Endothelium of the cerebral blood microvessels, which constitutes the major component of the blood-brain barrier, controls leukocyte and metastatic cancer cell adhesion and trafficking into the brain parenchyma. In this study, using rat primary brain microvascular endothelial cells (BMEC), we demonstrate that the vascular endothelial growth factor (VEGF), a potent promoter of angiogenesis, up-regulates the expression of the intracellular adhesion molecule-1 (ICAM-1) through a novel pathway that includes phosphatidylinositol 3 OH-kinase (PI3K), AKT, and nitric oxide (NO), resulting in the migration of BMEC. Upon VEGF treatment, AKT is phosphorylated in a PI3K-dependent manner. AKT activation leads to NO production and release and activation-deficient AKT attenuates NO production stimulated by VEGF. Transfection of the constitutive myr-AKT construct significantly increased basal NO release in BMEC. In these cells, VEGF and the endothelium-derived NO synergistically up-regulated the expression of ICAM-1, which was mediated by the PI3K pathway. This activity was blocked by the PI3K-specific inhibitor, wortmannin. Furthermore, VEGF and NO significantly increased BMEC migration, which was mediated by the up-regulation of ICAM-1 expression and was dependent on the integrity of the PI3K/AKT/NO pathway. This effect was abolished by wortmannin, by the specific ICAM-1 antibody, by the specific inhibitor of NO synthase, N(G)-l-monomethyl-arginine (l-NMMA) or by a combination of wortmannin, ICAM-1 antibody, and l-NMMA. These findings demonstrate that the angiogenic factor VEGF up-regulates ICAM-1 expression and signals to ICAM-1 as an effector molecule through the PI3K/AKT/NO pathway, which leads to brain microvessel endothelial cell migration. These observations may contribute to a better understanding of BMEC angiogenesis and the physiological as well as pathophysiological function of the blood-brain barrier, whose integrity is crucial for normal brain function.
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