Mechanisms of Hepatic Fibrosis

Liver fibrosis is a common sequel to a variety of liver diseases of varying causes. These include autoimmune disorders such as primary sclerosing cholangitis and primary biliary cirrhosis, gene defects such asα1-antitrypsin deficiency, cystic fibrosis and haemochromatosis, alcoholic liver disease, and viral hepatitis. The most common diseases that lead to the development of liver fibrosis in children are biliary atresia, cystic fibrosis, α1-antitrypsin deficiency and viral hepatitis. Liver fibrosis is a major health problem worldwide and in the United Kingdom alone accounts for approximately 6000 deaths annually. However, to date, there are few therapeutic strategies for treating liver fibrosis and liver cirrhosis, which represents the end stage of disease, frequently requires liver transplantation. In liver fibrosis, there is increased hepatic accumulation of extracellular matrix (ECM), most notably type I collagen, together with a wide variety of noncollagenous components such as proteoglycans and glycoproteins. Accumulation of ECM distorts the architecture of the normal liver, compromising its function, whereas qualitative changes in the ECM alter the normal function and phenotype of liver cells. Liver fibrosis may be viewed as a repair response to hepatocellular injury, and its underlying mechanisms are likely to share many features in common with scarring in other organs. There is now little doubt that the key event in liver fibrosis is the injury-induced activation of hepatic stellate cells (HSC, also known as lipocytes, fat-storing cells, or Ito cells) with their subsequent transformation to myofibroblastic cells that overproduce the complex array of ECM proteins ultimately deposited in fibrotic liver. Research in liver fibrosis has at its focal point the question of how these cells become activated after liver injury and the part played by these cells in shifting the homeostasis of ECM in the liver to the net deposition of ECM. In this article, the important aspects of the cell biology of liver fibrosis are reviewed, with particular concentration on recent advances in our understanding of the process of HSC activation. CELLS OF THE HEPATIC SINUSOID The hepatic sinusoid is of central importance to liver function. These small blood vessels permeate the liver and are fed at one end with blood from terminal branches of the portal and arterial blood supplies that flow through the sinusoids and feed into central venules. The lumen of the sinusoid contains Kupffer cells, liver-specific macrophages, that are important scavengers of microorganisms entering the liver in the portal circulation. Kupffer cells are key to the liver's response to injury and have a central role in the development of liver fibrosis. Hepatocytes, the cells of the liver that are responsible for the unique synthetic and metabolic capabilities of this organ, are arranged in palisades along sinusoids. This arrangement allows intimate contact between the brush borders of hepatocytes and the perisinusoidal space of Disse, which underlies the endothelium of the sinusoid. This endothelium differs importantly from that in normal capillaries, in that it is specialised to maximize exchange between hepatocytes and sinusoidal circulation. To this end, sinusoidal endothelial cells have multiple fenestrae through which macromolecules may freely pass between sinusoids and the space of Disse (1). Also, the basement membrane-like ECM underlying these endothelial cells, although having many components in common with typical basement membranes around capillaries (2), has a low-density structure that minimally impedes sinusoid-hepatocyte exchange. Hepatic stellate cells lie within the space of Disse and may be considered as liver-specific pericytes because they have cytoplasmic extensions encircling the adjacent sinusoid. These cells represent less than 5% of the total liver cells but are readily detectable by their cytoplasmic lipid granules, which contain more than 75% of the total body store of vitamin A sequestered as retinyl palmitate (3). During liver injury, HSCs proliferate and undergo activation with transformation to fibrogenic myofibroblasts, which deposit numerous ECM proteins into the space of Disse. As well as compromising macromolecular transfer between sinusoids and hepatocytes, fibrosis within the space of Disse has important consequences for cells surrounding this structure, as detailed later. CELLULAR ORIGINS OF LIVER MATRIX In keeping with their role as important structural components, the major types of ECM proteins (collagens, proteoglycans, glycoproteins) are widely distributed throughout the liver. Collagens type I, III, IV, V, and VI have all been found in liver. The interstitial or fibrillar collagens types I, III, and V are the major components that are localised to the liver capsule, the portal tracts, and surrounding large blood vessels (2,4-9). Collagen types I and VI have a sparse distribution in the lobule, and collagen type III is in periportal and centrilobular zones (4,5). Of the noncollagenous proteins, undulin, fibronectin, and laminin have been localised to portal tracts (10,11) and the lobule (2,11). Within the lobule, matrix is found mainly in the perisinusoidal space, which has an atypical low density basement membrane-like structure, a complex structure containing minor amounts of interstitial collagens, microfilamentous collagen type VI, collagen type IV (in human, but not in rat perisinusoidal space [2,12]), glycoconjugates (laminin, fibronectin, vitronectin, entactin), heparan sulphate proteoglycan, and hyaluronate (2,12-16). In several studies, investigators have examined the cellular origins of liver matrix using techniques applied to liver sections (immunocytochemistry, in situ hybridisation) or techniques such as immunoassay and Northern blot analysis, applied to isolated hepatic cell types maintained in culture. Endothelial cells isolated from rat liver synthesize collagen types I, III, IV, laminin, and fibronectin (17-21). Matrix synthesis has also been attributed to hepatocytes(22-24), although these cells are considered to play a minor role in liver matrix synthesis overall, and collagen type I originally detected in hepatocyte cultures may derive from contaminant HSCs (25). There is abundant evidence from several experimental approaches that activated HSCs elaborate a wide variety of the collagenous and noncollagenous ECM proteins that are known to accumulate in fibrotic liver (17,20,21,23,24,26-38). Cirrhotic liver contains approximately 6 times more ECM than does normal liver, and the relative proportions of ECM components are altered so that, although collagen types I, III and IV are all increased, the change for type I is disproportionate and its ratio to types III and IV therefore increases (2,27,39,40). This switch to collagen type I's synthesizing in excess of collagen type III is also a feature of HSCs activated in vitro(34). Because HSCs are confined to the space of Disse, it is not surprising that profound increases in collagenous and noncollagenous proteins occur in this region in liver fibrosis (2,4,7,11,41). There is an established temporal pattern of ECM deposition in the space of Disse during development of liver fibrosis: In early injury collagen types III and V and fibronectin accumulate(39), but in chronic injury there is increasing deposition of collagen types I and IV, undulin, elastin, and laminin(9). Hyaluronan, a minor component in normal liver space of Disse, is increased more than eightfold(42), and dermatan and chondroitin sulfate increase in proportion to heparan sulfate proteoglycans. This marked accumulation of basement membrane components and collagen type I in the space of Disse is accompanied by loss of fenestrae from sinusoidal endothelial cells(1,2), in an overall process termed capillarisation. Capillarisation of the space of Disse inhibits the transfer of macromolecules between the sinusoidal circulation and hepatocytes, and constricts and distorts the structure of the sinusoid, causing portal hypertension. In cirrhotic liver, the vascular structures at each end of the sinusoid become linked by dense ECM, which extends into the parenchyma, and the whole liver anatomy becomes infiltrated with collagen type I-rich bands easily identifiable in liver sections. At a microanatomic level, the gross qualitative and quantitative changes in matrix may contribute to the disease-associated alterations in the phenotype of hepatocytes, stellate cells, and endothelial cells, as described later. STELLATE CELL ACTIVATION-A PROCESS CENTRAL TO LIVER FIBROSIS Activation of HSCs is an invariant feature of liver diseases that ultimately lead to fibrosis. The process of HSC transformation has been intensively investigated in progressive liver fibrosis in animals. Administration of a single intraperitoneal bolus of CCI4 to rats induces hepatocyte injury and zonal necrosis together with a rapid increase within 48 to 72 hours of the desmin-positive cell population (desmin is a marker for rat HSCs) selectively in areas of injury, demonstrated by their increased incorporation of bromodeoxyuridine (43,44). Increased bromodeoxyuridine labelling has also been demonstrated in desmin-positive cells in rats subjected to chronic bile duct ligation (45,46). In acute CCI4 treatment, HSC numbers and liver histology return to normal within 1 week, but continuous treatment with CCI4 twice weekly results in sustained HSC proliferation and in appearance of activated HSC-expressing α-smooth muscle actin and various ECM products (17,26,46-49). Results in more limited studies with primates(50) and in human liver fibrosis confirm this pattern of expansion of the HSC population, which is transient in such acute insults as those in patients recovering from paracetamol overdose(38) but proceeds to subsequent transformation of HSC and ECM deposition in chronic disease (39,51). Proliferation of HSCs and activation in liver disease can be mimicked accurately in culture, and this model has been used extensively to study the cell biology of the activation process. Hepatic stellate cells freshly isolated by proteolytic digestion of normal liver have the same features as HSCs in intact liver, having a relatively compact phenotype and containing cytoplasmic granules rich in vitamin A esterified to fatty acids (retinyl palmitate) (52). Freshly isolated HSCs show little ECM synthesis or α-smooth muscle actin expression. However, during the first 7 days of culture in serum-containing medium, HSCs proliferate, release most of their vitamin A and gradually spread out on the plastic substrate as they transform to larger myofibroblastic cells that have greatly increased synthetic capacity for a wide variety of collagenous and noncollagenous matrix components (33,35,36,53-55). Activated HSCs also show reorganisation of the cytoskeleton, becoming positive for α-smooth muscle actin(47) and having increased content of fibrillar actin. FACTORS INFLUENCING MATRIX SYNTHESIS IN THE LIVER-IMPORTANCE OF TRANSFORMING GROWTH FACTOR BETA Studies of HSC activation in vitro have shown that this process is regulated by a wide variety of growth factors and cytokines derived from other liver cells (paracrine factors) or from HSCs themselves (autocrine factors). Damaged hepatocytes produce HSC-activating factors(56,57) including insulin-like growth factor(IGF) and IGF-binding proteins (58). Although binding of IGF to its receptors on HSCs is reduced during their activation(59), receptors for the potent HSC mitogen platelet-derived growth factor (60) are increasingly expressed during HSC activation in vivo and in vitro(61). Sinusoidal endothelial cells and platelets activated after liver injury are potential sources of this important HSC mitogen (62), as are HSCs themselves after activation(63). Infiltrating inflammatory cells also produce HSC mitogens such as interleukin-1 and tumour necrosis factor-α (TNF-α). Mast cells, which normally represent a minority cell type in normal liver, accumulate in fibrotic human liver (64) and in rat bile duct ligation model of fibrosis (65). In human liver, mast cell accumulation correlates with the extent of collagen deposition. These cells are a rich source of a variety of mediators, including the serine protease tryptase, heparin, histamine, and basic fibroblast growth factor (bFGF), which promote mitosis and increase collagen production by fibroblasts(66). Kupffer cells produce the HSC mitogens TNF-α, IL-1 and a transforming growth factor-α-related protein(67,68). They are also an important source of transforming growth factor-β1 (TGF-β1)(69): This protein is considered to play such an important role in fibrosis that it deserves detailed discussion. TGF-β mRNA in human and in rat liver after bile duct and and In rat injury, mRNA increases with a to the expansion of the Kupffer cell and HSC and mRNA is localised to these cells in fibrotic liver However, TGF-β are to by a wide variety of liver cell including endothelial cells, bile duct cells, hepatocytes, and HSCs TGF-β HSC of collagens types I, III, and IV, fibronectin and laminin and transformation of HSCs to Because activated HSCs produce and to it may be an important on these cells. The of TGF-β are by its of ECM from In inhibits production of the interstitial and by of of protease including of and HSCs I and for as well as the type III, the and the may TGF-β and its in Administration of to with experimental disease the development of fibrosis in this of an for into rats TGF-β in and reduced experimental fibrosis In show increased matrix deposition in liver and may also be in that their inhibits development of fibrosis in these the central role of in fibrosis. is as a of of an of a of TGF-β linked to a of A large of TGF-β in and is to proteoglycans within ECM. is therefore likely that the of TGF-β in is regulated mainly by activation The major of TGF-β activation in liver is considered to its through on the to the IGF proteolytic within the the of TGF-β The that are important in this are but the serine protease may play a derived from the of by such as As activated HSCs and produce they a for TGF-β activation. However, TGF-β activation by HSCs may be relatively in the of factors derived from other liver cells, such as sinusoidal endothelial cells the of transforming growth activation and its consequences for matrix insulin-like growth factor type TGF-β synthesis or activation an for of fibrosis, this protein has important in and in the proliferation and of cells, cells. important recent is that the than of may be by growth factor This protein is in cells in response to and in an to of ECM Studies of growth factor and its receptors are at an early and is known of their role in liver fibrosis. However, growth factor may be an therapeutic in this HSC advances in understanding the importance of in HSC activation detailed discussion. Hepatic stellate cells contain more than 75% of the total body store of vitamin A as retinyl The loss of cytoplasmic vitamin A is a feature of HSC and the liver content of vitamin A is during HSC activation in liver fibrosis and are known to be important in phenotype in several cell types and loss of vitamin A may play an role in HSC activation. The major of retinyl palmitate by HSCs is to HSCs in inhibits HSC proliferation in response to or platelet-derived growth and also their synthesis of ECM components These that loss of from HSCs may promote their activation. However, the that in which HSCs become with granules, is with their fibrosis this and to date, the role of vitamin A in liver is that between of within HSCs its is therefore that in recent studies investigators have shown that, after HSCs produce which to a of termed is a potent factor for but it has on HSCs has not been their on gene not but through binding to This of receptors has been into and which themselves are into of and During their HSCs show of in other cell is for of cell in may contribute to HSC proliferation by their normal to the of This be by loss of the cellular themselves during activation. for increasing of HSCs to or for the in their may be for liver fibrosis. MATRIX A FACTOR OF LIVER CELL There is a that the ECM is not a binding but that it also the phenotype and function of cells. During liver fibrosis, the major alterations in liver matrix are with changes in the phenotype and function of hepatocytes, endothelial cells, and The role of matrix in liver cell function has been in of hepatocytes on a basement membrane-like matrix or in their and of and but on collagen type I or plastic they become spread out on the substrate and show of these Endothelial cells also a basement membrane-like matrix for of their normal which are on collagen type I The of the on which HSCs are have on their Hepatic stellate cells, freshly isolated from normal liver and on a basement membrane-like matrix their matrix in normal liver, their compact rich in granules, and show production of collagen type In culture of HSCs on collagen type I or basement membrane components sulfate proteoglycan, laminin, collagen type results in their proliferation and transformation within days to the myofibroblastic phenotype collagen type I These that to the normal HSC matrix in early hepatic the normal to HSC HSC activation. 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