*1.2. Liver Fibrosis and Cell Types*

Liver damage leads to death of hepatocytes and cholangiocytes, which induces the release of pro-inflammatory mediators and stimulates phagocytosis of dead cell bodies by liver macrophages, mainly Kupffer cells and bone marrow-derived recruited monocytes [10]. Macrophages in the liver can also produce pro-inflammatory factors, such as reactive oxygen species (ROS), CC-chemokine ligand 2 (CCL2), tumor necrosis factor (TNF), interleukin-6 (IL-6), and 1β (IL-1β), thus triggering the wound-healing response, and stimulating the production of extracellular matrix components by myofibroblasts [11].

In ALD, the hepatocyte injury is mainly related to the oxidative metabolism of ethanol, whereas in NAFLD it depends on the lipotoxicity that induces cell death and lipo-apoptosis. When liver fibrosis is developed in an onset of ALD or NAFLD, the excessive deposition of ECM proteins is principally observed around the sinusoids (peri-sinusoidal fibrosis) and around groups of hepatocytes (peri-cellular fibrosis), and is mainly due to hepatic stellate cells (HSC) [4,12]. When fibrosis is developed in an onset of cholestasis, in addition to chronic damage to cholangiocytes, bile acids elicit hepatocyte injury and death [4,13,14]. In chronic diseases of the biliary tract, the excessive deposition of ECM proteins is principally observed around the injured bile ducts (biliary fibrosis pattern) and is mainly characterized by the proliferation of reactive ductular cells and myofibroblasts originated from portal fibroblast and HSC [4,15,16]. However, the contribution of portal fibroblasts to the development of fibrosis after cholestatic damage, compared to that of the HSC, is controversial [17].

As mentioned before, despite other minor cell sources (reviewed by [4]), HSC are the main sources of myofibroblasts in response to toxic liver injury [18,19]. In a healthy liver, HSC are in a quiescent state in which they accumulate retinoids. In response to toxic liver injury, HSC suffer a transdifferentiation process from a quiescent into an activated phenotype known as myofibroblasts [20]. These activated HSCs have a higher degree of proliferation and migration, hence repopulating the damaged liver, acquiring contractility by expressing alpha smooth muscle actin (α-SMA), expressing pro-inflammatory [(monocyte chemoattractant protein-1 (MCP-1), platelet-derived growth factor (PDGF), mouse stem cell factor (mSCF), CCL2, and CCL21, as well as IL-1β) and pro-fibrogenic markers (TGF-β)], and as well as increasing the synthesis of ECM proteins [collagen I (COL1A1) and III (COL1A3), fibronectin and tissue inhibitor of metalloproteinase (TIMP)], and of pro-angiogenic mediators [like vascular endothelial growth factor A (VEGFA), angiopoietin-1 or -2, and the homodimer (PDGF-BB)] [21,22]. One of the principal factors involved in HSC-induced proliferation is PDGF, which is upregulated in the fibrotic liver, whereas transforming growth factor (TGF-β) is the main profibrogenic factor and

contributes positively to the transdifferentiation process of HSCs into myofibroblasts. Briefly, TGF-β binds and activates TGF-β receptors (TβR), of which there are three different forms (TβRI, TβRII, and TβRIII). Smads are the effector proteins of the TGF superfamily ligands. There are 8 Smad proteins which include: receptor-regulated R-Smads (Smads 1, 2, 3, 5, and 8), common-mediator Co-Smads (Smad4), and inhibitory I-Smads (Smads 6, 7). When TβRI is activated, Smads are recruited to the receptor and phosphorylated, resulting in their activation and increased affinity for Smad4. Then the Smad2/3/4 heteromeric complex translocates to the nucleus, where it has an immediate effect on the gene expression of several hundred of genes. TGF-β signaling is terminated when the activated Smads are either dephosphorylated or degraded [23,24].
