**3.1.1 Angiogenesis in cholangiocarcinoma**

A recent immunohistochemical study of microvessel density and lymphatic microvessel density revealed that intrahepatic cholangiocarcinoma tumors demonstrated tumorassociated angiogenesis (Thelen et al. 2009). Tumors with increased microvessel density were correlated with a higher recurrence rate, lower 5-year survival rates and increased nodal spread which in turn influences patient survival (Thelen et al. 2009). Recent studies have also shown that the overexpression of the angiogenic factors nerve growth factor- (NGF-) and vascular endothelial growth factor-C (VEGF-C) occurred in approximately 57.1% and 46.4% of cholangiocarcinoma samples, respectively (Xu et al. 2010). A number of human cholangiocarcinoma cell lines and samples have also been shown to overexpress VEGF-A and VEGF receptors (VEGFRs), the angiogenic factors angiopoietin-1, -2, and thrombospondin-1, as well as EGF, EGF receptors (EGFR) and basic fibroblast growth factor (Ogasawara et al. 2001; Alvaro et al. 2006; Tang et al. 2006; Yoshikawa et al. 2008; Harder et al. 2009). Secretion of these factors may individually or co-ordinately bring about increased angiogenesis as demonstrated by increased microvessel density. For example, VEGF-A has been shown to play a role in the neovascularization of extrahepatic cholangiocarcinoma (Mobius et al. 2007).

The factors that drive angiogenesis have also been shown to have distinct effects on cholangiocyte and cholangiocarcinoma growth in an autocrine manner (Gaudio et al. 2006; Tang et al. 2006; Mobius et al. 2007; Yabuuchi et al. 2009; Yoshikawa et al. 2009; Glaser et al. 2010). Indeed, the proliferative effects of estrogen on cholangiocarcinoma cell lines have been attributed to a mechanism involving the upregulation of VEGF expression, as blocking VEGF ameliorates the estrogenic effects on proliferation (Mancino et al. 2009).

Taken together, these data suggest that agents that block angiogenesis (by blocking VEGF expression, for example) may also have a direct effect on cholangiocarcinoma cell proliferation in addition to their anti-angiogenic effects. In support of this notion, inhibition of VEGFR and EGFR signaling with vandetanib (ZD6474, a tyrosine kinase inhibitor) can be an important approach for the management of the subset of cholangiocarcinoma that lack KRAS mutations and/or have EGFR amplification (Yoshikawa et al. 2009). Furthermore, ZD1839 (IRESSA), an orally active, selective inhibitor of EGFR tyrosine kinase has clinical activity against cholangiocarcinoma by stabilizing the cell cycle inhibitor, p27Kip1 and enhancing radiosensitivity in cholangiocarcinoma cell lines (Yabuuchi et al. 2009). Curcumin, a natural phenol found in tumeric has recently been shown to suppress the expression of VEGF and decrease the microvessel density in a hamster model of cholangiocarcinoma (Prakobwong et al. 2011a). In parallel, curcumin also exerts antiproliferative and proapoptotic effects on cholangiocarcinoma cells independent of the effects on angiogenesis (Prakobwong et al. 2011a; Prakobwong et al. 2011b). Similar effects have been shown with inhibitors of histamine synthesis (Francis et al. 2011), H3 histamine receptor agonists (Francis et al. 2009), and Endothelin-1 (Fava et al. 2009) just to name a few. The interaction between angiogenesis, angiogenic factors and cholangiocarcinoma growth and progression is summarized in Figure 2.

Fig. 2. Schematic representation of the interactions between cholangiocarcinoma and angiogenic factors regulating cell proliferation and angiogenesis
