Thrombin up-regulates vascular endothelial growth factor in experimental gliomas

OBJECTIVE: Vascular endothelial growth factor is a potent stimulator of angiogenesis; activation of hypoxia inducible factor-1alpha and p44/42 mitogen-activated protein kinase pathways has been associated with vascular endothelial growth factor production. The present study examined if thrombin modulates vascular endothelial growth factor levels in gliomas via hypoxia inducible factor-1alpha and p44/42 mitogen-activated protein kinase pathways. METHODS: This study was divided into two parts: (1) adult male Fischer 344 rats had an intracaudatal implantation of C6 glioma cells. Rats were killed 6, 9 and 12 days later for Western blotting and immunohistochemistry. Hypoxia inducible factor-1alpha messenger ribonucleic acid was also determined; (2) cultured C6 cells were treated with vehicle, thrombin, thrombin plus YC-1, an inhibitor of hypoxia inducible factor-1alpha, or thrombin plus PD098059, an inhibitor of p44/42 mitogen-activated protein kinases. Cell culture medium was collected for vascular endothelial growth factor measurement using an enzyme-linked immunosorbent assay kit. RESULTS: In vivo, immunoreactivities for thrombin, vascular endothelial growth factor, hypoxia inducible factor-1alpha and activated p44/42 mitogen-activated protein kinases were detected in C6 gliomas. Thrombin immunoreactivity was co-localized with that of vascular endothelial growth factor, hypoxia inducible factor-1alpha or activated p44/42 mitogen-activated protein kinases. A time course showed that activated p44/42 mitogen-activated protein kinases and vascular endothelial growth factor protein levels increased in gliomas with time. Hypoxia inducible factor-1alpha protein levels were higher in C6 gliomas than those in the contralateral brain tissue. In vitro, thrombin exposure increased the levels of vascular endothelial growth factor, hypoxia inducible factor-1alpha and activated p44/42 mitogen-activated protein kinases in C6 cells. Thrombin-induced vascular endothelial growth factor up-regulation was blocked by YC-1 and PD098059. DISCUSSION: Thrombin activates hypoxia inducible factor-1alpha and p44/42 mitogen-activated protein kinase pathways, and up-regulates vascular endothelial growth factor in gliomas. Inhibiting hypoxia inducible factor-1alpha and p44/42 mitogen-activated protein kinase pathways reduces the thrombin-induced up-regulation of vascular endothelial growth factor. These results suggest thrombin can increase vascular endothelial growth factor levels via activating hypoxia inducible factor-1alpha and p44/42 mitogen-activated protein kinase pathways in gliomas.