proc Natl Acad Sci USA 1995;92:47254727. 32. Alata W, Ye Y, St-Amour I, et al. Human apolipoprotein E varepsilon4 expression impairs cerebral vascularization and blood-brain barrier function in mice. J Cereb Blood Flow Metab 2015;35:86-94. 33. Charnay Y, Imhof A, Vallet PG, et al. Clusterin in neurological disorders: molecular perspectives and clinical relevance. Brain Res Bull 2012;88:434-443. 34. Wehrli P, Charnay Y, Vallet P, et al. Inhibition of post-ischemic brain injury by clusterin overexpression. Nat Med 2001;7:977-979. 35. Imhof A, Charnay Y, Vallet PG, et al. Sustained astrocytic clusterin expression improves remodeling after brain ischemia. Neurobiol Dis 2006;22:274-283. 36. Kim HJ, Yoo EK, Kim JY, et al. Protective role of clusterin/apolipoprotein J against neointimal hyperplasia via antiproliferative effect on vascular smooth muscle cells and cytoprotective effect on endothelial cells. Arterioscler Thromb Vasc Biol 2009;29:1558-1564. 37. Riwanto M, Rohrer L, Roschitzki B, et al. Altered activation of endothelial anti- and proapoptotic pathways by high-density lipoprotein from patients with coronary artery disease: role of high-density lipoprotein-proteome remodeling. Circulation 2013;127:891-904. 38. Takagi Y, Kikuta K, Sadamasa N, et al. Caspase-3dependent apoptosis in middle cerebral arteries in patients with moyamoya disease. Neurosurgery 2006;59:894-900, discussion 900-891.
MD, PhD,
Haruto Uchino,
MD,
and Satoshi Kuroda,
MD, PhD
Background and Purpose: Genetic factors are closely involved in the etiology of moyamoya disease (MMD). However, its postgenomic mechanisms are still unknown. This study was aimed to identify specific biomarkers in the cerebrospinal fluid (CSF) of patients with MMD, using quantitative proteome technique. Methods: This study included 10 patients with MMD and 4 controls. The CSF was collected without blood contamination during surgery. A comparative 2-dimensional gel electrophoresis study (2D-PAGE) was performed. Protein spots that showed significant differences between moyamoya patients and controls were selected for further analysis by mass spectrometry. Results: On 2D-PAGE, 2 proteins were significantly upregulated, and 2 other proteins were downregulated in the CSF of MMD. Further mass spectrometry analysis revealed that haptoglobin and α-1-Bglycoprotein (A1BG) were upregulated. On the other hand, apolipoprotein-E (apoE), apoE precursor, and apolipoprotein-J (apoJ) were significantly downregulated in the CSF of MMD. The observed probability-based MOWSE score was 72 for haptoglobin (P < .05), 521 for A1BG (P < .05), 62 for apoE (P < .05), 72 for apoE precursor (P < .05), and 112 for apoJ (P < .05). Conclusion: Although the role of A1BG in the central nervous system is still unknown, the overexpressed haptoglobin may indicate the inflammation and/or angiogenesis in MMD. The downregulation of apoE and apoJ strongly suggests a critical role of lipid metabolism in the development and progression of MMD. These proteins may be novel biomarkers in shedding light on the pathogenesis of MMD, although further studies would be warranted. Key Words: Apolipoprotein—cerebrospinal fluid—moyamoya disease—proteome analysis. © 2017 National Stroke Association. Published by Elsevier Inc. All rights reserved.
From the Department of Neurosurgery, Graduate School of Medicine and Pharmaceutical Science, University of Toyama University, Toyama, Japan. Received June 22, 2017; revision received July 18, 2017; accepted July 25, 2017. Grant support: This study was supported by a grant from the Research Committee on Moyamoya Disease, sponsored by the Ministry of Health, Labour and Welfare, Japan. Address correspondence to Daina Kashiwazaki, MD, Department of Neurosurgery, Graduate School of Medicine and Pharmaceutical Science, University of Toyama University, 2630 Sugitani, Toyama 930-0194, Japan. E-mail: [email protected]. 1052-3057/$ - see front matter © 2017 National Stroke Association. Published by Elsevier Inc. All rights reserved. https://doi.org/10.1016/j.jstrokecerebrovasdis.2017.07.028
Moyamoya disease (MMD) is an uncommon cerebrovascular disease characterized by a progressive occlusion of the terminal portion of the internal carotid artery and its main branches, resulting in the development of a fine vascular network (moyamoya vessels) at the base of the brain.1,2 Recent studies have disclosed the important role of genetic factors in the pathogenesis of MMD.3,4 In spite of extensive research, however, the precise mechanism of MMD is still unknown. On the other hand, the diagnosis of MMD largely depends on radiological examinations; thus, it is obscure to identify definite MMD. Therefore, it is essential to identify the novel biomarkers that can be used to improve the diagnosis, predict the disease progression, improve our understanding of the pathology, or serve as therapeutic targets for MMD.
Journal of Stroke and Cerebrovascular Diseases, Vol. 26, No. 12 (December), 2017: pp 2981–2987
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Cerebrospinal fluid (CSF) is regarded as an excellent source in identifying the biomarkers for neurological diseases affecting the central nervous system (CNS). Previous studies have shown that the CSF in MMD has the increased concentrations of basic fibroblast growth factor, hepatocyte growth factor, transforming growth factorβ1, soluble vascular cell adhesion molecule type 1, intercellular adhesion molecule-1, E-selectin, and cellular retinoic acid–binding protein.5-9 These proteins are known to be involved in angiogenesis and/or inflammation. However, unknown pathognomonic proteins may still exist because a majority of previous studies are performed using the ELISA technique. Based on these observations, this study was aimed to identify unknown proteins that may be upregulated or downregulated in the CSF of patients with MMD. For this purpose, the quantitative proteomics technique was applied to analyze the whole proteins expressed in the CSF of patients with MMD, using 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry. 2D-PAGE is a powerful technique for analyzing complex protein mixtures by separating numerous numbers of proteins on a single gel according to their relative molecular mass (Mr) and isoelectric point (pI). The technology enables us to differentiate and identify complex protein mixtures extracted from cells, tissues, and other biological samples.
Materials and Methods Sample Collection In this study, the CSF sample was obtained from 10 patients with MMD and 4 controls. All of them met the guideline for the diagnosis set by the Research Committee on Moyamoya Disease of the Ministry of Health, Labor and Welfare of Japan.10 There were 3 males and 7 females. Their mean age was 26.1 ± 17.9 years, ranging from 6 to 48 years. There were 5 children (<20 years) and 5 adults. All 10 patients had a history of transient ischemic attack. None of them had cerebral infarction on magnetic resonance imaging. All of them underwent superficial temporal artery to middle cerebral artery anastomosis and encephalo-duro-myo-arterio-pericranial synangiosis on the involved hemispheres. The surgical procedures for MMD were performed at least after 3 weeks from the last ischemic event. Among 4 control patients, there were 2 males and 2 females. Their mean age was 53.2 ± 10.5 years ranging from 44 to 61 years. CSF was obtained just after the opening of the dura mater. Under a surgical microscope, the small incision was made on the arachnoid membrane over the Sylvian fissure. A 2.5-mL syringe was employed to collect the CSF from the Sylvian cistern without blood contamination. Control specimen was collected from 3 patients with unruptured aneurysms and 1 patient with arteriosclerosis using the same technique during clipping surgery
and superficial temporal artery to middle cerebral artery anastomosis.
2D-PAGE Samples were mixed with lysis buffer (5 M urea, 2 M thiourea, 2% CHAPS, 2% SB3-10, and 1% dithiothreitol) and were subjected individually to ultrafiltration (5 KDa cutoff) for desalting and concentrating proteins. Protein concentrations of these samples were measured by the Protein Assay system (Bio-Rad Laboratories, USA). Aliquots containing 90 micrograms of proteins were applied overnight to Immobiline Drystrip (GE Healthcare BioScience, Japan) by in-gel rehydration.11,12 The rehydrated gels were then gently dried with a filter paper to remove excess fluid, and isoelectric focusing was performed in a Pharmacia Hoefer Multiphor II electrophoresis chamber (GE Healthcare Bio-Science) according to the manufacturer’s instruction. Two-dimensional SDS-PAGE was performed in 9%-18% of acrylamide gradient gels using an IsoDalt electrophoresis chamber (Thermo Fisher Scientific, Japan). The 2-dimensional gels were stained with SYPRO Ruby (Invitrogen, USA) under the manufacturer’s protocols.13 The background subtraction was performed to measure the density of each spot. The SYPRO Rubystained proteins were detected using the Molecular Imager FX (Bio-Rad Laboratories) and were subjected to in-gel digestion. Image analysis and database management were done using the Image Master Platinum image analysis software (GE Healthcare Bio-Science). Differentially regulated proteins were identified below 67 of percent volume from control as downregulated and above 133% of control as upregulated. These proteins were chosen for subsequent analysis to identify the specific proteins.
In-Gel Digestion and Mass Spectrometry Precise procedures were essentially described elsewhere.14 Briefly, protein spots were excised from the dried silver stained 2-dimensional gels and rehydrated for 20 minutes in 100-mM NH4HCO3. The gel spots were then destained for 20 minutes in a solution of 15-mM potassium ferricyanide and 50-mM thiosulfate, rinsed twice in Milli-Q water, and finally dehydrated in 100% acetonitrile until they turned opaque white.15 The spots were then dried in a vacuum centrifuge and subsequently rehydrated in a digestion solution consisting of 50-mM NH4HCO3, 5-mM CaCl2, and .1-µg/µL modified sequence-grade trypsin (Promega, Japan). After overnight incubation at 37°C, the digestion was terminated in 5% trifluoroacetic acid for 20 minutes. Peptides were extracted 3 times (20 minutes each) with 5% trifluoroacetic acid in 50% acetonitrile, and the extracted peptides were pooled and dried in a vacuum centrifuge. The peptides were purified with ZipTip (Millipore, USA) under the manufacturer’s protocol and analyzed by Ultraflex tof/tof (Bruker Daltonics) MALDI mass spectrometer and MASCOT database software (Matrix
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Figure 1. Representative 2-dimensional polyacrylamide gel electrophoretograms of moyamoya patients and control show 5 differentially displayed proteins: haptoglobin (1), α-1-B-glicoprotein (2), apolipoprotein-E (3), apolipoprotein-E precursor (4), and apolipoprotein-J (5). Abbreviation: MMD, moyamoya disease.
Science). The proteins were identified on the basis of m/z values obtained for each protein spot. Database: NCBInr 20101104 (12171356 sequences; 4157546526 residues; taxonomy: Homo sapiens [human] [232072 sequences]) was used to identify the aforementioned proteins. Probability-based MOWSE scores were estimated as ion scores −10*Log(P), where P is the probability that the observed match is a random event. Individual ion scores > 39 indicate identity or extensive homology (P < .05). Protein scores are derived from ions scores as a nonprobabilistic basis for ranking protein hits.
Results The 2D-PAGE pattern of CSF showed the differential expression of 5 proteins between moyamoya patients and controls. The display of 2 proteins (1 and 2) was found to be significantly high in the CSF of MMD. The other
Figure 2. A portion of 2-dimensional gels showing 5 differentially displayed proteins: haptoglobin (1), α-1B-glicoprotein (2), apolipoprotein-E (3), apolipoprotein-E precursor (4), and apolipoprotein-J (5). Abbreviation: MMD, moyamoya disease.
3 proteins (3, 4, and 5) were significantly less in the CSF of moyamoya patients in comparison with controls (Figs 1, 2). The Mr and pI of each spot are shown in Table 1. Comparative electrophoretograms showed that the percent volume of spot 1 was .079 ± .024 (mean ± standard deviation) in moyamoya patients, being significantly higher than .053 ± .013. Likewise, the percent volume of spot 2 showed a significantly increased level (moyamoya patients versus controls: .084 ± .070 versus .053 ± .020). Similarly, spot 3 (moyamoya patients versus controls: .158 ± .063 versus .384 ± .29), spot 4 (moyamoya patients versus controls: .073 ± .025 versus .156 ± .100), and spot 5 (moyamoya patients versus controls: .279 ± .081 versus .420 ± .130) were less represented in moyamoya patients when compared with controls (Fig 3). The protein spots (1-5), which exhibited a significant change in the expression, were identified by MALDI mass spectrometry. As a result, the overdisplayed proteins 1
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Table 1. The proteins significantly changed in the cerebrospinal fluid of patients with moyamoya disease Spot number 1 2 3 4 5
Protein name Haptoglobin Alpha-1-B-glycoprotein ApoE precursor ApoE Apolipoprotein-J
Observed Observed Mr (kDa) pI 18.7 75 32.5 30.1 34.4
6.1 5.4 5.9 5.7 5.4
Abbreviation: apoE, apolipoprotein-E.
and 2 were identified as haptoglobin and α-1-B-glicoprotein (A1BG), respectively. Thus, the less abundant proteins 3, 4, and 5 were identified as apolipoprotein-E (apoE), apoE precursor, and apolipoprotein-J (apoJ). The observed probability-based MOWSE score was 72 for haptoglobin (P < .05), 521 for A1BG (P < .05), 62 for apoE (P < .05), 72 for apoE precursor (P < .05), and 112 for apoJ (P < .05; Fig 4).
Discussion Using the proteome analysis technique, this study identified 5 novel proteins in the CSF, which may be associated with the pathogenesis and/or pathophysiology of MMD. They included haptoglobin, A1BG, apoE, apoE precursor, and apoJ. None of them have been evaluated in previous ELISA studies before. Using the same technique, some investigators have previously tried to detect the proteins specific for MMD. Thus, Hojo et al (1999)
identified an overdisplayed protein with an Mr of 12 kDa and a pI of 5.35 on 2D-PAGE but could not characterize it on SWISS-PROT databases at that time. 16 Subsequently, Kim et al (2003) reported that one similar polypeptide spot (Mr = 13 to 15 kDa, pI = 5 to 5.5) was differentially expressed in the CSF samples of moyamoya patients and identified it as a cellular retinoic acid– binding protein using MALDI mass spectrometry.6 Very recently, Araki et al (2010) employed surface-enhanced laser desorption/ionization time-of-flight mass spectrometry and analyzed the differentially expressed proteins in the CSF. As a result, they found 34 candidates of single biomarker proteins within the range of 1-50 kDa but could not characterize them.17 In this study, 2 proteins are overexpressed in the CSF of patients with MMD. Of these, haptoglobin is a plasma α2-glycoprotein consisting of an alpha and a beta polypeptide chain of 16-20 kDa and 45 kDa. Haptoglobin is produced in the liver. The well-known biological function of haptoglobin is as a carrier of free hemoglobin. Haptoglobin is recognized as an acute-phase protein that acts as an antioxidant by binding free hemoglobin, and one possible mechanism is through its effect on angiogenesis. 18 Haptoglobin is also known as an inflammation-sensitive protein and is induced by proinflammatory cytokines such as IL-6.19 More importantly, haptoglobin acts as an angiogenic factor. Persistent elevation of serum haptoglobin in the setting of inflammation and ischemia is considered to stimulate angiogenesis and tissue repair.20 Therefore, haptoglobin may be involved in the development of collateral circulation through moyamoya vessels and external carotid arteries in MMD.
Figure 3. A bar diagram showing changes in 5 differentially displayed proteins: haptoglobin (1), α-1-Bglicoprotein (2), apolipoprotein-E (3), apolipoprotein-E precursor (4), and apolipoprotein-J (5). The values are expressed as mean ± standard deviation. Abbreviation: MMD, moyamoya disease.
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Figure 4. MASCOT search results and probability plots corresponding to haptoglobin, α-1-B-glicoprotein, apolipoprotein-E (apoE), ApoE precursor, and apolipoprotein-J (apoJ). The m/z-values obtained for each protein are used to identify the protein by searching in the protein database. High probability scores are used to identify the proteins.
A1BG is a 54.3-kDa glycoprotein in human plasma, but its function is still unknown. A1BG has been reported to be overexpressed in pancreatic and hepatocellular cancers.21 There is no information on its role in the CNS, but Rithidech et al (2009) reported that 12 proteins in total, including A1BG, were upregulated in the plasma of pediatric patients with multiple sclerosis.22 Future studies would clarify the significance of A1BG overexpression in the CSF of MMD. On the other hand, this study first demonstrates that apoE, apoE precursor, and ApoJ (clusterin) are downregulated in the CSF of patients with MMD. Interestingly, Koh et al (2010) reported that the apoE precursor is also downregulated in the serum of moyamoya patients.23 Apolipoproteins are proteins that bind lipids to form lipoproteins. They transport the lipids through the lymphatic and circulatory systems. There are 2 major types of apolipoproteins. Apolipoprotein B forms low-density lipoprotein particles, and other apolipoproteins form highdensity lipoprotein particles. Apolipoproteins are known
to play an important role in the transport and metabolism of lipids in the CNS. Specifically, the 3 apolipoprotein genes (APOD, APOE, CLU) are expressed at high levels in the brain, and their expression is dramatically affected by age. Thus, APOD and CLU levels increase 5- to 10-fold from neonatal to adult ages, while APOE levels drop by about 50% over the same period.24 Lipid metabolism is quite important in the CNS because the CNS is the most cholesterol-rich organ in the body. All cholesterol in the CNS is synthetized de novo in the CNS and is efficiently recycled within the CNS, having a long half-life of 1-5 years. Astrocytes are the major source of apoE and apoJ in the CSF.24 ApoE is a 34-kDa glycoprotein that transports cholesterol and other lipids in the plasma and the CNS by binding to cell-surface apoE receptors. The concentration is known to be highest in the liver and the brain. ApoE is the most abundant CSF apolipoprotein.25 ApoE is produced by astrocytes, and its synthesis is stimulated in the event of CNS injury.26 Thus, apoE is an essential
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mediator in maintaining and repairing cell membrane in the CNS and in regulating the synaptic remodeling during or after CNS injury.27 A very recent study has reported that apoE protects the astrocytes against hypoxiainduced apoptosis in a dose-dependent manner.28 Sheng et al (1999) have shown that apoE deficiency accelerates brain damage after global cerebral ischemia.29 ApoE genotype is also a major prognostic factor in patient outcome after aneurysmal subarachnoid hemorrhage.30 Furthermore, apoE is known to represent the most important genetic risk factor for sporadic Alzheimer’s disease.31 Very recently Alata et al reported that the expression of human APOE4 in the mouse was found to lead to a collection of morphological, functional, and molecular changes in cerebral blood vessels, consistent with impaired blood perfusion, and BBB dysfunction.32 These data suggested that apoE may be associated with the pathogenesis of MMD. Our data might be a result of the consumption of apoE. Therefore, the downregulation of apoE and its precursor may provoke cerebrovascular dysfunction and progress occlusive changes in the terminal portion of the internal carotid artery. It may also be involved in neuronal vulnerability against ischemia. In this study, another apolipoprotein, apoJ, is also less abundant in the CSF of moyamoya patients. Human apoJ, also known as clusterin, is a 75- to 80-kDa glycoprotein that serves as both a lipid-transport protein and a molecular chaperone in the cellular stress response.24 ApoJ is ubiquitously produced in the mammalian tissues and is found in all body fluids, including plasma, seminal, milk, urine, and CSF. ApoJ is dramatically upregulated in response to cellular stress, such as cerebral ischemia, and binds to stabilize a diverse range of misfolded proteins. In extracellular space, apoJ binds these proteins and inhibits an inflammatory response.24,33 In fact, apoJ may play a neuroprotective role against cerebral ischemia and contribute to the remodeling of the CNS.34,35 Interestingly, apoJ is induced in the vascular smooth muscle cells during atherosclerosis and neointimal hyperplasia. ApoJ deficiency also augments vascular smooth muscle cell proliferation in vitro and accelerates neointimal hyperplasia in vivo.36 ApoJ prevents endothelial apoptosis by inhibiting caspase-3 activation.37 Thus, apoJ may play a crucial role in keeping a balance between cell proliferation and death. Based on these observations, the downregulation of apoJ may activate caspase-3-involved apoptosis pathway of endothelial cells in MMD. In fact, Takagi et al (2006) found the caspase-3 activation and apoptosis in the arterial specimen of moyamoya patients.38 Endothelial dysfunction and injury may be closely related to the initiation and progression of MMD. There are some limitations in this study. Although the result may indicate the relation between MMD and alteration in the expression level of 5 factors, it is not clear which is the cause or the result. The sample size of this
study is small, so a study with a larger number of samples is warranted.
Conclusion Using the proteome analysis technique, this study demonstrates that the CSF of MMD includes 2 upregulated proteins, haptoglobin and A1BG, and 3 downregulated proteins, apoE, apoE precursor, and apoJ. Although the role of A1BG in the CNS is still unknown, the overexpressed haptoglobin may indicate the inflammation and/or angiogenesis in MMD. Downregulated apoE may be involved in NVU dysfunction and neuronal vulnerability against cerebral ischemia. The decreased content of apoJ may induce the apoptosis of endothelial cells. Altogether, apolipoproteins may be novel biomarkers for shedding light on the pathogenesis of MMD to develop new therapeutic strategies from the viewpoints of lipid metabolism in the intracranial arteries and the brain.
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