Identification of the soluble form of tyrosine kinase receptor Axl as a potential biomarker for intracranial aneurysm rupture

Intracranial aneurysms (IAs) are a common type of cerebrovascular disorder with a high prevalence of 1-5% in the general population [1]. Although only 1-2% of IAs rupture annually, such a rupture can cause subarachnoid hemorrhage (SAH), a devastating clinical consequence with high mortality (25-40%) and morbidity (~50%) constituting a major public health problem [2,3]. In contrast, IAs that are repaired before they rupture by microsurgical clipping or by endovascular coiling have a reported mortality as low as 0.6% and morbidity < 5% [4,5]. Therefore, it would be advantageous to discover reliable predictive markers for screening individuals at risk for the development or rupture of an IA.

Recently, much work has focused on identifying susceptibility genes using genome-wide DNA linkage analyses or examining the single-nucleotide polymorphisms of individual genes that might be related to the development of IAs and their subsequent rupture [6-8]. Unfortunately, these studies have frequently yielded conflicting results and associations have not been replicated in other populations despite decades of research. These discrepancies may be due to genetic heterogeneity and a lack of knowledge of the molecular pathogenesis of IA formation, progression, and rupture. The molecular alterations within an aneurysm i.e. the dynamic molecular environment of the vasculature controlled by gene expression, may prove to be more predictive in distinguishing individuals at high risk of IA development or rupture. A series of human and animal studies of IA tissues using gene expression microarrays have highlighted the molecular changes resulting in endothelial dysfunction, the inflammatory response and degeneration of the vascular wall [9]. Although discovering the molecular markers in IA tissue has shed light on pathogenesis, it is neither practical nor feasible to use these molecules as biomarkers for screening individuals at risk for the development or IA rupture unless they can be confirmed in body fluids such as plasma and cerebrospinal fluid (CSF), which are easily accessible and ideal sources for biomarker discovery.

CSF is an important target for proteomic discovery of the disease-specific biomarkers of many brain disorders such as neurodegenerative diseases and traumatic brain injury [10,11]. Several markers have been identified in CSF that could predict susceptibility to aneurysmal SAH and post-SAH cerebral vasospasm [12-14]. However, the comprehensive characterization of CSF is still challenging because of its complexity and the high dynamic range of protein concentrations. To circumvent this difficulty, we focused on one subproteome of CSF: glycosylated proteins. Protein glycosylation is an abundant and biologically important post-translational modification. It is most commonly associated with membrane and secreted proteins. Indeed, many clinical markers and therapeutic targets such as Her2/neu (breast cancer) and prostate-specific antigen (prostate cancer) are glycoproteins [15,16]. In this study, we used a complementary proteomic approach which integrated the lectin-affinity column for glycoprotein enrichment and a multiplex quantitative proteomic method called iTRAQ (isobaric Tagging for Relative and Absolute protein Quantification), to simultaneously quantify relative changes in the glycoproteins of CSF obtained from patients with ruptured IAs (RIAs) and unruptured IAs (UIAs) compared to healthy controls (HCs) and disease controls (DCs). First, we identified a number of proteins that displayed quantitative changes unique to RIA, UIA or IA (discovery cohort, n = 40). Then we confirmed the proteomic results in both CSF and plasma from a larger and independent cohort (validation cohort, n = 80). Finally, we tested the specificity and sensitivity of the candidate marker in CSF and plasma to evaluate whether it could be directly applied as a biomarker of IA rupture.

Extracellular proteins, including secreted, cell surface and transmembrane proteins are mostly glycosylated and likely to enter the body fluids to serve as potential biomarkers. Lectin-based approaches such as lectin affinity chromatography have been widely used in the discovery of glycoprotein biomarkers in diseases such as hepatocellular carcinoma [18], ovarian tumors [19] and prostate cancer [20]. In the present study, we applied lectin-affinity enrichment of glycoproteins coupled with quantitative iTRAQ-labeling proteomic techniques to discover candidate biomarkers for predicting the risk of development or rupture of IA. Using this quantitative glycoproteomics, we identified CSF proteins either distinctively changed in RIA or UIA or commonly changed in RIA and UIA. The former could be used as candidate biomarkers to predict the risk of IA rupture, while the latter could be used to distinguish individuals at risk of IA formation.

A total of 73 CSF proteins that were differentially expressed in IA compared to controls were identified. These candidate markers fall into all 11 functional categories: 1) immune/inflammatory response, 2) cell adhesion, 3) ECM, 4) protein synthesis or protein degradation, 5) transport, 6) apoptosis and anti-apoptosis, 7) cell proliferation/differentiation/migration, 8) signal transduction, 9) metabolism, 10) cell structure/cytoskeleton, and 11) unknown functions. Among these proteins, some have been implicated in the pathogenesis of IA in previous studies, such as the complement components C1, C3 and C5 [21,22] and the ECM remodeling-related proteins ECM1 and TIMP1 [23]. Furthermore, we also found a number of proteins that have never been reported but are involved in the injury and/or repair of vascular epithelial cells, cell adhesion and migration, and angiogenesis, such as pigment epithelium-derived factor (PEDF) [24], nectin-like protein 2 (NECL2) [25], ephrin A1 [26], neogenin [27], acheron [28], lumican [29] and Axl [30-33].

Of these novel potential biomarkers, receptor protein kinase Axl was distinctively increased in RIA and its differential expression was confirmed in an independent cohort not only in CSF but also in the paired plasma. Axl, which was originally cloned from cancer cells, is broadly expressed in a variety of cells including vascular epithelial cells, smooth muscle cells and fibroblasts [32,33]. Overexpression and an increase of Axl activity have been found in cancer, chronic immune disorders and cardiovascular diseases [30,32,34,35]. Axl-dependent signaling regulates various functions, including survival, growth, aggregation, migration and anti-inflammation, in multiple cell-types. The mechanisms of Axl receptor activation/inactivation are not completely understood. Typically, growth-arrest-specific protein 6 (Gas6), the ligand with the highest affinity for Axl, binds to Axl receptor to form an initial 1:1 Gas6-Axl complex followed by the dimerization of two 1:1 Gas6-Axl complexes, which is a ligand-dependent activation that usually occurs under physiological conditions [32]. During pathophysiological conditions with increased oxidative stress and experimental receptor overexpression, Axl can be activated and autophosphorylated via ligand-independent homophilic dimerization [36]. Furthermore, release of a soluble form of Axl (sAxl), the extracellular domain of Axl, is another important feature of Axl receptor biology. A specific proteinase that mediates the cleavage of sAxl has yet to be identified. Axl signaling has mainly been implicated in cancer. However, a rapid increase in publications supports the importance of Axl in other chronic pathological conditions [34,35]. A correlation of plasma concentrations of Gas6 and sAxl with disease activity in systemic lupus erythematosus has recently been reported [31]. Ekam et al. found that plasma Gas6 correlated positively and sAxl correlates negatively with abdominal aortic aneurysms (AAA) size and the Gas6/sAxl ratio may be useful as an AAA biomarker [37]. Nevertheless, more studies are required to determine the role of Axl in the pathogenesis of IA formation, progression, and rupture, and Axl may be a promising biomarker for predicting the risk of IA rupture upon further validation in larger cohorts.

There are some potential limitations in our study. First, we used patients who had completely recovered from head trauma for at least six months as HCs. They may not be considered as “really healthy” controls even if all the results of the physical and neurological examinations and laboratory tests were within the normal range. The longest time period reported in previous studies to analyze inflammatory factors or cytokines in CSF was 22 days and they had returned the normal range by this time [38]. Most of the control patients we recruited had recovered from head trauma for about one year, in order to reduce the effect of disease as much as possible. Second, the CSF samples from the RIA group were collected one month after endovascular embolization or a neurosurgical operation, since contamination of CSF with blood could significantly confound the interpretation of quantitative or qualitative proteomic analysis of CSF. This might not be the ideal time point to collect CSF for RIA. However, considering the inability to collect RIA CSF samples before rupture, it is still reasonable at the discovery step. Moreover, the blood samples of RIA were taken within 2 h after the patients were admitted to hospital. Our results demonstrated that the sAxl level was not only elevated in CSF but also in plasma, which excluded the chance that the increase of sAxl was a result of a restorative process after aneurysm rupture. Third, since most of our UIA patients were treated with coiling or clipping, it is possible that some of them had a risk of rupture. It is interesting that two follow-up UIA patients with ophthalmic artery aneurysms had a relatively low concentration of CSF sAxl (0.076 nM and 0.043 nM). Therefore, next step we will measure the dynamic sAxl levels in CSF and plasma from patients harboring incidental IA and follow-up with CTA every 6 months looking for a correlation between the sAxl levels and the anatomical and/or morphological changes in the aneurysm as signs of impending rupture. So far, we have not recruited enough patients to draw conclusions. Finally, the sensitivity and specificity of plasma sAxl is probably not sufficient to identify RIA patients although the overall expression levels of sAxl from RIA were significantly higher than UIAs and controls. On the one hand, a larger population is needed for further validation. On the other hand, more candidate biomarkers need to be validated.

In summary, we successfully used lectin-affinity purification and iTRAQ quantitative proteomics approaches to identify candidate biomarkers for predicting the risk of development or rupture of IAs. One candidate biomarker, sAxl, was confirmed to be elevated both in CSF and plasma of RIA patients. The sAxl levels in CSF discriminated RIA from UIA with relatively high sensitivity and specificity, indicating that sAxl might serve as a promising biomarker to predict the rupture of IA.