Morphological Observation and In Vitro Angiogenesis Assay of Endothelial Cells Isolated From Human Cerebral Cavernous Malformations

Abstract

Little is known about the role of endothelial cells (ECs) in the pathogenesis of cerebral cavernous malformation because of the difficulties to obtain highly pure ECs. Thus, this study attempted to establish a reliable procedure to isolate and culture ECs from human cerebral cavernous malformation lesions. The biological features and the angiogenic potential of the cultured ECs were also investigated. A modified protocol was developed to isolate and culture cerebral cavernous malformation endothelial cells (CECs)from surgically resected human specimens. The biological features of CECs were investigated by electron microscope, immunostaining, real-time polymerase chain reaction, fluorescence-activated cell sorter, and Western blotting. The tube formation by CECs was examined in an in vitro angiogenesis model with or without the addition of vascular endothelial growth factor. CECs from the specimens unaffected by the intraoperative bipolar coagulation were cultivated successfully with higher than 95% purity. Comparing to the ECs from control brain tissue, CECs presented primitive nucleus in ultrathin section, expressed higher levels of vascular endothelial growth factor receptor-1 and vascular endothelial growth factor receptor-2, and spontaneously formed tube structures in a 3-dimensional collagen matrix. The tube formation by CECs was significantly promoted by vascular endothelial growth factor treatment. A modified protocol for the attainment of purified CECs and the first in vitro angiogenesis model of CECs were successfully established. We provided initial evidence that CECs had enhanced angiogenic potential and showed increased responsiveness to vascular endothelial growth factor.