## 4 Angiogenic Factors
### Vascular Endothelial Growth Factors (VEGFs) and Placental Growth Factor (PGF)  
It is well known that VEGF is overexpressed in many types of tumors (i.e., colorectal, lung, and breast cancer) other than in CCA and it appears closely correlated with increased microvessel density (MVD) and with tumor stage [64; 80; 81; 82; 83]. VEGF is a family of molecules which includes different isoforms (VEGF-A, -B, -C, -D, -E, and placenta growth factor) secreted by several cells such as endothelial cells, neutrophils and platelets [84]. The specific function of each subunit of VEGF is mediated by the interaction between the ligand and its corresponding receptor VEGFR (vascular endothelial growth factor receptor). VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1) are two tyrosine kinase receptors mainly involved in angiogenesis and vasculogenesis processes while the third form VEGFR-3 (Flt-4) acts principally during lymphogenesis [72]. Several in vivo studies documented the role of VEGF in CCA and its ability to induce vascular permeability, cell migration and vasodilation, which promote angiogenesis in tumor parenchyma [85; 86; 87; 88]. [PERSON] et al. investigated the expression of VEGF-A and the MVD in patients with extrahepatic cholangiocarcinoma to evaluate neovascularization and its prognostic role in this tumor, demonstrating an improved prognosis in patients with low MVD compared to patients with high MVD [86]. No correlation between VEGF-A levels and survival has been found, in accordance to previously research conduct by [PERSON] et al. in which VEGF expression resulted lower in CCA respect to control patients [89]. In contrast with these data, patients affected by extrahepatic biliary tract carcinomas showed an overall significantly worse perspective of survival with a high VEGF positivity compared to patients in which this factor is undetectable [90]. [PERSON] et al. analyzed the VEGF levels in both intrahepatic and extrahepatic CCA, demonstrating that their expression is higher in both tumors compared to the control and also proved that high VEGF levels clinically correlated with intrahepatic metastasis [87]. Most recently, other authors analyzed the expression of cyclooxygenase 2 (COX2) and VEGF-C in different stages of CCA to elucidate their clinical (based on tumor infiltration) and pathological (based on cell differentiation) involvement in this disease [91]. COX2 and VEGF-C result overexpressed in a late clinical and pathological stage of CCA indicating their contribution in tumor cell differentiation and in metastatic process [91]. [PERSON] et al. evaluated the expression of VEGF/VEGF and their interaction with TGF-\(\beta\)/TGF-\(\beta\) receptor pathway on human CCAs after surgical resection and they found that these two growth factors are up-regulated and could thus be responsible for neoangiogenesis [85]. VEGF expression has been detected and correlated with advanced disease stage and poor prognosis of CCA. The addition of an antiangiogenic therapy to the chemotherapy induces a synergistic effect. The upregulation of the angiogenic pathway underlines the potential possible use of an antiangiogenic therapy associated with the chemotherapy. Bevacizumab, a recombinant humanized monoclonal antibody against VEGF, is an important therapeutic agent exploited against cancers. The normalization of the tumor vascularization has been suggested as a new objective to increase survival. In a phase II non-comparative study, the addition of bevacizumab to gemcitabine and oxaliplatin regimen (GEMOX) is associated with a seven-month median progression-free survival (PFS) with tolerable safety in metastatic carcinomas of the biliary tract [92]. Moreover, in vitro experiments show a time-dependent increase of VEGF levels in response to a paracrine/autocrine TGF-\(\beta\) stimulation, suggesting that TGF-\(\beta\) induces VEGF activation via transcription factor Sp1. Therefore, in addition to canonical TGF-\(\beta\) target genes, VEGF represents the first proangiogenic factor regulated by TGF-\(\beta\)1 through Sp1.