Abstract
Intracranial vascular malformations manifest on a continuum ranging from predominantly arterial to predominantly venous in pathology. Cerebral cavernous malformations (CCMs) are capillary malformations that exist at the midpoint of this continuum. The axon guidance factor Ephrin B2 and its receptor EphB4 are critical regulators of vasculogenesis in the developing central nervous system. Ephrin B2/EphB4 dysregulation has been implicated in the pathogenesis of arterial-derived arteriovenous malformations and vein-based vein of Galen malformations. Increasing evidence supports the hypothesis that aberrant Ephrin B2/EphB4 signaling may contribute to developing vascular malformations, but their role in CCMs remains largely uncharacterized. Evidence of Ephrin dysregulation in CCMs would be important to establish a common link in the pathogenic spectrum of EphrinB2/Ephb4 dysregulation. By studying patient-derived primary CCM endothelial cells (CCMECs), we established that CCMECs are functionally distinct from healthy endothelial cell controls; CCMECs demonstrated altered patterns of migration, motility, and impaired tube formation. In addition to the altered phenotype, the CCMECs also displayed an increased ratio of EphrinB2/EphB4 compared to the healthy endothelial control cells. Furthermore, whole exome sequencing identified mutations in both EphrinB2 and EphB4 in the CCMECs. These findings identify functional alterations in the EphrinB2/EphB4 ratio as a feature linking pathophysiology across the spectrum of arterial, capillary, and venous structural malformations in the central nervous system while revealing a putative therapeutic target.
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Data Availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
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Acknowledgements
Be Brave for Life, Kids@Heart Fund, Chae Fund, Irving Fund, Lucas Warner Research Fund. We thank Dr. Keith Ligon, Cecilia Sousa, and Jayne Vogelzang from the Pediatric Neuro-Oncology/Neuro-Pathology at Dana Farber Cancer Institute and the Pathology Department at Boston Children's Hospital for providing the CCM patient tissue samples. We thank Caitlin Edwards and the Brigham and Women’s Hospital Imaging Core for their assistance with the histopathology staining. We thank Psomagen for performing and analyzing the WES.
Funding
This work was funded by Be Brave for Life, Kids@Heart Fund, Chae Fund, Irving Fund, Lucas Warner Research Fund.
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KF and ERS: designed the study and provided funding. JS, AG, JD, and TM: conducted the experiments and acquired the data. JS, AG, and AL: analyzed the data. SA: provided the patient samples. KF, ERS, and JS: wrote and reviewed the manuscript.
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10571_2023_1447_MOESM1_ESM.tif
Supplementary file1 (TIF 4729 KB) Supplementary Figure 1 These images represent five different CCM patient cell lines at confluency. Images were taken on the ECHO Rebel-18 Microscope at 4X objective. Scale bar added for size determination.
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Supplementary file2 (TIF 1696 KB) Supplementary Figure 2 Immunoblot representing the full western blots that are depicted in Fig. 4. A–C are representations of the same immunoblot with different protein expressions. A Shows the EphrinB4 band between 100 and150 kD. B Shows the Ephrin B2 band at 50 kD and the αSMA band at 42 kD. C Shows the GAPDH band at 37kD.
10571_2023_1447_MOESM3_ESM.tiff
Supplementary file3 (TIFF 1045 KB) Supplementary Figure 3 Immunoblots representing the expression of CD31 and alpha-SMA in normal endothelial cells (HUVECs and HBMVEC) and in CCM patient cells. GAPDH is used as a loading control.
10571_2023_1447_MOESM4_ESM.xlsx
Supplementary file4 (XLSX 17 KB) Supplemental File 1 Analyses using nonparametric testing, the test statistic (Z), and the p-value from the Wilcoxon rank sum test are reported. Z values and p values are shown in the results tables.
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Sesen, J., Ghalali, A., Driscoll, J. et al. Discovery and Characterization of Ephrin B2 and EphB4 Dysregulation and Novel Mutations in Cerebral Cavernous Malformations: In Vitro and Patient-Derived Evidence of Ephrin-Mediated Endothelial Cell Pathophysiology. Cell Mol Neurobiol 44, 12 (2024). https://doi.org/10.1007/s10571-023-01447-0
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DOI: https://doi.org/10.1007/s10571-023-01447-0