Dysregulation of the EphrinB2−EphB4 ratio in pediatric cerebral arteriovenous malformations is associated with endothelial cell dysfunction in vitro and functions as a novel noninvasive biomarker in patients

Abstract

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.

Non-invasive Urinary Biomarkers in Moyamoya Disease

“…Our laboratory has extensive experience with the identification and validation of urinary biomarkers in cerebrovascular disease, cancer and the central nervous system (CNS), including the first report of successfully applying this novel methodology specifically to brain tumors in a multicenter trial ( 3 – 10 ). Current data from our lab and others supports the hypothesis of angiogenesis and extracellular matrix (ECM) remodeling as essential processes in many CNS disorders, including moyamoya disease, involving vascular endothelial growth factor (VEGF), matrix metalloproteinases (MMPs), a multigene family of degradative enzymes and neutrophil gelatinase associated lipocalin (NGAL), an enzyme that binds with MMP-9 to protect it from degradation—and elevated levels of MMPs and VEGF have been reported in the serum of moyamoya disease patients ( 2 , 4 , 5 , 22 – 30 ).…”

“…In addition, some markers—like the ones in this study—may be elevated immediately after stroke, but typically normalize after a short window of a few weeks, making the timing of biomarker sampling important and which we have addressed in the selection of our patient cohort to minimize confounding ( 11 , 13 – 15 ). Diagnostic specificity can be improved by looking at a broader range of molecules and multiplexing several proteins in combination to create a biomarker “fingerprint” that is unique to a given disease, and we have done this for other CNS vascular and tumor cohorts, including moyamoya disease ( 3 – 5 , 10 , 49 ). In particular, we anticipate that molecules that regulate mechanisms of vascular morphogenesis and arteriogenesis (including, but not limited to, axon guidance factors, for example), may be useful as future areas of investigation to enhance the functionality of biomarker profiling in moyamoya disease.…”

“…First, we subjected the urinary data to rigorous statistical analysis, performed by a biostatistician, in order to ensure the robustness of our results. Second, we obtained matched CSF samples from the same patients, in order to establish that the biomarker profile in the CNS matched the same pattern seen in the urine, a technique validated in the literature and—to our knowledge—unique in its application to moyamoya disease as described here ( 3 – 5 ). Third, we provide longitudinal data that demonstrates that the urinary biomarker profile changes in direct response to changes in disease status after treatment, as corroborated by radiographic studies and also with precedent in the literature ( 3 – 6 , 10 , 49 ).…”

“…Our laboratory has had a longstanding interest in the development of non-invasive biomarkers designed to aid in the diagnosis, prognosis and therapy of tumors and cerebrovascular disease, including biomarker “fingerprints” that can distinguish between central nervous system tumors, moyamoya disease and arteriovenous malformations ( 3 – 10 ). Here we present initial proof-of-principle data demonstrating that a novel, non-invasive panel of urinary biomarkers can identify the presence of moyamoya disease and that the biomarkers track from the central nervous system in cerebrospinal fluid (CSF) to urine.…”

Adverse effects of erenumab on cerebral proliferative angiopathy: A case report

“…This is unlike brain AVMs, which are characterized by higher flow than CPA, with more brisk arteriovenous shunting, and which do not demonstrate intervening brain parenchyma within their boundaries) (1). Evidence suggests that endothelial cell function and the normal blood brain barrier (BBB) may be dysregulated in these dynamic, congenital vascular lesions (2,3). These data have led to the exclusion of patients with suspicion of cerebral vascular malformation from the Phase 2 and 3 studies of erenumab, a new anti-calcitonin gene related protein (CGRP) receptor monoclonal antibody (anti-CGRPr-mAbs) approved by the FDA for the treatment of chronic migraine.…”