*5.1. Selective Targeting of Mast Cells in Liver Fibrosis*

"Personalized medicine" and "precision medicine" are keywords when it comes to discussions about successful patient-tailored treatment of diseases; such an endeavor first requires thorough and sophisticated diagnostic tools. On a different level, this also concerns pharmacological regulation of cellular activation states depending on the (patho-)physiological tissue niches, as well as patient comorbidities. The more sensitive cells are to environmental conditions, the more complicated the choice of beneficial pharmacological substances might be. This applies in particular to MCs, whose task is to recognize changes in micro-environmental conditions and occurrence of biological mediators and/or chemical substances. As already indicated in Section 1.2., MCs are endued with enormous heterogeneity, as well as fascinating plasticity; thus, depending on their localization and prevailing conditions, MCs as a whole express hundreds of mediators and surface receptors, complicating the choice of pharmacological treatment in a given pathophysiological setting. Hence, returning to the keyword of personalized medicine, deciphering the complexity of MC phenotypes in a given (patho-)physiological situation might enhance the ability to use MCs as "drug targets."

Two different pharmacological approaches, not necessarily mutually exclusive, could be followed to address and inhibit MC activity in pathophysiological situations like liver fibrosis: i) MC stabilizers, such as disodium cromoglycate (cromolyn sodium), Tranilast, ketotifen, or many others (Figure 4); and/or ii) inhibitors of MC-selective enzymes (e.g., tryptase and chymase), and antagonists for receptors of typical MC mediators, such as histamine (receptors). These mediators importantly add to the pleiotropic inflammatory functions of MCs (cf. Figure 2).

**Figure 4.** Naturally occurring and synthetic mast cell stabilizers with highly variant structures. Interestingly, some of these drugs, such as Tranilast, Luteolin, Curcumin, Theanine, Nilotinib, and others showed highly beneficial effects in experimental hepatic fibrosis [62]. Most of their activities are attributed to their antioxidant activity, capacity to prevent hepatic infiltration with circulating blood cells, potential to target pro-inflammatory and/or pro-fibrotic signaling pathways, or to influence extracellular matrix generation or turnover. However, none of these substances have been tested systematically regarding the modulation of mast cell stability in models of hepatic fibrogenesis. All structure images were prepared with Jmol (http://jmol.sourceforge.net/) using the following PubChem Compound Identification (CID) numbers: Bromoenol lactone (5940264), Curcumin (969516), disodium cromoglycate (27503), Hypothemicin (9929643), Janex-1 (3794), K1 (25211416), Ketofen (3827), Lodoxamide (44564), Luteolin (5280445), Nilotinib (644241), Pathenolide (7251185), Pemirolast (57697), RO 20-1724 (5087), Scopoletin (5280460), Theanine (439378), Tranilast (5282230), Vacuolin-1 (9661141). More details about the structure and functions of representative MC stabilizers can be found elsewhere [178].

Already in 1972, results from a controlled study of cromolyn sodium in patients with asthma came to the conclusion that this drug is a useful adjunct to the pharmacological treatment of asthma [179]. Since it was shown in rat peritoneal MCs that cromolyn sodium is able to attenuate IgE-mediated histamine release [180], MCs were seen as the cellular targets of this drug, fitting to their well-known detrimental role in anaphylaxis and asthma. In addition to using cromolyn sodium in patients and experimental rat models, researchers were increasingly using this drug in mouse models to prove the contributions of MCs. However, in a previous study by Galli and coworkers, in which MC-related effects of cromolyn sodium in vitro and in vivo were thoroughly compared in rat and mouse models, the authors came to the conclusion that cromolyn sodium's effectiveness and selectivity as a MC stabilizer in mice is questionable [159,181]. Nevertheless, in an in vitro model, they could demonstrate the positive effect of histamine secreted from MCs on the proliferation and activation of cholangiocytes, as well as HSCs [181]. In a model of bleomycin-induced pulmonary fibrosis, another MC stabilizer, Tranilast, was shown to be effective in suppressing fibrosis in genetically MC-deficient mice [182], raising doubt about the MC selectivity of this drug, at least in mice.

Nowadays, there are many reports testing mast cell stabilizers, such as luteolin and curcumin in vivo for their efficacy at protecting against inflammation, disease-associated apoptosis, tumorigenesis, and overshooting oxidative stress in the liver [183–187]. Unfortunately, in all these studies, the impact of these stabilizers on mast cell activity was not analyzed systematically. With respect to the mitigation of liver fibrosis, convincing studies have been published with respect to the role of MC proteases, tryptase and chymase. Tryptase β forms non-covalent ring-like tetramers and has trypsin-like activity [188,189]. Tetramerization appears to be required for proper activity and selectivity. The central pore containing the active sites of the four tryptase β molecules prohibits the access of large substrates and also suppresses inhibition by most protease inhibitors [190]. In a model of BDL-induced hepatic fibrosis in rats, treatment with the tryptase inhibitor APC366 resulted in a reduced hepatic fibrosis score, attenuated HSC proliferation, collagen content, and serum biochemical parameters [146]. In contrast to tryptase β, tryptase α is proteolytically inactive; nevertheless, it is conserved throughout evolution and significantly expressed in MCs. Very recently, Le et al. have reported the in vivo existence of biologically functional α/β tryptase heterotetramers; however, their contribution to fibrotic diseases has not been investigated yet [191].

In addition to certain soluble substrates, the G-protein-coupled receptor PAR-2 is cleaved by tryptase and appears to be its main substrate [192]. The tryptase-mediated, N-terminal cleavage of PAR-2 generates an intramolecular ligand activating PAR-2. PAR-2 has been shown to be expressed, amongst others, on fibroblasts and HSCs, and thus, in addition to tryptase inhibitors, PAR-2 antagonists might be beneficial in the treatment of liver fibrosis. Concerning liver fibrosis, rat HSCs express PAR-2 and the expression increases with the transition of stellate cells to myofibroblasts. The PAR-2 agonists, tryptase and the peptide SLIGRL, induced proliferation and collagen secretion [145], suggesting that tryptase as a PAR-2 agonist could sustain liver fibrosis. Indeed, analyzing CCl4-induced liver fibrosis in PAR-2-deficient compared to wild-type mice, Knight et al. found a reduced progression of liver fibrosis, hepatic collagen gene expression, hydroxyproline content, TGF-β expression, and matrix metalloproteinase 2 gene expression in the absence of PAR-2. Additionally, PAR-2 activation stimulated proliferation, collagen production, and TGF-β production by human HSCs [144]. This indicated the pro-fibrogenic action of hepatic PAR-2 activation and strongly proposed a role for hepatic MCs in liver fibrosis. Interestingly, the recently identified α/β tryptase heterotetramer was also shown to cleave and activate PAR-2 [191].

Pepducins are cell-penetrating peptides acting as intracellular modulators of signal propagation from receptors of the G-protein-coupled receptor family to the associated G proteins [193]. Intriguingly, the PAR-2 pepducin PZ-235 significantly suppressed hepatic steatosis and inflammation in the experimental methionine-choline-deficient diet model in mice [194]. Moreover, PZ-235 repressed CCl4-induced liver fibrosis, even with delayed treatment. PZ-235 also inhibited production of reactive oxygen species and hence, enhanced viability of hepatocytes in vitro [194], which might imply a

reduction of necrosis-driven sterile inflammation in the course of liver fibrosis. Thus, PAR-2 pepducin inhibitors have the potential to be efficient in the treatment of liver fibrosis.

Whereas tryptase forms heparin-stabilized ring-like tetramers, which restrict the access of substrates to the active centers of the protease monomers, chymase is active as a monomeric protein, though in a macromolecular complex with heparin [195]. Accordingly, its structure sterically allows for activity against a bigger number of substrates. For instance, chymase can generate angiotensin II by cleaving the non-bioactive peptide angiotensin I [148], and cleave/activate MMP-9 [196] and TGF-β [197], all of which are associated with liver inflammation and/or fibrosis. Furthermore, human chymase was found to cleave type I pro-collagen and hence initiate collagen fibril formation [152]. Isolated rat HSCs were treated with chymase and it was shown that the proliferation and expression of α-smooth muscle actin and TGF-β1 protein were significantly enhanced in a dose-dependent manner. This implied a potential role of chymase in the development of liver fibrosis [151]. Moreover, the effect of the chymase inhibitor TY-51469 in hamsters fed a methionine- and choline-deficient (MCD) diet, which developed a marked hepatic steatosis and fibrosis, was analyzed. Both non-alcoholic steatohepatitis (NASH) and fibrosis were significantly attenuated by chymase inhibition [198,199]. Likewise, in rats fed a high-fat and high-cholesterol diet, the chymase inhibitor TY-51469 significantly attenuated all parameters associated with NASH and fibrosis [200]. Furthermore, CCl4-induced liver fibrosis in hamsters was significantly reduced in TY-51469-treated compared to placebo-treated animals [201]. Finally, immunohistochemical analysis, as well as enzymatic activity measurements, of liver biopsies from 49 patients with chronic hepatitis revealed increased chymase levels and activity correlating with the severity of the disease. This suggested that hepatic chymase is implicated in liver fibrosis [202]. A correlative investigation of 77 patients with the aim to elucidate the function of chymase as an angiotensin-converting enzyme for the progression of liver fibrosis suggested an important role of this MC protease in the hepatic fibrosis of patients with cirrhosis [147].
