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. 2001 Jan;48(1):87–96. doi: 10.1136/gut.48.1.87

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

Z von Marschall 1, T Cramer 1, M Hocker 1, G Finkenzeller 1, B Wiedenmann 1, S Rosewicz 1
PMCID: PMC1728160  PMID: 11115828

Abstract

BACKGROUND/AIMS—Vascular endothelial growth factor (VEGF) plays a key role in regulation of tumour associated angiogenesis. In the current study we analysed expression of VEGF and its receptors in human hepatocellular carcinoma (HCC) and investigated the molecular mechanisms of VEGF regulation by hypoxia.
METHODS—VEGF, kinase domain region (KDR)/fetal liver kinase 1 (flk-1), and flt-1 expression were examined by immunohistochemistry and in situ hybridisation in 15 human HCC tissues. Expression of VEGF and regulation by hypoxia were assessed in three human HCC cell lines using a quantitative competitive reverse transcription-polymerase chain reaction, ELISA, and a series of 5' deletion reporter gene constructs of the human VEGF promoter in transient transfection assays.
RESULTS—We observed over expression of VEGF mRNA and protein in HCC compared with cirrhosis or normal liver. Expression of VEGF in tumour cells was strongly increased in areas directly adjacent to necrotic/hypoxic regions. Both VEGF receptors were detected in vascular endothelia of HCC while only KDR/flk-1 receptors were detected in endothelial cells of cirrhotic livers. Expression of VEGF was observed in all human HCC cell lines examined. Hypoxia (1% oxygen) resulted in profound upregulation of VEGF mRNA and protein levels. Furthermore, hypoxia treatment resulted in a doubling of VEGF mRNA stability. Deletion analysis of the human VEGF 5' flanking region −2018 and +50 demonstrated induction of VEGF promoter activity under hypoxic conditions 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 activation 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.


Keywords: hepatocellular carcinoma; angiogenesis; vascular endothelial growth factor; hypoxia

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Figure 1  .

Figure 1  

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Expression of vascular endothelial growth factor (VEGF) in human hepatocellular carcinoma (HCC). Immunostaining with polyclonal VEGF antibody (A, C) and in situ hybridisation with 35S labelled antisense cRNA for VEGF (B, D). VEGF was expressed by HCC cells (A, B) and epithelial cells of bile ducts (C, D). Exposure time for autoradiography was 14 days. The arrow indicates necrosis. Original magnification: A, C, and D ×40; B ×20.

Figure 2  .

Figure 2  

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Expression of vascular endothelial growth factor (VEGF) receptors kinase domain region (KDR)/fetal liver kinase 1 (flk-1) and fms-like tyrosine kinase 1 (flt-1) in human hepatocellular carcinoma (HCC). Immunostaining with anti-CD31 antibody (A) and anti-flt-1 (B). In situ hybridisation with 35S labelled antisense cRNA for flt-1 (C) and KDR/flk-1 (E) or sense probes, respectively (D, F). Flt-1 (B, C) and KDR/flk-1 (E) expression was observed exclusively in endothelial cells of HCC tissue. Hybridisation with sense cRNAs for flt-1 (D) and KDR/flk-1 (F) did not reveal any significant hybridisation signal. Exposure time for autoradiography was 14 days. Original magnification: A, B, E, and F ×40; C and D ×20.

Figure 3  .

Figure 3  

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Expression of vascular endothelial growth factor (VEGF) in human hepatocellular carcinoma (HCC) cell lines. (A) Analysis of VEGF mRNA transcripts by reverse transcription-polymerase chain reaction (RT-PCR) using primers specific for all known isoforms. The size of the indicated bands of 403 and 535 bp was determined by a 100 bp ladder and correspond to VEGF121 and VEGF165, respectively. RT−, PCR without reverse transcription. (B) Expression of VEGF protein: a VEGF specific ELISA was used to determine VEGF concentrations in supernatants and cell extracts of the indicated cell lines and normalised to protein content. Mean (SEM) of three experiments, each performed in triplicate.

Figure 4  .

Figure 4  

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Hypoxia mediated induction of vascular endothelial growth factor (VEGF) protein in human hepatocellular carcinoma (HCC) cell lines. VEGF specific ELISA was used to determine VEGF concentrations in supernatants (A) and cell extracts (B) of the indicated cell lines incubated either under normoxic (21% O2 ) or hypoxic (1% O2 ) conditions for 16 hours and normalised to protein content. Mean (SEM) of three experiments, each performed in triplicate.

Figure 5  .

Figure 5  

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Effects of hypoxia on vascular endothelial growth factor (VEGF) mRNA expression in hepatocellular carcinoma (HCC) cell lines. VEGF mRNA expression was measured by quantitative competitive reverse transcription-polymerase chain reaction (RT-PCR). Total RNA was isolated from cells incubated for 16 hours under normoxic or hypoxic conditions and subjected to RT. A constant amount of competitor and threefold serial dilutions of cDNAs were used as templates for PCR using VEGF165 or β-actin specific primers. (A) Representative image of ethidium bromide stained gels for HepG2 cells. (B) Quantitative analysis. VEGF165 expression was calculated as the molar ratio of VEGF to β-actin copies. Mean (SEM) values of three independent experiments determined as fold induction by hypoxia.

Figure 6  .

Figure 6  

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Hypoxia increased vascular endothelial growth factor (VEGF) mRNA half life in HepG2 cells. Cells were incubated for 16 hours under normoxic or hypoxic conditions and subsequently incubated with 150 µM DRB to block transcription. Cells were harvested for RNA after 0, 30, 60, 120, 180, 240, and 360 minutes. VEGF mRNA was then determined by quantitative competitive reverse transcription-polymerase chain reaction as described in materials and methods. Mean (SEM) values of three independent experiments.

Figure 7  .

Figure 7  

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Effects of hypoxia on vascular endothelial growth factor (VEGF) gene transcription. (A) Schematic diagram of the VEGF promoter-Luc reporter constructs. The nucleotide sequence -2018 to +50 bp of the human VEGF gene promoter was cloned in front of a luciferase gene, and serial 5' deletion constructs were generated between positions −2018 and −52 bp. The transcription start is indicated by an arrow. Potential consensus binding sites for HIF-1 are indicated (•). (B) Hypoxia mediated transactivation in HepG2 cells. HepG2 cells were transiently cotransfected with VEGF-Luc and β-gal plasmids and incubated for 16 hours with 21% O2 or 1% O2. Luciferase activity was determined and normalised for transfection efficiency. Mean (SEM) values of four independent experiments, each performed in triplicate, and determined as fold induction by hypoxia.*Significantly different from control (p<0.001).

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