Involvement of the Transcription Factor NF-κB in Tubular Morphogenesis of Human Microvascular Endothelial Cells by Oxidative Stress

Abstract

Oxygen radicals are induced under various pathologic conditions associated with neovascularization. Oxygen radicals modulate angiogenesis in cultured human microvascular endothelial cells by an unknown mechanism. Treatment of human microvascular endothelial cells for 15 min with 0.1 to 0.5 mM hydrogen peroxide (H₂O₂) or 100 U of tumor necrosis factor alpha per ml induced tubular morphogenesis in type I collagen gels. Gel shift assays with nuclear extracts demonstrated that H₂O₂ increases the binding activities of two transcription factors, NF-κB and AP-1, but not of Sp1. Tumor necrosis factor alpha increased the binding activities of all three factors. A supershift assay with specific antibodies against JunB, JunD, and c-Jun (Jun family) showed that the antibody against c-Jun supershifted the AP-1 complex after H₂O₂ treatment. Coadministration of the antisense sequence of NF-κB inhibited H₂O₂-dependent tubular morphogenesis, and the antisense c-Jun oligonucleotide caused partial inhibition. The angiogenic factor responsible for H₂O₂-induced tubular morphogenesis was examined. Cellular mRNA levels of vascular endothelial growth factor and interleukin-8 (IL-8), but not those of transforming growth factor α, were increased after treatment with 0.5 mM H₂O₂. Coadministration of anti-IL-8 antibody inhibited tubular morphogenesis enhanced by H₂O₂, and IL-8 itself also enhanced the formation of tube-like structures. Treatment with antisense NF-κB oligonucleotide completely blocked H₂O₂-dependent IL-8 production by endothelial cells. The tubular morphogenesis of vascular endothelial cells after treatment with oxidative stimuli and its possible association with NF-κB and IL-8, is examined.