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). Post-treatment 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.
Highlights
Cerebral arteriovenous malformations (AVMs) are vascular anomalies with an average hemorrhage risk of2–4% per year cumulative over a patient’s lifetime, and a 25% fatality risk with each bleed[1]
We found that the level of AVM-derived endothelial cells (AVMECs) mesenchymal impurity was comparable to that of commercially available endothelial cell populations, and it was similar to the human brain microvascular endothelial cells (HBMVECs) control line
The research described here increases the understanding of the role of axon guidance factors (AGFs) signaling in AVM pathology, establishes the importance of a specific EphrinB2:EphB4 ratio in vasculogenesis, and reveals a relative increase in EphrinB2 in AVMs as a result of imbalances in the Ephrin signaling ratio
Summary
Cerebral arteriovenous malformations (AVMs) are vascular anomalies with an average hemorrhage risk of2–4% per year cumulative over a patient’s lifetime, and a 25% fatality risk with each bleed[1]. Cerebral arteriovenous malformations (AVMs) are vascular anomalies with an average hemorrhage risk of. Fehnel et al Experimental & Molecular Medicine (2020) 52:658–671 techniques and novel therapeutic targets to reduce risk and improve patient outcomes, in the pediatric population. Ephrins are cell surface proteins that are readily quantifiable and potentially amenable to manipulation; they play a critical role in normal cerebrovascular development, proliferation, migration, and adhesion, through complex bidirectional signaling (forward through EphB4 receptor, reverse through EphrinB2 ligand). Aggregate binding can alter the strength of signaling with downstream effects influenced by the ratio of ligand to receptor. These data suggest that a balance of ligand to receptor is critical to influencing downstream vascular fate
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