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Vascular endothelial growth factor (VEGF), a potent mediator of endothelial proliferation and migration, has an important role also in brain edema formation during hypoxia and ischemia. VEGF binds to the tyrosine kinase receptors Flt-1 and Flk-1. Yet, their relative importance for hypoxia-induced hyperpermeability is not well understood. We used an in vitro blood-brain barrier (BBB) model consisting of porcine brain microvascular endothelial cells (BMEC) to determine the role of Flt-1 in VEGF-induced endothelial cell (EC) barrier dysfunction. Soluble Flt-1 abolished hypoxia/VEGF-induced hyperpermeability. Furthermore, selective antisense oligonucleotides to Flt-1, but not to Flk-1, inhibited hypoxia-induced permeability changes. Consistent with these data, addition of the receptor-specific homolog placenta-derived growth factor, which binds Flt-1 but not Flk-1, increased endothelial permeability to the same extent as VEGF, whereas adding VEGF-E, a viral VEGF molecule from the orf virus family activating Flk-1 and neuropilin-1, but not Flt-1, did not show any effect. Using the carcinoma submandibular gland cell line (CSG), only expressing Flt-1, it was demonstrated that activation of Flt-1 is sufficient to induce hyperpermeability by hypoxia and VEGF. Hyperpermeability, induced by hypoxia/VEGF, depends on activation of phosphatidylinositol 3-kinase/Akt (PI3-K/Akt), nitric oxide synthase (NOS) and protein kinase G (PKG). The activation of the PI3-K/Akt pathway by hypoxia was confirmed using an in vivo mice hypoxia model. These results demonstrate that hypoxia/VEGF-induced hyperpermeability can be mediated by activation of Flt-1 independently on the presence of Flk-1 and indicate a central role for activation of the PI3-K/Akt pathway, followed by induction of NOS and PKG activity.

In vivo, ischemia is known to damage the blood-brain barrier (BBB) leading to the development of vasogenic brain edema. Hypoxia-induced vascular endothelial growth factor (VEGF) has been shown to be a key regulator of these permeability changes. However, the signaling pathways that underlie VEGF-induced hyperpermeability are incompletely understood. In this study, we demonstrate that hypoxia- and VEGF-induced permeability changes depend on activation of phospholipase Cgamma (PLCgamma), phosphatidylinositol 3-kinase/Akt (PI3-K/Akt), and protein kinase G (PKG). Inhibition of mitogen-activated protein kinases (MAPK) and of the protein kinase C (PKC) did not affect permeability at all. Paralleling hypoxia- and VEGF-induced permeability changes, localization of the tight junction proteins occludin, zonula occludens-1 (ZO-1), and ZO-2 along the cell membrane changed from a continuous to a more discontinuous expression pattern during hypoxia. In particular, localization of ZO-1 and ZO-2 expression moved from the cell membrane to the cytoplasm and nucleus whereas occludin expression remained at the cell membrane. Inhibition of PLCgamma, PI3-kinase, and PKG abolished these hypoxia-induced changes. These findings demonstrate that hypoxia and VEGF induce permeability through rearrangement of endothelial junctional proteins which involves activation of the PLCgamma and PI3-K/AKT pathway leading to the activation of PKG.