Basic FGF augments hypoxia induced HIF-1-alpha expression and VEGF release in T47D breast cancer cells.

AIM: Both hypoxia inducible factor 1 (HIF-1) and basic fibroblast growth factor (bFGF) play important roles in tumour angiogenesis. This study was designed to clarify the cooperative effect of these two mediators in induction of vascular endothelial cell growth factor (VEGF) release from breast cancer and probe possible mechanisms involved. METHODS: Release of VEGF from a breast cancer cell line (T47D) was quantitated by enzyme linked immunosorbent assay (ELISA). Expression of HIF-1 and ERK was assayed using Western blotting. Transient transfection and dual luciferase reporter assay were used to study HIF-1 transactivity. RESULTS: The data showed that hypoxia induced the expression of HIF-1alpha protein, the transactivity of HIF-1 and the release of VEGF. bFGF further augmented these hypoxic inductions. The PI3K pathway was required for these processes as demonstrated by application of PI3Kinase inhibitor (LY294002) or mutant construct transfections. In contrast, the MEK1 inhibitor PD98059 showed no effect on either activation of HIF-1 or VEGF release, which is in agreement with our finding that ERK1/2 was not activated by hypoxia. Under hypoxic conditions, bFGF activated the MEK1/ERK pathway. PD98059 blocked the activation of ERK1/2 and suppressed bFGF-induced HIF-1 transactivity, yet the protein expression of HIF-1alpha or VEGF release was not affected by PD98059. CONCLUSION: bFGF augments hypoxia induced VEGF release mainly through the PI3K pathway and partly depending on HIF-1 activity. Elucidation of this mechanism may provide a new target for anti-angiogenesis in cancer therapy.

Alphastatin, a 24-amino acid fragment of human fibrinogen, is a potent new inhibitor of activated endothelial cells in vitro and in vivo.

Angiogenesis, the development of new blood vessels from existing vasculature, is crucial for the development and metastasis of solid tumors. Here, we show for the first time that a 24-amino acid peptide derived from the amino terminus of the alpha chain of human fibrinogen (termed "alphastatin") has potent antiangiogenic properties, inhibiting both the migration and tubule formation of human dermal microvascular endothelial cells in response to vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (bFGF) in vitro. Moreover, alphastatin markedly inhibits the growth of tumors in a syngeneic murine model. Tumors from mice receiving daily injections of alphastatin for 12 days exhibited large areas of intravascular disruption and thrombosis with substantial cellular necrosis. Importantly, alphastatin administration had no detectable effect on vessels in such normal tissues as liver, lungs, and kidney. Taken together, these data indicate that alphastatin is a potent new antiangiogenic agent in vitro and antivascular agent in vivo.

Hypoxia-induced gene expression in human macrophages: implications for ischemic tissues and hypoxia-regulated gene therapy.

Macrophages accumulate in ischemic areas of such pathological tissues as solid tumors, atherosclerotic plaques and arthritic joints. Studies have suggested that hypoxia alters the phenotype of macrophages in a way that promotes these lesions. However, the genes up-regulated by macrophages in such hypoxic tissues are poorly characterized. Here, we have used cDNA array hybridization to investigate the effects of hypoxia on the mRNAs of 1185 genes in primary human monocyte-derived macrophages. As shown previously in other cell types, mRNA levels for vascular endothelial growth factor (VEGF) and glucose transporter 1 (GLUT-1) were up-regulated by hypoxia. However, the mRNAs of other genes were also up-regulated including matrix metalloproteinase-7 (MMP-7), neuromedin B receptor, and the DNA-binding protein inhibitor, Id2. The promoters of GLUT-1 and MMP-7 confer hypoxic inducibility on a reporter gene in RAW 264.7 macrophages, indicating that the hypoxic up-regulation of these mRNAs may occur, at least in part, at the transcriptional level. GLUT-1 and MMP-7 mRNA were also shown to be up-regulated in hypoxic macrophages in vitro by real-time RT-PCR, and these proteins were elevated in hypoxic macrophages in vitro and in hypoxic areas of human breast tumors. The hypoxia up-regulated genes identified could be important for the survival and functioning of macrophages in hypoxic diseased tissues, and their promoters could prove useful in macrophage-delivered gene therapy.

Expression of HIF-1alpha by human macrophages: implications for the use of macrophages in hypoxia-regulated cancer gene therapy.

Large numbers of monocytes extravasate from the blood into human tumours, where they differentiate into macrophages. In both breast and prostate carcinomas, these cells accumulate in areas of low oxygen tension (hypoxia), where they respond to hypoxia with the up-regulation of one or more hypoxia-inducible factors (HIFs). These then accumulate in the nucleus and bind to short DNA sequences called hypoxia-response elements (HREs) near or in such oxygen-sensitive genes as that encoding the pro-angiogenic factor vascular endothelial growth factor (VEGF). This stimulates gene expression and could explain why, in part, macrophages express abundant VEGF only in avascular, hypoxic areas of breast carcinomas. It also suggests that macrophages could be used to deliver HRE-regulated therapeutic genes specifically to hypoxic tumour areas. A recent study suggested that hypoxic macrophages accumulate HIF-2 rather than HIF-1, prompting the search for HRE constructs that optimally bind HIF-2 for use in macrophage-based gene therapy protocols. However, the present study shows that human macrophages accumulate higher levels of HIF-1 than HIF-2 when exposed to tumour-specific levels of hypoxia in vitro; that macrophages in human tumours express abundant HIF-1; and that expression from HRE-driven reporter constructs in the human macrophage-like cell line MonoMac 6 correlates more closely with HIF-1 than with HIF-2 up-regulation under hypoxia. Taken together, these findings suggest that HIF-1 may be the major hypoxia-inducible transcription factor in macrophages and that HIF-1-regulated constructs are likely to be effective in macrophage delivery of hypoxia-regulated gene therapy to human tumours.