Apelin is a bioactive peptide and endogenous ligand for the

Apelin is a bioactive peptide and endogenous ligand for the APJ receptor (APLNR), a member of the G protein coupled receptor family that shares a similar sequence as the angiotensin type-1 receptor (AT1) [5]. Early studies demonstrated that the apelin/APLNR receptor axis plays a significant role in blood pressure regulation and cardiovascular disease by regulating angiogenesis and the response of endothelial SB269970 HCl to hypoxic injury [6], [7], [8], [9]. APLNR signaling is also essential for embryonic angiogenesis and has been shown to regulate blood vessel diameter [8], [10]. More recent studies have shown that the apelin/APLNR axis also regulates angiogenesis and growth of certain malignancies, potentially serving as a novel therapeutic target [5], [11]. Additionally, apelin has been shown to promote lymphangiogenesis and lymph node metastasis, further emphasizing its importance in cancer physiology [12]. Berta et al. demonstrated that apelin expression is up regulated in human non-small cell lung cancer, expression is associated with poor overall survival, and apelin stimulates tumor growth and microvessel densities in their in vivo model [13]. Additionally, an apelin/APLNR autocrine loop has been identified in colon adenocarcinoma tumors that regulates tumor growth, but is inhibited by administration of an APLNR antagonist [14]. Within the liver, Muto et al. demonstrated that the apelin-APLNR system induces hepatocellular carcinoma angiogenesis [15]. In this study we present the novel findings that the apelin/APLNR axis is up regulated in CCA cells lines and human CCA tissues. We demonstrate that apelin promotes CCA proliferation and angiogenesis, a process that is inhibited by co-treatment with an APLNR antagonist. Additionally, we show that CCA growth is inhibited in vivo by intravenous administration of ML221, an apelin receptor antagonist, in our nu/nu mouse xenograft model.
Materials and methods
Results
Discussion These results further show that the apelin receptor and its cognate peptide ligand, apelin, are important for tumor growth and angiogenesis. Sorli et al. demonstrated that apelin is a potent activator of neoangiogenesis, which in turn regulates tumor growth, using mouse mammary carcinoma cell clones (TS/A-apelin) [11]. Their data from a human cancer-profiling array shows that the apelin gene is expressed in a variety of benign and malignant tissues, however, the frequency of gene up regulation was high in carcinomas of the colon, skin and pancreas [11]. They did not see an increase of apelin gene expression in tumors of liver origin, however, carcinomas of the pancreatic head and extrahepatic CCA share similar features, including embryologic origin and several phenotypic characteristics [34]. Our in vitro data showed significant up regulation of apelin and APLNR in intrahepatic and extrahepatic cell lines. Not all tumors in our CCA tissue array showed increased APLNR expression and the array did not make a distinction between intrahepatic or extrahepatic tumors, so we are unable to determine whether or not expression of APLNR is dependent on tumor location. More research about the heterogeneity of apelin and APLNR expression in CCA is needed to determine if anatomical location changes expression of this axis, which would further impact potential therapeutic strategies. Furthermore, in normal liver samples APLNR expression was primarily located in cholangiocytes. Our CCA tissue array staining suggests that hepatocyte APLNR expression also increases in the presence of an adjacent CCA tumor. It is possible that the tumor microenvironment promotes up regulation of the apelin/APLNR axis; however, additional studies are needed to investigate these findings. These results parallel other studies in which hepatic APLNR expression is minimal in normal conditions but significantly up regulated in the setting of liver fibrosis and cirrhosis [35].