Meena B. Bansal

Reversal of hepatic fibrosis -- fact or fantasy?

The prospect of reversing hepatic fibrosis has generated great interest now that basic science advances are being translated into promising new antifibrotic therapies. It is appropriate to recognize both the historical advances that created the framework for these successes, and the important role that Hepatology has played in disseminating them. A sense of urgency underlies this effort as the epidemics of HCV and NASH are becoming associated with advancing fibrosis. To maintain progress and minimize confusion among investigators and clinicians it is essential to standardize terms referring to fibrosis ‘reversal’ and ‘regression.’ There must also be rapid optimization of non‐invasive markers of fibrosis to relieve this current bottleneck to conducting clinical trials. Progress in identifying genetic determinants of fibrosis could further refine patient selection for clinical trials and shorten their duration, as well as unearthing new directions of scientific inquiry. Realistic expectations for successful anti‐fibrotic therapies reflect solid evidence of fibrosis regression in patients treated effectively for viral liver disease, as well as growing clarity in the understanding mechanisms of extracellular matrix production and degradation. The paradigms of stellate cell activation and apoptosis remain valuable frameworks for understanding pathways of hepatic fibrogenesis and fibrosis regression, respectively. Continued progress is essential in order to identify the determinants and dynamics of fibrosis reversibility, to discover additional targets for anti‐fibrotic therapy, and to develop customized multi‐drug regimens. These advances are sure to be captured in the next 25 years by Hepatology , and to profoundly impact the prognosis of patients with chronic liver disease. (Hepatology 2006;43:S82–S88.)

Features and Outcomes of 899 Patients With Drug-Induced Liver Injury: The DILIN Prospective Study

BACKGROUND & AIMS The Drug-Induced Liver Injury Network is conducting a prospective study of patients with DILI in the United States. We present characteristics and subgroup analyses from the first 1257 patients enrolled in the study. METHODS In an observational longitudinal study, we began collecting data on eligible individuals with suspected DILI in 2004, following them for 6 months or longer. Subjects were evaluated systematically for other etiologies, causes, and severity of DILI. RESULTS Among 1257 enrolled subjects with suspected DILI, the causality was assessed in 1091 patients, and 899 were considered to have definite, highly likely, or probable DILI. Ten percent of patients died or underwent liver transplantation, and 17% had chronic liver injury. In the 89 patients (10%) with pre-existing liver disease, DILI appeared to be more severe than in those without (difference not statistically significant; P = .09) and mortality was significantly higher (16% vs 5.2%; P < .001). Azithromycin was the implicated agent in a higher proportion of patients with pre-existing liver disease compared with those without liver disease (6.7% vs 1.5%; P = .006). Forty-one cases with latency ≤7 days were caused predominantly by antimicrobial agents (71%). Two most common causes for 60 DILI cases with latency >365 days were nitrofurantoin (25%) or minocycline (17%). There were no differences in outcomes of patients with short vs long latency of DILI. Compared with individuals younger than 65 years, individuals 65 years or older (n = 149) were more likely to have cholestatic injury, although mortality and rate of liver transplantation did not differ. Nine patients (1%) had concomitant severe skin reactions; implicated agents were lamotrigine, azithromycin, carbamazepine, moxifloxacin, cephalexin, diclofenac, and nitrofurantoin. Four of these patients died. CONCLUSIONS Mortality from DILI is significantly higher in individuals with pre-existing liver disease or concomitant severe skin reactions compared with patients without. Additional studies are needed to confirm the association between azithromycin and increased DILI in patients with chronic liver disease. Older age and short or long latencies are not associated with DILI mortality.

Human immunodeficiency virus (HIV)-1 infects human hepatic stellate cells and promotes collagen I and monocyte chemoattractant protein-1 expression: Implications for the pathogenesis of HIV/hepatitis C virus–induced liver fibrosis†

Patients coinfected with human immunodeficiency virus (HIV) and hepatitis C virus (HCV) develop more rapid fibrosis than those infected with HCV only. In HIV/HCV‐coinfected patients, fibrosis progression correlates with HIV RNA levels, suggesting a direct role of HIV in liver fibrogenesis. Chemokine (C‐C motif) receptor 5 (CCR5) and cysteine‐X‐cysteine receptor 4 (CXCR4), the two major coreceptors required for HIV entry into cells, are expressed on activated hepatic stellate cells (HSCs), the principle fibrogenic cell type in the liver. We therefore examined whether HIV can infect HSCs, explored the potential mechanisms of viral entry, and assessed the impact of infection as reflected by the ability of HSCs to transfer virus to T lymphocytes and elicit a proinflammatory and profibrogenic response. We report that the laboratory‐adapted viruses HIV‐IIIB (CXCR4‐tropic or X4) and HIV‐BaL (CCR5‐tropic or R5) and primary HIV isolates can infect both a human stellate cell line, LX‐2, and primary human HSCs. HIV entry and gene expression in HSCs was confirmed using HIV–green fluorescent protein (GFP) expression viral constructs in the presence or absence of the reverse‐transcriptase inhibitor azidothymidine. CD4 expression on a subset of primary HSCs was demonstrated using fluorescence‐activated cell sorting and immunofluorescence staining. Blocking experiments in the presence of anti‐CD4, anti‐CXCR4, and anti‐CCR5 revealed that HIV entry into HSCs is predominantly CD4/chemokine coreceptor–independent. HIV infection promoted HSC collagen I expression and secretion of the proinflammatory cytokine monocyte chemoattractant protein‐1. Furthermore, infected LX‐2 cells were capable of transferring GFP‐expressing virus to T lymphocytes in a coculture system. Conclusion: Taken together, our results suggest a potential role of HIV in liver fibrosis/inflammation mediated through effects on HSCs. The role of early highly active antiretroviral therapy initiation in patients with HIV/HCV coinfection warrants further investigation. (HEPATOLOGY 2010)

Hepatic Stellate Cells Express Functional CXCR4: Role in Stromal Cell–Derived Factor-1α–Mediated Stellate Cell Activation

Chemokine interactions with their receptors have been implicated in hepatic stellate cell (HSC) activation. The hepatic expression of CXCR4 messenger RNA is increased in hepatitis C cirrhotic livers and plasma levels of its endogenous ligand, stromal cell–derived factor‐1α (SDF‐1α), correlate with increased fibrosis in these patients. The expression of CXCR4 by HSCs has not been reported. We therefore examined whether HSCs express CXCR4 in vivo and in vitro and explored whether SDF‐1α/CXCR4 receptor engagement promotes HSC activation, fibrogenesis, and proliferation. The hepatic protein expression of both CXCR4 and SDF‐1α is increased in hepatitis C cirrhotic livers and immunoflourescent and immunohistochemical staining confirms that HSCs express CXCR4 in vivo. Immortalized human stellate cells as well as primary human HSCs express CXCR4, and cell surface receptor expression increases with progressive culture‐induced activation. Treatment of stellate cells with recombinant SDF‐1α increases expression of α‐smooth muscle actin and collagen I and stimulates a dose‐dependent increase in HSC proliferation. Inhibitor studies suggest that SDF‐1α/CXCR4‐dependent extracellular signal‐regulated kinase 1/2 and Akt phosphorylation mediate effects on collagen I expression and stellate cell proliferation. Conclusion: HSCs express CXCR4 receptor in vivo and in vitro. CXCR4 receptor activation by SDF‐1α is profibrogenic through its effects on HSC activation, fibrogenesis, and proliferation. Extracellular signal‐regulated kinase 1/2 and phosphoinositide 3‐kinase pathways mediate SDF‐1α–induced effects on HSC expression of collagen I and proliferation. The availability of small molecule inhibitors of CXCR4 make this receptor an appealing target for antifibrotic approaches. (HEPATOLOGY 2009.)

X4 Human Immunodeficiency Virus Type 1 gp120 Promotes Human Hepatic Stellate Cell Activation and Collagen I Expression through Interactions with CXCR4

Background & Aims Patients coinfected with HIV-1 and HCV develop more rapid liver fibrosis than patients monoinfected with HCV. HIV RNA levels correlate with fibrosis progression implicating HIV directly in the fibrotic process. While activated hepatic stellate cells (HSCs) express the 2 major HIV chemokine coreceptors, CXCR4 and CCR5, little is known about the pro-fibrogenic effects of the HIV-1 envelope protein, gp120, on HSCs. We therefore examined the in vitro impact of X4 gp120 on HSC activation, collagen I expression, and underlying signaling pathways and examined the in vivo expression of gp120 in HIV/HCV coinfected livers. Methods Primary human HSCs and LX-2 cells, a human HSC line, were challenged with X4 gp120 and expression of fibrogenic markers assessed by qRT-PCR and Western blot +/− either CXCR4-targeted shRNA or anti-CXCR4 neutralizing antibody. Downstream intracellular signaling pathways were evaluated with Western blot and pre-treatment with specific pathway inhibitors. Gp120 immunostaining was performed on HIV/HCV coinfected liver biopsies. Results X4 gp 120 significantly increased expression of alpha-smooth muscle actin (a-SMA) and collagen I in HSCs which was blocked by pre-incubation with either CXCR4-targeted shRNA or anti-CXCR4 neutralizing antibody. Furthermore, X4 gp120 promoted Extracellular signal-regulated kinase (ERK) 1/2 phosphorylation and pretreatment with an ERK inhibitor attenuated HSC activation and collagen I expression. Sinusoidal staining for gp120 was evident in HIV/HCV coinfected livers. Conclusions X4 HIV-1 gp120 is pro-fibrogenic through its interactions with CXCR4 on activated HSCs. The availability of small molecule inhibitors to CXCR4 make this a potential anti-fibrotic target in HIV/HCV coinfected patients.

Effect of fibrosis on adverse events in patients with hepatitis C treated with telaprevir

Data about adverse events are needed to optimise telaprevir‐based therapy in a broad spectrum of patients.

Inhibition of the CXCL12/CXCR4 chemokine axis with AMD3100, a CXCR4 small molecule inhibitor, worsens murine hepatic injury

Activation of hepatic stellate cells and development of chronic inflammation are two key features in the progression of hepatic fibrosis. We have shown that in vitro activated stellate cells increase their expression of CXCL12 as well as the receptor CXCR4 and that receptor engagement promotes a profibrogenic phenotype. Furthermore, injury promotes increased hepatic expression of CXCL12 and a massive infiltration of CXCR4‐expressing leukocytes, granulocytes and myeloid cells. The primary site of inflammatory cell accumulation is around the CXCL12‐rich portal tracts and within fibrotic septae, indicating a role for CXCR4 during injury. In order to characterize the relevance of the CXCR4/CXCL12 chemokine axis during hepatic injury we inhibited the axis using AMD3100, a CXCR4 small molecule inhibitor, in models of chronic and acute liver injury.

CXCL12 induces hepatic stellate cell contraction through a calcium-independent pathway

Liver fibrosis, with subsequent development of cirrhosis and ultimately portal hypertension, results in the death of patients with end-stage liver disease if liver transplantation is not performed. Hepatic stellate cells (HSCs), central mediators of liver fibrosis, resemble tissue pericytes and regulate intrahepatic blood flow by modulating pericapillary resistance. Therefore, HSCs can contribute to portal hypertension in patients with chronic liver disease (CLD). We have previously demonstrated that activated HSCs express functional chemokine receptor, CXCR4, and that receptor engagement by its ligand, CXCL12, which is increased in patients with CLD, leads to further stellate cell activation in a CXCR4-specific manner. We therefore hypothesized that CXCL12 promotes HSC contraction in a CXCR4-dependent manner. Stimulation of HSCs on collagen gel lattices with CXCL12 led to gel contraction and myosin light chain (MLC) phosphorylation, which was blocked by addition of AMD3100, a CXCR4 small molecule inhibitor. These effects were further mediated by the Rho kinase pathway since both Rho kinase knockdown or Y-27632, a Rho kinase inhibitor, blocked CXCL12 induced phosphorylation of MLC and gel contraction. BAPTA-AM, a calcium chelator, had no effect, indicating that this pathway is calcium sensitive but not calcium dependent. In conclusion, CXCL12 promotes stellate cell contractility in a predominantly calcium-independent fashion. Our data demonstrates a novel role of CXCL12 in stellate cell contraction and the availability of small molecule inhibitors of the CXCL12/CXCR4 axis justifies further investigation into its potential as therapeutic target for portal hypertension.

Hepatic stellate cells: fibrogenic, regenerative or both? Heterogeneity and context are key

AbstractSince their original identification, our understanding of the role of hepatic stellate cells in both health and disease continues to grow. Numerous studies have delineated the role of stellate cell activation in contributing to the pool of myofibroblasts responsible for liver fibrosis, and these have resulted in the development of a number of anti-fibrotic strategies targeting this cell. However, their potential role in liver regeneration, both initiation and termination, is also emerging and needs to be contemplated when considering targeted therapy. Perhaps what is most striking is the increasing recognition that this is not just one cell, but rather, a heterogenous population made up of a number of different subsets of cells, each with differentiated and specific functions. The tools are emerging for this dissection and are greatly needed to truly develop targeted therapies that will inhibit fibrosis while promoting liver regeneration and repair.

Interleukin-15 receptor α on hepatic stellate cells regulates hepatic fibrogenesis in mice

BACKGROUND & AIMS Interleukin-15 (IL-15) and its high affinity receptor interleukin-15 receptor alpha (IL-15Rα) are widely expressed in immune cells and hepatic resident cells. IL-15 signaling has important functions in homeostasis of natural killer (NK), natural killer T (NKT) and cytotoxic T (CD8(+) T) cells, and in liver regeneration. We hypothesized that IL-15 has a protective role in liver fibrosis progression by maintaining NK cell homeostasis. METHODS Fibrosis was induced using two mechanistically distinct models. Congenic bone marrow transplantation was used to evaluate the contribution of IL-15 signaling from various compartments to NK, CD8(+) T and NKT cell homeostasis and fibrogenesis. The gene expression profile of hepatic stellate cell (HSC) from IL-15Rα knockout (IL-15RαKO) mice and wild-type mice were captured using microarray analysis and validated in isolated HSC. Quantitative real-time PCR was used to assess repressors of collagen transcription. RESULTS IL-15RαKO mice exhibited more fibrosis in both models. IL-15 signaling from specific types of hepatic cells had divergent roles in maintaining liver NK, CD8(+) T and NKT cells, with a direct and protective role on radio-resistant non-parenchymal cells beyond the control of NK homeostasis. HSCs isolated from IL-15RαKO mice demonstrated upregulation of collagen production. Finally, IL-15RαKO HSC with or without transforming growth factor beta (TGF-β) stimulation exhibited increased expression of fibrosis markers and decreased collagen transcription repressors expression. CONCLUSIONS IL-15Rα signaling has a direct anti-fibrotic effect independent of preserving NK homeostasis. These findings establish a rationale to further explore the anti-fibrotic potential of enhancing IL-15 signaling in HSCs. LAY SUMMARY We investigated how a cellular protein, Interleukin-15 (IL-15), decreases the amount of scar tissue that is formed upon liver injury. We found that IL-15 and its receptor decrease the amount of scar tissue that is created by specialized liver cells (called stellate cells) and increase the number of a specific subgroup of immune cells (natural killer cells) that are known to eliminate stellate cells. TRANSCRIPT PROFILING ACCESSION NUMBER GSE45612, GSE 68001 and GSE 25097.