**7. Involvement of the Apelin System in the Pathogenesis of Liver Fibrosis**

The apelin system has certain therapeutic abilities, but it may also play a role in disease depending on the cellular and molecular milieu. A clear example of this is the role of apelin in tissue fibrosis. Apelin displays anti-fibrotic actions counteracting AII in models of cardiac fibrosis [173,174] and renal fibrosis [175,176]. In contrast, the scenario and role of the apelin system are completely different in liver disease. Apelin is a hepatic pro-fibrotic agent, in part by mediating some of the fibrogenic effects triggered by AII and ET-1 in the activation of HSC occurring in liver fibrosis [118]. These outcomes highlight the intriguing difference between myofibroblastic-cell types in liver compared to myofibroblasts in heart, kidney, or skin in which apelin acts by decreasing myofibroblast accumulation and activity [137,176–178]. In vitro, apelin has also demonstrated a significant potential to promote liver fibrosis. It acts directly on LX-2 cells (a cell line used as a reliable in vitro model of HSC) through Erk signalling [179], stimulating cell survival and the synthesis of PDGF-β receptor and collagen-I in these cells [118]. In turn, PDGF-β and LPS can stimulate the expression of APJ, expanding and perpetuating HSC activation [180]. Therefore, apelin may, in theory, induce HSC to a pro-fibrogenic profile and prolong its stimulation autocrinally during all stages of liver fibrosis in chronic liver disease (Figure 6). In fact, some data have revealed that the inhibition of APJ using F13A (an APJ antagonist) prevents fibrosis progression in rats under a non-discontinued fibrosis induction program using CCl4 [104].

**Figure 6.** Overview of the roles of the apelin system in liver fibrosis.

Several investigations have uncovered the close and integrated relationship between pathological angiogenesis and fibrosis [12,181–186]. As mentioned above, apelin is a powerful angiogenic agent through the activation of endothelial APJ and different downstream signalling pathways. Two reports have associated the inhibition of APJ with a reduction in angiogenesis and with a concomitant drop in fibrosis in CH and fibrotic rats [18,104]. Although a direct relationship was not established between the two phenomena, there are evidence pointing out that APJ activation by apelin stimulates the expression of a well-known pro-angiogenic factor, angiopoietin-1 (from the angiopoietin family involved in pathological angiogenesis in chronic liver disease) [185,187] in LX-2 cells [118]. This suggests that fibrogenic cells such as HSC may participate in hepatic angiogenesis by secreting angiogenic factors such as angiopoietin 1. Indeed, the inhibition of HSC-secreted angiopietin-1 has shown to drastically reduce pathological angiogenesis and liver fibrosis induced in mice by either CCl4 or BDL [187].

Aside from HSC, hepatocytes have also been related to contribute to both phenomena, pathological angiogenesis and liver fibrosis, by releasing pro-angiogenic and pro-fibrogenic factors [186]. There is a growing belief that local hypoxia is the link interconnecting both pathological angiogenesis and liver fibrogenesis, orchestrating the harmonic and coordinated activation of HSC, hepatocytes and other hepatic cells [188]. Detection of hypoxic areas is a common trait at any stage of chronic liver disease, expanding progressively from early injury to the development of cirrhosis [189]., Through the action of hypoxia-inducible factors (HIF), hepatic hypoxia up-regulates the expression of a wide array of growth factors and mediators of liver repair and angiogenesis [189]. However, pathological angiogenesis can be inefficient due to the immaturity and permeability of vascular endothelial growth factor (VEGF)-induced new vessels [190] and, consequently, the liver may be unable to reduce hypoxia. Hypoxia up-regulates in vitro the expression of APJ in LX-2 (HSC) and in HepG2 (hepatocytes) [124]. Interestingly, the pro-inflammatory and pro-fibrogenic agents TNF-α and AII also induce the expression of APJ in HepG2 cells [124]. APJ activation in HepG2 cells triggers the expression of VEGF-A and PDGF-β, factors that in turn may promote angiogenesis and activation of HSC and, consequently, liver fibrosis [15,191]. According to these data, hypoxia, inflammation and pro-fibrogenic factors up-regulate APJ in HSC and hepatocytes, which can release different pro-angiogenic and pro-fibrogenic factors such as apelin (from activated HSC) together to perpetuate fibrosis while injury and these stimuli remain. In vivo, APJ is upregulated preferentially in hepatocytes and HSC, while apelin levels are increased and localized in HSC in cirrhotic rats and in patients with liver cirrhosis caused by hepatitis C virus or ethanol [18,124]. High apelin levels and liver damage have also been observed in human non-alcoholic fatty liver disease [192]. A contemporary clinical investigation revealed that circulating apelin levels are associated with histological and hemodynamic features of chronic liver disease [193], but more clinical studies are needed to confirm the major relevance of apelin system in human liver fibrosis.

One study suggested that apelin may play a different role in liver fibrosis by being an initiator of hepatic injury instead of merely a HSC activator following exogenous injury [194]. In this study, the authors described that the apelin system may stimulate Fas-induced liver injury via the phosphorylation of Jnk in mice intraperitoneally injected with an agonistic anti-Fas antibody. Similar results have been obtained when acute liver injury was promoted by hepatectomy. Blockade of the apelin system resulted in mouse liver regeneration via activation of Kupffer cells and by increasing TNF-α and IL-6 levels in hepatectomized mice [195]. These results suggest that the apelin system may interfere with hepatocyte proliferation after partial hepatectomy in mice. However, a recent study has shown that the administration of a long-acting apelin fusion protein resulted in attenuated hepatocyte damage, diminished apoptosis and ROS production in a mouse model of LPS-induced liver injury [196]. Altogether, these findings suggest that the apelin system may be involved in the processes of hepatic injury and regeneration, but these specific aspects need further investigation.
