Changing the Pathogenetic Roadmap of Liver Fibrosis? Where Did It Start; Where Will It Go?

Olav A. Gressner; Mohamed S. Rizk; Evgeniya Kovalenko; Ralf Weiskirchen; Axel M. Gressner


J Gastroenterol Hepatol. 2008;23(7):1024-1035. 

In This Article

Where Is It Now?

Experimental studies over the last 20 years or so have provided a detailed knowledge on structure and composition of ECM in normal and fibrotic liver tissue,[27,28] of the cellular origin of the various matrix components,[29] of cytokine- and growth factor-regulated stimulation of ECM synthesis (fibrogenesis) and regulation of matrix degradation (fibrolysis),[30,31,32] of several genetic conditions predisposing to fibrogenic reactions,[33,34] and of multiple, experimentally successful therapeutic approaches.[35]

Accordingly, fibrosis of the liver is now characterized (i) by an up to 10-fold increase of ECM that comprises several types of collagens, structural glycoproteins, sulfated proteoglycans (glycosaminoglycans), and hyaluronan; (ii) by a histological redistribution with preferred initial matrix deposition in the perivenular zone 3 of the acinus along the subendothelial space of Disse leading to the formation of an incomplete subendothelial basement membrane creating additional diffusion barriers between hepatocytes and the liver sinusoid ('capillarization of sinusoids');[36] (iii) by changes to the ECM-profile; and (iv) by changes to the fine structure of collagens (e.g. degree of hydroxylation of proline and lysine), glycoproteins (variations of the carbohydrate structure), and proteoglycans (changes of the degree of sulfation of the glycosaminoglycan side chains) combined with certain splice variants of ECM molecules[27] (Figure 1).

Figure 1.

Major components of the extracellular matrix (connective tissue) of the liver and the four most important targets of changes of the fibrotic matrix. The bindings of glycosaminoglycans (GAG) to the respective core proteins of proteoglycans (PG) are shown. BM, basement membrane; FACIT, fibril-associated collagens with interrupted triple helices.

The development of fibrosis is an active biosynthetic process, which is attributed to stimulated matrix production in portal or peribiliary fibroblasts and, in particular, in contractile myofibroblasts (MFB) localized initially in the subendothelial space of Disse.[37] HSC are liver pericytes, which display a dendritic morphology.[16] As liver-resident stellate cells, they are members of a diffuse stellate cell system in the body with cells in various tissues having a similar phenotype and cytoskeletal architecture.[26] In the liver, HSC embrace with thorn-like microprojections of 20-39 µm length the endothelial cell layer of the sinusoids providing physical contact not only to sinusoidal endothelial cells, but also with the cell body to the hepatocytes.[38] They constitute about one-third of the non-parenchymal cell population (Kupffer cells, endothelial cells, HSC), 1.4% of liver volume, and about 15% of total liver resident cells including hepatocytes. The 'hepatic stellate cell index', i.e. the number of HSC per 1000 hepatocytes was estimated to be 109 in the healthy rat liver.[39] The spindle-like cell body of HSC of about 700 µm3 contains multiple triglyceride-rich vacuoles, in which vitamin A metabolites (retinoids) are dissolved and stored.[40] About 85% of the vitamin A of the liver is found in HSC. Additional functions of these cells have been recently discovered: (i) their role as antigen presenting cells (APC);[41,42,43] (ii) their role as CD133+ progenitor cells with the ability to differentiate to progenitor endothelial cells and hepatocytes suggesting important roles in liver regeneration and repair;[44] (iii) their involvement in endocytosis of apoptotic parenchymal cells;[45,46] (iv) secretion of matrix metalloproteinases (MMPs), the respective MMP-inhibitors (TIMPs),[47,48] and growth factors[29] indicating a role both in matrix remodelling and degradation (fibrolysis); (v) support of liver regeneration through promotion of hepatocyte proliferation involving the neurotrophin receptor p75,[49] (vi) regulation of angiogenesis and vascular remodelling through secretion of angiogenic factors such as VEGF, endothelin-1, IGF-II, neurotrophins, and erythropoietin;[50] and (vii) hemodynamic functions as evidenced by activated HSC contraction stimulated with thromboxane, prostaglandin F2, angiotensin II, vasopressin, and endothelin-1 leading to sinusoidal constriction.[51,52,53,54] Some of these functions, however, are not expressed in the quiescent state of HSC, but are triggered following their activation. The generation of MFB from HSC follows a multistep sequence, which is initiated by liver cell necrosis induced by toxic and immunologic agents and mechanisms[55,56] (Figure 2). As a consequence, HSC,[10,16] which are localized in the immediate vicinity of hepatocytes, are activated (Figure 3). Activation of HSC leads to the expression of α-smooth-muscle actin, desmin, and gelsolin, and a concomitant decrease of glial fibrillary acidic protein (GFAP) and a loss of the volume density of lipid droplets <20% combined with a decrease of retinoids, but increased contractility and expression and secretion of a broad spectrum of matrix components.[29] The activation process includes proliferation and phenotypic transdifferentiation of HSC to MFB, but both processes are not causally related. This mechanism, designated as the 'canonical principle' of fibrogenesis, is believed to be the central pathogenetic event in the development of fibrosis. The HSC-derived MFB have the core competency not only for matrix synthesis, but also for the expression and secretion of numerous pro- and anti-inflammatory cytokines and growth factors (Figure 4). MFB have features of a highly active biosynthetic phenotype characterized by a hypertrophic rough endoplasmic reticulum containing membrane-bound ribosomes necessary for the synthesis of export proteins. The mechanism of fibrogenic activation and transdifferentiation of HSC to MFB can be summarized in a three-step cascade model,[57] which is initiated by the pre-inflammatory phase due to direct paracrine activation of HSC by necrotic (apoptotic?) hepatocytes with release of activating cytokines supplemented by loss of mito-inhibitory cell surface heparan sulfate of hepatocytes.[58,59,60,61,62] The growth promoting activity of hepatocytes, partially due to IGF-1 and respective IGF-binding proteins,[56] is released from damaged cells and parallels the elevation of LDH and AST as known leakage enzymes of hepatocytes.[63] In the following inflammatory phase, the preactivated HSC are further stimulated in a paracrine mode by invaded leukocytes and thrombocytes,[64] by activated Kupffer cells,[60,65,66,67,68] sinusoidal endothelial cells, and hepatocytes[56,58,61] to transdifferentiate to MFB. The consecutive post-inflammatory phase is characterized by the secretion of fibrogenic cytokines from MFB and interacting matrix components. Some of these cytokines can stimulate MFB in an autocrine way and HSC in a paracrine fashion. Thus, the postinflammatory phase may contribute to the perpetuation of the fibrogenic process, even after elimination or reduction of the pre-inflammatory and inflammatory phases. Activation and transdifferentiation of HSC are the result of extensive interactions with liver-resident and non-resident cells (Figure 5). Most relevant mediators are reactive oxygen species (hydroxyl radicals, oxygen radicals, superoxide anions, hydrogen peroxide) produced by activated Kupffer cells[65,69] and leukocytes, the stimulated NAD(P)H oxidase activity of HSC,[70] which phagocytose apoptotic bodies,[46] the cytochrome P4502E1 (CYP2E1) pathway of ethanol-metabolizing hepatocytes,[71] and subsets of leukocytes.[72] In addition, acetaldehyde of ethanol-exposed hepatocytes[73,74,75,76] and tissue hypoxia[77] promote the activation of HSC.[78] Among the peptide mediators, TGF-ß is the profibrogenic master cytokine.[79,80,81] Additional cytokines and growth factors involved in fibrogenesis are PDGF-B and PDGF-D, endothelin-1, several fibroblast growth factors (FGFs), insulin-like growth factor I, tumor necrosis factor-alpha (TNF-α), adipocytokines (leptin, adiponectin), and others, which are partly bound as 'crinopectins'[82] to the extracellular matrix.[83] The matrix serves as a sponge for several of these growth factors fixed in a covalent or non-covalent manner to fibronectin, proteoglycans, and collagens. TGF-ß is secreted in a high molecular (large) latent form (Figure 6) by HSC/MFB, sinusoidal endothelial cells, and Kupffer cells, and released by thrombocytes and hepatocytes.[84,85] It initiates not only the transdifferentiation of HSC to MFB, but also enhances matrix gene expression, decreases their degradation by down-regulation of MMPs and up-regulation of their specific inhibitors (i.e. TIMPs), induces apoptosis of hepatocytes,[86,87,88,89] and inhibits (together with activin A) liver cell proliferation.[90,91] Extracellular activation of latent TGF-ß by proteases, oxygen radicals, thrombospondin type I, and ανß1, ανß8, ανß1 integrins is an important step in the regulation of TGF-ß bioavailability.[92] Antagonism of TGF-ß[93] or inhibition of its intracellular Smad-signaling cascade by specific inhibitors[94] results in a significant retardation or even inhibition of HSC activation and, thus, to a sustained antifibrotic effect. Interestingly, TGF-ß response and signaling are modulated during transdifferentiation of HSC to MFB leading to partial TGF-ß insensitivity of MFB.[95] This observation suggests that TGF-ß plays a role in the initiation of HSC activation in vivo but TGF-ß seems to be less important for the entire transdifferentiation process.[96] Current research is focused on transcriptional control of HSC activation.[97] A growing number of transcriptional mediators are implicated and epigenetic mechanisms (histone acetylation, promoter methylation) are recognized as major determinants of the activation process. The activation of HSC to MFB in the chronically inflamed liver is partially mimicked by primary cultures of HSC, if these cells are plated on plastic surfaces instead of ECMs allowing no integrin anchorage.[98,99] The model was previously suggested as a valuable tool for studying the role of HSC in chronic liver disease.[100] Accordingly, this cell culture system is quite extensively used for the testing of potentially antifibrotic drugs, e.g. PPAR-γ agonists,[101] trichostatin A, pirfenidone, halofuginone, scavengers of reactive oxygen species (α-tocopherol, resveratrol, quercetin, curcumine), protease inhibitors, cytokines (hepatocyte growth factor (HGF), IL-10, and interferon-γ) and antagonists to receptors of endothelin, cannabinoid receptor CB1, and angiotensin.[29] However, a comparison of the gene expression profiles of HSC activated in vivo by bile-duct ligation or CCl4-injury with that of culture-activated HSC established major differences.[102] Thus, culture activation does not properly reflect genetic reprogramming of disease-driven HSC activation. Due to morphological and functional intralobular (zonal) heterogeneity of HSC,[103,104,105] the processes of activation and transdifferentiation in situ are topographically different, which is also dependent on the different zonal vulnerability of hepatocytes.[106] Accordingly, hepatocytes around the central vein (perivenuous acinus zone 3) are most sensitive and fibrogenesis, i.e. in alcoholic liver injury starts there first.[107] The heterogeneity of HSC or MFB is not confined to their topographic localization, but can also result from their different origins. Morphological and functional criteria and the response to growth factors point to different sources of MFB.[108] As an example, HSC express the neural marker GFAP, vascular cell adhesion molecule 1, and the cytoskeleton protein desmin, which are almost absent in MFB. MFB, however, almost exclusively synthesize the matrix protein fibulin-2.[109] The ETM protein reelin, which is present in quiescent and activated HSC but not detectable in myofibroblasts was suggested as an additional marker to differentiate of HSC from other liver myofibroblasts.[110] Using a dual-reporter gene transgenic mouse model of secondary biliary fibrosis (bile duct ligation) it could be shown that peribiliary, parenchymal, and vascular fibrogenic cells expressed both transgenes (α-smooth muscle actin and collagen α1[I], respectively,) differentially indicating functional heterogeneity.[111] Taken together, there is considerable uncertainty on the relation between HSC and MFB, suggesting several distinct myofibroblast-like cell types. Their composition and functional role might be dependent on the nature of the underlying disorder[112] and point to various sources of MFB beside transdifferentiating HSC.

Figure 2.

Pathogenetic cascade of liver fibrosis (fibrogenesis). The 'canonical principle' of fibrogenesis starts with necrosis or apoptosis of hepatocytes and inflammation-connected activation of hepatic stellate cells (HSC), their transdifferentiation to myofibroblasts (MFB) with enhanced expression and secretion of extracellular matrix (ECM) and matrix deposition (fibrosis). The latter is a precondition for cirrhosis. New pathogenetic mechanisms concern the influx of bone marrow-derived cells (fibrocytes) and of circulating monocytes and their TGF-ß-driven differentiation to fibroblasts in the damaged liver tissue. A further new mechanism is epithelial-mesenchymal transition (EMT) of bile duct epithelial cells (BEC) and potentially of hepatocytes (PC). All three complementary mechanisms enlarge the pool of matrix-synthesizing (myo-)fibroblasts in the damaged liver. Important fibrogenic mediators are transforming growth factor-beta (TGF-ß), platelet-derived growth factor (PDGF), insulin-like growth factor 1 (IGF-1), endothelin-1 (ET-1), reactive oxygen species (ROS including hydroxyl radicals, superoxid anions), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), thrombin, and leptin. ASH, alcoholic steatohepatitis; NAFLD, nonalcoholic fatty liver disease.

Figure 3.

Schematic presentation of hepatic stellate cells (HSC) located in the vicinity of adjacent hepatocytes (PC) beneath the sinusoidal endothelial cells (EC) in normal livers. In fibrotic livers HSC are converted to matrix (ECM)-producing myofibroblasts (MFB). On the right site morphological changes of primary cultures of HSC at the first and fourth day after seeding and MFB (3rd day of secondary culture) are shown. S, liver sinusoids; KC, Kupffer cells; BM, basement membrane; D, space of Disse.

Figure 4.

Most important components of extracellular matrix (ECM) and of cytokines synthesized by activated hepatic stellate cells (HSC) shown in the center by α-smooth muscle staining. Arrows indicate their interaction. CF, colony-stimulating factor; ET, endothelin; HBV, hepatitis B virus; HCV, hepatitis C virus; HGF, hepatocyte growth factor; IGF, insulin-like growth factor; KGF, keratinocyte growth factor; LTBP, latent TGF-ß binding protein; MCP, monocyte chemotactic peptide; MIP, macrophage inflammatory protein; NAFLD, nonalcoholic fatty liver disease; PAF, platelet activating factor; PDGF, platelet-derived growth factor; PGF, prostaglandin F; SF, scatter factor; TGF, transforming growth factor; VEGF, vascular endothelial growth factor.

Figure 5.

Synopsis of cellular interactions of resident liver cells (yellow) and immigrated inflammatory cells (red) with hepatic stellate cells (HSC) in the process of activation and transdifferentiation to myofibroblasts. The most important paracrine mediators are given, among which TGF-ß has a high priority. ECM, extracellular matrix; IGFBP, IGF binding protein; AcAld, acetaldehyde; ICAM, intercellular adhesion molecule; HNE, hydroxynonenal; CTGF, connective tissue growth factor; α2M, α2-macroglobulin.

Figure 6.

Schematic presentation of the compartments of TGF-ß synthesis, secretion, and extracellular immobilization via tissue transglutaminase-dependent fixation of the large latent TGF-ß binding protein (LTBP) to extracellular matrix, release by proteases, and activation of the latent TGF-ß complex by reactive oxygen species (ROS), specific integrins, thrombospondin-1 (TSP-1), or proteases with release of the active TGF-ß homodimer, which binds to TGF-ß receptors (TßR) III, II, and I to initiate the intracellular signaling cascade by Smad phosphorylation. Regulation of the bioactivity of TGF-ß occurs at the transcriptional level and, most importantly, by extracellular activation. LAP, latency associated peptide; TIMP, tissue inhibitor of metalloproteinases.


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