Angiopoietin switching regulates angiogenesis and progression of human hepatocellular carcinoma

Abstract

Aim: Angiopoietin 1 (Ang-1) and its antagonist, angiopoietin 2 (Ang-2), are novel ligands that regulate the Tie2 receptor. The Ang-2 gene is upregulated in the hypervascular type of human hepatocellular carcinoma (HCC). To gain a better understanding of the role of the Ang-Tie2 system in HCC the expression of these genes was investigated in a series of human HCCs.

Methods: The expression of the angiopoietin and Tie2 proteins was investigated in nine normal liver tissues and 52 surgically resected HCCs. In addition, the effects of hypoxic stimuli on Ang-1, Ang-2, vascular endothelial growth factor (VEGF), and erythropoietin (EPO) expression was investigated in Hep3B cells.

Results: Ang-1, rather than Ang-2, was more frequently expressed in the normal liver. Ang-1 was expressed in 68% of HCCs, whereas Ang-2 was expressed in 81%, and was significantly higher in poorly differentiated HCCs characterised by high vascularity (p = 0.02), and in tumours with a peliotic change (p = 0.02). Strong expression of Tie2 was seen in tumour vessels in accordance with Ang-2 expression. In Hep3B cells, hypoxic stimuli upregulated VEGF and EPO, but not Ang-1 or Ang-2.

Conclusions: These data support the evidence that the reversal of Ang-1 and Ang-2 expression plays an important role in the angiogenic and dedifferentiation processes in HCC. The hypoxic stimuli were not responsible for Ang-2 upregulation, unlike that of VEGF, in human HCC cells.

Figure 1

Immunohistochemistry of angiopoietins in normal human liver tissue. (A) Angiopoietin 1 (Ang-1) shows positive staining in normal hepatocytes. (B) Ang-2 is negative in normal hepatocytes. Original magnification, ×35.

Figure 2

Immunohistochemistry of angiopoietin 2 (Ang-2) in hepatocellular carcinoma (HCC). HCC cells show (A) negative, (B) partially positive, and (C) diffusely positive cytoplasmic staining for Ang-2. Original magnification, ×40.

Figure 3

Relation between angiopoietin (Ang) expression and histology of normal liver tissue and hepatocellular carcinoma (HCC). Well HCC, well differentiated hepatocellular carcinoma, mod/poor HCC, moderately and poorly differentiated hepatocellular carcinoma. *p < 0.01; **p = 0.02.

Figure 4

Immunohistochemistry of Tie2 and CD31 in hepatocellular carcinoma (HCC). (A) Both pericytes and endothelial cells of arterial tumour vessels are positive for Tie2; (B) only endothelial cells are CD31 positive in the same sample. Original magnification, ×40.

Figure 5

(A) Reverse transcription polymerase chain reaction analysis and (B) semiquantitative analysis of angiopoietin (Ang), vascular endothelial growth factor (VEGF), and erythropoietin (EPO) mRNA in Hep3B cells. Under hypoxic conditions, VEGFs (VEGF189, VEGF165, and VEGF121) and EPO are upregulated approximately 2.5 to 6 fold, but Ang-1 and Ang-2 mRNA are not detected. M, DNA markers; N, normoxia; 16, 16 hours of hypoxia; 24, 24 hours of hypoxia; C, control hepatocellular carcinoma samples.