Angiotensin-converting enzyme 2 and AMPK/mTOR pathway in the treatment of liver fibrosis: Should we consider further implications?

Abstract

This editorial contains comments on the article by Zhao et al in print in the World Journal of Gastroenterology. The mechanisms responsible for hepatic fibrosis are also involved in cancerogenesis. Here, we recapitulated the complexity of the renin-angiotensin system, discussed the role of hepatic stellate cell (HSC) autophagy in liver fibrogenesis, and analyzed the possible implications in the development of hepatocarcinoma (HCC). Angiotensin-converting enzyme inhibitors and angiotensin receptor blockers definitively contribute to reducing hepatic fibrogenesis, whereas their involvement in HCC is more evident in experimental conditions than in human studies. Angiotensin-converting enzyme 2 (ACE2), and its product Angiotensin (Ang) 1-7, not only regulate HSC autophagy and liver fibrosis, but they also represent potential targets for unexplored applications in the field of HCC. Finally, ACE2 overexpression inhibits HSC autophagy through the AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway. In this case, Ang 1-7 acts binding to the MasR, and its agonists could modulate this pathway. However, since AMPK utilizes different targets to suppress the mTOR downstream complex mTOR complex 1 effectively, we still need to unravel the entire pathway to identify other potential targets for the therapy of fibrosis and liver cancer.

Key Words: Renin-angiotensin system, Liver fibrosis, Hepatic stellate cells, Autophagy, Hepatocellular carcinoma

Core Tip: In the light of clarifying the link between liver fibrosis and the development of hepatocellular carcinoma, we discussed the renin-angiotensin system involvement in liver fibrosis and hepatocarcinoma development, the specific mechanisms by which angiotensin-converting enzyme 2 (ACE2) regulates hepatic stellate cell (HSC) autophagy and consequently fibrosis, and the ACE2-dependent upstream signals modulating the AMP-activated protein kinase/mammalian target of the rapamycin pathway implicated in the regulation of HSC activation.

INTRODUCTION

The article by Zhao et al[1] discussing the role of angiotensin-converting enzyme 2 (ACE2) on liver fibrosis prompted further comments on this hot topic since liver fibrosis, inflammation, and hepatocarcinogenesis share some physiopathological mechanisms. In this editorial article, I will discuss the current research status and future directions on the involvement of renin-angiotensin system (RAS) and liver fibrogenesis, focusing on the potential links with hepatocarcinogenesis.

Advanced liver fibrosis is associated with portal hypertension, progressive liver function deterioration, and an increased risk of hepatocellular carcinoma. The mechanisms responsible for hepatic fibrosis and cancerogenesis are related to chronic necroinflammation, compensatory regeneration, and activation of non-parenchymal cells, together with an altered immune response[2,3]. Recent experimental data demonstrate that ACE2 can reduce liver fibrosis and hepatic sinusoidal remodeling by regulating hepatic stellate cell (HSC) autophagy[4]. The molecular mechanism by which ACE2 would regulate HSC autophagy would be mediated by the AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway, opening up a new opportunity to develop new potential therapies for the prevention/cure of liver fibrogenesis[4].

Zhao et al[1] have further discussed the role of ACE2 in the reduction of liver fibrosis by implementing the literature data on the role of HSCs and underlining some specific effects of ACE2 overexpression on RAS. They conclude that the pathway described by Wu et al[4] is not entirely independent from the pathway associated with RAS, which could explain alternative mechanisms for liver fibrogenesis reduction.

Here, the role of RAS in liver fibrogenesis, the ACE2-mediated mechanisms regulating autophagy, and the upstream signals regulating the AMPK/mTOR pathway will be further discussed, in the light of the possible link between liver fibrosis and the development of hepatocellular carcinoma.

THE RENIN-ANGIOTENSIN SYSTEM

The RAS can be considered a hormonal (tissue-to-tissue), paracrine (cell-to-cell), and intracrine (intracellular/nuclear) system, which is responsible for the control of multiple biological/functional activities. It consists of several components that include angiotensinogen (AGT), renin, ACE, angiotensin I (Ang I), Ang II, AT1R and AT2R, which all together constitute the classical RAS pathway, and ACE-2, angiotensin 1-7 (Ang 1-7), MAS Receptor (MasR), and Mas-related G-protein-coupled receptor D (MrgD), which constitute the alternative so called RAS protective pathway (Figure 1) (for a more comprehensive description of all RAS[5,6]).

Figure 1 Renin-angiotensin system main pathways. AGT: Angiotensinogen; Ang I: Angiotensin I; ACE: Angiotensin-converting enzyme; ACE2: Angiotensin-converting enzyme 2; ARBs: Angiotensin receptor blokers; AT1R: Angiotensin II type 1 receptor; AT2R: Angiotensin II type 2 receptor; Ang 1-7: Angiotensin 1-7; MasR: G-protein-coupled Mas receptor; MrgD: Mas-related G-protein-coupled receptor D; AMPK: AMP-activated protein kinase; RAS: Renin-angiotensin system.

As shown in Figure 1, renin is the rate-limiting enzyme and the first recognized component of RAS[7]. AGT, the only precursor of all angiotensin peptides, is a member of the non-inhibitory serine protease inhibitor superfamily, and it is mainly synthesized and released from the liver[8,9].

Ang I is generated from AGT under the action of renin[10]. Angiotensin-converting enzyme is an evolutionarily conserved zinc-metallopeptidase that is expressed on the surface of endothelial and epithelial cells in a wide variety of tissues[11] and is responsible for the formation of Ang II[5], which is the major effector peptide of RAS[12]. The biological function of Ang II is mediated by two receptors with antagonist functions, namely angiotensin type 1 receptors (AT1R) and type 2 receptor (AT2R), the former common in adult tissues such as liver, brain, and kidney, and the latter predominant in fetal tissues, ovary, and uterus, and practically absent in the liver[5,13,14]. AT1R is the dominant receptor that activates intracellular signaling pathways implicated in several biological activities, including inflammation and fibrosis[15]. The hepatic AT1R would be the main target by which Ang II stimulates the proliferation of HSCs and their synthesis of extracellular matrix proteins through induction of transforming growth factor beta (TGF-β)[6,16]. AT2R is practically absent in the liver, and its activation by Ang II has opposite effects, including vasodilation and anti-fibrotic effects[15].

ACE2 represents the initial step for the alternative protective RAS pathway by promoting the degradation of Ang II in Ang 1-7, which in turn becomes a negative regulator of AT1R and AT2R activation. ACE2 is present in two different catalytic forms, the type I transmembrane protein and the soluble form that does not contain the cytosolic and transmembrane domains[17]. Its transmembrane form is expressed by a wide variety of human tissues (alveolar epithelial cells, esophageal epithelial cells, small intestinal epithelial cells, vascular endothelial cells) and at low concentration in the liver, especially in HSCs[18,19]. Ang 1-7 binds to the G protein-coupled receptor named MasR to elicit its cellular responses. It has been shown that MasR can heterooligomerize with the AT1R receptor and inhibit the actions of Ang II, which makes it physiological antagonist of AT1R[20]. Although the knowledge of the signaling mechanisms associated with MasR is essential for therapeutic purposes, there is little data in the literature on this topic, mainly in experimental models hyperexpressing MasR[21]. This receptor is distributed in various tissues and HSCs[22,23].

Overall, the ACE2/Ang- (1-7)/MasR pathway has anti-fibrotic and anti-inflammatory properties[22,15]. In attrition to its activity towards AT1R, Ang 1-7 can reduce inflammation by decreasing the expression of the proinflammatory cytokines, including tumor necrosis factor-alpha (TNF-α) and interleukins (IL)-6[24], whereas its binding with MrgD seems to induce hemodynamic responses[6,25].

AUTOPHAGY AND AMPK mTOR PATHWAY

Autophagy is a cellular process that degrades damaged organelle or protein aggregation, safeguarding cells against damage or death by eliminating damaged organelles and proteins. However, an excess of autophagy can produce damage, as observed in HSCs where it promotes their activation, which increases liver fibrosis. In fact, autophagy can antagonize apoptosis, can regulate cell death independently from apoptosis, or it can cooperate with apoptosis to promote cell death[26].

Starvation, hypoxia, oxidative stress, protein aggregation, and endoplasmic reticulum stress are the main signals that activate the autophagic process. Autophagy is mainly downregulated by mTOR and its downstream signal, the mTOR complex 1 (mTORC1). The latter is a protein complex regulated by growth factors, energy levels, oxygen, and amino acids. TORC1 controls different cell functions, including inhibition of autophagy, and it can be inhibited by rapamycin[27].

One of the three upstream signaling pathways regulating mTORC1 activation is represented by the AMPK, a Ser/Thr kinase that exists as a heterotrimer composed of catalytic α and regulatory β and γ subunits. AMPK is the key sensor of the AMP:ATP ratio within the cell, which is deputed to transduce this information to mTOR. In the absence of AMP, the autoinhibitory domain of AMPKα maintains the kinase in an inactive conformation. Upon ATP consumption, AMP/ADP directly binds to AMPKγ and promotes the catalytic AMPK subunit phosphorylation, which increases its activity approximately 100 times. AMPK phosphorylation is also mediated by the TGF-β-activated kinase 1[28,29] and several other physiological signals, including cytokines and growth factors[30]. AMPK inhibits mTORC1 both directly and indirectly, while activated mTORC1 inhibits autophagy; therefore, AMPK indirectly leads to the induction of autophagy by inhibiting mTORC1[28]. Interestingly, the main product of ACE2 is Ang 1-7, which seems to have the ability to increase the activation of AMPK[29].

LIVER FIBROSIS AND HEPATOCARCINOMA

To discuss the link between liver fibrosis, hepatocarcinogenesis, and RAS, we will consider three distinct aspects: The potential antineoplastic use of ACE inhibitors or Angiotensin receptor blokers (ARBs) to reduce the risk of hepatocellular carcinoma through the inhibition of the classical RAS pathway (Ang II/AT1R), the upstream mechanism of HSCs autophagy regulated by ACE2 potentially involving Ang 1-7 agonists, and the downstream mechanism that occurs intracellularly in HSC involving the AMPK/mTOR pathway in the reduction of hepatic fibrosis. In this case, the target for downregulation of autophagy would be the modulation of AMPK activity or the increase of mTORC1 activity.

Interestingly, 15%-20% of all cancer deaths are associated with infections. In addition, the incidence and mortality from cancer decrease in patients treated with both steroidal and non-steroidal anti-inflammatory drugs.

Some of the molecules involved in cancer-related inflammation are IL-1β, IL-6, IL-23 and TNF-α. In the liver, the activation of Kupffer cells increases the of IL-6 production and promotes inflammation, tissue damage, compensatory cell proliferation, and, ultimately, the formation of liver tumors[31].

The mechanism by which the RAS pathway would be involved in liver carcinogenesis would depend essentially on the Ang II/AT1R. The chronically damaged liver increases the production of AGT and Ang II, which stimulates the contraction and proliferation of HSCs[32] and increases the TGF-β expression through AT1R[6,16].

The use of RAS inhibitors in experimental models of liver cancer and some studies on human hepatocellular carcinoma have provided evidence that long-term use of ACE inhibitors and ARBs may protect against cancer[5].

However, although the interest in the potential beneficial effects of RAS inhibitors against HCC started two decades ago, we must wait until 2009 for the first study in humans. Some observational studies, including ours, showed better results with ARBs compared to RAS inhibitors[33,34]. More recently, it has been reported that the potential RAS blockade can be used for cancer metastasis treatment. This antitumoral activity would depend on blockade of AT1R with a reduction of VEGF, angiopoietin 2, fibroblast growth factor, and platelet-derived growth factor, all factors involved in cell proliferation and/or angiogenesis and inhibitory effects on tumor-associated macrophages, cancer-associated fibroblasts, and Kupffer cell activation[14]. Moreover, a large retrospective study showed that ACE inhibitors/ARBs were associated with a lower risk of HCC and cirrhotic complications in patients with non-alcoholic fatty liver disease[35], an effect most likely linked to the antifibrotic activity of these drugs[36]. Interestingly, another possible mechanism linking the antifibrogenic and antitumoral activity of ARBs comes from the experimental study of Gu et al[37]. They demonstrated that losartan ameliorates the immunological response against HCC by reducing peritumoral fibrosis and enhancing HCC infiltration by effector CD8+ T cells. However, it should be considered that two studies report a tumor-promoting effect of ARBs in human lung cancer and melanoma. In the former type of cancer, the authors report that ARBs are associated with a modest increase in risk for a new lung cancer occurrence[38], whereas, in the latter, the authors report that RAS has both oncogenic and tumor suppressor functions in melanoma[39]. Finally, a more recent meta-analysis confirms that treatment with ARBs and ACE inhibitors reduces the risk of hepatocellular carcinoma[40].

The ACE2-regulated HSCs autophagy represents another link between liver fibrosis and hepatocellular carcinoma since ACE2 would increase the production of Ang 1-7, which inhibits the proliferation of activated HSCs. The involvement of ACE2 induced Ang 1-7 up-regulation in the reduction of HCC risk is supported by the study of Barbosa et al[41] in which they describe a novel Ang 1-7 agonist, the A-1317, able to increase both Mas, MRGD, and AMPK mRNA gene expression in the liver, with positive effects on metabolic syndrome-related disorders (altered liver biochemical parameters, increased liver AGT), and AT1R, ACE mRNA gene expression.

To complete our comment on the link between liver fibrosis and carcinogenesis, we are considering the recent study of Wu et al[4] on the involvement AMPK/mTOR pathway in HSC autophagy downregulation to improve liver fibrosis[42]. In their experimental study, they used a liver-specific recombinant viral vector to increase the expression of ACE2 in the liver, and they found reduced fibrogenesis associated with a reduced number of activated HSCs (HSCs contained a lower number of phagosomes compared to controls), and an increased number of apoptotic HSCs. These histological aspects were associated with a reduction of activated AMPK and an increase of p-mTOR. Based on these findings, they concluded that ACE2 overexpression inhibits the expression of HSC autophagy in the liver through the AMPK/mTOR pathway.

Some studies suggest that Ang 1-7 is an activator of AMPK through binding to the MasR[43,44]. Once again, these findings suggest that a possible intervention on this pathway could be mediated by Ang 1-7 agonists. Finally, since AMPK utilizes different targets to suppress mTORC1, i.e., the mTOR downstream complex[28], we must still to unravel the complete pathway to identify other potential targets for therapy.

CONCLUSION

The studies on the relationship between RAS and fibrogenesis can stimulate new possible therapeutic targets for antifibrotic agents and can also contribute to a deeper comprehension of the mechanisms promoting and inhibiting liver carcinogenesis. It is important to keep in mind that in addition to the mechanisms previously illustrated, angiotensin inhibitors can directly reduce the expression of important mediators of fibrogenesis and inflammation such as TGF-β[6,16,42] and TNFα[22], or indirectly induce downregulation of proinflammatory Th1 and Th17 cytokines, and the production of Treg/Th2 cytokines[45].