Section 2 -

Update on Hepatic Stellate Cells and Other Fibrogenic Cells Paulette Bioulac-Sage Service d'Anatomie Pathologique - Hôpital Pellegrin - CHUBordeaux Groupe de Recherche pour l'Etude du Foie / INSERM E362, Université Bordeaux 2 FRANCE

Introduction Although there are various types of chronic injury in the liver (alcohol abuse, viral hepatitis, drugs, metabolic and autoimmune diseases, congenital abnormalities…), all of them may lead to liver fibrosis [1] which is the main complication of many known chronic liver diseases. Liver fibrosis is defined as the abnormal accumulation of extracellular matrix in the liver and its endpoint is cirrhosis. Whatever the cause and the initial site of injury, the accumulation of extracellular matrix observed in fibrosis and cirrhosis is due to the activation of liver fibrogenic cells, which acquire a myofibroblastic phenotype. Myofibroblasts appear to be a morphological and functional intermediate between fibroblasts and the smooth muscle cells. Myofibroblasts contain cytoplasmic bundles of microfilaments or stress fibres, which play a role in contraction, via mechanisms similar but not identical to those in smooth muscle cells. Myofibroblasts are surrounded by an irregular basal membrane, and are connected to each other by gap junctions and to the extracellular matrix by focal contacts. Myofibroblasts are the main cellular type involved in extracellular matrix deposition during tissue repair, but they are also responsible for synthesising enzymes involved in matrix degradation, tissue remodelling, and scar formation. Myofibroblasts express different sets of cytoskeletal proteins that can be used as markers of cell differentiation; the study of their expression allows the characterization of fibroblastic phenotypic modifications corresponding to functional changes occurring during physiological and pathological repair [2]. Myofibroblasts, absent from normal liver, are produced by the activation of precursor cells, such as hepatic stellate cells (HSC) [3] ; however, it is now well admitted that liver fibrogenic cells are heterogeneous, and that different subpopulations may participate in liver fibrogenesis [4, 5, 6]. There are Different Types of Fibrocompetent Cells in the Liver. 1 – The Hepatic Stellate Cells - HSC in their sinusoidal microenvironment Previously called perisinusoidal or Ito cell, HSC which account for about 5-8% of cells in the normal liver, are one of the four sinusoidal cells, forming special capillaries or liver sinusoids laying in between each hepatocellular plate. The 3 other types of sinusoidal cells are fenestrated endothelial cells limiting the sinusoidal lumen, Kupffer cells which are specialized liver macrophages, often in contact with liver-associated-lymphocytes inside the sinusoidal lumen. HSC are characterized by a perisinusoidal distribution in the Disse space and their long, thin processes extending along and around sinusoids, between the hepatocyte plates. Eight to 10 HSC lie along each sinusoid, between the centrolobular vein and the portal tract ; there are approximately 5 to 20 HSC per 100 hepatocytes. The relationship of the HSC in the sinusoidal environment is one of the most important features of these cells. They are organized into a sheath surrounding the sinusoid network. Indeed, the close association of HSC with endothelial cells resembles that of pericytes in capillaries. However, in normal liver, the endothelium is discontinuous and presents multiple fenestrations without diaphragms, allowing the rapid transport of solutes across the subendothelial space; furthermore, a basal lamina-like substance separates the two cell types but there is no true basement membrane [7, 8]. The subendothelial processes of the HSC terminate as very thin thorn-like microprojections. These microprojections, also seen on the cytoplasmic processes of cultured HSC, are unique and represent characteristic structures of these cells; they make contacts with the plasma membrane of the hepatocyte microvilli. Thus the HSC adhere to the sinusoidal endothelial cells and also to the hepatocytes with these numerous hairs [7]. These hepatocyte-HSC interactions are probably important for the understanding of the role of HSC in the early phase of liver regeneration [9, 10]. - Morphology and functions of HSC: from a "quiescent" to an "activated" state HSC constitute the largest cellular reservoir of vitamin A in the body and play a crucial role in vitamin A metabolism. In their quiescent phenotype, HSC store vitamin A, incorporated in the form of retinol-RBP complex and released as retinol to peripheral target tissues, according to the needs of the organism. Thus, the cytoplasm of quiescent HSC contains a variable number of lipid droplets, present in most (75%) HSC in normal liver [11, 12] intermingled with usual organelles and a more or less abundant cytoskeleton. We have recently shown that cellular retinol binding protein-1 (CRBP-1) is the most useful marker which permit to identify quiescent HSC [13] (see below). HSC belong to the myofibroblast family. They can be activated and change their phenotype under conditions of stress or injury, often losing their lipid droplets upon "activation". The biological markers of these cells also change according to activation level (see below) ; they acquire a myofibroblastic phenotype with the expression of a -smooth muscle actin (SMA). It is generally thought that the activated HSC are responsible for the extracellular matrix accumulation [14]. In normal conditions, HSC synthesize collagens (Co) of different types (Co I, III, IV, V, VI ...), other glycoproteins (such as fibronectin, laminin, tenascin, entactin...) and various kinds of proteoglycans and glycosaminoglycans as well as matrix-degradating enzymes (metalloproteinases such as collagenases, gelatinases, stromelysins...) and some tissue inhibitors of metalloproteinases (TIMPs). All these secretions are increased, in variable proportions, during the process of hepatic fibrosis, by activated HSC. They do highly express a-SMA and exhibit marked morphological and functional modifications through a state of «transitional cells» characterized by the progressive decrease of lipids and increase in RER and filamentous skeleton towards the final state of myofibroblastic cell [15]. In those conditions of fibrosis, capillarisation of sinusoids also occurs (with a continuous endothelium underlined by a true basal lamina and exhibiting CD34 expression by immunohistochemistry). HSC have been shown to migrate in vitro [16], suggesting that they may migrate and take part in the repair process of a lesion [17]. Furthermore, it has been suggested that several subclasses of HSC are present in the liver parenchyma [18]. - Several morphological and functional arguments support the role of HSC in the hemodynamic regulation of sinusoidal blood flow: location in the Disse space; stellate shape; contiguity with nerve endings [19, 20]; contractility in response to different vasoactive substances (such as prostaglandin, thromboxone A2, endothelin-1, substance P, angiotensin II....), as observed in culture. In the context of fibrotic liver, activated HSC have thicker processes and increase their contractile properties. 2 – The Portal Fibroblasts Portal area exhibited the presence of portal fibroblasts in the connective tissue, mainly around vessels and biliary structures; they have a typical appearance of quiescent fibroblasts presenting few cytoplasmic processes. The portal connective tissue of normal liver contains only quiescent fibroblasts, but no myofibroblasts which appeared only when a lesion occurs in the portal zone. It has been shown that the proliferation of biliary structures induced by common bile duct ligation in rats is accompanied by a proliferation of portal fibroblasts, which acquire a myofibroblastic phenotype and are involved in the early deposition of extracellular matrix in the portal zones [6, 21]. 3 – Other Fibrogenic Cells Second-layer cells located around centrolobular veins [22], fibroblasts present in the Glisson capsule surrounding the liver, and vascular smooth muscle cells may also be involved in fibrogenesis, probably by modulation to myofibroblastic cells. Progenitor fibroblastic cells have never been detected in the liver. However, it has been suggested that the canals of Hering and the surrounding stromal cells may constitute a niche for hepatic stem cells [23]. In addition to resident fibrogenic cells, circulating cells could play a role in fibrogenesis ; for example, it has been suggested that circulating fibrocytes represent a major source of fibroblasts during the healing of extensive burn wounds; however, no role for fibrocytes in the development of liver fibrosis has yet been demonstrated. Immunohistochemical Markers Contribute to Differentiate These Liver (Myo)fibroblastic Cell Populations. Many markers have been used to identify the fibroblastic cells involved in liver fibrosis. HSC express classical mesenchymal markers, such as desmin in rats, and vimentin in humans and rats. They also display neural/neuroendocrine features such as neural-cell adhesion protein, glial fibrillary acidic protein, nestin, synaptophysin, neurotrophins, and neurotrophin receptors [24]. The embryonic origin of HSC could be of mesenchymal, endodermal, or neuroectodermal origin [25]. Recent data have shown that HSC most probably do not derive from the neural crest [26]. CRBP-1, involved in vitamin A metabolism, mediating the esterification of retinol to retinyl esters and the oxidation of retinol to retinal and retinoic acid [27] has been shown to be a useful marker for identifying quiescent HSC in normal human liver [13], whereas portal fibroblasts do not [28]. During different model of fibrogenesis leading to myofibroblast differentiation, HSC lose their stores of retinol but maintain CRBP-1 expression, whereas portal fibroblasts begin to express CRBP-1. This suggests that CRBP-1 expression is correlated with myofibroblast differentiation. In addition, it is well known that activated HSC, as well as other fibrogenic cells acquire a-SMA expression, a feature of myofibroblastic differentiation. Double immunostaining for CRBP-1 and a-SMA can identify, in pathological conditions, a wide range of HSC phenotypes and allows the characterisation of different (myo)fibroblast-like sub-populations [13]. In the cirrhotic liver, CRBP-1 is expressed in both septal myofibroblasts and HSC. The (Myo)fibroblastic Cell Subpopulations in Pathological Conditions. The data now available suggest that there is a need to reconsider the role of HSC and the other types of fibrogenic cells in the development of liver fibrosis, as well as in other pathological conditions. In fibrogenesis process, depending on the site of primary aggression, fibrosis is initially portal, central or perisinusoidal. Therefore, different types of diseases (such as chronic viral hepatitis, alcoholic hepatitis and non-alcoholic steatohepatitis, chronic bile duct obstruction…) may lead to different patterns of fibrosis during disease progression [29, 30]. Thus, different types of fibrogenic cells could be predominantly involved in the different types of fibrotic disease. The pericyte-like characteristics of HSC including their close relationships with endothelial cells and their expression of smooth muscle markers when activated have been extensively discussed [31]. HSC are probably "resting pericytes" that may be rapidly activated (e.g., in response to changes in blood pressure), acquiring contractile properties and functioning as "liver-specific pericytes", participating in the regulation of local sinusoidal blood pressure. HSC have been shown to be activated early in transplantation, as demonstrated by the acute, transient production of a-smooth muscle actin [32]. Moreover, ex vivo liver perfusion induces the early activation of HSC, which begin to produce a-SMA and significant changes in the perisinusoidal extracellular matrix [33]. Furthermore, it has been suggested that activated HSC can revert to quiescence [34, 35], and thus that they have a plastic phenotype. Both myofibroblastic HSC and hepatic epithelial progenitors accumulate in damaged livers. Surprisingly, it was recently shown HSC and hepatic epithelial progenitors both co-express epithelial and mesenchymal markers, providing evidence that epithelial- mesenchymal transition occurs in adult liver cells [36]. Conclusion It is increasingly accepted that in the liver, as in many other organs (e.g., the kidney with the mesangial cells and the interstitial cells), different fibrogenic cell populations are involved in repair processes leading, in situations of chronic injury, to an excessive deposition of extracellular matrix. This better knowledge of fibrogenic cells could lead to find new effective treatment specifically preventing the progression of fibrosis/cirrhosis. Putative anti-fibrogenic drugs could prevent excessive matricial deposition by decreasing myofibroblastic differentiation and activation, by inducing myofibroblast apoptosis, or by increasing extracellular matrix degradation. New strategies involve drug targeting or selective gene delivery [3]. Furthermore, in order to correctly investigate liver fibrosis (development and reversibility), and even if liver biopsy - despite its well known limits- remains the current gold standard, non invasive new methods of investigation are now available [37]. In addition to blood tests such as the FibroTest and ActiTest combination, a new FibroScan® technique has been developed, based on ultrasound elastography and providing an instantaneous, totally non-invasive assessment of hepatic fibrosis [38]. 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