Hepatic Stellate Cells and Liver Fibrosis

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1 Juan E. Puche, 1,2 Yedidya Saiman, 1 and Scott L. Friedman *1 ABSTRACT Hepatic stellate cells are resident perisinusoidal cells distributed throughout the liver, with a remarkable range of functions in normal and injured liver. Derived embryologically from septum transversum mesenchyme, their precursors include submesothelial cells that invade the liver parenchyma from the hepatic capsule. In normal adult liver, their most characteristic feature is the presence of cytoplasmic perinuclear droplets that are laden with retinyl (vitamin A) esters. Normal stellate cells display several patterns of intermediate filaments expression (e.g., desmin, vimentin, and/or glial fibrillary acidic protein) suggesting that there are subpopulations within this parental cell type. In the normal liver, stellate cells participate in retinoid storage, vasoregulation through endothelial cell interactions, extracellular matrix homeostasis, drug detoxification, immunotolerance, and possibly the preservation of hepatocyte mass through secretion of mitogens including hepatocyte growth factor. During liver injury, stellate cells activate into alpha smooth muscle actin-expressing contractile myofibroblasts, which contribute to vascular distortion and increased vascular resistance, thereby promoting portal hypertension. Other features of stellate cell activation include mitogen-mediated proliferation, increased fibrogenesis driven by connective tissue growth factor, and transforming growth factor beta 1, amplified inflammation and immunoregulation, and altered matrix degradation. Evolving areas of interest in stellate cell biology seek to understand mechanisms of their clearance during fibrosis resolution by either apoptosis, senescence, or reversion, and their contribution to hepatic stem cell amplification, regeneration, and hepatocellular cancer. C 2013 American Physiological Society. Compr Physiol 3: , Introduction Liver fibrosis is a common outcome of virtually all chronic hepatic insults including viral hepatitis (i.e., hepatitis B and C), alcoholic or obesity-associated steatohepatitis (i.e., nonalcoholic steatohepatitis (NASH)), parasitic disease (i.e., schistosomiasis), metabolic disorders (i.e., Wilson s), hemochromatosis and other storage diseases, congenital abnormalities, autoimmune and chronic inflammatory conditions (i.e., sarcoidosis), and drug toxicity, among others (102). Fibrosis was described in 1951 as a passive process with no direct evidence of fibrous tissue proliferation but the suggestion of connective tissue appearance just by stromal condensation due to hepatocyte cell collapse. However, in 1978, a growing body of evidence prompted the World Health Organization to update its definition of fibrosis as the presence of excess collagen due to new fiber formation (3). It is virtually axiomatic now that liver fibrosis results from enhanced production of extracellular matrix (ECM) due to accumulation and activation of myofibroblasts in the context of ongoing or repetitive liver damage. Early studies focused on methods to isolate and grow primary hepatic stellate cells (HSCs) in culture establishing them as one of the main sources of myofibroblasts in liver parenchymal disease resulting from hepatocyte as oppsed to biliary injury (49, 71). Immediately thereafter, the concept of stelllate cell activation emerged, which represents a transdifferentiation of the cell during liver injury from a quiescent state that is rich in vitamin A droplets, to one that is proliferative fibrogenic and contractile (62, 82, 83, 234, 241, 289). While HSCs are a major source of myofibroblasts, mounting evidence also implicates portal fibroblasts as a source when the injury is to bile ducts rather than hepatocytes (53, 162). From early studies focusing on the role of the stellate cells solely as a source of ECM during liver injury, a sustained effort has subsequently uncovered broadening roles of the cell type as a source of regenerative cytokines, an immunomodulatory cell with a range of activities and one that serves roles far beyond those envisioned at the time of its isolation 35 years ago (65). From these studies realistic hopes have emerged for exploiting features of stellate cell activation and hepatic inflammation in devising effective antifibrotic and regenerative therapies for patients with chronic liver disease and fibrosis. This article builds upon a comprehensive review of stellate cell biology in 2008 by one of the authors (S.L.F.) (65), * Correspondence to scott.friedman@mssm.edu 1 Division of Liver Diseases, Icahn School of Medicine at Mount Sinai Hospital, New York, New York 2 University CEU-San Pablo, School of Medicine, Institute of Applied Molecular Medicine (IMMA), Madrid, Spain Published online, October 2013 (comprehensivephysiology.com) DOI: /cphy.c Copyright C American Physiological Society. Volume 3, October

2 Figure 1 Appearance of hepatic stellate cells and the sinusoidal microenvironment in normal and injured liver. In normal liver, stellate cells (shown in blue) are laden with perinuclear retinoid droplets and preserve the differentiated function of surrounding cells, including hepatocytes and sinusoidal endothelial cells. In liver injury, the cells multiply, lose vitamin A and become embedded within dense extracellular matrix. This leads to deterioration of hepatocyte function manifested as loss of microvilli, and decreased size and number of endothelial fenestrations. Reprinted, with permission, from (68). by highlighting the accelerating pace of progress and increasingly nuanced understanding of a cell type that is unique not only in liver, but throughout mammalian biology. The fascination with stellate cells may explain why the published literature contains more than 2800 articles about this cell type only in the last ten years (Pubmed search using the keyword hepatic stellate cell from ). Hepatic Stellate Cell Biology, Origin and Ultrastructure HSCs are resident nonparenchymal cells located in the subendothelial space of Disse, between the basolateral surface of hepatocytes and the antiluminal side of sinusoidal endothelial cells (Fig. 1). This privileged location, together with their dendritic cytoplasmic processes, facilitates their direct contact with hepatocytes, endothelial cells, other stellate cells, and Kupffer cells up to 140 μm away (86, 126). This intimate contact between stellate cells and their neighboring cell types may facilitate intercellular transport of soluble mediators and cytokines. In addition, stellate cells are directly adjacent to nerve endings (19, 299), which is consistent with reports identifying neurotrophin receptors (36, 137, 284, 333), and with functional studies confirming neurohumoral responsiveness of stellate cells (158, 203, 249). Interestingly, apart from the different patterns of distribution (pericentral vs. periportal predominances) among species (28, 65, 305), stellate cells only represent 10% of the total number of resident cells in normal liver (86) and 1.5% of total liver volume, a low proportion of the total cell number in contrast to their remarkably divergent functions in normal and diseased liver. Prominent dendritic cytoplasmic processes from stellate cells contact hepatocytes and endothelial cells (65, 266, 302, 304). These subendothelial processes have three cell surfaces: inner, outer, and lateral. The inner one is smooth and adheres to the adluminal (basal) surface of the liver sinusoidal endothelial cells (LSECs) while the outer surface, facing the space of Disse, has numerous micro-projections which contact with hepatocytes and may function in detecting chemotactic signals, and transmitting them to the cell s mechanical apparatus to generate a contractile force that regulates blood flow (180). Actin filaments and microtubules are distributed in both the periphery and the core of the cell s processes, respectively, and could be responsible for their extension and retraction (115, 148, 204, 246, 252). While no electron dense basement membrane can be identified within the perisinusoidal space of Disse, basement membrane proteins, including type IV collagen, nidogen, and laminin, have been identified, which are thought be functionally important in preserving differentiated hepatocellular function and stellate cell quiescence (20, 29, 70). HSCs were first described by Kupffer in 1876 (303), using a gold chloride method for detection of neuronal components in the liver. Their star-shaped characteristics led Kupffer to call them sternzellen ( star cell, in German). However, in 1898, a misinterpretation based on India ink staining led him to conclude that they were actually special endothelial cells of the sinusoids with phagocytic capacity, thereby confusing them with macrophages (now often called Kupffer cells (304)). Ironically, recent studies have indeed established that stellate cells have phagocytic properties (35, 129, 190, 301). Since Kupffer first identified stellate cells nearly 150 years ago (303), a number of new techniques for their detection and isolation have been developed based on their lipid droplet content, cytoskeletal features, and cell surface markers (288). These approaches have aided in further defining their ultrastructure and enhancing their purity during isolation, for example, by using vitamin A fluorescence to isolate the cells using flow cytometry (45). In normal liver, stellate cells have spindle-shaped cell bodies with perikaryons within recesses between neighboring parenchymal cells, with oval or elongated nuclei. Vitamin A lipid droplets are conspicuous features of the cytoplasm. Stellate cells also have 1474 Volume 3, October 2013

3 Hepatic Stellate Cells and Liver Fibrosis well-developed juxtanuclear small Golgi complex and rough endoplasmic reticulum spaces indicating active biosynthesis of secreted polypeptides or proteins (56). The presence of active lysosomes in stellate cell cytoplasm described for decades (33, 129) is now recognized to indicate a high capacity for the cells to undergo autophagy during cellular activation (104, 105). Endosomes and multivesicular bodies are also present in HSCs (267) and contribute to the generation of vitamin A-containing lipid droplets. In contrast to the well-established origins of other liver populations (endoderm-derived hepatocytes and cholangiocytes, mesoderm-derived endothelial cells and fibroblasts, and ectoderm-derived neurons), the ontogeny of this enigmatic cell has been controversial for decades, because the cells express a pattern of cytoskeletal markers reflecting a range of origins including ectoderm (e.g., glial fibrillary acidic protein (GFAP), nestin, neurotrophins and their receptors, nerve growth factor (NGF), brain-derived neurotrophic factor, synaptophysin, and N-CAM) and mesoderm (e.g., vimentin, desmin, alpha smooth muscle actin, hematopoietic markers) (80, 186). More recently, elegant developmental studies have established that stellate cells are traced to mesothelial cells (likely derived from septum transversum mesenchyme), which appear to give rise to cells that invade the hepatic bud (8, 11, 165). However, due to the limited labeling efficiency of the mesothelium, the proportion of stellate cells derived from the mesothelium is not known and other cellular sources may also be possible. There is ample evidence that HSCs are remarkably heterogeneous in their content of retinoid, cytoskeletal phenotype, potential for activation, and even their capacity to revert to a quiescent state after liver injury resolves (45, 80, 143, 170). For example, there is a subpopulation of stellate cells that may lack typical cytoskeletal markers (16, 235, 257) depending on the lobular location. Pericentral areas are rich in stellate cells with longer cytoplasmic processes (i.e., more astrocytic morphology), predominant GFAP expression (instead of desmin), decreased number and size of lipid droplets, and more differentiated. Periportal stellate cells are typically desmin positive with shorter cytoplasmic processes (with a more contractile phenotype), contain more and larger lipid droplets and may be less differentiated (82, 195, 304, 305). While the origin of stellate cells is becoming less controversial, still uncertain is the possibility that stellate cells are pluripotent and can give rise to multiple cell lineages. This phenomenon has been reported in at least two studies (154, 324), but based only on cell culture findings, where modulation of cellular phenotype is notoriously promiscuous and may not reflect events in vivo. On the other hand, it is increasingly clear that HSCs are intimately associated with the progenitor cell niche and typically surround cells that have stem cell-like properties (39, 40, 89, 219, 311). These findings indicate a strong likelihood that stellate cells support stem cell expansion, although underlying mechanisms and mediators that drive this interaction are not known. Extrahepatic stellate cells have also been described throughout many organs in particular pancreas, where they retain their characteristic shape and markers (267). Stellate cells in other tissues typically store vitamin A and synthesize and secrete ECM components. Best characterized among these are pancreatic stellate cells, which clearly contribute to pancreatic fibrosis and tumor stroma (4, 5, 57). Pancreatic stellate cells reportedly generate stem cells that may also directly contribute to liver regeneration through differentiation into hepatocytes and duct-forming cholangiocytes across tissue boundaries, but this observation requires confirmation (153). Role of Hepatic Stellate Cells in Normal Liver Because of their recognized role in hepatic fibrosis, most studies of HSCs have focused primarily on their behavior during liver injury, but have neglected their contribution to normal liver homeostasis. With an increased availability of tools to selectively express transgenes in stellate cells, their role in normal liver development and function is now being illuminated (Fig. 2). Contribution of stellate cells to liver development As noted above, stellate cells can be identified within the progenitor cell niche (near the canals of Hering) in normal, developing, and regenerating liver (248, 326). Additionally, murine fetal liver-derived Thy1 + cells, which express classical markers of HSCs (α-sma, desmin, and vimentin), promote maturation of hepatic progenitors through cell-cell contact in culture (12). Pleiotrophin, a morphogen secreted by stellate cell precursors (ALCAM + submesothelial cells) during liver development, may contribute to liver organogenesis and regeneration (9, 10). Quiescent stellate cells also express epimorphin, a mesenchymal morphogenic protein involved Vasoregulation Preservation of hepatocyte mass Figure 2 Extracellular matrix homeostasis Quiescent stellate cell Drug metabolism and detoxification Normal liver development Roles of hepatic stellate cells in normal liver. Retinoid metabolism Volume 3, October

4 in differentiation of rat hepatic stem-like cells by a putative epithelial-mesenchymal contact that promotes bile duct epithelial morphogenesis (184), which involves the RhoA and C/EBPβ pathways. These findings complement evidence of paracrine interactions between bile duct epithelium and either stellate cells or portal fibroblasts both in culture (156, 166) and in vivo (141, 156, 285). While not limited to stellate cellbile duct crosstalk, components of the Notch (254) and Wnt pathways (178, 330), purinergic signaling (52), chemokines (156) and the Dlk1 protein (298, 330) are also important in hepatic development. Stellate cell precursors, isolated from fetal liver based on UV fluorescence in flow cytometry (157), display extensive proliferative activity, and a high capacity to express hepatocyte growth factor (HGF), CXCL12, and homeobox transcription factors, supporting their potential contributions to both hepatic development and hematopoiesis (157). A recent study has identified stellate cells in zebrafish using a reporter gene driven by the Hand2 promoter (325). Use of this model will further clarify the stellate cell s contributions to hepatic development and homeostasis. Retinoid metabolism (see section on Perpetuation, Section V, below) Vitamin A (retinoid) is primarily stored in the liver in mammals, and among liver cell types stellate cells are the primary cellular depot (303). Normally, dietary retinoids are absorbed by the gut and transported in chylomicron remnants as retinyl esters to hepatocytes, where they are hydrolyzed into free retinol. Retinol is then transferred to stellate cells, where they are reesterified (101). Importantly, these droplets contain not only retinoids, but also triglycerides, phospholipids, cholesterol, and free fatty acids, among others (188, 320). Recent studies have identified a family of proteins that coat lipid droplets known as perilipins (27, 28, 253). One perilipin, adipose-differentiation-related protein, is expressed by stellate cells and its levels are reduced as the cells activate and lose retinoid droplets (161). The contributions of these lipid droplets go beyond the simple storage of Vitamin A, and extend to the regulation of stellate cell activation (206, 207), possibly through the impact of lipids in fueling autophagy (103, 104). The importance of these retinoid mechanisms in fibrosis however has been challenged, however, as mice deficient in lecithin retinol acetyl transferase (LRAT), the enzyme that catalyzes the esterification of retinol into retinyl esters nonetheless undergo fibrosis (144) following toxic liver injury, perhaps indicating an alternate or more complex role for retinoid metabolism in hepatic and stellate cell homeostasis. Extracellular matrix homeostasis In normal liver, the ECM comprises 0.5% of the total liver weight (245) and is distributed between portal triads, central veins and Glisson s capsule, with only a small portion present in the space of Disse (81). Normal ECM components include collagens (type I, III, IV, V, VI, XIV, and XVIII), elastin, structural glycoproteins (laminin, fibronectin, nidogen/ entactin, tenascin, osteopontin, and secreted acidic proteins rich in cysteine), proteoglycans (heparan sulfate, chondroitin 4-sulfate, chondroitin 6-sulfate, and dermatan sulfate, syndecan, biglycan, and decorin), and the free glycosaminoglycan hyaluronan (74, 136, 259). While quantitatively modest in the latter location, ECM in the space of Disse has an important role in preserving liver homeostasis and has a unique spatial expression pattern. Type IV collagen is primarily located between LSECs and stellate cells while type I and III fibrillar collagens are located between HCSs and hepatocytes in normal liver (20, 70). Primarily, the ECM composition can affect the behavior of surrounding liver cells through cell surface receptors, especially integrins, of which stellate cells express α1β1, α1β2 (227), αvβ6 (222), α5 β1 (110), αvβ6 (216, 222), as well as integrin linked kinase (269). The three cell types surrounding the space of Disse (hepatocytes, endothelial cells, and stellate cells) each produce ECM components in normal liver. While all of them express collagen type I, hepatocytes mainly produce fibronectin (233), endothelial cells express collagen IV, and quiescent stellate cells secrete laminin and collagen types III and IV (83, 171), among several other ECM proteins. The maintenance of ECM homeostasis requires turnover in which production of new components is offset by parallel rates of degradation. Matrix metalloproteinases (MMPs) are the primary effectors of ECM degradation, whose activity is regulated in turn by tissue inhibitors of metalloproteinases (TIMPs) (6, 145). Several liver cell populations (i.e., Kupffer cells, myofibroblast, and hepatocytes) can produce both MMPs and TIMPs (6, 145), however a subgroup of this family, the A Disintegrin and Metalloproteinase-domain proteins (ADAMs) may elude TIMP action and contribute to transforming growth factor beta (TGF-β) activation (26), the most potent stimulus for collagen I production by stellate cells (91, 116, 296). While most ADAMs are expressed by more than one liver cell type, at least two (ADAM 13 and 28) are produced solely by stellate cells (194, 261, 273, 314). Secretion of mediators While stellate cell-derived molecules are a major driving force in hepatic fibrosis they may also play an important role in preserving liver homeostasis and promoting regeneration, although the data do not fully support such a role yet. The specific spatial and temporal expression patterns of these molecules may therefore be important to promoting proper hepatic development and regeneration after injury. In steady state conditions, stellate cells are reported to secrete a range of molecules detailed in the following sections. Growth factors HGF is the most potent mitogen for hepatocytes (256). Quiescent stellate cells can produce HGF, but interestingly, during 1476 Volume 3, October 2013

5 Hepatic Stellate Cells and Liver Fibrosis liver damage the expression of this growth factor is downregulated in stellate cells by the action of transforming growth factor β (TGF-β) (232). This temporal expression of HGF may, therefore, explain the decreased rate of hepatic regeneration in a fibrotic/injured liver. TGF-β is among most potent cytokines that regulate stellate cell phenotype (21, 51). In normal liver TGF-β isoform expression (TGF-β 1,2,3 ) is shared between hepatocytes, Kupffer cells and stellate cells (21). While TGF-β 1 is more highly expressed by Kupffer cells than HSCs, TGF-β 3 is only expressed by stellate cells (48). Regardless, TGF-β is secreted in its latent form, and requires a further activation of the latent molecule to exert its action. The predominant pathways of TGF-β activation diverge among different tissues. In liver, integrins, fibrinogen, and urokinase-type plasminogen activator, among others, can activate latent TGF-β during liver injury, which eventually induces stellate cell activation (25, 227, 278). On the other hand, it can be inactivated by binding to the proteoglycan decorin (15). The recent elucidation of the latent TGF-β structure (271) could yield important new approaches to selectively blocking its activation in vivo. Vascular endothelial growth factor (VEGF) is also expressed by quiescent stellate cells (316). Its potent mitogenic effect toward sinusoidal and endothelial cells underscores the key role that stellate cells play in communication and control of liver homeostasis. This important paracrine signaling pathway between stellate cells and sinusoidal endothelium, mediated by VEGF and soluble guanylate cyclase, may be critical for sinusoidal homeostasis in normal liver and regeneration (309, 310, 319). Other growth factors synthesized by stellate cells are insulin-like growth factors (IGF-I and IGF-II), transforming growth factor α (TGF-α), EGF, stem cell factor, and fibroblast growth factors (both acidic and basic FGF), although their contributions may be more critical during liver development and regeneration (39, 40, 75, 177, 182, 191, 247, 298, 332). Neurotrophins and their receptors NGF, brain-derived neurotrophin, neurotrophin 3, neurotrophin 4/5, the low-affinity NGF receptor p75 and the highaffinity tyrosine kinase receptors B and C are all expressed by HSCs (36), and/or their precursors (284). A number of potential functions of these pathways are suggested by their activities in other tissues; however, to date, their best known function in liver is in contributing to stellate cell activation and tissue repair (36, 137, 215). Other mediators Endothelin-1 (ET-1) is a potent vasoconstrictor produced primarily by endothelial cells in normal liver (322), but also by stellate cells. Interestingly, during liver injury, endothelial cells decrease their production and stellate cells become the dominant source of ET-1, which correlates with stellate cell activation (138, 221, 242, 270) highlighting the complex interplay of ET-1 between stellate and endothelial cells. A carefully regulated pathway exists for activation of latent endothelin in stellate cells (138, 164). Human stellate cells have also functional receptors for adrenomedullin (ADM), a peptide produced by most contractile cells, which modulates the contractile effect of ET-1 (88). Moreover, cultured human stellate cells secrete ADM in baseline conditions, and its production is markedly increased by cytokines (88), a surprising finding given that stellate cell activation promotes cellular contraction. ADM can also attenuate activation of stellate cells by inhibiting TGF-β1 production and TGF-β-induced MMP-2 expression partially through the ERK pathway (312). These results suggest that ADM regulates stellate cell activation and contractility in an autocrine manner. A more comprehensive assessment of protein production by stellate cells was generated by an unbiased proteomic analysis of the cells and their surrounding ECM (13, 127, 236). In initial work by Kristensen, the patterns of protein expression were compared between quiescent, in vivo activated and in vitro activated stellate cells, by two-dimensional-gel electrophoresis. From the 300 identified proteins, 83 were found to be secreted, including collagen α1(i), α1 (III), andα2 (I);α1- antitrypsin; calcyclin, calgizzarin, and galectin-1; proteases including plasminogen activator inhibitor-1 and cathepsin A, B, and D; ganglioside GM2; among others. A more recent analysis by Ji et al. has emphasized the importance of stellate cells in also generating immunoregulatory molecules, consistent with their function in conferring immune tolerance in liver (130, 290, 318). Drug metabolism and detoxification HSCs express both alcohol- and acetaldehydedehydrogenases, but not cytochrome P450-2E1 (34) and it is likely that their contribution to ethanol detoxification is minimal compared to hepatocytes. Apart from P450-2E1, other isoforms of cytochrome p450 are expressed by stellate cells, and are downregulated during cellular activation (321); however, their roles in cellular quiescence and activation are unknown. Some cytochrome p450 isoforms have been identified in stellate cells (160), implicating their participation in xenobiotic detoxification and oxidant stress response. Role of Hepatic Stellate Cells in Liver Injury and Fibrosis The framework for understanding stellate cell activation was established several years ago (63), and remains a practical and relevant template for characterizing the cell s response to injury. A common consequence of liver injury is parenchymal damage with an increase in apoptotic bodies, Kupffer cell activation, production of oxidative species, and ECM remodeling (65), which all function as triggers for cellular activation. Activation of stellate cells comprises two well-established phases: initiation (also called preinflammatory stage ) and perpetuation, which can be followed by a Volume 3, October

6 potential third phase, resolution, if the liver injury resolves (58, 64, 69). During resolution of fibrosis, loss of activated stellate cells occurs through numerous pathways and there is now evidence that stellate cells can not only undergo apoptosis, but are also able to either become senescent or revert to a quiescent phenotype (67, 143, 294). The elucidation of molecular mechanisms underlying these events may accelerate the discovery of potential antifibrotic drug targets. Initiation of stellate cell activation Stellate cell initiation promotes changes in gene expression and phenotype that render the cells susceptible to the changing environment and stimuli in the injured liver, thereby promoting the transition to the perpetuation phase. The earliest signals triggering the initiation of stellate cell activation result from paracrine stimulation by neighboring cell populations (endothelial cells, platelets, immune cells, and hepatocytes) and changes in its surrounding ECM (Fig. 3). As endothelial cells comprise the vascular lining of the liver s sinusoids, they play a vital role in these early stages. They induce the activation of stellate cells by secreting fibronectin (125) and by activating latent transforming TGF-β (149), as well as through secretion of a range of mediators that modulate inflammation and participate in cellular crosstalk (275, 319). Fibronectin s effects are largely promigratory and not fibrogenic, suggesting that stellate cell migration is an important first step in responding to injury (210). Platelets also contribute by secreting TGF-β, as well as EGF and plateletderived growth factor (PDGF), the most potent stellate cell mitogen identified (14, 24). Overall however, the resident hepatic macrophage population may be the main source of PDGF, as well as other paracrine mediators that drive stellate cell activation (106, 287, 308). There is an increasingly nuanced understanding of how inflammatory and immune cells regulate stellate cell responses and activation. In particular, T cells, dendritic cells (DC), and macrophage subsets all have well defined interactions with stellate cells [see (76, 131, 152, 179, 313) for reviews]. Among these, recent studies have characterized a specific macrophage subset in rodents, Ly-6C lo, that are vital for regression of hepatic fibrosis (231). On the other hand, different macrophages can drive stellate cell function including stimulation of matrix synthesis, cell proliferation, and retinoid release by secreting TGF-β, TNF-α, and MMP-9, and production of reactive oxygen species (ROS) and lipid peroxides (230). Moreover, ROS produced by hepatic macrophages can initiate downstream signals that include osteopontin, an ECM protein that can induce collagen (300) and perpetuate the activated stellate cell phenotype. Recent studies further implicate an inhibitory role of platelets in blocking stellate cell activation based on the following: (i) transgenic thrombocytopenic mice develop exacerbated liver fibrosis, with increased expression of type I collagen α1 and α2, during cholestasis (147); (ii) in vitro experiments reveal that, upon exposure to stellate cells, platelets became activated, released HGF, and then inhibited stellate cell expression of the type I collagen gene in a Met signal-dependent manner (147); and, (iii) activation of human stellate cells in culture is suppressed by human platelets or platelet-derived ATP via the adenosinecamp signaling pathway (114). Hepatocytes are the main target for most forms of liver injury including viral infection, alcohol, and obesity, among others (198). Following injury, damaged hepatocytes become a major source of lipid peroxides and apoptotic bodies that initiate stellate activation through a process mediated by Fas and TRAIL (32). The contribution of hepatocytederived apoptotic bodies to stellate cell activation is independent of the inflammatory response, since in cultured stellate cells, addition of hepatocyte apoptotic fragments are directly fibrogenic (33), and can also activate Kupffer cells (31). Hepatocytes also express P450-2E1, an important enzyme involved in the metabolism of xenobiotics as ethanol, and a potent source of ROS (193) that can stimulate stellate cell fibrogenesis (193). Changes in the composition and stiffness of ECM also impact on stellate cell responses (84, 121, 315) suggesting a feed-forward loop where stellate cell mediated changes in ECM further drive stellate cell activation. Early changes in transcription factor activity in response to ECM, as well as soluble signals set the stage for a broad phenotypic transition of the cells, and a large number of nuclear factors have been implicated [see (173) for review]. Additionally, pathways of translational, transcriptional and posttranscriptional regulatory control (including epigenetic pathways and mir- NAs) contribute to this process (18, 41, 99, 163, 172, 174, 218, 243, 297). While the initial presumption that transcription factors largely stimulate stellate cell activation, it appears equally true that other factors repress activation, and their activity is downregulated during cellular activation. Three key examples include Lhx2 (306), KLF6 (85), and Foxf1 (133), in which each contribute to preservation of a quiescent phenotype, such that their loss or downregulation derepresses the activation program. Perpetuation of stellate cell activation mechanisms and implications After the initial liver injury, stellate cells initiate activation followed by a process of perpetuation, leading to accumulation of ECM and culminating in the formation of scar tissue. Perpetuation of stellate cell activation is a tightly orchestrated process that includes a number of functional responses including proliferation, fibrogenesis, chemotaxis, contractility, matrix degradation, retinoid loss, and cytokine/chemokine expression (Fig. 4). Proliferation PDGF is most potent stellate cell mitogen during liver injury. An increase in available PDGF and stellate cell responsiveness due to increased expression of PDGF receptor 1478 Volume 3, October 2013

7 Hepatic Stellate Cells and Liver Fibrosis (A) Normal liver Portal triad Bile duct Hepatocytes HSC Sinusoidal space of Disse Portal vein Sinusoidal endothelial cells KC Terminal hepatic vein Hepatic arteriole (B) Fibrotic liver HSC activation and proliferation Loss of endothelial fenestrations Loss of hepatocyte microvilli Distortion of veins Increase in fibril-forming collagen in space of Disse Fibril-forming collagens (types I, III, and V) Basement membrane collagens (type IV and VI) Glycoconjugates (laminin, fibronectin, glycosaminoglycans, and tensacin) Hernandez-Gea V, Friedman SL Annu. Rev. Pathol. Mech. Dis. 6: Figure 3 Matrix and cellular alteration in hepatic fibrosis. Normal liver parenchyma contains epithelial cells (hepatocytes) and nonparenchymal cells: fenestrated sinusoidal endothelium, hepatic stellate cells (HSCs), and Kupffer cells (KCs). (A) Sinusoids are separated from hepatocytes by a low-density basement membrane-like matrix confined to the space of Disse, which ensures metabolic exchange. Upon injury, the stellate cells become activated and secrete large amounts of extracellular matrix (ECM), resulting in progressive thickening of the septa. (B) Deposition of ECM in the space of Disse leads to the loss of both endothelial fenestrations and hepatocyte microvilli, which results in both the impairment of normal bidirectional metabolic exchange between portal venous flow and hepatocytes and the development of portal hypertension. Volume 3, October

8 Initiation Perpetuation Injury Oxidative stress Apoptotic bodies LPS Paracrine stimuli Proliferation PDGF VEGF FGF ET 1 Contractility NO TGF-β1/ CTGF Fibrogenesis Reversion MMP-2&9; PDGF Chemokines TIMP-1,2 MT-1-MMP Altered matrix degradation Apoptosis Resolution TIMP-1,2 TRAIL Fas Chemokines TLR ligands Inflammatory signaling Adenosine T cells B cells NK cells NK-T cells HSC chemotaxis Figure 4 Pathways of hepatic stellate cell activation and loss during liver injury and resolution. Features of stellate cell activation can be distinguished between those that stimulate initiation and those that contribute to perpetuation. Initiation is provoked by soluble stimuli that include oxidant stress signals (reactive oxygen intermediates), apoptotic bodies, lipopolysaccharide (LPS), and paracrine stimuli from neighboring cell types including hepatic macrophages (Kupffer cells), sinusoidal endothelium, and hepatocytes. Perpetuation follows, characterized by a number of specific phenotypic changes including proliferation, contractility, fibrogenesis, altered matrix degradation, chemotaxis, and inflammatory signaling. During resolution of hepatic fibrosis, there is both programmed cell death (apoptosis) to clear fibrogenic cells, as well as reversion to a more quiescient phenotype. FGF, fibroblast growth factor; ET-1, endothelin-1; NK, natural killer; NO, nitric oxide; MT, membrane type. Reprinted, with permission, from (66). results in rapid proliferation and an overall increase in the absolute number of stellate cells with a profibrogeneic phenotype (24, 220, 317). Tumor necrosis factor (TNF) alpha signaling also contributes to PDGF-mediated stellate cell proliferation primarily through the TNF receptor 1 (291). Stellate cells are also responsive to a wide array of factors including, VEGF (327), thrombin, EGF, keratinocytre growth factor (282), and bfgf (328). Among those, VEGF is one of the major cytokines secreted by activated HSCs, which drives both angiogenesis and fibrogenesis, as described above (44, 135, 159). VEGF production is dependent on the overexpression of COX-2 protein via phospho-p42/44 MAP kinase activation (329). Overall, HSCs contribute to both wound healing and tumor growth, a conclusion underscored by several studies implicating this cell type in the development and growth of both primary and metastatic tumors (47, 134, 209). Tissue inhibitors of matrix metalloproteinases (TIMPs) are profibrogenic by inhibiting matrix degradation, and promoting stellate cell survival. Increased TIMP-1 expression by stellate cells also drives stellate cell proliferation in an AKT-dependent manner (61). This mechanism might contribute to the antiproliferative effects of activated Vitamin D, 1,25(OH)(2)D(3) (1). Vitamin D receptor is expressed by quiescent stellate cells (79), and its expression is downregulated with activation. Consequently, treatment of stellate cells with 1,25(OH)(2)D(3) dampens proliferation via cyclin D1 suppression and decreases expression of type I collagen and TIMP-1 while simultaneously increasing MMP-9 expression. Patients with liver disease exhibit vitamin D deficiency, and appropriate supplementation might prove to be a useful therapeutic intervention (225). MicroRNAs are small noncoding RNA sequences that can regulate posttransciptional gene expression by sequence-specific binding to the 3 -UTR of mrnas to promote their degradation. The role of mirnas in fibrosis progression is being clarified, as mirna levels change in livers of patients with fibrotic disease and in stellate cells during activation (93, 99, 112, 196, 197, 205, 243, 307). In stellate 1480 Volume 3, October 2013

9 Hepatic Stellate Cells and Liver Fibrosis cells, mirnas control proliferation and fibrogenesis by regulating protein expression of proproliferative and profibrogenic signaling pathways. In particular, mir-27a and -27b and mir29b are upregulated in activated stellate cells, whereas their suppression leads to decreased proliferation and an increase in lipid droplets indicative of the quiescent phenotype (205, 244). mir-27a -27b directly target the 3 -UTR of retinoid X receptor α (RXRα) to inhibit its expression. RXRα can decrease DNA synthesis, leading to growth arrest in stellate cells (100). Furthermore, RXRα regulates adipogenesis by activation of peroxisome proliferator-activated receptor γ (PPARγ) which is a master regulator of stellate cell activation (297). RXRα expression is decreased in stellate cell activation and its expression in activated stellate cells increases with inactivation of mir-27a -27b, indicating a direct interaction between RXRα and mir27a -27b (128). In contrast, mir- 195 is downregulated during stellate cell proliferation and its expression is induced upon treatment with IFN-β, which exhibits antifibrotic effects independent of its antiviral activity. Treatment with IFN-β downregulates cyclin E1 and upregulates p21 in a mir-195 specific manner thereby promoting cell cycle arrest and decreased stellate cell proliferation (265). Fibrogenesis Production of ECM, in particular collagen type I, is a hallmark of activated stellate cells. Production of collagen type I by stellate cells is regulated both transcriptionally and posttransciptionally (2, 37, 73, , 167, 175, 214, 238, 279, 280, 295). TGF-β1 is major driver of this process through autocrine and paracrine stimulation of ECM production (see above). The other well-characterized fibrogenic cytokine towards stellate cells is connective tissue growth factor (CTGF/CCN2). Levels of CTGF are increased in liver injury and the cytokine promotes a range of profibrotic activities toward stellate cells, mediated by a G-coupled protein receptor (78, 92, 111). CTGF represents a very appealing target for antifibrotic therapy, since unlike antagonism of TGF-β1, CTGF inhibition should have no impact on hepatocyte growth or confer a risk of carcinogenesis. There is a growing list of other factors that contribute to fibrogenesis, including signaling molecules, chemokines, and cellular stressors (250). For example, osteopontin, an ECM cytokine expressed by stellate cells, activates collagen I expression via integrin α(v)β(3) engagement and activation of the PI3K/pAkT/NFκB signaling pathways (300). Furthermore, the recent identification of receptors to profibrogenic chemokines on stellate cells including CXCR4 (108), CCR1, CCR5 (262), CXCR2 (281), and CCR2 (263), add to the repertoire of signals promoting stellate cell activation. The targetability of chemokine receptors with small molecule inhibitors makes them ideal candidates for antifibrotic therapies. Recently, blockade of IL-17A has been proposed as a potential strategy for cirrhosis treatment due to its induction (together with its receptor) in response to liver injury. IL-17A may promote fibrosis by activating inflammatory and liver resident cells and inducing collagen type I production in HSCs through engagement of the signal transducer and activator of transcription 3 signaling pathway (181). As discussed above, (see Proliferation ) mirnas play a significant role in stellate cell biology and can modulate collagen synthesis. MiR-29b binds directly to the 3 -UTR of collagen-iαi and -IV, thereby inhibiting its translation. mir- 29b expression is repressed by TGF-β, and its overexpression inhibits TGF-β induced collagen expression via a SMADindependent mechanism, while HGF, which exhibits antifibrotic effects in stellate cells, induces mir-29b expression (244, 266). MiR-21, whose expression is enhanced during fibrotic disease, is also controlled by TGF-β signaling. TGFβ functions via 2 distinct mechanisms to increase mir-21 production. Smad3 induces mir-21 transcription, while both Smad2 and Smad3 enhance mir-21 maturation. Chemotaxis Stellate cell chemotaxis is an important event in the generation of fibrotic septae by allowing activated cells to align within regions of injury. Stellate cells migrate primarily towards chemoattractant cytokines (chemokines), and they express a range of chemokine receptors and their cognate chemokine ligands. Notably, stellate cells migrate towards PDGF (113, 142), VEGF, Ang-1 (199), TGF-β 1, EGF (323), b-fgf (59), CCL2 (176), and CXCR4 (254), and CXCR3 specific ligands (23). The mechanism of chemotaxis includes a cytoskeletal remodeling with cell spreading at the tip, movement of the cell body towards the stimulus, and retraction of trailing protrusions (180). Oxidant signaling contributes to these responses. Specifically, numerous chemoattractant signals (PDGF, VEGF, and CCL2) increase NADPH oxidasedependent intracellular ROS and activation of the ERK1/2 and JNK1/2 pathways (50, 212). Furthermore, generation of intracellular superoxide anion or H 2 O 2 by treatment with menadione promotes cell migration even in the absence of specific chemoattractants (198). Hypoxia is another broad activator of stellate cell migration, which functions via two distinct mechanisms. After induction of hypoxic conditions, mitochondrial-generated ROS activate the ERK1/2 and JNK1/2 pathways, driving migration. Sustained hypoxia leads to a HIF-1α-dependent increased production and secretion of VEGF by stellate cells, promoting their mobility (200). Since stellate cell mobilization is also required for tissue wound healing, it has been reported that the space of Disse microenvironment, per se, is another key factor in regulating the migratory behavior of stellate cells. ECM components including MMP-2 and type I collagen are able to induce stellate cell migration (208, 323). Cellular fibronectin containing an alternatively spliced domain A (EIIA) is upregulated during liver injury. Signaling specifically by the EIIA fibronectin variant though integrin receptor α(9)β(1) on stellate cells promotes formation of lamellipodia and cellular motility, further implicating ECM signaling in stellate cell Volume 3, October

10 biology (210). The hyaluronic acid receptor (CD44) is also increased in liver injury and repair, promoting both activation and migration of stellate cells (140). Interestingly, a specific splice variant (CD44v6) is responsible for up to 50% of this migration, confirming the idea that activated stellate cells may depend, to some degree, on CD44v6 and hyaluronic acid for migration. Contraction Stellate cell contraction is thought to be a primary determinant of portal hypertension in patients with end-stage liver disease. The factors leading to portal hypertension include increased blood flow, increased intrahepatic resistance, and disrupted liver architecture. During injury, the hepatic sinusoids undergo both morphological and functional changes mediated by HSCs. Dramatic remodeling occurs, characterized by deposition of collagen matrix, loss of fenestrations and increased density of contractile HSCs (293). Additionally, there is an imbalance of vasoactive forces characterized by deficient nitric oxide production and an increase in vasoconstrictive substances including ET-1, angiotensinogen II, eicosanoids, atrial natriuretic peptide, somatostatin, and carbon monoxide (237, 239, 240, 292). Together, these factors lead to an increase in sinusoidal resistance and portal hypertenstion. The final step in the induction of cellular contraction is phosphorylation of MLC-2, resulting from a calciumdependent and a calcium-sensitive pathway. In the calciumdependent pathway, typically in skeletal and cardiac muscle, release of Ca 2+ from the endoplasmic/sarcoplasmic reticulum leads to activation of myosin light-chain kinase (MLCK) by calmodulin and subsequent phosphorylation of MLC. Alternatively, in the Ca 2+ -sensitive pathway, the dominant pathway in stellate cell mediated contraction and smooth muscle cells, Rho-kinase inactivates the myosin binding subunit by phosphorylation, thereby preventing it from dephosphorylating MLC and inhibiting contraction. The net effect of Rho-kinase activation is increased phosphorylated-mlc and contraction (168). Adenosine plays an important role in stellate cell differentiation, proliferation, and type I collagen production. Despite these profibrotic effects, however, adenosine inhibits stellate cell contraction (as well as chemotaxis) via loss of actin stress fibers. Engagement of the A2a receptor by adenosine promotes PKA activity and Rho A inhibition (276), which establishes a rationale for Rho antagonism as a strategy for treatment of portal hypertension (132). Adenosine therefore promotes both injurious and protective effects on stellate cells and understanding these different functions will be important in the development of antifibrotic agents. Interestingly, the antifibrotic activities of caffeine, now validated in several large cohorts (43, 187, 211), may reflect the compound s effect in reducing adenosine signaling in stellate cells (38). Retinoid loss Recently, there has been a renewed focus on the role of retinoid loss in stellate cell activation and collagen production. Stellate cell activation is characterized by the loss of perinuclear retinoid droplets (65, 70); however, their function in activation and fibrogenesis is only now being revealed. Stellate cells are the largest reserve of retinoids in the body ( 60%) and conversion of retinol into retinyl ester is a hallmark of stellate cell activation. The abundance of vitamin A in stellate cells is heterogenous depending on the intralobular position of the cell (80) and may be indicative of alternate activation states. Quiescent stellate cells that are isolated based on their collagen 1 expression display an increase in CYP251 retinoid catabolizing cytochrome, a decrease in retinyl esters, and a more activated phenotype compared to cells isolated based on their buoyancy in gradient centrifugation (45). LRAT, which catalyzes the esterification of retinol into retinyl ester, is the sole acyltransferase found in the liver. With stellate cell activation, LRAT expression is lost. Additionally, treatment with IL-1 promotes decreased LRAT expression (139). Despite its apparent role in stellate cell activation, mice deficient in LRAT neither display spontaneous fibrogenesis, nor do they exhibit increased fibrogenesis in liver injury models, indicating that perhaps retinoid loss is a marker of activation, but is not crucial for stellate cell activation (144). However, treatment with retinoic acid can decrease stellate cell activation as reflected in reduced collagen I, MMP-9, and α-sma by inhibiting expression of TGF-β (98). In contrast to the lack of dependence on LRAT for fibrogenesis by stellate cells, mice deficient in LRAT are protected from chemical hepatocarcinogenesis. An increase in active retinoids due to their lack of conversion to retinol storage form leads to an overall antiproliferative effect, increased p21 levels, and inhibition of tumor progression (144, 272). An additional link between retinoid metabolism to stellate cell activation has emerged through the recognition that this process requires cellular autophagy (104). Specifically, hydrolysis of retinyl esters liberates fatty acids that are metabolized by β-oxidation, generating the substrates that are essential for fueling the energy-intensive pathways of cellular activation. The free retinol can be detected in the extracellular milieu under these conditions (72), but pathways enabling its cellular egress are not known. Similar to stellate cells, autophagy contributes to the intracellular catabolism of lipids in hepatocytes, fibroblasts (274), and neurons (150, 151). Moreover, inhibition of autophagy in hepatocytes leads to reduced rates of β-oxidation and marked lipid accumulation in cytosolic lipid droplets (274). Matrix degradation Fibrosis is a dynamic process of matrix production and degradation. Fibrotic progression is characterized by the replacement of normal basement membrane, collagen type IV, with scar forming collagen type I. Early matrix degradation is an important step in fibrosis and may be essential for stellate cell 1482 Volume 3, October 2013