In vivo there is a close topographical relationship between the site of inflammation and the development of fibrosis. Although there are a substantial number of leukocytes in the resting liver, liver injury results in a massive accumulation of recruited inflammatory cells, with contemporaneous activation of the resident inflammatory cells. There is evidence that inflammation promotes fibrosis through a number of mechanisms and cell mediators. Foremost among these is TGF-β1, which has been identified as the most profibrotic cytokine, promoting HSC expression of collagen I, HSC transition to a myofibroblast-like phenotype, and HSC inhibition of ECM degradation through the expression of TIMPs (44
). In parallel, PDGF has emerged as the most potent pro-proliferative cytokine for HSCs (45
). For both of these cytokines, a mechanistic role for these mediators in the fibrotic process has been demonstrated in animal models (46
). Connective tissue growth factor (CTGF) (48
) has also recently emerged as a potential mediator of fibrogenesis.
The cell culture model of HSC activation has also facilitated the detailed study of nonsoluble regulatory factors present in the fibrotic microenvironment. Among these are cell-cell and cell-ECM interactions. The process of inflammation and repair results in substantial and potentially rapid changes in the ECM content adjacent to activated HSCs. In the normal liver, HSCs are close to a non–electron-dense basement membrane–like ECM (50
). Following injury and fibrosis, this is degraded and replaced with an ECM rich in fibrillar collagens and other noncollagen ECM molecules and integrin ligands (50
). An accumulating body of evidence now indicates that HSC-matrix interactions exert a profound influence on HSC behavior, regulating their activation, proliferation, survival, and cell cycle arrest (51
). If culture-activated HSCs are replated on a basement membrane–like matrix, a reversal of activation is observed, and several of the markers of activation become downregulated, including expression of collagen and TIMPs (51
). Plating freshly isolated HSCs on a basement membrane–like matrix has the effect of preventing spontaneous activation. By contrast, the activated phenotype is promoted by plating quiescent HSCs on collagen I (51
). Other experiments blocking specific molecular HSC-ECM interactions have identified HSC expression of discoidin domain receptors and integrins as crucial for the effects of ECM on HSC behavior (52
An emerging area of interest is the role of ECM stiffness (as opposed to composition) as a mediator of HSC behavior. In a series of elegant studies, Wells and her group have developed culture models that demonstrate that HSC activation can be linked to the rigidity of the subcellular matrix (56
). This mechanism of HSC activation might be particularly germane early in injury because tissue edema (which increases tissue rigidity) is a characteristic of inflammation.
Animal models have also proven to be highly effective in demonstrating the role of specific components of the inflammatory system in the development of fibrosis (for detailed review, see ref. 57
). A brief summary of studies in which individual inflammatory cell types have been depleted, and the subsequent effect of this on the development of fibrosis, is given in Tables 1 and 2. Macrophages have been demonstrated to promote fibrosis in a series of studies, are a potent source of the HSC activator TGF-β1, and can regulate the HSC response to PDGF (58
). NK cells might also profoundly affect the fibrotic response (63
). However, other cells of the innate immune system that might be expected to provoke fibrosis, such as mast cells and neutrophils, seem to exert a less profound influence on experimental fibrosis (64
). One of the main potential sources of PDGF in liver injury is, of course, the platelet. Remarkably, the role of platelets in liver fibrosis has not been studied in detail in vivo to date. Models of wound healing elsewhere in the body have successfully employed platelet depletion without dramatic results, but this work merits extension to models of liver injury (70
). The role of the clotting cascade has received much attention in studies of pulmonary fibrosis (72
), and recent studies in liver fibrosis have implied a role, particularly for thrombin, in promoting the fibrotic response. Indeed, the procoagulant state associated with factor V Leiden is also associated with the progression of fibrosis in chronic HCV infection (73
Animal models demonstrate that both B and T cells can regulate the fibrotic response in vivo (74
). Indeed, one of the most interesting observations to arise from studies of the differential susceptibility of mouse strains to fibrosis was that a Th2 cell response drives fibrosis more effectively than a Th1 cell response (76
). It is tempting to speculate that the response to liver infection with parasites, which is characterized as a Th2 cell response, might have evolved to promote the development of an aggressive intrahepatic scarring process that in the short term might be expected to wall off and compartmentalize the parasite but that in the long term leads to progressive fibrosis (76
). Additionally, soluble factors have been shown to have a role in the pathogenesis of fibrosis in studies using gene knockout mice. These include adipokines, vasoactive substances, interleukins, and IFN-γ (reviewed in ref. 5
Tissue culture models have also demonstrated that HSCs can themselves regulate fibrosis and inflammation, both through autocrine expression of specific cytokines and through direct response to non-cytokine components of the inflamed microenvironment (78
). The ingestion of apoptotic hepatocytes by HSCs, for example, leads to their increased secretion of TGF-β1 in a manner analogous to that seen for macrophages (78
). HSCs also participate in the innate immune response directly, through expression of TLR4 (80
). These data emphasize the dynamic role played by the activated HSC regulating the inflammatory and fibrotic responses in addition to mediating fibrogenesis.
Non-HSC origins of myofibroblasts in the fibrotic liver.
Very recently, evidence has emerged from both animal models and human studies that liver myofibroblasts can be derived from BM stem cells (81
). Additionally, there is evidence that periportal fibroblasts and myofibroblasts derived by epithelial-mesenchymal transition (EMT) might make up part of the fibrogenic cell population (see below) (Figure ) (84
Diagramatic representation of the possible sources of liver myofibroblasts.
Each portal tract contains a population of portal myofibroblasts that probably contribute to fibrotic diseases, such as viral hepatitis and autoimmune conditions, with a portal component. Intriguingly, comparative tissue culture studies suggest that activated HSCs proliferate more rapidly than portal myofibroblasts and might therefore represent the dominant resident liver myofibroblast cell population during fibrotic injury (89
). Additionally it has been postulated that hepatic myofibroblasts might arise by EMT (88
). It seems probable that, in the near future, sophisticated lineage tracking will be employed to define the contribution of EMT to liver fibrosis.
Recently there has been substantial interest in the role of stem cells in the pathogenesis of liver fibrosis. Although initiated by the observation in human liver that hepatocytes might be derived from BM (90
), it seems increasingly probable that fusion of stem cells and hepatocytes accounts for this finding (90
). Lineage tracking of cells in humans is impossible in most situations, although it has been possible in a series of male patients with sex-mismatched liver transplants, who subsequently developed graft disease (i.e., fibrosis), and a single female patient who developed cirrhosis after receiving a BM transplant from a male (81
). After Forbes and colleagues identified these individuals, they used Y chromosome tracking to identify the origin of the cells participating in liver fibrosis (81
). Substantial numbers of scar-associated myofibroblasts in fibrotic areas were found to be BM derived (81
). Subsequently, using a mouse CCl4
-intoxication model of liver fibrosis in which sex-mismatched BM transplants were undertaken, the same group observed clear evidence of a BM contribution to the myofibroblasts within fibrotic scars (82
). Additionally, there was evidence that the BM contributed to both the macrophage and HSC populations within the injured liver (82
). This work was subsequently reproduced in a BDL model of liver fibrosis (83
). Mouse models using sex-mismatched BM transplantation have dissected the bone marrow stem cell–liver axis in greater detail (82
). By subfractionating the BM stem cell compartment, it has been demonstrated that although hematopoietic stem cells contribute to the inflammatory cell infiltrate, the myofibroblast-like cells derived from the BM are of mesenchymal stem cell origin (82
). Intriguingly, BM-derived cells are widely distributed within the scar in advanced fibrosis. That is, whatever the origin and topography of the injury in chronic disease, BM-derived myofibroblasts begin to replace local recruitment of myofibroblasts over time.
Evidence of a functional role for BM-derived myofibroblasts was provided by transplanting BM from genetically-modified mice into wild-type mice before inducing fibrosis with CCl4
). When BM was transplanted from mice bearing a reporter transgene for collagen, the recruited myofibroblasts were shown to transcribe this gene. Moreover, when wild-type mice were transplanted with BM from a transgenic mouse that develops a characteristic pattern of liver scarring because it expresses a form of collagen I not susceptible to degradation by MMPs, CCl4
administration induced the development of liver scarring with characteristics similar to those seen in the BM donor mouse (82
). Therefore, transfer of genetically modified BM altered the phenotype of the liver fibrosis to reflect the genotype of the BM donor rather than the recipient mouse. Additionally, this study provided hard evidence that the recruited cells contribute directly to fibrosis through the expression, synthesis, and secretion of collagen I. These studies did not analyze the specific mechanisms by which the BM-derived cells are recruited to the liver and did not identify whether the cells are recruited to directly become myofibroblasts or whether they require a transition through, say, an HSC phenotype. Some evidence from the BDL model, however, suggests that the cells might be recruited as CD45+
fibrocytes that enjoy a relatively widespread lymphoid organ distribution in injury but that transform to myofibroblasts in the liver in the presence of TGF-β1 (83
Plasticity of the liver myofibroblast.
A further level of complexity in our understanding of the liver myofibroblast comes from recent studies aimed at monitoring the expression of characteristics perceived to define activated HSCs and myofibroblasts, namely, collagen I and α-SMA. In a study using a dual reporter transgenic mouse in which expression of collagen I and α-SMA could be detected independently, strong evidence emerged for functional differences between the periportal myofibroblasts (which were shown to express collagen I but not α-SMA) and the myofibroblasts derived from HSCs (which were shown to express both collagen I and α-SMA) (93
). Moreover, after extracting HSCs from the liver of these transgenic mice, there was evidence in tissue culture for temporal changes in the expression pattern of individual genes, suggesting that there might be day-to-day variation in the expression of genes used to define the myofibroblast phenotype (93
Taken together, these recent observations suggest that we need to be open minded about the origin of the fibrogenic cells in the liver, an origin that might change with the topography and duration of the pathology. Furthermore, perhaps rather than make assumptions about cell behavior and function on the basis of (oftentimes presumed) lineage, we should aim to define cells on the basis of function, in situ and in vivo.
Although fibrosis was previously thought to be at best irreversible and at worst relentlessly progressive, data from animal models and human studies have recently challenged these ideas (7
). Cell culture studies have provided clues to the mechanism underlying both ECM accumulation and degradation in liver fibrosis. As described above, the activation of HSCs and their transition to a myofibroblast-like phenotype is associated with increased expression of collagen I. The mechanisms and chronology of gene expression during activation are easily studied in tissue culture. For example, it has been demonstrated that in addition to enhanced transcription, changes in mRNA stability mediated by a 5′ loop in the mature transcript encoding collagen I are responsible for the increased amounts of mRNA encoding collagen I in activated HSCs (94
). During activation in tissue culture, rodent HSCs demonstrate a distinct pattern of MMP expression. Early during the activation process, MMP-13 (also known as collagenase 3) and MMP-3 (also known as stromelysin) are transiently expressed (30
). As HSCs become more activated, expression of MMP-13 and MMP-3 decreases, whereas expression of MMP-2 (also known as gelatinase A), MMP-9 (also known as gelatinase B), and MMP-14 (also known as MT1-MMP) increases (97
). Therefore, in terms of target substrates, HSCs undergoing activation express a true collagenase in combination with a promiscuous enzyme with degradative activity against several ECM components. By contrast, fully activated myofibroblast-like HSCs express MMPs that act against type IV collagen, the main component of the basement membrane–like matrix, degradation of which would be expected to further promote activation (see above). Expression of MMP-2 can also be important in mediating HSC proliferation, potentially by regulating ECM turnover (54
), and in combination with MMP-14 might confer degradative activity against collagen I (101
). Studies of whole human and rat liver indicate that MMPs that act against several ECM components are expressed in end-stage cirrhosis (95
). However, during the process of HSC activation and before collagen I expression is increased, HSC expression of TIMP1 and TIMP2 is markedly increased (30
). Indeed, it is possible to demonstrate that the secreted MMPs, MMP-2 and MMP-9, are inhibited more than 20-fold by HSC-derived TIMP1 (103
). Subsequent studies using several animal models of progressive fibrosis and studies of explanted human liver have confirmed that fibrosis is characterized by an upregulation of TIMP1 and TIMP2 (30
These data led to the hypothesis that TIMPs regulate the pattern of ECM degradation that characterizes liver fibrosis by holding in check the activity of concurrently secreted MMPs (100
). The implication of this observation is that liver fibrosis is potentially reversible and that ECM degradation should occur if the TIMP-MMP balance is altered to favor ECM degradation. Studies of BDL- and CCl4
-mediated liver fibrosis were undertaken to determine the reversibility of fibrosis and to confirm this prediction. Following withdrawal of CCl4
after four weeks of intoxication, an established fibrosis will undergo spontaneous resolution with remodeling of the ECM (104
) (Figure ). A return to a virtually normal liver architecture ensues. A similar sequence of events occurs in BDL-mediated fibrotic livers after bilio-jejunal anastamosis (105
Liver cirrhosis is an example of dynamic wound healing.
By studying multiple time points during the recovery process, it can be shown that the amounts of TIMP1 and TIMP2 decrease after the insult that induced fibrosis is withdrawn. In association with this decrease, hepatic collagenase activity increases and net ECM degradation occurs (102
). In parallel, the myofibroblasts are lost from the receding hepatic scar by apoptosis. The identification of myofibroblast apoptosis as a major feature of spontaneous resolution of fibrosis has led to substantial interest in how this process is regulated, with the aim of manipulating the hepatic scar (for detailed review, see ref. 106
As one might expect from this data, subsequent mechanistic studies to modulate the TIMP-MMP balance have confirmed the powerful influence this ratio has on the development and resolution of fibrosis. Overexpression of TIMP1 in mice was not associated with fibrosis in the absence of injury. However, following CCl4
intoxication, TIMP1 overexpression enhanced fibrosis and prevented spontaneous resolution (107
). Adenovirus-mediated overexpression of MMP-8 (also known as neutrophil collagenase) in mice is associated with decreased fibrosis (109
), and administration of neutralizing TIMP1-specific antibody decreases the collagen content in CCl4
-induced fibrosis (110
). A particularly ingenious approach to dysregulating the TIMP-MMP balance has been taken by Roeb and colleagues. They have engineered a nonfunctional form of MMP-9 that binds TIMPs and sequesters the MMP inhibitory activity (111
), thereby unharnessing the ECM-degrading potential present in a tissue. This TIMP1-scavenging tool has been successfully deployed in CCl4
-induced fibrosis to decrease collagen levels in the fibrotic tissue and to increase apoptosis of activated HSCs.
Taken together, these data indicate that during progressive fibrosis in wild-type animals, TIMPs are expressed at levels that are sufficiently high to prevent net ECM loss from the liver. Nevertheless, controlled ECM turnover might still occur, particularly at the cell surface, where the concentration of active MMPs is likely to be greatest. Furthermore, the degradative activity of activated HSCs is likely to be most effective against the normal basement membrane–like matrix, and as a result, any degradation will promote activation of HSCs, thus perpetuating the fibrotic response.