Systems biology methods help develop a better understanding of hepatitis C virus-induced liver injury
Authors: Ragunath Singaravelu, Neda Nasheri, Allison Sherratt, John Paul Pezacki
Journal: Hepatology (2012)

See Articles on Page 17 and 28 Recently, systems biology methods have risen to the forefront of techniques for elucidating the molecular details of disease progression.1 There has been a transition from an emphasis on individual genes to more comprehensive analyses, as technologies continue to evolve for quantitative high-throughput molecular detection. This has led to a variety of studies that have integrated different "omics" approaches to identify key factors in complex host-pathogen molecular networks that are potentially clinically relevant targets for therapy.2 These multidisciplinary strategies continue to enhance our understanding of disease progression and identify prognostic markers; specifically, the identification of key factors linked to disease progression susceptibility and therapy response. To this end, several studies have been performed aiming to link hepatitis C virus (HCV) disease progression and/or therapy outcome to both genetic and proteomic markers.3-5 Genome-wide association and profiling studies have identified human single nucleotide polymorphisms and several interferon stimulated genes, respectively, with varying predictive values.6 In the current issue of HEPATOLOGY, Katze and colleagues have published seminal studies using systems biology methods to gain a better understanding of how HCV reinfection in liver transplant patients can lead to the rapid development of liver disease. DAA, directed anti-viral agent; DEG, differentially expressed gene; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; HDAC, histone deacetylase; HSC, hepatic stellate cells; MF, myofibroblast; OLT, orthotopic liver transplantation; RT, retransplantation; SVD-MDS, singular value decomposition initialized multidimensional scaling. HCV infection remains the leading cause of orthotopic liver transplantation (OLT). For those suffering from chronic HCV infection, OLT remains one of the last recourses. Viremia recurrence is essentially universal in the graft posttransplant; however, the rate of disease progression varies, with 5%-30% of patients developing cirrhosis ∼5 years posttransplant due to accelerated rates of fibrosis.7 The accelerated requirement for retransplantation (RT) in this subset poses an additional economic burden, with patient and graft survival rates post-RT being lower than after primary OLT.8 Serial liver biopsy remains the best way of monitoring disease progression. However, this technique is invasive and risks the possibility of misdiagnosis. Reliable and noninvasive prognostic markers of HCV-associated disease progression are currently being evaluated, with the goal to improve allograft survival. Thus far, significant factors influencing disease progression that have been identified include HCV RNA levels pre- and post-OLT, viral genetics, donor age, and donor/recipient genetics.9 There has been limited work, however, investigating molecular signatures that predict clinical disease progression prior to histological evidence of fibrosis. One study, examining gene expression of recurrent HCV-infected biopsies 1 year post-OLT, reported an increase in myofibroblast (MF) and MF-like cell markers and a decrease in retinoid-related proteins. These observations suggest decreased hepatic stellate cell (HSC) quiescence is correlated with rapid fibrosis progression.10 Another proteomics study linked an up-regulation of genes associated with oxidative stress and mitochondrial dysfunction to later stages of fibrosis (Batts-Ludwig stage 3-4).11 These studies emphasized a correlation between oxidative stress, HSC activation, and fibrosis. A more recent study by Mas et al.12 examined gene expression in biopsies at the time of HCV recurrence to develop a prognostic signature, which, based on nine differentially expressed genes, was capable of distinguishing mild and severe fibrosis at 3 years post-OLT. This report highlighted the potential for identifying predictors of fibrosis in gene expression early after OLT. In this issue of HEPATOLOGY, alternate avenues for diagnosing liver fibrosis, along with characterizing prognostic signatures indicative of rapid disease progression, are explored.13, 14 These two companion studies utilized systems biology approaches to characterize early molecular signatures that correlated with rapid progression in both liver disease and fibrosis in HCV-infected transplant patients. These reports make use of the novel singular value decomposition initialized multidimensional scaling (SVD-MDS) analysis to separate the prognostic markers of interest from the biological noise in their high-dimensional data, which impedes analysis in traditional clustering strategies. In a longitudinal study, Rasmussen et al.14 report transcriptional changes in 57 chronic HCV-infected transplant patients from 111 liver biopsy specimens taken over 110 months post-OLT. Based on reinfection kinetics and homogeneity of sample distribution, biopsies were separated into early, intermediate, and late timepoints post-OLT. The cohort was further separated based on progressors, who exhibited adverse clinical outcomes ∼4-7 years post-OLT (i.e., Batts-Ludwig stage 3-4 of hepatic fibrosis, symptoms of cirrhosis, or graft failure/death), or nonprogressors. Although there was no histological evidence of Batts-Ludwig stage 3-4 fibrosis evident at the time of biopsy for any of the profiled samples, transcriptional changes pointed to possible markers for clinical outcome. This promising study revealed 400 differentially expressed genes (DEG; P < 0.01) 0-3 months post-OLT between progressive patients who suffered adverse clinical outcomes and nonprogressors. Notably, genes associated with immune and inflammatory responses (e.g., leukocyte antigen genes), cell cycle progression (e.g., cell cycle arrest, DNA damage checkpoint, and apoptosis), and metabolic function (e.g., lipid biosynthesis/transport, vitamin and mineral metabolism) were down-regulated in patients who progressed to severe liver disease at an accelerated rate. This is the first study to suggest that patients who develop progressive liver disease experience a more dramatic immunosuppression and HCV-induced reprogramming of both mitotic and metabolic functions within the first 3 months post-OLT. The authors suggest that this reprogramming may allow for enhanced HCV infection leading to disease progression, and that this 0- to 3-month timepoint post-OLT may represent a crucial period for prognostics (Fig. 1). Systems biology approach to predict severe liver disease progression in HCV-infected patients and extend graft survival through personalized therapeutic intervention. HCV reinfection occurs in all newly transplanted livers, with a subset of patients progressing to severe fibrosis and liver disease at an accelerated rate. Progressors can be identified for therapeutic intervention using transcriptomics within the first 3 months posttransplant, where gene expression alterations appear to reprogram hepatic cells for enhanced viral replication. Prognostic gene expression profiles detected within the first 3 months posttransplant include repression of immune responses (e.g., antigen presentation, inflammation) and cell proliferation (e.g., mediators of cell cycle arrest, DNA damage checkpoint control, apoptosis) and could provide key targets of personalized medicine. Without therapeutic intervention, progressors experience elevated oxidative stress and increased HSC activation with proteomic and metabolomic markers detected before histological evidence of liver damage. A companion study in this issue of HEPATOLOGY by Diamond et al.13 identified a proteomic signature indicative of elevated oxidative stress in patients who develop severe liver fibrosis prior to any histological evidence of severe liver injury. The authors performed detailed proteome analysis on liver biopsies for 15 HCV-infected transplant patients taken 6 or 12 months post-OLT. Patients were segregated into progressors or nonprogressors based on histologic evidence of severe liver disease at 1 year post-OLT (Batts-Ludwig stages 3-4 fibrosis). The authors found that transplant patients with rapid fibrosis progression exhibited an altered protein signature, comprising 250 differentially expressed proteins, which can distinguish the progressors from nonprogressors based on SVD-MDS analysis. The authors demonstrated this signature was robust against clinically confounding variables. This is the first study to show that fibrosis proteome markers can be identified weeks to months before there is histological evidence of liver damage. In this study, rapid progression to liver disease was linked with increased oxidative stress, activated HSCs, and HCV-induced epigenetic alterations. Hepatoprotective activities such as superoxide, cysteine, gluthathione, and xenobiotic metabolism and oxidation stress response pathways were down-regulated, suggesting that progressors were experiencing increased oxidative stress (Fig. 1), consistent with previous studies of HCV-associated liver fibrosis.11 This was further emphasized by an up-regulation of genes involved in counteracting protein and DNA modifications due to oxidizing species. In addition to increased oxidative stress, the authors found significant differential expression of genes associated with fibrogenesis, including a general up-regulation of cytokines and genes associated with the proliferative and contractile phenotype of activated HSCs. Finally, protein network analysis identified protein kinase A RII alpha (PRKAR2A) as one of 10 bottlenecks among the differentially expressed genes. This protein's down-regulation has been linked to histone deacetylase (HDAC) activity15 and reinforces a potential link between HCV-induced epigenetic alterations and HSC activation contributing to HCV-associated liver injury.16 With key markers identified in liver biopsy proteomes, the Diamond et al. study provides further evidence for the involvement of oxidative stresses through serum metabolite profiling on an independent cohort of 60 HCV-infected liver transplant patient biopsies taken ∼2-4 years post-OLT. The authors noted increased levels of gamma-glutamyl peptides and a decline in cysteine levels—both consistent with disrupted glutathione homeostasis and increased oxidative stress. Future investigations should analyze these metabolite levels along with the differentially expressed protein candidates that have been found in blood at earlier timepoints, based on the Rasmussen et al.14 study, and evaluate their potential as prognostic markers of fibrosis in progressive patients. Although susceptibility to genetic, dietary, and environmental influences may affect the ability of metabolite screening to predict fibrosis, serum biomarkers may serve as a useful measure to identify high-risk patients, leaving subsequent needle biopsies as confirmation. This strategy would reduce the number of patients subjected to this painful procedure. Taken together, these studies suggest that a prognostic signature, based on a combination of messenger RNA (mRNA), protein, and metabolite markers may allow for identification of patients at high risk of severe disease progression post-OLT. This signature could enable the development of personalized therapies for transplant patients deemed high risk. Considering that early repression of immune responses and cell cycle regulation may lead to increased HCV infection and rapid progression to fibrosis, treatments post-OLT with interferon, ribavirin, or the newly clinically approved directed antiviral agents (DAAs) may rescue innate immunity and intervene in the accelerated liver disease, as supported by a recent study.17 The importance of oxidative stress in disease progression suggests that the use of antioxidants may help delay fibrosis in transplant patients, as previously examined in combination with standard therapy against primary infection.18 Alternatively, the suggested link between oxidative stress, HDAC activity, and HSC activation warrants the investigation of HDAC inhibitors as antivirals to extend patient and graft survival. Previous studies already suggest that HDAC may be an ideal therapeutic target for fibrotic disorders, to promote retention of the quiescent phenotype of HSCs.19 HDAC inhibitors may also have an added benefit as anti-hepatocellular carcinoma (HCC) drugs.20 It should be noted that any preemptive therapy would be limited by low drug tolerance posttransplantation. For example, a recent study suggests that interferon therapy posttransplant may induce acute cellular rejection.21 However, this risk may be justified in patients with data suggesting an unfavorable disease progression. In conclusion, these seminal systems biology studies by Katze and colleagues show evidence that, prior to histological evidence, differential expression at both the protein and mRNA level exists. A model of progressive disease is illustrated (Fig. 1) in patients who exhibit early hepatic immunosuppression (0-3 months posttransplantation). This initial suppression enables higher viral loads and leads to an increased inflammatory response at later timepoints (after 6 months post-OLT). The higher viral load increases oxidative stress and subsequently depletes mitochondrial antioxidants,22 which leads to activation of HSCs and subsequent secretion of collagen scar tissue.23 These molecular mechanisms culminate in accelerated liver fibrosis. Early markers of disease progression could be the basis for a future clinical prognostic test for HCV-recurrent transplant patients that are more likely to have progressive disease and help to direct personalized therapy. The studies also represent a significant step toward the development of a noninvasive index for predicting liver fibrosis, based on serum protein and metabolite profiling. It will be of interest to see if any of these observed markers draw any parallels in the molecular signature of nontransplant patients. Finally, it is exciting to consider other avenues of systems biology that have not been exploited for the development of additional prognostic markers, including lipidomics,24 noncoding transcriptomics, and activity-based protein profiling.25, 26 These approaches have already revealed novel markers of viral infection.27-30 The development of a robust prognostic signature would allow for patient-tailored therapeutic schemes post-OLT, and, undoubtedly, systems biology will play a major role in both driving the signature's definition and assessment. The companion studies within this issue represent a large step forward in achieving these goals.