The results of our in

The results of our in vivo experiments provide promising evidence that the apelin/APLNR axis is implicated in CCA growth and that targeting this axis with a receptor specific antagonist may help develop effective, tumor directed therapies. Not only do we show decreased proliferation and angiogenesis in ML221 treated tumors, but we also demonstrate decreased 20187 of vimentin, MMP-9 and MMP-3. Previous studies in CCA have shown that vimentin expression is induced by epithelial-mesenchymal transition (EMT) and is associated with progressive tumor growth and a poor prognosis [55]. MMP-9 and MMP-3 have also been implicated in cancer proliferation, angiogenesis and the induction of EMT [56]. These results are similar to previously mentioned studies in lung and colon cancer [14], [43]. We did not identify any side effects to ML221 treatment in our xenograft model, however, since apelin signaling also regulated blood pressure and cardiac activity, it is possible significant side effects could develop in more advanced therapeutic trials. Furthermore, apelin signaling has been shown to be organ protective in specific circumstances such as cardiac ischemia/reperfusion injury and hemorrhagic shock [57], [58]. Additionally, Chen et al. demonstrated that intranasal apelin treatment following an ischemic stroke was neuroprotective and induced angiogenesis in mice [59]. Additional studies focusing on dose optimization and potential systemic side effects are necessary to determine if the therapeutic benefits of an APLNR antagonist outweigh the potential risks. This study does have some limitations to address. The amount of human data in this study is limited due to the availability of human tissues in our laboratory. Our data suggests that not all CCA tumors over-express apelin and its receptor. We are unable to make accurate predictions into the percentage of CCA tumors that over-express components of the apelin signaling pathway. The potential therapeutic benefit of an APLNR antagonist is tumor specific and may not be applicable to all patients with CCA. Additionally, our in vivo studies in immunocompromised mice provide a useful model, however, there is a degree of variability in tumor measurements and drug administration due to technical error. We attempted to minimize this error by having one person perform all measurements and treatments throughout the study period. Also, the design of our xenograft model allowed for frequent tumor measurements and ease of tumor collection, however, ML221 dosing, administration frequency, and treatment efficacy have to be considered in other models. Additionally, we only used one cell line to conduct our in vivo experiments. Our experience has shown that Mz-ChA-1 cells produce the most reliable tumors in our xenograft model and we have not been able to consistently grow tumors using other cell lines [27], [60], [61], [62], [63].
Funding This work was supported by the Dr. Nicholas C. Hightower Centennial Chair of Gastroenterology from Scott & White, a VA Research Career Scientist Award, a VA Merit award to Dr. Alpini (5I01BX000574), a VA Merit Award (5I01BX002192) to Dr. Glaser, and the NIH grants DK58411, DK07698, and DK062975 to Drs. Alpini, and Glaser. This material is the result of work supported by resources at the Central Texas Veterans Health Care System. The views expressed in this article are those of the authors and do not necessarily represent the views of the Department of Veterans Affairs.
Introduction Apelin is one of two cognate peptide hormone ligands for a single class A G-protein coupled receptor (GPCR) called the apelin receptor (AR, or APLNR, initially known as APJ) [1]. Apelin-AR binding and activation regulates multiple biological systems. These include the cardiovascular system, where apelin is one of the most potent physiological inotropic agents, and the central nervous system, where apelin regulates fluid homeostasis [1]. Apelin-AR binding also causes vasodilation or vasoconstriction in an eNOS-dependent manner [1]. This system also has links to various pathological states, including cardiovascular diseases, diabetes, and obesity [1], [2]. Taken together, all these factors make apelin-AR signalling of great interest for increased understanding of disease aetiology and potential treatment of many diseases.