Early HCV-associated stellate cell activation in aggressive recurrent HCV: What can liver allografts teach about HCV pathogenesis?

Abstract

Two remarkably similar studies by Russo et al.1 and Gawrieh et al.2 in this issue of Liver Transplantation tested the following hypothesis: can activation of hepatic stellate cells (HSCs), as determined by α smooth muscle actin (αSMA) immunostaining in liver allograft biopsies obtained at 4 months after transplantation, predict development of bridging fibrosis 1 or 2 years later? This hypothesis is based on the traditional model of hepatitis C virus (HCV) pathogenesis: fibrogenesis is the primary process, and HSC activation is the initial event in that process.3, 4 The goal is to identify the liver allograft recipients early after transplantation who are destined to develop aggressive recurrent disease and intervene aggressively with antiviral and/or antifibrogenic therapy.3 HSC, hepatic stellate cell; SMA, smooth muscle actin; HCV, hepatitis C virus. In the normal liver, HSCs, also referred to as lipocytes, perisinusoidal cells, or Ito's cells, are a heterogeneous group of relatively quiescent mesenchymal cells, probably derived from the neural crest, that store retinoids and extend dendritic cytoplasmic projections to optimize contact with neighboring cells, such as endothelial cells, Kupffer's cells, and hepatocytes.3 Classical HSCs are situated primarily in the space of Disse, with some predilection for periportal areas. HSCs are facultative myofibroblasts and are viewed as key participants in the formation of fibrous septae. After local injury, HSCs are thought to transform into myofibroblasts via a two-step process called “activation.”3 The “initiation,” or proinflammatory, stage refers to early paracrine-mediated changes in gene expression and phenotype that render the HSCs responsive to other cytokines and stimuli.3 Examples of early initiators include reactive oxygen species, cellular fibronectin, Toll-like receptor-4,5 active transforming growth factor-β, lipid peroxides, altered extracellular matrix, platelet-derived growth factors, and epidermal growth factor.3 During the second, “perpetuation” stage, HSCs transform into cells that can proliferate, migrate, produce and degrade extracellular matrix, lose retinoids, and release leukocyte chemoattractants and various growth factors and cytokines.3 They also acquire the ability to contract, which can be detected by staining for αSMA. It is important to realize that other portal- and perivenule-based stromal cells, including fibroblasts, myofibroblasts, and smooth muscle cells, also participate in liver fibrogenesis.6 These mesenchymal cells can also behave as facultative myofibroblasts and normally express αSMA or can upregulate αSMA expression after injury.6 The functional and proliferative abilities, relative contributions to fibrous septae, and developmental and functional relationships between these other mesenchymal cells and classical HSCs have not yet been clarified.3, 6 Thus, the reader should keep an open mind about the nature and origin of the αSMA+ cells in these studies.1, 2 As early as 4 months after transplantation, it seems more plausible that activation of other portal-based mesenchymal cells occurs, rather than detection of classical HSCs that have migrated to the portal tracts. Nevertheless, the designs, methods, and results of these two studies1, 2 are remarkably similar to each other (Table 1) and to a previous report showing early and more prevalent αSMA staining of portal and septal HSCs in liver allografts with recurrent HCV than in HCV cases from the general population.7 Importantly, pathologists scoring the biopsies were “blinded” to the clinical course, subsequent outcome, and liver injury test profile. Although the extent of “blinding” is not given in detail, it is assumed that the slide reviews and scoring were independent of absolutely all other data. This includes whether different biopsies were obtained from the same patient or any temporal relationship between the samples. The major shortcoming of both studies1, 2 is that they were conducted retrospectively. The authors try to overcome this deficiency through their “blinded” analysis, but it would be more ideal to prospectively evaluate and score HSC activation at the time the biopsy was obtained. The data should then be stored on an ongoing basis along with other histopathologic, clinical, and laboratory data.8 Another, less significant, drawback is that immunohistochemistry adds a potential source of significant variability and error.9 The variability arises from the method and time of tissue fixation, antigen retrieval and stain amplification techniques, natural variability in reagents, and αSMA expression by portal and other mesenchymal cells in normal liver.6, 9 Finally, fibrosis itself is a risk factor for HCV progression, and progression is not linear: once fibrosis is present, HCV can progress from stage to stage more quickly.4 Liver allografts can be injured simultaneously by several processes that cause portal fibrosis. For example, moderate to severe preservation injury can cause marked portal expansion by 1-2 months after transplantation; superimposed recurrent HCV can then develop more rapidly. The same is true for suboptimal biliary drainage in patients with HCV. It is assumed that these other complications were reasonably excluded. The ability of αSMA staining at 4 months to predict aggressive disease recurrence suggests that early after transplantation something triggers a cascade of events that begins with mesenchymal and/or HSC activation and inexorably leads to the development of bridging fibrosis. What that event might be is certainly open to speculation. Immunosuppression and subsequently increased viral loads certainly must play an important role because HCV certainly progresses more rapidly in allograft than in native livers. Analysis of risk factors might provide some clues. Risk factors for severe recurrent HCV disease include advanced donor age,10, 11 HCV genotype 1,12-15 high HCV RNA levels before and after transplantation,16, 17 hepatic necroinflammatory activity or fibrosis after transplant,17-20 concurrent cytomegaloviral infection,13, 21 T lymphocyte-depleting immunosuppression,11, 22 number of allograft rejection episodes,18, 23, 24 and weaning from immunosuppression.22 In these studies,1, 2 the association between HSC activation score and development of fibrosis was independent of the necroinflammatory activity in the 4-month biopsies, as determined by hepatitis activity index scoring.25, 26 This suggests that factors other than inflammation may be responsible for HSC or stromal cell activation. Possibilities would include various viral proteins,27, 28 viral nucleic acid, and soluble mediators secreted by infected hepatocytes or other cells such as dendritic cells, endothelial cells, Kupffer's cells, and bile duct cells or cholangiocytes, which have been largely neglected in pathogenic models of liver fibrosis. In general, there is wide variation in viral loads after transplantation, and there is not a strong correlation between viral load and severity of liver disease.29 However, like hepatitis B viral infection, extreme HCV RNA levels that usually occur as a consequence of overimmunosuppression and major histocompatibility complex nonidentity between the donor and recipient are definitely associated with a very aggressive fibrosing cholestatic HCV.30-33 In fibrosing cholestatic HCV, a tissue wedge comprised of cholangiocytes and surrounding mesenchymal cells emerges from portal tracts and grows toward other portal tracts to form fibrous bridges.34-36 This extreme example is akin to “forced overexpression” of HCV and suggests that viral replication and cholangiocytes are of importance in disease pathogenesis. Tillmann et al.4 recently compared the traditional model of HCV pathogenesis to the telomere model. The traditional model invokes fibrogenesis as the predominant process, which is induced by tissue damage and begins with HSC activation. In contrast, the telomere model suggests that continual hepatocyte damage and hepatocyte telomere shortening is the predominant process.4 Telomere shortening exhausts the ability of hepatocytes to replicate and thereby fosters fibrogenesis. The telomere model has also been used to explain the rapid progression of fibrosis in chronic allograft nephropathy.37 However, our group showed that irreversible telomere-based mechanisms need not be invoked.38, 39 Instead, reversible replicative senescence induced by inflammation or other hepatocyte stressors, such as viral replication,40 iron,41 steatosis,42 and age42 can also produce the same inhibition. A series of recent publications suggests direct relevance to HCV disease pathogenesis. Marshall et al.43 showed that the HCV core protein could enhance the gene transactivation activity of the tumor suppressor p53, which in turn could enhance the expression of p21waf1/Cip1. Kobayashi et al.40 showed that HCV replication upregulated hepatocyte p21. Transforming growth factor-β, a key activator of HSCs3, 4 and liver stromal cells, can also increases hepatocyte p21 expression.44 Using p21-deficient mice and human liver tissue, we recently showed that stress-induced hepatocyte nuclear p21 expression leads to a replicative disadvantage for hepatocytes.39 Altogether these influences lead to accelerated architectural distortion because of an enhanced ductular reaction at the interface zone, which includes activation of portal-based myofibroblasts and/or HSCs.39 In a recent collaborative study with Clouston et al.,45 we also showed that progression of fibrosis in HCV+ livers with coexistent steatosis was directly proportional to p21-mediated inhibition of hepatocyte proliferation and an enhanced ductular reaction at the interface zone. Similar to the telomere model, a stress-induced model of p21-mediated hepatocyte mitoinhibition emphasizes the importance of a replicative disadvantage for hepatocytes.39 However, an important difference is that hepatocyte inhibition is potentially reversible if stressors are minimized or eliminated. In contrast, when stressors predominate, an enhanced ductular reaction and portal-based stromal and/or HSC activation is provoked. This results in formation of a tissue wedge that more rapidly distorts the liver architecture. This model better fits observations about disease pathogenesis. It explains why any hepatocyte stressors, such as steatosis, iron, inflammation, HCV replication, or spontaneously increased 21 expression, such as occurs with aging, can accelerate liver disease progression.39 It also suggests that the process is potentially reversible and brings to the forefront an important role for cholangioles in the development of classical cirrhosis.