Dual mechanism of vascular endothelial growth factor upregulation by hypoxia in human hepatocellular carcinoma

Background/aims-Vascular endothelial growth factor (VEGF) plays a key role in regulation of tumour associated angiogenesis. In the current study we an alysed expression of VEGF and its receptors in human hepatocellular carcino ma (HCC) and investigated the molecular mechanisms of VEGF regulation by hy poxia. Methods-VEGF, kinase domain region (KDR)/fetal liver kinase 1 (flk-1), and flt-1 expression were examined by immunohistochemistry and in situ hybridis ation in 15 human HCC tissues, Expression of VEGF and regulation by hypoxia were assessed in three human HCC cell lines using a quantitative competiti ve reverse transcription-polymerase chain reaction, ELISA, and a series of 5' deletion reporter gene constructs of the human VEGF promoter in transien t transfection assays. Results-We observed over expression of VEGF mRNA and protein in HCC compare d with cirrhosis or normal liver. Expression of VEGF in tumour cells was st rongly increased in areas directly adjacent to necrotic/hypoxic regions. Bo th VEGF receptors were detected in vascular endothelia of HCC while only KD R/flk-1 receptors were detected in endothelial cells of cirrhotic livers. E xpression of VEGF was observed in all human HCC cell lines examined. Hypoxi a (1% oxygen) resulted in profound upregulation of VEGF mRNA and protein le vels. Furthermore, hypoxia treatment resulted in a doubling of VEGF mRNA st ability. Deletion analysis of the human VEGF 5' flanking region -2018 and 50 demonstrated induction of VEGF promoter activity under hypoxic condition s which was significantly decreased following deletion of the region -1286 and -789 suggesting a substantial contribution of the -975 putative hypoxia inducible factor 1 binding site to hypoxia mediated transcriptional activa tion of the VEGF gene. Conclusion-These data suggest hypoxia as a central stimulus of angiogenesis in human HCC through upregulation of VEGF gene expression by at least two distinct molecular mechanisms: activation of VEGF gene transcription and an increase in VEGF mRNA stability.