The blood-brain barrier (BBB) is essential for maintaining brain homeostasis and low permeability. BBB maintenance is important in the central nervous system (CNS) because disruption of the BBB may contribute to many brain disorders, including Alzheimer disease and ischemic stroke. The molecular mechanisms of BBB development remain ill-defined, however. Here we report that src-suppressed C-kinase substrate (SSeCKS) decreases the expression of vascular endothelial growth factor (VEGF) through AP-1 reduction and stimulates expression of angiopoietin-1 (Ang-1), an antipermeability factor in astrocytes. Conditioned media from SSeCKS-overexpressing astrocytes (SSeCKS-CM) blocked angiogenesis in vivo and in vitro. Moreover, SSeCKS-CM increased tight junction proteins in endothelial cells, consequently decreasing [3H]sucrose permeability. Furthermore, immunoreactivity to SSeCKS gradually increased during the BBB maturation period, and SSeCKS-expressing astrocytes closely interacted with zonula occludens (ZO)-1-expressing blood vessels in vivo. Collectively, our results suggest that SSeCKS regulates BBB differentiation by modulating both brain angiogenesis and tight junction formation.

Angiogenesis is an important mediator of tumor progression. As tumors expand, diffusion distances from the existing vascular supply increases, resulting in hypoxia in the cancer cells. Sustained expansion of a tumor mass requires new blood vessel formation to provide rapidly proliferating tumor cells with an adequate supply of oxygen and nutrients. The key regulator of hypoxia-induced angiogenesis is the transcription factor known as hypoxia-inducible factor (HIF)-1. HIF-1alpha is stabilized by hypoxia-induced reactive oxygen species (ROS) and enhances the expression of several types of hypoxic genes, including that of the angiogenic activator known as vascular endothelial cell growth factor (VEGF). In this study, we found that melatonin, a small lipophilic molecule secreted primarily by the pineal gland, destabilizes hypoxia-induced HIF-1alpha protein levels in the HCT116 human colon cancer cell line. This destabilization of HIF-1alpha resulted from the antioxidant activity of melatonin against ROS induced by hypoxia. Moreover, under hypoxia, melatonin suppressed HIF-1 transcriptional activity, leading to a decrease in VEGF expression. Melatonin also blocked in vitro tube formation and invasion and migration of human umbilical vein endothelial cells induced by hypoxia-stimulated conditioned media of HCT116 cells. These findings suggest that melatonin could play a pivotal role in tumor suppression via inhibition of HIF-1-mediated angiogenesis.

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We investigated a molecular mechanism underlying quercetinmediated amelioration of colonic mucosal injury and analyzed chemical structure contributing to the quercetin's effect. Quercetin up-regulated vascular endothelial growth factor (VEGF), an ulcer healing factor, not only in colon epithelial cell lines but also in the inflamed colonic tissue. VEGF derived from quercetin-treated colon epithelial cells promoted tube formation. The VEGF induction was dependent on quercetin-mediated hypoxia-inducible factor-1 (HIF-1) activation. Quercetin delayed HIF-1␣ protein disappearance, which occurred by inhibiting HIF-prolyl hydroxylase (HPH), the key enzyme for HIF-1␣ hydroxylation and subsequent von Hippel Lindau-dependent HIF-1␣ degradation. HPH inhibition by quercetin was neutralized significantly by an elevated dose of iron. Consistent with this, cellular induction of HIF-1␣ by quercetin was abolished by pretreatment with iron. Two iron-chelating moieties in quercetin, -OH at position 3 of the C ring and/or -OH at positions 3Ј and 4Ј of the B ring, enabled the flavonoid to inhibit HPH and subsequently induce HIF-1␣. Our data suggest that the clinical effect of quercetin may be partly attributed to the activation of an angiogenic pathway HIF-1-VEGF via inhibiting HPH and the chelating moieties of quercetin were required for inhibiting HPH.