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Quantitative proteomics analysis of differentially expressed proteins in ruptured and unruptured cerebral aneurysms by iTRAQ
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Quantitative proteomics analysis of differentially expressed proteins in ruptured and unruptured cerebral aneurysms by iTRAQ Pengjun Jianga,b, Jun Wua,b, Xin Chena,b, Bo Ningc, Qingyuan Liua,b, Zhengsong Lia,b, ⁎ Maogui Lia,b, Fan Yanga,b, Yong Caoa,b, Rong Wanga,b, Shuo Wanga,b, a
Department of Neurosurgery, Beijing Tiantan Hospital, Capital Medical University, Beijing, PR China China National Clinical Research Center for Neurological Diseases, Beijing, PR China c Department of neurosurgery, Guangzhou Red Cross Hospital, Jinan University, Guangzhou, Guangdong Province, PR China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Intracranial aneurysm Rupture iTRAQ Proteomics analysis
The underlying pathophysiological mechanisms involved in cerebral aneurysms rupture remain unclear. This study was performed to investigate the differentially expressed proteins between ruptured and unruptured aneurysms using quantitative proteomics. The aneurysmal walls of six ruptured aneurysms and six unruptured aneurysms were collected during the surgical operation. The isobaric tags for relative and absolute quantification (iTRAQ) were used to identify the differentially expressed proteins and western blotting was performed to validate the expression of the proteins of interest. Bioinformatics analysis of the differentially expressed proteins was also performed using the KEGG database and GO database. Between ruptured and unruptured aneurysms, 169 proteins were found differently expressed, including 74 up-regulated proteins and 95 down-regulated proteins with a fold change ≥ 2 and p value ≤ .05. KEGG pathway analysis revealed that phagosome, focal adhesion and ECM-receptor interaction were the most common pathways involved in aneurysm rupture. In addition, the differential expressions of ITGB3, CRABP1 and S100A9 were validated by western blotting. Through the iTRAQ method, we found that inflammatory responses and cell-matrix interactions may play a significant role in the rupture of cerebral aneurysms. These findings provide a basis for better understanding of pathophysiological mechanisms associated with aneurysm rupture. Biological significance: Intracranial aneurysm is the leading cause of life-threating subarachnoid hemorrhage which can cause 45% patients die within 30 days and severe morbidity in long-term survivors. With a high prevalence ranging from 1% to 5% in general population, cerebral aneurysm has become a widespread health hazard over past decades. Though great advances have been achieved in the diagnosis and treatment of this disease, the underlying pathophysiological mechanisms of aneurysm rupture remains undetermined and a lot of uncertainty still exists surrounding the treatment of unruptured cerebral aneurysms. Clarifying the mechanism associated with aneurysm rupture is important for estimating the rupture risk, as well as the development of new treatment strategy. Some previous studies have analyzed the molecular differences between ruptured and unruptured IAs at gene and mRNA levels, but further comprehensive proteomic studies are relatively rare. Here we performed a comparative proteomics study to investigate the differentially expressed proteins between ruptured IAs (RIAs) and unruptured IAs (UIAs). Results of our present study will provide more insights into the pathogenesis of aneurysm rupture at protein level. With a better understanding of pathophysiological mechanisms associated with aneurysm rupture, some noninvasive treatment strategies may be developed in the future.
1. Introduction Intracranial aneurysm (IA) is a common cerebrovascular disease characterized by degeneration and abnormal dilation of focal cerebral artery. The estimated prevalence is 3.2% in general population and overall risk of rupture is about 1% [1,2]. The rupture of an intracranial
⁎
aneurysm leads to aneurysmal subarachnoid hemorrhage (aSAH), which may produce a devastating outcome in up to 40–65% of patients and significant morbidity in 50% patients who survive [3–5]. With the development of neuroimaging techniques, patients with incidental intracranial aneurysms are detected more frequently. Management of incidental intracranial aneurysms continues to be
Corresponding author at: Department of neurosurgery, Beijing Tiantan Hospital, Capital Medical University, Beijing 100050, PR China. E-mail address: [email protected] (S. Wang).
https://doi.org/10.1016/j.jprot.2018.05.001 Received 2 January 2018; Received in revised form 1 April 2018; Accepted 2 May 2018 1874-3919/ © 2018 Elsevier B.V. All rights reserved.
Please cite this article as: Jiang, P., Journal of Proteomics (2018), https://doi.org/10.1016/j.jprot.2018.05.001
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2.3. iTRAQ labeling
controversial, because of a inherent risk of complications carried by preventive treatments and the high case fatality and morbidity caused by aSAH [6]. Many factors such as hypertension, smoking, larger aneurysm size and irregular morphology have been demonstrated to be associated with increased risk of aneurysm rupture [7]. Nevertheless, some limitations still exist in those predictive models due to the incompletely understanding of true molecular mechanisms responsible for aneurysm rupture. More insight into the pathomechanism of aneurysm rupture will facilitate clinical management choice and promote the development of new noninvasive treatment strategies in the future. Many previous studies have analyzed the biological and molecular differences between ruptured and unruptured IAs [8–10]. An accumulating body of evidence indicates that inflammatory response may play a critical role in the progression and rupture of IA [11–13]. Several microarray-based whole-genome gene expression analyses have also investigated the differential expressed genes in ruptured versus unruptured aneurysms and pathways involved in immune response and the lysosome pathway were found to be enriched in ruptured aneurysms [14,15]. Further comprehensive proteome analysis will provide more knowledge and deeper understanding of the mechanisms underlying aneurysm rupture. Currently, few proteomic studies have been performed in intracranial aneurysm and only differential proteins in normal artery versus aneurysm were identified [16,17]. Here we performed this study to compared the differentially expressed proteins between ruptured IAs (RIAs) and unruptured IAs (UIAs) using iTRAQ quantitative proteomics, which will provide more insight into the pathogenesis of aneurysm rupture at protein level.
After precipitation with acetone, the proteins (200 μg) of each pool were dissolved with 1 M DTT for 1 h at 37 °C and kept in the dark with 1 M indole-3-acetic acid (IAA) for 1 h at room temperature. Samples were dissolved and centrifuged twice with 120 μL UA (8 M urea in 0.1 M Tris.HCl, pH 8.5), and then re-dissolved and centrifuged three times with 100 M lautyltrethylammonium bromide (LTEAB). The proteins (2–4 μg) were digested with trypsin (trypsin: protein = 1:50; Sigma) and incubated at 37 °C overnight. Each peptide pool was then passed through a 0.2 μm centrifugal filter for 20 min with 10,000 g at 20 °C. Peptide mixture samples were labeled using a 4-Plex iTRAQ Reagent Kit from AB SCIEX. The ruptured and unruptured aneurysmal samples were labeled with 114 and 115 iTRAQ tags, respectively. 2.4. Nano-LC-MS/MS Using the EASY-nLC 1000 system (Thermo Scientific, USA), the labeled peptide mixtures were separated and then were trapped on a PepMap100 C18, 3 μm, 75 μm × 2 cm column (Thermo Scientific, USA). The peptides were then separated on a PepMap100 RSLC C18, 2 μm, 75 μm × 15 cm analytic column by a 70-min mobile phase gradient from 5% to 35%. Mass spectra were recorded on a Q Exative mass spectrometer configured with a Nano-ESI source (Thermo Scientific, USA). Full scan MS spectra were acquired in the m/z range 300–1800 at a resolution of 70,000, the top 10 precursors were selected for high energy collision-induced dissociation (HCD) with collision energy of 27%, and the product ions were detected at a resolution of 17,500 on data-dependent acquisition mode.
2. Materials and methods 2.5. iTRAQ data analysis 2.1. Patients and samples Protein identifications were performed using the MASCOT search engine (version 2.4.1; Matrix Science, London, UK) embedded into Proteome Discoverer 1.3 (Thermo Electron, San Jose, CA, USA). The search parameters were as follows: (1) database, uniprot; (2) taxonomy, Homo sapiens; (3) Enzyme, trypsin; (4) fixed modifications, carbamidomethyl of C, iTRAQ 4plex(N-term), iTRAQ 4plex(K); (5) variable modifications, oxidation of M; (6) max missed cleavages, 2; (7) peptide charges state, +2, +3, and + 4; (8) peptide mass tolerance, 20 ppm; (9)mass/mass tolerance, ± 0.05 Da. Differentially expressed proteins were defined as those reached to 2-fold changes between two groups. The data analysis was supported by Wayen Biotechnologies Co., Ltd. (Shanghai, China).
Twelve patients with saccular IAs (6 ruptured and 6 unrptured) underwent microsurgical clipping in our neurosurgical department were enrolled between October 2015 to June 2017. The exclusion criteria included: (1) patients aged over 70 years old or younger than 18 years old; (2) patients with cerebral infarction and other cerebrovascular disease, including arteriovenous malformation, arteriovenous fistula and cavernous hemangioma; (3) the mean time between SAH and the operation was longer than one month; (4) patients with current or previous history of receiving aspirin treatment. Clinical data was gathered from medical records. The aneurysm tissue samples were harvested during operation after placement of the clip. The mural thrombus was removed and only aneurysmal wall tissues were collected. Tissue samples were transferred to liquid nitrogen immediately and stored at −80 °C until protein extraction. The study was approved by the Ethics Committee of the Department of Medicine, Beijing Tiantan Hospital, Capital Medical University, and all patients gave informed consent to participate in this study.
2.6. Bioinformatics analysis Pathway analysis was performed by the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Gene Ontology (GO) database was used to facilitate the biological interpretation of the identified proteins in these studies. The differentially expressed proteins of GO were divided into 3 categories as follows: biological process (BP), molecular function (MF) and cellular component (CC). CytoScape was used to generate network maps (http://www.cytoscape.org) using the protein-protein interaction (PPI) data from STRING database.
2.2. Protein extraction Each tissue sample was rinsed twice with ice-cold PBS to remove residual blood cells in the aneurysmal wall. The samples were homogenized with RIPA buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1 mM PMSF, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 2% SDS). The homogenates were held on ice for 30 min and centrifuged at 12,000g for 30 min to remove insoluble components. Bicinchoninic acid (BCA) assay was performed to quantify the protein concentration of each sample. The UIA and RIA samples were pooled using the strategy described in previous report [18]. 200 μg of proteins from each group were reduced, alkylated and digested in the centrifugal unit. After digested at 37 °C overnight, the peptide solutions were centrifuged and the filtrates were collected. The digested peptides were dried by vacuum centrifugation and stored at −80 °C until future use.
2.7. Western blotting To validate the accuracy of iTRAQ results, western blotting analysis was performed to examine the relative abundance of some differently expressed proteins again. Proteins extracted from ruptured aneurysms and unruptured aneurysms were separated by SDS-PAGE and then electrotransferred to a PVDF membrane. The membranes were blocked for 1 h at room temperature in PBST containing 5% nonfat dry milk and then incubated with rabbit monoclonal anti-integrin β3 antibody (Cell Signaling Technology), rabbit monoclonal anti-CRABP1 antibody 2
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(Abcam, Cambridge, UK) and rabbit monoclonal anti-S100A9 antibody (Proteintech, Wuhan, China) overnight at 4 °C. Membranes were incubated with secondary antibodies for 1 h at room temperature after washed three times with PBST. Blots were visualized using an ECL detection system and proteins were quantified using a ChemiDoc XRS+ image analyzer (Bio-Rad, Hercules, CA, USA). Densitometric analysis was used to evaluate the expression levels of target proteins. Ratios of densitometric measurements of target proteins relative to β-actin were compared between ruptured and unruptured group. Three experimental replicates were performed and independent Student's t-test was used for assess the statistical significance between the two groups.
Table 2 Comparison of clinical features between ruptured and unruptured group. Characteristics Sex Age Hypertension Smoking history
Male Female Yes No Yes No
Size
RIAs
UIAs
p-Value
2 4 55 ± 11.2 5 1 0 6 9.1 ± 2.4
3 3 44 ± 10.5 2 4 2 4 14 ± 9.6
1.0 0.15 0.24 0.43 0.25
3.2. Identification of differently expressed proteins between RIA and UIA tissues
2.8. Statistical analysis The measurement data are expressed as means and standard deviation for three independent WB experiments. For continuous variables, independent Student's t-test was used for comparisons between the two groups. Categorical variables were compared using the Pearson's ×2 test or Fisher's exact test. P < .05 was considered statistically significant. All statistical analyses were performed by using the statistical software SPSS (version 22, SPSS, Chicago, Illinois, USA).
Six ruptured cerebral aneurysmal tissues and six unruptured cerebral aneurysmal tissues collected from 12 patients were used for iTRAQ assessment. Using the LC-MS/MS method, a total of 2638 proteins were identified and 169 proteins were found statistically differently expressed between RIAs and UIAs. Among these proteins, 74 proteins were significantly up-regulated and 95 proteins were down-regulated in ruptured aneurysm samples with a fold change ≥ 2 and p value ≤ .05. The 20 most significantly up-regulated and 20 most significantly downregulated proteins were shown in Table 3 and Table 4.
3. Results
3.3. Classification of differently expressed proteins
3.1. Clinical information
The PANTHER (protein annotation through evolutionary relationship) classification system was used to further analyze the biological significance of upregulated proteins based on their molecular function, cellular component and biological process. The distribution of the biological processes of the upregulated proteins was shown in Fig. 1A. Classification analyses of cellular components and molecular functions were shown in Fig. 1B and C. A protein-Protein interaction (PPI) network of the 169 differently expressed proteins was constructed by STRING and was shown in Fig. 2. The PPI network analysis revealed that CRABP1 and S100A9 participated in various biological processes such as inflammation while integrin β3 (ITGB3) participate in cell adhesion and signal transduction, which may play an important role in the pathogenesis of many cerebrovascular diseases.
Six patients with ruptured aneurysms and six patients with unruptured were included in this study. The clinical information of all these cases was summarized in Table 1. The male to female ratio was 1:1.4 (5 males and 7 females). The average size of aneurysms was 11.6 mm (ranged from 4.3 to 32.1 mm). The analysis of group homogeneity was shown in Table 2, no significant differences were shown between the two groups. All patients were free of aspirin and statins treatment before surgery. Among those with unruptured aneurysms, the mean observation time before surgery was 3.3 months (ranged from 0.5 to 10 months) and no aneurysm growth was observed during this period. The mean interval from SAH-confirmed diagnosis to treatment was 17.5 days in patients with ruptured aneurysms. No special preoperative therapy was applied in patients with unruptured aneurysms. For those with ruptured aneurysms, nimodipine was commonly used in 5 patients to prevent the occurrence of vasospasm and the average Hunt-Hess score grade at presentation was 2, with a range from 1 to 4 (Table 1). Postoperative ischemic changes were observed in two patients (both with ruptured aneurysms), leaving those two patients with bad prognosis (mRS ≥ 3) at last follow up (ranged from 5 to 24 months).
3.4. KEGG pathway analysis of differently expressed proteins KEGG pathway enrichment analysis was performed to determine the biological processes participating in aneurysm rupture. The top ten most significantly enriched pathways were shown in Table 5. Phagosome, focal adhesion and ECM-receptor pathway were among the top three pathways identified by the pathway analysis.
Table 1 Clinical characterization of 12 patients. Cases
Comorbidities
Cigarette
HH score
Sex
Age (years)
Location
Size (mm)
Ruptured/unruptured
1 2 3 4 5 6 7 8 9 10 11 12
HP HP HP – HP HP Bradycardia – – HP – HP
No No No No No No Yes No No No Yes No
1 2 1 3 4 1 0 0 0 0 0 0
Male Male Female Female Female Female Male Female Female Female Male Male
60 69 58 35 61 48 42 48 28 42 51 59
R-MCA R-PcoA L-MCA AcoA R-ICA R-ICA AcoA L-ICA R-ICA R-ICA AcoA L-MCA
8.2 11.1 10.3 6.4 12.2 6.5 4.3 12.6 32.1 11.7 8.0 15.4
Ruptured Ruptured Ruptured Ruptured Ruptured Ruptured Unruptured Unruptured Unruptured Unruptured Unruptured Unruptured
HH score: Hunt-Hess score, HP: hypertension. 3
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Table 3 Proteins significantly unregulated in RIAs. No.
Protein
Accession no.
Gene name
Protein function
Fold change
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Eosinophil cationic-related protein (Fragment) Protein S100-P Eosinophil peroxidase Resistin Apolipoprotein E Arachidonate 12-lipoxygenase, 12S-type Platelet glycoprotein Ib beta chain Lactoferrin Neutrophil defensin 3 Truncated lactoferrin Myelin P2 protein Thrombocidin-1 antimicrobial variant (Fragment) Integrin beta Integrin alpha-IIb Neutrophil cytosol factor 4 Eosinophil cationic protein cDNA FLJ57557 Alpha-2-antiplasmin Putative neutrophil cytosol factor 1C Ras-related C3 botulinum toxin substrate 2
Q12762 P25815 P11678 Q9HD89 Q8TCZ8 P18054 P13224 B3VMW0 P59666 B2MV14 P02689 D3JV43 L7UUZ7 P08514 Q15080 P12724 B7Z5A7 P08697 A8MVU1 P15153
ECRP S100P EPX RETN APOE ALOX12 GP1BB N/A DEFA3 LTF PMP2 N/A ITGB3 ITGA2B NCF4 RNASE3 N/A SERPINF2 NCF1C RAC2
nucleic acid binding cadherin binding peroxidase activity hormone activity unclassified arachidonate 12-lipoxygenase activity transmembrane signaling receptor activity serine-type endopeptidase activity protein homodimerization activity serine-type endopeptidase activity cholesterol binding chemokine activity receptor activity extracellular matrix binding ARF guanyl-nucleotide exchange factor activity endonuclease activity substrate-specific transmembrane transporter activity endopeptidase inhibitor activity phosphatidylinositol binding protein kinase regulator activity
9.74 8.22 4.99 4.88 4.43 4.23 3.88 3.87 3.66 3.48 3.41 3.40 3.24 3.24 3.22 3.12 3.12 3.12 3.11 3.09
rupture in long period [22–25]. Many previous studies have performed at genome and transcriptome level to identify the related pathophysiological processes underlying aneurysm rupture. It is well known that both expression and function of the related proteins can be influenced by post-translational protein modifications and protein-protein interaction, so studies at proteome level may give a more realistic view of the pathological changes associated with aneurysm rupture. In the present study, we identified 169 proteins differently expressed in ruptured aneurysm tissues compared to unruptured aneurysm tissues. Gene ontology (GO) enrichment analysis revealed that most of the upregulated proteins in ruptured aneurysms belong to cell parts and mainly participate in the cellular process. KEGG pathway analysis revealed that phagosome, focal adhesion and ECM-receptor interaction pathway are the main signaling pathways activated in ruptured aneurysms, suggesting that inflammatory responses and cellmatrix interactions may play a significant role in the rupture of cerebral aneurysms. Several representative differential proteins (ITGB3, CRABP1 and S100A9) were further confirmed by western blotting.
3.5. Validation of differently expressed proteins identified by iTRAQ The expressions of ITGB3, CRABP1 and S100A9 were further evaluated by immunoblotting to validate the accuracy of iTRAQ and LCMS/MS results. As shown in Fig. 3, ITGB3 and S100A9 were unregulated while CRABP1 was down-regulated in the RIAs.
4. Discussion Cerebral aneurysm is a cerebrovascular disorder result from a complex interaction of inherited and acquired factors. The mechanisms underling aneurysm genesis and rupture are very complex and many factors such as shear stress induced nitric oxide production, phenotype change and apoptosis of smooth muscle cell, fluid-structure interaction and genetic factors have been implicated in aneurysm formation and rupture [19–21]. Though many risk factors associated aneurysm rupture have been identified, the specific molecular mechanisms responsible for aneurysm rupture remains poorly understood and this has hampered the development of medical approaches to prevent aneurysm Table 4 Proteins significantly downregulated in RIAs. No.
Protein
Accession no.
Gene name
Protein function
Fold change
1 2 3 4
HLA class I histocompatibility antigen, B-44 alpha chain cDNA FLJ58762 Putative uncharacterized protein DKFZp686K06110 Alpha-2-HS-glycoprotein
P30481 B4DDD5 Q6MZK8 P02765
HLA-B N/A DKFZp686K06110 AHSG
0.13 0.19 0.19 0.20
5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Coagulation factor VII (Fragment) MHC class I antigen Dermatopontin Putative uncharacterized protein DKFZp686H1812 Cellular retinoic acid-binding protein 1 Olfactomedin-like protein 1 Apolipoprotein E Asporin Carnitine O-acetyltransferase (Fragment) Prostaglandin-H2 D-isomerase Olfactomedin-like protein 3 Biglycan cDNA FLJ51023 Carbohydrate sulfotransferase 14 Basement membrane-specific heparan sulfate proteoglycan core protein variant (Fragment) Stromal cell-derived factor 1
A3RKG7 C5IWY0 Q07507 Q5HYE3 P29762 Q6UWY5 P02649 Q9BXN1 H0Y4Z7 P41222 Q9NRN5 A6NLG9 B4DPC8 H0YN65 Q59EG0
N/A HLA-A DPT DKFZp686H1812 CRABP1 OLFML1 APOE ASPN CRAT PTGDS OLFML3 N/A N/A CHST14 N/A
Peptide antigen binding Unclassified Calcium ion binding Cysteine-type endopeptidase inhibitor activity Serine-type endopeptidase activity Immune response Cell adhesion Unclassified Retinoic acid binding Unclassified Cholesterol transporter activity Calcium ion binding Transferase activity Transporter activity Multicellular organism development Collagen fiber assembly Serine-Type Endopeptidase Activity Sulfotransferase activity Calcium ion binding
0.24 0.24 0.25 0.26 0.28 0.28 0.30 0.31 0.31 0.31 0.32 0.32 0.32 0.33 0.33
P48061
CXCL12
Chemoattractant activity
0.34
20
4
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Fig. 1. Classification of upregulated proteins in RIAs by PANTHER program. (A) Biological Process; (B) Celluar Component; (C) Molecular Function.
subarachnoid space [32,33]. The mRNA expression levels of inflammation-related cytokines were significantly increased in experimental SAH models. Delayed cerebral ischaemia (DCI) and angiographic vasospasm may be results of direct damage caused by inflammatory cells infiltration. Previous pathological study revealed that macrophages are the main inflammatory cells infiltrated into the wall. Continuous aneurysm wall remodeling and chronic inflammatory reaction mediated by macrophages may participate in the healing process after aneurysm rupture [34,35]. From this point of view, it is possible that the changed proteins identified in our present study, among which many proteins are associated with inflammatory responses, may be the secondary response caused by subarachnoid hemorrhage. Though above-mentioned opinion may be a possible explanation for the differentially expressed proteins between ruptured and unruptured aneurysms. We should still keep in mind that roles of inflammatory reaction in the initiation and rupture of cerebral aneurysm are not negligible. In a pathological study performed by Kataoka et al. [36], invasion of macrophages and leukocytes in the wall was commonly observed in ruptured aneurysms. But no relationship was observed between the degree of inflammatory responses and the length of time elapsed between rupture onset and specimen harvest, suggesting that inflammatory reaction may exist before aneurysm rupture. Another study performed by Crompton [37] also revealed that inflammatory infiltration was commonly associated with fibrosis, which is usually considered as the end stage of chronic inflammatory response. Recent animal studies also revealed that some pro-inflammatory molecules may act as potential targets for rupture prevention aneurysm intervention [38,39]. Though more studies are needed to confirm the association between inflammation and aneurysm rupture, results of the present study shed new light on the molecular mechanisms underlying the rupture process.
4.1. Pathways involving aneurysm rupture There is growing evidence suggested that the pathobiology of RIAs is substantially distinct from that of UIAs. Different signaling pathways were proved to be involved in the development and progression of cerebral aneurysms. Using microarray technique, Shi et al. found that differentially expressed genes in aneurysm wall participated in focal adhesion, extracellular matrix receptor interaction and cell communication [26]. Consistent with that result, our present study further revealed involvement of focal adhesion and ECM-receptor interaction pathway in aneurysm rupture at the protein levels. Though the exact mechanism underling aneurysm rupture is still unknown, degenerative remodeling characterized by de-endothelializatioin, thinning of aneurysm wall and macrophage infiltration has been widely accepted as the hallmark associated with aneurysm rupture [27,28]. Among these pathologic changes, infiltration of macrophages into aneurysmal walls is increasingly recognised as having a central role in the progression of human cerebral aneurysms [29,30]. A recent study has demonstrated that the expression and activity of focal adhesion kinase (FAK) are markedly enhanced in macrophage which infiltrated to abdominal aortic aneurysm tissue [31]. Their results also suggested that FAK activity is essential to macrophage chemotaxis in response to monocyte chemoattractant protein-1 (MCP-1) and FAK may be a new potential therapeutic target. Thus it is reasonable to conclude that focal adhesion pathway may also plays a crucial role in the inflammatory responses in intracranial aneurysm rupture. 4.2. Association between inflammatory responses and aneurysm rupture As a cause-effect relationship between the proteomic profile change and aneurysm rupture is hard to be determined by a cross-sectional study, it is reasonable to speculate that proteomic profile changes in the aneurysmal wall may result from the early brain injury following subarachnoid hemorrhage. Many pathological processes can be initiated by SAH due to release of haemoglobin and erythrocyte contents to the 5
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Fig. 2. Protein-Protein interaction (PPI) network of 169 differently expressed proteins between ruptured and unruptured aneurysms. Proteins selected for further western blot validation were indicated with filled circles.
confirmed by western blotting, indicating that these molecules participate in the occurrence of aneurysms rupture and may work as potential therapeutic targets in the future. ITGB3 is an important subunit of transmembrane integrin receptor which can mediate signal transduction between cell and extracellular microenvironment [40]. As aneurysms development and rupture are mechanobiological processes influenced by hemodynamic stress and wall strength, receptors that connect ECM to intracellular signaling molecules may play a central role in the progress. By combing with extracellular ligands such as fibronectin, laminin and matrix metalloproteinase-2, integrins can activate RhoA/RhoKinase and MAPK signaling pathways and may participate in the wall remodeling changes [41,42]. Further investigations are needed to elucidate their precise biological mechanisms. The S100 calcium-binding protein A9 (S100A9), a member of the S100 calcium binding protein family, is a recently identified member of the damage associated molecular pattern (DAMP) family [43]. S100A9 is a cytosolic protein mainly produced by myeloid cells and plays a
Table 5 Top ten significantly enriched pathways identified by KEGG pathway analysis. No.
Term
ID
Count
%
1 2 3 4 5 6 7 8 9 10
Phagosome Focal adhesion ECM-receptor interaction Platelet activation Cell adhesion molecules (CAMs) Leukocyte transendothelial migration Complement and coagulation cascades Hematopoietic cell lineage Staphylococcus aureus infection Asthma
hsa04145 hsa04510 hsa04512 hsa04611 hsa04514 hsa04670 hsa04610 hsa04640 hsa05150 hsa05310
13 12 11 11 10 8 7 7 6 5
10.2 9.4 8.6 8.6 7.8 6.2 5.5 5.5 4.7 3.9
4.3. Differential expressed proteins between ruptured and unruptured aneurysms Alternations in expression levels of interested proteins were 6
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during surgery. In addition, further investigations are needed to validate the other proteins and pathways identified in our present study before drawing definitive conclusions. 5. Conclusion Our results showed that inflammatory responses and cell-matrix interactions may play a significant role in the rupture of cerebral aneurysms. ITGB3, CRABP1 and S100A9 were differentially expressed between ruptured and unruptured aneurysm and they may participate the pathological process of aneurysm rupture. The present study provides a more comprehensive view of protein expression difference between RIAs and UIAs, and the pathway analysis allows a better understanding of pathophysiological mechanisms associated with aneurysm rupture, which will shed light on the prevention of cerebral aneurysm rupture. Conflict of interest All the authors have approved the enclosed manuscript and declare that there is no conflict of interests regarding this article. Acknowledgments This work was supported by the National Key Research and Development Program of China (Grant No. 2016YFC1301800) and the projects of National Natural Science Foundation of China (Grant No. 81471210 and 81671129).
Fig. 3. Western blot validation of selected proteins that differentially expressed between ruptured intracranial aneurysms and unruptured intracranial aneurysms. The expression levels of ITGB3, CRABP1 and S100A9 revealed by western blotting analysis. B. Quantitative assessment of protein expression using densitometric analysis. Error bars represent the standard deviation for three experimental replicates and asterisk indicates p < .05.
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prominent role in the regulation of inflammatory response. S100 protein plasma levels were found to be significantly higher in patients suffering from SAH and be associated with early neurological deficit [44]. Recent study also revealed that gene expression level of S100A9 was significantly upregulated in ruptured cerebral aneurysms and it was assumed that S100A9 could be an important contributor to the rupture of intracranial aneurysms [45]. In agreement with previous studies, our present study further demonstrated that the expression of S100A9 protein increased in ruptured aneurysms. Similar to other S100 proteins identified as DAMPs, S100A9 is an intracellular protein involved in inflammatory process and immune response and is closely related to the development and severity of cardiovascular disease (CVD) [46]. S100A9 also plays a critical role in the occurrence of atherothrombosis by amplificating the inflammatory reaction, and S100A8 and S100A9 gene-deleted mice showed an significant decrease in atherosclerotic lesions. These properties suggested that S100A9 could be an important moderator of aneurysms and may be a possible target for therapy against human intracranial aneurysms. Cellular retinoic acid-binding protein 1 (CRABP1) is a small cytosolic protein encoded by CRABP1 gene and it plays a dominant role in the modulation of retinoic acid (RA) signaling pathway [47]. Because of the potential role of RA in regulating cell growth and differentiation, retinoic acid and retinoid receptors expression have been reported to be associated with initiation and progression of several human tumors [48]. Our present study revealed that CRABP1 was down-regulated remarkably in ruptured aneurysm tissues, therefore it is possible that CRABP1 may participate in the progression of cerebral aneurysms through abnormal growth regulation. Several limitations exist in the present study. First, the total number of included aneurysm samples is relatively small because of difficulties in collecting IA samples. Sample selection bias may exist in our present study due to only relatively large aneurysm samples could be obtained 7
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Contents lists available at ScienceDirect
Journal of Proteomics journal homepage: www.elsevier.com/locate/jprot
Quantitative proteomics analysis of differentially expressed proteins in ruptured and unruptured cerebral aneurysms by iTRAQ Pengjun Jianga,b, Jun Wua,b, Xin Chena,b, Bo Ningc, Qingyuan Liua,b, Zhengsong Lia,b, ⁎ Maogui Lia,b, Fan Yanga,b, Yong Caoa,b, Rong Wanga,b, Shuo Wanga,b, a
Department of Neurosurgery, Beijing Tiantan Hospital, Capital Medical University, Beijing, PR China China National Clinical Research Center for Neurological Diseases, Beijing, PR China c Department of neurosurgery, Guangzhou Red Cross Hospital, Jinan University, Guangzhou, Guangdong Province, PR China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Intracranial aneurysm Rupture iTRAQ Proteomics analysis
The underlying pathophysiological mechanisms involved in cerebral aneurysms rupture remain unclear. This study was performed to investigate the differentially expressed proteins between ruptured and unruptured aneurysms using quantitative proteomics. The aneurysmal walls of six ruptured aneurysms and six unruptured aneurysms were collected during the surgical operation. The isobaric tags for relative and absolute quantification (iTRAQ) were used to identify the differentially expressed proteins and western blotting was performed to validate the expression of the proteins of interest. Bioinformatics analysis of the differentially expressed proteins was also performed using the KEGG database and GO database. Between ruptured and unruptured aneurysms, 169 proteins were found differently expressed, including 74 up-regulated proteins and 95 down-regulated proteins with a fold change ≥ 2 and p value ≤ .05. KEGG pathway analysis revealed that phagosome, focal adhesion and ECM-receptor interaction were the most common pathways involved in aneurysm rupture. In addition, the differential expressions of ITGB3, CRABP1 and S100A9 were validated by western blotting. Through the iTRAQ method, we found that inflammatory responses and cell-matrix interactions may play a significant role in the rupture of cerebral aneurysms. These findings provide a basis for better understanding of pathophysiological mechanisms associated with aneurysm rupture. Biological significance: Intracranial aneurysm is the leading cause of life-threating subarachnoid hemorrhage which can cause 45% patients die within 30 days and severe morbidity in long-term survivors. With a high prevalence ranging from 1% to 5% in general population, cerebral aneurysm has become a widespread health hazard over past decades. Though great advances have been achieved in the diagnosis and treatment of this disease, the underlying pathophysiological mechanisms of aneurysm rupture remains undetermined and a lot of uncertainty still exists surrounding the treatment of unruptured cerebral aneurysms. Clarifying the mechanism associated with aneurysm rupture is important for estimating the rupture risk, as well as the development of new treatment strategy. Some previous studies have analyzed the molecular differences between ruptured and unruptured IAs at gene and mRNA levels, but further comprehensive proteomic studies are relatively rare. Here we performed a comparative proteomics study to investigate the differentially expressed proteins between ruptured IAs (RIAs) and unruptured IAs (UIAs). Results of our present study will provide more insights into the pathogenesis of aneurysm rupture at protein level. With a better understanding of pathophysiological mechanisms associated with aneurysm rupture, some noninvasive treatment strategies may be developed in the future.
1. Introduction Intracranial aneurysm (IA) is a common cerebrovascular disease characterized by degeneration and abnormal dilation of focal cerebral artery. The estimated prevalence is 3.2% in general population and overall risk of rupture is about 1% [1,2]. The rupture of an intracranial
⁎
aneurysm leads to aneurysmal subarachnoid hemorrhage (aSAH), which may produce a devastating outcome in up to 40–65% of patients and significant morbidity in 50% patients who survive [3–5]. With the development of neuroimaging techniques, patients with incidental intracranial aneurysms are detected more frequently. Management of incidental intracranial aneurysms continues to be
Corresponding author at: Department of neurosurgery, Beijing Tiantan Hospital, Capital Medical University, Beijing 100050, PR China. E-mail address: [email protected] (S. Wang).
https://doi.org/10.1016/j.jprot.2018.05.001 Received 2 January 2018; Received in revised form 1 April 2018; Accepted 2 May 2018 1874-3919/ © 2018 Elsevier B.V. All rights reserved.
Please cite this article as: Jiang, P., Journal of Proteomics (2018), https://doi.org/10.1016/j.jprot.2018.05.001
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2.3. iTRAQ labeling
controversial, because of a inherent risk of complications carried by preventive treatments and the high case fatality and morbidity caused by aSAH [6]. Many factors such as hypertension, smoking, larger aneurysm size and irregular morphology have been demonstrated to be associated with increased risk of aneurysm rupture [7]. Nevertheless, some limitations still exist in those predictive models due to the incompletely understanding of true molecular mechanisms responsible for aneurysm rupture. More insight into the pathomechanism of aneurysm rupture will facilitate clinical management choice and promote the development of new noninvasive treatment strategies in the future. Many previous studies have analyzed the biological and molecular differences between ruptured and unruptured IAs [8–10]. An accumulating body of evidence indicates that inflammatory response may play a critical role in the progression and rupture of IA [11–13]. Several microarray-based whole-genome gene expression analyses have also investigated the differential expressed genes in ruptured versus unruptured aneurysms and pathways involved in immune response and the lysosome pathway were found to be enriched in ruptured aneurysms [14,15]. Further comprehensive proteome analysis will provide more knowledge and deeper understanding of the mechanisms underlying aneurysm rupture. Currently, few proteomic studies have been performed in intracranial aneurysm and only differential proteins in normal artery versus aneurysm were identified [16,17]. Here we performed this study to compared the differentially expressed proteins between ruptured IAs (RIAs) and unruptured IAs (UIAs) using iTRAQ quantitative proteomics, which will provide more insight into the pathogenesis of aneurysm rupture at protein level.
After precipitation with acetone, the proteins (200 μg) of each pool were dissolved with 1 M DTT for 1 h at 37 °C and kept in the dark with 1 M indole-3-acetic acid (IAA) for 1 h at room temperature. Samples were dissolved and centrifuged twice with 120 μL UA (8 M urea in 0.1 M Tris.HCl, pH 8.5), and then re-dissolved and centrifuged three times with 100 M lautyltrethylammonium bromide (LTEAB). The proteins (2–4 μg) were digested with trypsin (trypsin: protein = 1:50; Sigma) and incubated at 37 °C overnight. Each peptide pool was then passed through a 0.2 μm centrifugal filter for 20 min with 10,000 g at 20 °C. Peptide mixture samples were labeled using a 4-Plex iTRAQ Reagent Kit from AB SCIEX. The ruptured and unruptured aneurysmal samples were labeled with 114 and 115 iTRAQ tags, respectively. 2.4. Nano-LC-MS/MS Using the EASY-nLC 1000 system (Thermo Scientific, USA), the labeled peptide mixtures were separated and then were trapped on a PepMap100 C18, 3 μm, 75 μm × 2 cm column (Thermo Scientific, USA). The peptides were then separated on a PepMap100 RSLC C18, 2 μm, 75 μm × 15 cm analytic column by a 70-min mobile phase gradient from 5% to 35%. Mass spectra were recorded on a Q Exative mass spectrometer configured with a Nano-ESI source (Thermo Scientific, USA). Full scan MS spectra were acquired in the m/z range 300–1800 at a resolution of 70,000, the top 10 precursors were selected for high energy collision-induced dissociation (HCD) with collision energy of 27%, and the product ions were detected at a resolution of 17,500 on data-dependent acquisition mode.
2. Materials and methods 2.5. iTRAQ data analysis 2.1. Patients and samples Protein identifications were performed using the MASCOT search engine (version 2.4.1; Matrix Science, London, UK) embedded into Proteome Discoverer 1.3 (Thermo Electron, San Jose, CA, USA). The search parameters were as follows: (1) database, uniprot; (2) taxonomy, Homo sapiens; (3) Enzyme, trypsin; (4) fixed modifications, carbamidomethyl of C, iTRAQ 4plex(N-term), iTRAQ 4plex(K); (5) variable modifications, oxidation of M; (6) max missed cleavages, 2; (7) peptide charges state, +2, +3, and + 4; (8) peptide mass tolerance, 20 ppm; (9)mass/mass tolerance, ± 0.05 Da. Differentially expressed proteins were defined as those reached to 2-fold changes between two groups. The data analysis was supported by Wayen Biotechnologies Co., Ltd. (Shanghai, China).
Twelve patients with saccular IAs (6 ruptured and 6 unrptured) underwent microsurgical clipping in our neurosurgical department were enrolled between October 2015 to June 2017. The exclusion criteria included: (1) patients aged over 70 years old or younger than 18 years old; (2) patients with cerebral infarction and other cerebrovascular disease, including arteriovenous malformation, arteriovenous fistula and cavernous hemangioma; (3) the mean time between SAH and the operation was longer than one month; (4) patients with current or previous history of receiving aspirin treatment. Clinical data was gathered from medical records. The aneurysm tissue samples were harvested during operation after placement of the clip. The mural thrombus was removed and only aneurysmal wall tissues were collected. Tissue samples were transferred to liquid nitrogen immediately and stored at −80 °C until protein extraction. The study was approved by the Ethics Committee of the Department of Medicine, Beijing Tiantan Hospital, Capital Medical University, and all patients gave informed consent to participate in this study.
2.6. Bioinformatics analysis Pathway analysis was performed by the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Gene Ontology (GO) database was used to facilitate the biological interpretation of the identified proteins in these studies. The differentially expressed proteins of GO were divided into 3 categories as follows: biological process (BP), molecular function (MF) and cellular component (CC). CytoScape was used to generate network maps (http://www.cytoscape.org) using the protein-protein interaction (PPI) data from STRING database.
2.2. Protein extraction Each tissue sample was rinsed twice with ice-cold PBS to remove residual blood cells in the aneurysmal wall. The samples were homogenized with RIPA buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1 mM PMSF, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 2% SDS). The homogenates were held on ice for 30 min and centrifuged at 12,000g for 30 min to remove insoluble components. Bicinchoninic acid (BCA) assay was performed to quantify the protein concentration of each sample. The UIA and RIA samples were pooled using the strategy described in previous report [18]. 200 μg of proteins from each group were reduced, alkylated and digested in the centrifugal unit. After digested at 37 °C overnight, the peptide solutions were centrifuged and the filtrates were collected. The digested peptides were dried by vacuum centrifugation and stored at −80 °C until future use.
2.7. Western blotting To validate the accuracy of iTRAQ results, western blotting analysis was performed to examine the relative abundance of some differently expressed proteins again. Proteins extracted from ruptured aneurysms and unruptured aneurysms were separated by SDS-PAGE and then electrotransferred to a PVDF membrane. The membranes were blocked for 1 h at room temperature in PBST containing 5% nonfat dry milk and then incubated with rabbit monoclonal anti-integrin β3 antibody (Cell Signaling Technology), rabbit monoclonal anti-CRABP1 antibody 2
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(Abcam, Cambridge, UK) and rabbit monoclonal anti-S100A9 antibody (Proteintech, Wuhan, China) overnight at 4 °C. Membranes were incubated with secondary antibodies for 1 h at room temperature after washed three times with PBST. Blots were visualized using an ECL detection system and proteins were quantified using a ChemiDoc XRS+ image analyzer (Bio-Rad, Hercules, CA, USA). Densitometric analysis was used to evaluate the expression levels of target proteins. Ratios of densitometric measurements of target proteins relative to β-actin were compared between ruptured and unruptured group. Three experimental replicates were performed and independent Student's t-test was used for assess the statistical significance between the two groups.
Table 2 Comparison of clinical features between ruptured and unruptured group. Characteristics Sex Age Hypertension Smoking history
Male Female Yes No Yes No
Size
RIAs
UIAs
p-Value
2 4 55 ± 11.2 5 1 0 6 9.1 ± 2.4
3 3 44 ± 10.5 2 4 2 4 14 ± 9.6
1.0 0.15 0.24 0.43 0.25
3.2. Identification of differently expressed proteins between RIA and UIA tissues
2.8. Statistical analysis The measurement data are expressed as means and standard deviation for three independent WB experiments. For continuous variables, independent Student's t-test was used for comparisons between the two groups. Categorical variables were compared using the Pearson's ×2 test or Fisher's exact test. P < .05 was considered statistically significant. All statistical analyses were performed by using the statistical software SPSS (version 22, SPSS, Chicago, Illinois, USA).
Six ruptured cerebral aneurysmal tissues and six unruptured cerebral aneurysmal tissues collected from 12 patients were used for iTRAQ assessment. Using the LC-MS/MS method, a total of 2638 proteins were identified and 169 proteins were found statistically differently expressed between RIAs and UIAs. Among these proteins, 74 proteins were significantly up-regulated and 95 proteins were down-regulated in ruptured aneurysm samples with a fold change ≥ 2 and p value ≤ .05. The 20 most significantly up-regulated and 20 most significantly downregulated proteins were shown in Table 3 and Table 4.
3. Results
3.3. Classification of differently expressed proteins
3.1. Clinical information
The PANTHER (protein annotation through evolutionary relationship) classification system was used to further analyze the biological significance of upregulated proteins based on their molecular function, cellular component and biological process. The distribution of the biological processes of the upregulated proteins was shown in Fig. 1A. Classification analyses of cellular components and molecular functions were shown in Fig. 1B and C. A protein-Protein interaction (PPI) network of the 169 differently expressed proteins was constructed by STRING and was shown in Fig. 2. The PPI network analysis revealed that CRABP1 and S100A9 participated in various biological processes such as inflammation while integrin β3 (ITGB3) participate in cell adhesion and signal transduction, which may play an important role in the pathogenesis of many cerebrovascular diseases.
Six patients with ruptured aneurysms and six patients with unruptured were included in this study. The clinical information of all these cases was summarized in Table 1. The male to female ratio was 1:1.4 (5 males and 7 females). The average size of aneurysms was 11.6 mm (ranged from 4.3 to 32.1 mm). The analysis of group homogeneity was shown in Table 2, no significant differences were shown between the two groups. All patients were free of aspirin and statins treatment before surgery. Among those with unruptured aneurysms, the mean observation time before surgery was 3.3 months (ranged from 0.5 to 10 months) and no aneurysm growth was observed during this period. The mean interval from SAH-confirmed diagnosis to treatment was 17.5 days in patients with ruptured aneurysms. No special preoperative therapy was applied in patients with unruptured aneurysms. For those with ruptured aneurysms, nimodipine was commonly used in 5 patients to prevent the occurrence of vasospasm and the average Hunt-Hess score grade at presentation was 2, with a range from 1 to 4 (Table 1). Postoperative ischemic changes were observed in two patients (both with ruptured aneurysms), leaving those two patients with bad prognosis (mRS ≥ 3) at last follow up (ranged from 5 to 24 months).
3.4. KEGG pathway analysis of differently expressed proteins KEGG pathway enrichment analysis was performed to determine the biological processes participating in aneurysm rupture. The top ten most significantly enriched pathways were shown in Table 5. Phagosome, focal adhesion and ECM-receptor pathway were among the top three pathways identified by the pathway analysis.
Table 1 Clinical characterization of 12 patients. Cases
Comorbidities
Cigarette
HH score
Sex
Age (years)
Location
Size (mm)
Ruptured/unruptured
1 2 3 4 5 6 7 8 9 10 11 12
HP HP HP – HP HP Bradycardia – – HP – HP
No No No No No No Yes No No No Yes No
1 2 1 3 4 1 0 0 0 0 0 0
Male Male Female Female Female Female Male Female Female Female Male Male
60 69 58 35 61 48 42 48 28 42 51 59
R-MCA R-PcoA L-MCA AcoA R-ICA R-ICA AcoA L-ICA R-ICA R-ICA AcoA L-MCA
8.2 11.1 10.3 6.4 12.2 6.5 4.3 12.6 32.1 11.7 8.0 15.4
Ruptured Ruptured Ruptured Ruptured Ruptured Ruptured Unruptured Unruptured Unruptured Unruptured Unruptured Unruptured
HH score: Hunt-Hess score, HP: hypertension. 3
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Table 3 Proteins significantly unregulated in RIAs. No.
Protein
Accession no.
Gene name
Protein function
Fold change
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Eosinophil cationic-related protein (Fragment) Protein S100-P Eosinophil peroxidase Resistin Apolipoprotein E Arachidonate 12-lipoxygenase, 12S-type Platelet glycoprotein Ib beta chain Lactoferrin Neutrophil defensin 3 Truncated lactoferrin Myelin P2 protein Thrombocidin-1 antimicrobial variant (Fragment) Integrin beta Integrin alpha-IIb Neutrophil cytosol factor 4 Eosinophil cationic protein cDNA FLJ57557 Alpha-2-antiplasmin Putative neutrophil cytosol factor 1C Ras-related C3 botulinum toxin substrate 2
Q12762 P25815 P11678 Q9HD89 Q8TCZ8 P18054 P13224 B3VMW0 P59666 B2MV14 P02689 D3JV43 L7UUZ7 P08514 Q15080 P12724 B7Z5A7 P08697 A8MVU1 P15153
ECRP S100P EPX RETN APOE ALOX12 GP1BB N/A DEFA3 LTF PMP2 N/A ITGB3 ITGA2B NCF4 RNASE3 N/A SERPINF2 NCF1C RAC2
nucleic acid binding cadherin binding peroxidase activity hormone activity unclassified arachidonate 12-lipoxygenase activity transmembrane signaling receptor activity serine-type endopeptidase activity protein homodimerization activity serine-type endopeptidase activity cholesterol binding chemokine activity receptor activity extracellular matrix binding ARF guanyl-nucleotide exchange factor activity endonuclease activity substrate-specific transmembrane transporter activity endopeptidase inhibitor activity phosphatidylinositol binding protein kinase regulator activity
9.74 8.22 4.99 4.88 4.43 4.23 3.88 3.87 3.66 3.48 3.41 3.40 3.24 3.24 3.22 3.12 3.12 3.12 3.11 3.09
rupture in long period [22–25]. Many previous studies have performed at genome and transcriptome level to identify the related pathophysiological processes underlying aneurysm rupture. It is well known that both expression and function of the related proteins can be influenced by post-translational protein modifications and protein-protein interaction, so studies at proteome level may give a more realistic view of the pathological changes associated with aneurysm rupture. In the present study, we identified 169 proteins differently expressed in ruptured aneurysm tissues compared to unruptured aneurysm tissues. Gene ontology (GO) enrichment analysis revealed that most of the upregulated proteins in ruptured aneurysms belong to cell parts and mainly participate in the cellular process. KEGG pathway analysis revealed that phagosome, focal adhesion and ECM-receptor interaction pathway are the main signaling pathways activated in ruptured aneurysms, suggesting that inflammatory responses and cellmatrix interactions may play a significant role in the rupture of cerebral aneurysms. Several representative differential proteins (ITGB3, CRABP1 and S100A9) were further confirmed by western blotting.
3.5. Validation of differently expressed proteins identified by iTRAQ The expressions of ITGB3, CRABP1 and S100A9 were further evaluated by immunoblotting to validate the accuracy of iTRAQ and LCMS/MS results. As shown in Fig. 3, ITGB3 and S100A9 were unregulated while CRABP1 was down-regulated in the RIAs.
4. Discussion Cerebral aneurysm is a cerebrovascular disorder result from a complex interaction of inherited and acquired factors. The mechanisms underling aneurysm genesis and rupture are very complex and many factors such as shear stress induced nitric oxide production, phenotype change and apoptosis of smooth muscle cell, fluid-structure interaction and genetic factors have been implicated in aneurysm formation and rupture [19–21]. Though many risk factors associated aneurysm rupture have been identified, the specific molecular mechanisms responsible for aneurysm rupture remains poorly understood and this has hampered the development of medical approaches to prevent aneurysm Table 4 Proteins significantly downregulated in RIAs. No.
Protein
Accession no.
Gene name
Protein function
Fold change
1 2 3 4
HLA class I histocompatibility antigen, B-44 alpha chain cDNA FLJ58762 Putative uncharacterized protein DKFZp686K06110 Alpha-2-HS-glycoprotein
P30481 B4DDD5 Q6MZK8 P02765
HLA-B N/A DKFZp686K06110 AHSG
0.13 0.19 0.19 0.20
5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Coagulation factor VII (Fragment) MHC class I antigen Dermatopontin Putative uncharacterized protein DKFZp686H1812 Cellular retinoic acid-binding protein 1 Olfactomedin-like protein 1 Apolipoprotein E Asporin Carnitine O-acetyltransferase (Fragment) Prostaglandin-H2 D-isomerase Olfactomedin-like protein 3 Biglycan cDNA FLJ51023 Carbohydrate sulfotransferase 14 Basement membrane-specific heparan sulfate proteoglycan core protein variant (Fragment) Stromal cell-derived factor 1
A3RKG7 C5IWY0 Q07507 Q5HYE3 P29762 Q6UWY5 P02649 Q9BXN1 H0Y4Z7 P41222 Q9NRN5 A6NLG9 B4DPC8 H0YN65 Q59EG0
N/A HLA-A DPT DKFZp686H1812 CRABP1 OLFML1 APOE ASPN CRAT PTGDS OLFML3 N/A N/A CHST14 N/A
Peptide antigen binding Unclassified Calcium ion binding Cysteine-type endopeptidase inhibitor activity Serine-type endopeptidase activity Immune response Cell adhesion Unclassified Retinoic acid binding Unclassified Cholesterol transporter activity Calcium ion binding Transferase activity Transporter activity Multicellular organism development Collagen fiber assembly Serine-Type Endopeptidase Activity Sulfotransferase activity Calcium ion binding
0.24 0.24 0.25 0.26 0.28 0.28 0.30 0.31 0.31 0.31 0.32 0.32 0.32 0.33 0.33
P48061
CXCL12
Chemoattractant activity
0.34
20
4
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Fig. 1. Classification of upregulated proteins in RIAs by PANTHER program. (A) Biological Process; (B) Celluar Component; (C) Molecular Function.
subarachnoid space [32,33]. The mRNA expression levels of inflammation-related cytokines were significantly increased in experimental SAH models. Delayed cerebral ischaemia (DCI) and angiographic vasospasm may be results of direct damage caused by inflammatory cells infiltration. Previous pathological study revealed that macrophages are the main inflammatory cells infiltrated into the wall. Continuous aneurysm wall remodeling and chronic inflammatory reaction mediated by macrophages may participate in the healing process after aneurysm rupture [34,35]. From this point of view, it is possible that the changed proteins identified in our present study, among which many proteins are associated with inflammatory responses, may be the secondary response caused by subarachnoid hemorrhage. Though above-mentioned opinion may be a possible explanation for the differentially expressed proteins between ruptured and unruptured aneurysms. We should still keep in mind that roles of inflammatory reaction in the initiation and rupture of cerebral aneurysm are not negligible. In a pathological study performed by Kataoka et al. [36], invasion of macrophages and leukocytes in the wall was commonly observed in ruptured aneurysms. But no relationship was observed between the degree of inflammatory responses and the length of time elapsed between rupture onset and specimen harvest, suggesting that inflammatory reaction may exist before aneurysm rupture. Another study performed by Crompton [37] also revealed that inflammatory infiltration was commonly associated with fibrosis, which is usually considered as the end stage of chronic inflammatory response. Recent animal studies also revealed that some pro-inflammatory molecules may act as potential targets for rupture prevention aneurysm intervention [38,39]. Though more studies are needed to confirm the association between inflammation and aneurysm rupture, results of the present study shed new light on the molecular mechanisms underlying the rupture process.
4.1. Pathways involving aneurysm rupture There is growing evidence suggested that the pathobiology of RIAs is substantially distinct from that of UIAs. Different signaling pathways were proved to be involved in the development and progression of cerebral aneurysms. Using microarray technique, Shi et al. found that differentially expressed genes in aneurysm wall participated in focal adhesion, extracellular matrix receptor interaction and cell communication [26]. Consistent with that result, our present study further revealed involvement of focal adhesion and ECM-receptor interaction pathway in aneurysm rupture at the protein levels. Though the exact mechanism underling aneurysm rupture is still unknown, degenerative remodeling characterized by de-endothelializatioin, thinning of aneurysm wall and macrophage infiltration has been widely accepted as the hallmark associated with aneurysm rupture [27,28]. Among these pathologic changes, infiltration of macrophages into aneurysmal walls is increasingly recognised as having a central role in the progression of human cerebral aneurysms [29,30]. A recent study has demonstrated that the expression and activity of focal adhesion kinase (FAK) are markedly enhanced in macrophage which infiltrated to abdominal aortic aneurysm tissue [31]. Their results also suggested that FAK activity is essential to macrophage chemotaxis in response to monocyte chemoattractant protein-1 (MCP-1) and FAK may be a new potential therapeutic target. Thus it is reasonable to conclude that focal adhesion pathway may also plays a crucial role in the inflammatory responses in intracranial aneurysm rupture. 4.2. Association between inflammatory responses and aneurysm rupture As a cause-effect relationship between the proteomic profile change and aneurysm rupture is hard to be determined by a cross-sectional study, it is reasonable to speculate that proteomic profile changes in the aneurysmal wall may result from the early brain injury following subarachnoid hemorrhage. Many pathological processes can be initiated by SAH due to release of haemoglobin and erythrocyte contents to the 5
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Fig. 2. Protein-Protein interaction (PPI) network of 169 differently expressed proteins between ruptured and unruptured aneurysms. Proteins selected for further western blot validation were indicated with filled circles.
confirmed by western blotting, indicating that these molecules participate in the occurrence of aneurysms rupture and may work as potential therapeutic targets in the future. ITGB3 is an important subunit of transmembrane integrin receptor which can mediate signal transduction between cell and extracellular microenvironment [40]. As aneurysms development and rupture are mechanobiological processes influenced by hemodynamic stress and wall strength, receptors that connect ECM to intracellular signaling molecules may play a central role in the progress. By combing with extracellular ligands such as fibronectin, laminin and matrix metalloproteinase-2, integrins can activate RhoA/RhoKinase and MAPK signaling pathways and may participate in the wall remodeling changes [41,42]. Further investigations are needed to elucidate their precise biological mechanisms. The S100 calcium-binding protein A9 (S100A9), a member of the S100 calcium binding protein family, is a recently identified member of the damage associated molecular pattern (DAMP) family [43]. S100A9 is a cytosolic protein mainly produced by myeloid cells and plays a
Table 5 Top ten significantly enriched pathways identified by KEGG pathway analysis. No.
Term
ID
Count
%
1 2 3 4 5 6 7 8 9 10
Phagosome Focal adhesion ECM-receptor interaction Platelet activation Cell adhesion molecules (CAMs) Leukocyte transendothelial migration Complement and coagulation cascades Hematopoietic cell lineage Staphylococcus aureus infection Asthma
hsa04145 hsa04510 hsa04512 hsa04611 hsa04514 hsa04670 hsa04610 hsa04640 hsa05150 hsa05310
13 12 11 11 10 8 7 7 6 5
10.2 9.4 8.6 8.6 7.8 6.2 5.5 5.5 4.7 3.9
4.3. Differential expressed proteins between ruptured and unruptured aneurysms Alternations in expression levels of interested proteins were 6
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during surgery. In addition, further investigations are needed to validate the other proteins and pathways identified in our present study before drawing definitive conclusions. 5. Conclusion Our results showed that inflammatory responses and cell-matrix interactions may play a significant role in the rupture of cerebral aneurysms. ITGB3, CRABP1 and S100A9 were differentially expressed between ruptured and unruptured aneurysm and they may participate the pathological process of aneurysm rupture. The present study provides a more comprehensive view of protein expression difference between RIAs and UIAs, and the pathway analysis allows a better understanding of pathophysiological mechanisms associated with aneurysm rupture, which will shed light on the prevention of cerebral aneurysm rupture. Conflict of interest All the authors have approved the enclosed manuscript and declare that there is no conflict of interests regarding this article. Acknowledgments This work was supported by the National Key Research and Development Program of China (Grant No. 2016YFC1301800) and the projects of National Natural Science Foundation of China (Grant No. 81471210 and 81671129).
Fig. 3. Western blot validation of selected proteins that differentially expressed between ruptured intracranial aneurysms and unruptured intracranial aneurysms. The expression levels of ITGB3, CRABP1 and S100A9 revealed by western blotting analysis. B. Quantitative assessment of protein expression using densitometric analysis. Error bars represent the standard deviation for three experimental replicates and asterisk indicates p < .05.
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prominent role in the regulation of inflammatory response. S100 protein plasma levels were found to be significantly higher in patients suffering from SAH and be associated with early neurological deficit [44]. Recent study also revealed that gene expression level of S100A9 was significantly upregulated in ruptured cerebral aneurysms and it was assumed that S100A9 could be an important contributor to the rupture of intracranial aneurysms [45]. In agreement with previous studies, our present study further demonstrated that the expression of S100A9 protein increased in ruptured aneurysms. Similar to other S100 proteins identified as DAMPs, S100A9 is an intracellular protein involved in inflammatory process and immune response and is closely related to the development and severity of cardiovascular disease (CVD) [46]. S100A9 also plays a critical role in the occurrence of atherothrombosis by amplificating the inflammatory reaction, and S100A8 and S100A9 gene-deleted mice showed an significant decrease in atherosclerotic lesions. These properties suggested that S100A9 could be an important moderator of aneurysms and may be a possible target for therapy against human intracranial aneurysms. Cellular retinoic acid-binding protein 1 (CRABP1) is a small cytosolic protein encoded by CRABP1 gene and it plays a dominant role in the modulation of retinoic acid (RA) signaling pathway [47]. Because of the potential role of RA in regulating cell growth and differentiation, retinoic acid and retinoid receptors expression have been reported to be associated with initiation and progression of several human tumors [48]. Our present study revealed that CRABP1 was down-regulated remarkably in ruptured aneurysm tissues, therefore it is possible that CRABP1 may participate in the progression of cerebral aneurysms through abnormal growth regulation. Several limitations exist in the present study. First, the total number of included aneurysm samples is relatively small because of difficulties in collecting IA samples. Sample selection bias may exist in our present study due to only relatively large aneurysm samples could be obtained 7
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