Induction Of IGF Binding Protein-6 In Vascular Endothelial Cells Is A Conserved Negative Feedback Mechanism In Hypoxia-induced Angiogenesis

Hypoxia stimulates tumor angiogenesis by inducing the expression of angiogenic factors. The negative feedback mechanisms in hypoxia-induced tumor angiogenesis, however, are not well understood. Here we report that prolonged hypoxia induced the expression of insulin-like growth factor binding protein-6 (IGFBP-6), a tumor repressor, in human and rodent vascular endothelial cells (VECs) via a HIF-1-mediated mechanism. Addition of human IGFBP-6 to cultured human VECs inhibited angiogenesis in vitro. An IGFBP-6 mutant with at least 10,000-fold lower binding affinity for IGFs was an equally potent inhibitor of angiogenesis, suggesting that this action of IGFBP-6 is IGF-independent. The in vivo role of IGFBP-6 in angiogenesis was tested in flk1:GFP zebrafish embryos, which exhibit green fluorescence in vascular endothelium, permitting visualization of the developing blood vessels. Injection of human IGFBP-6 mRNA reduced the number of embryonic inter-segmental blood vessels by ~40%. This anti-angiogenic activity is conserved because forced expression of a zebrafish IGFBP-6 had similar effects. To determine the anti-angiogenic effect of IGFBP-6 in a tumor model, Rh30 humanrhabdomyosarcoma cells stably transfected with IGFBP-6 were inoculated into athymic BALB/c nude mice. Vessel density was 52% lower in IGFBP-6-transfected xenografts than in vector control xenografts. These results suggest that the induction of IGFBP-6 expression by hypoxia in VECs is an important component of a conserved negative feedback mechanism in hypoxia induced tumor angiogenesis. Hypoxia is widely recognized as an important driving force for tumor angiogenesis. As tumors grow, the original vasculature can become insufficient to supply oxygen to the growing tumor cells, and local hypoxia develops. Hypoxia stimulates new blood vessel formation via the induction of angiogenic factors. This neovascularization is critical for tumor progression as it provides tumor cells with oxygen, nutrients, and growth factors. A key player in the hypoxia response is hypoxia-inducible factor-1 (HIF-1), which is a heterodimeric transcription factor composed of HIF-1αand HIF-1β. While HIF-1β(also known as ARNT) is constitutively expressed, HIF-1αis sensitive to O2 tension. When O2 levels are high, HIF-1αis hydroxylated by prolyl hydroxylase domain-containing proteins (PHDs), then ubiquitinated and targeted for proteosomal degradation. In hypoxic conditions, HIF-1αhydroxylation is inhibited, and it enters the nucleus where it forms a dimer with HIF-1β. The dimerized HIF-1 complex binds to hypoxia response elements (HREs) of its target genes and promotes their transcription. HREs are present in many angiogenic genes including vascular endothelial growth factor (VEGF), a major angiogenic gene, and its receptor, Flk1. Recent studies suggest that there are also negative feedback mechanisms that inhibit angiogenesis during prolonged hypoxia RGC32, which inhibits VEGF- and hypoxia-induced angiogenesis, is up-regulated by hypoxia. Likewise, hypoxia induces the expression of GRS5, which inhibits VEGF-induced angiogenesis through p38 MAPK, via HIF-1 activation. Compared to our relatively comprehensive understanding of the induction of angiogenic genes by hypoxia/HIF-1, however, the negative feedback mechanisms in hypoxia-induced tumor angiogenesis are not well understood.Insulin-like growth factor binding proteins (IGFBP) 1-6 bind IGFs with high affinity in extracellular fluids and regulate IGF actions at target tissues. Some IGFBPs also possess distinct biological actions that are independent of IGFs. IGFBP-6 is unique among the six IGFBPs because of its 50-fold greater affinity for IGF-II, making it a relatively specific inhibitor of IGF-II actions. Recent studies suggest that IGFBP-6 is a tumor suppressor that inhibits the growth of a number of IGF-II-dependent tumors. In addition to its ability to bind and sequester IGF-II, recent in vitro studies suggest that IGFBP-6 also has IGF-independent action. An early report showed that hypoxia increased IGFBP-6 mRNA levels in bovine endothelial cells, but expression of IGFBP-6 and its relationship to hypoxia have not been studied in human endothelial cells. More importantly, the role of IGFBP-6 in normal and tumor angiogenesis, if any, is unknown. The objective of the present study was therefore to test the hypothesis that hypoxia induces IGFBP-6 gene expression in vascular tissues, and that this induction is part of a negative feedback mechanism in hypoxia-induced tumor angiogenesis.