Cholangiocarcinoma (CCC) is relatively hypovascular, in contrast to hepatocellular carcinoma (HCC), which is often highly vascular. We investigated if the diminished vascularity of CCC is related to altered expression of thrombospondin-1 (TSP-1), an antiangiogenic factor, and/or vascular endothelial growth factor (VEGF), a potent angiogenic factor, comparing the relationships with those of high-and low-vascular HCC. We also investigated the relationship between the mutation of the p53 gene and TSP-1 expression or VEGF expression. Northern blot analysis and immunohistochemical staining were performed on surgically resected human CCC and HCC. The ratios of TSP-1 mRNA level in cancer cells versus adjacent noncancerous cells (T/N ratios) were significantly higher in CCC (n β«Ψβ¬ 11) than in HCC with high vascularity (n β«Ψβ¬ 15). In contrast, T/N ratios of VEGF mRNA level in CCC (n β«Ψβ¬ 11) were comparable with those in HCC with low vascularity (n β«Ψβ¬ 5). In CCC, the cancer cells and fibroblasts were positively stained with anti-TSP-1 antibody. We observed that T/N ratios of VEGF mRNA level, but not those of the TSP-1 mRNA level, were significantly correlated with vascularity in HCC. The relative increase in TSP-1 and the relative decrease in VEGF in tumors compared with normal tissue may underlie the limited angiogenesis of CCC. The p53 gene did not affect the expression of TSP-1 in CCC or VEGF in HCC. (HEPATOLOGY 1998;28:1512-1517.) Physiological angiogenesis results from the balance of angiogenesis activators and inhibitors. 1 Rapid growth of malignant tumors is accompanied by increased angiogenesis regulated through either increased production of activators or decreased production of inhibitors. Tumors and their surrounding tissues produce various factors that affect angiogenesis. Angiogenic factors include vascular endothelial growth factor (VEGF), basic fibroblast growth factor, transforming growth factor β£, and interleukin-8; antiangiogenic factors include thrombospondin-1 (TSP-1), interferon alfa/beta, platelet factor-4, angiostatin, and endostatin. 1,2 TSP-1, a multifunctional matrix protein, influences tumor growth through its inhibition of angiogenesis. It inhibits vascular endothelial cell migration and adhesion in an in vitro model, 3,4 and also inhibits basic fibroblast growth factorinduced angiogenesis in the rabbit cornea. 5,6 The expression of TSP-1 increases in newly formed microvessels, proliferating vascular endothelial cells, activated macrophages, as well as stroma surrounding highly vascularized tumors. 7 TSP-1 and its receptors are highly expressed in the desmoplastic stroma and basement membrane associated with tumors, including breast cancer and fibroadenoma, 7 and is important in the inhibition of angiogenesis in vitro and in vivo. 8 Overexpression of TSP-1 in TSP-1 cDNA-transfected endothelial cells inhibits tumorigenesis. 9 VEGF is mitogenic for endothelial cells, 3,4 and its tyrosine kinase receptors, flk-1 and flt-1, are specifically expressed in endothelial cells. 10,11 It is expressed in kidney epithelial cells and other cell types, and it increases in glioblastoma, renal cancer, breast cancer, and other malignancies in humans and animals. 3,4 Moreover, interference with VEGF or its receptors by VEGF antibodies or by dominant-negative VEGF receptors induces a dramatic inhibition of tumor growth. [12][13][14] In this study, we asked if the hypovascularity of cholangiocarcinoma (CCC) was associated with the expression of VEGF and/or TSP-1. We also compared the VEGF and TSP-1 expressions in CCC with those in hepatocellular carcinoma (HCC) having a high and low vascularity, because the vascularity of HCC is closely associated with the expression of the VEGF gene. 15,16 Because the wild-type p53 tumor suppressor gene modulates the production of VEGF 17 and TSP-1, 5 we examined the mutation status of p53 in CCC and HCC.
Methods
Samples. Twenty patients with HCC and 11 patients with CCC underwent hepatectomy in the Second