We investigated (1) EphrinB2 and EphB4 receptor expression in cerebral AVMs, (2) the impact of an altered EphrinB2: EphB4 ratio on brain endothelial cell function and (3) potential translational applications of these data. The following parameters were compared between AVM endothelial cells (AVMECs) and human brain microvascular endothelial cells (HBMVECs): quantified EphrinB2 and EphB4 expression, angiogenic potential, and responses to manipulation of the EphrinB2:EphB4 ratio via pharmacologic stimulation/inhibition. To investigate the clinical relevance of these in vitro data, Ephrin expression was assessed in AVM tissue (by immunohistochemistry) and urine (by ELISA) from pediatric patients with AVM (n = 30), other cerebrovascular disease (n = 14) and control patients (n = 29), and the data were subjected to univariate and multivariate statistical analyses. Compared to HBMVECs, AVMECs demonstrated increased invasion (p = 0.04) and migration (p = 0.08), impaired tube formation (p = 0.06) and increased EphrinB2:EphB4 ratios. Altering the EphrinB2:EphB4 ratio (by increasing EphrinB2 or blocking EphB4) in HBMVECs increased invasion (p = 0.03 and p < 0.05, respectively). EphrinB2 expression was increased in AVM tissue, which correlated with increased urinary EphrinB2 levels in AVM patients. Using the optimal urinary cutoff value (EphrinB2 > 25.7 pg/μg), AVMs were detected with high accuracy (80% vs. controls) and were distinguished from other cerebrovascular disease (75% accuracy). Posttreatment urinary EphrinB2 levels normalized in an index patient. In summary, AVMECs have an EphrinB2:EphB4 ratio that is increased compared to that of normal HBMVECs. Changing this ratio in HBMVECs induces AVMEC-like behavior. EphrinB2 is clinically relevant, and its levels are increased in AVM tissue and patient urine. This work suggests that dysregulation of the EphrinB2:EphB4 signaling cascade and increases in EphrinB2 may play a role in AVM development, with potential utility as a diagnostic and therapeutic target.

OBJECTIVE The authors report the use of urinary biomarkers as a novel, noninvasive technique to detect juvenile pilocytic astrocytomas (JPAs), capable of distinguishing JPAs from other CNS diseases, including other brain tumors. Preliminary screening of an array of tumors implicated proteases (including matrix metalloproteinases [MMPs]) and their inhibitors (tissue inhibitors of metalloproteinase [TIMPs]) as well as growth factors (including basic fibroblast growth factor [bFGF]) as candidate biomarkers. These data led the authors to hypothesize that tissue inhibitor of metalloproteinase 3 (TIMP3) and bFGF would represent high-probability candidates as JPA-specific biomarkers. METHODS Urine was collected from 107 patients, which included children with JPA (n = 21), medulloblastoma (n = 17), glioblastoma (n = 9), arteriovenous malformations (n = 25), moyamoya (n = 14), and age- and sex-matched controls (n = 21). Biomarker levels were quantified with enzyme-linked immunosorbent assay, tumor tissue expression was confirmed with immunohistochemical analysis, and longitudinal biomarker expression was correlated with imaging. Results were subjected to univariate and multivariate statistical analyses. RESULTS Using optimal urinary cutoff values of bFGF > 1.0 pg/μg and TIMP3 > 3.5 pg/μg, multiplexing bFGF and TIMP3 predicts JPA presence with 98% accuracy. Multiplexing bFGF and MMP13 distinguishes JPA from other brain tumor subtypes with up to 98% accuracy. Urinary biomarker expression correlated with both tumor immunohistochemistry and in vitro tumor levels. Urinary bFGF and TIMP3 decrease following successful tumor treatment and correlate with changes in tumor size. CONCLUSIONS This study identifies 2 urinary biomarkers—bFGF and TIMP3—that successfully detect one of the most common pediatric brain tumors with high accuracy. These data highlight potential benefits of urinary biomarkers and support their utility as diagnostic tools in the treatment of children with JPA.

Introduction: Diagnosis of moyamoya requires imaging which include risks associated with anesthesia and angiography. We describe the novel application of non-invasive urinary biomarkers to assess preoperative ischemia, development of transdural collaterals and predict 1-year angiographic outcomes from preoperative samples. Hypothesis: We tested the hypothesis that urinary levels of netrin-1 and vascular endothelial growth factor (VEGF) correlate with disease severity at diagnosis and prospectively identify patients better able to grow surgical collaterals, as quantified by Matsushima grade. Methods: Preoperative urine samples were collected from patients (age<22 years). Radiographic data (Suzuki stage, presence/location of preoperative transdural collaterals and postoperative Matushima grade) were performed by neuroradiologists and obtained from clinical records. Quantitative analysis of protein levels was performed with ELISA, normalizing for protein and subjected to statistical comparison between groups. Results: A total of 132 patients with moyamoya had samples and imaging for analysis. Urinary VEGF and netrin-1 levels were significantly elevated in moyamoya patients compared to matched controls (6.6 vs. 4.4pg/ug and 0.6 vs. 2.3pg/ug, p<0.05 respectively). In patients without transdural collaterals, VEGF and netrin-1 urinary levels were higher than in those with collaterals (n=68 without, 64 with, p= 0.03 netrin, NS VEGF) Higher preoperative urine levels of both VEGF and netrin-1 correlated with better postoperative Matsushima grade (n=38,29,16 for Matsushima A,B,C, p<0.01 for VEGF and p<0.05 for netrin-1). Conclusions: Urinary levels of VEGF and netrin-1 are elevated in moyamoya patients and may correlate with an increased angiogenic drive that is higher in patients without spontaneous collaterals (who may be more ischemic than those who have already established collaterals) and whose presence may aid in development of surgical collaterals after indirect revascularization. These experiments provide proof-of-principle data that urinary biomarkers may have utility in augmenting clinical decision-making.

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