Reversal of Liver Fibrosis: A Review

Liver fibrosis is the final pathway for most chronic liver disease and is the main reason for increased mortality in affected patients. The extent of liver fibrosis displays great individual variation, even after controlling for age (at infection), gender & exogenous factors. Thus, host genetic factors are likely to play an important role in the process of liver scarring. (Weber et al., 2008). Inflammation is strongly associated with chronic liver injury, and fibrosis is part of the liver’s wound-healing response. Inflammation represents the driving force for the progressive accumulation of extracellular matrix (ECM) components, eventually leading to liver cirrhosis and hepatic failure. Although even acute injury will activate mechanisms of fibrogenesis, the sustained signals of inflammation associated with chronic liver disease caused by infection, drugs, metabolic disorders, or immune attacks are required for significant fibrosis to accumulate. Cirrhosis is the result of many liver diseases and consists of fibrosis and regenerating nodules. Clinical presentations vary from asymptomatic to advanced end stage liver diseases with complications. In addition, a significant need exists for developing safe and accurate noninvasive technique for detecting progression or regression of hepatic fibrosis in patients with chronic liver disease (Fallowfield JA et al., 2006). The past few years have seen remarkable progress in the field of liver fibrosis, including understanding the crosstalk between innate immunity and inflammatory cells & pathways regulating fibrosis regression. This article will review recent advances in this field as well as imaging and diagnostic test of liver fibrosis.


Introduction
Liver fibrosis is the final pathway for most chronic liver disease and is the main reason for increased mortality in affected patients.The extent of liver fibrosis displays great individual variation, even after controlling for age (at infection), gender & exogenous factors.Thus, host genetic factors are likely to play an important role in the process of liver scarring.(Weber et al., 2008).Inflammation is strongly associated with chronic liver injury, and fibrosis is part of the liver's wound-healing response.Inflammation represents the driving force for the progressive accumulation of extracellular matrix (ECM) components, eventually leading to liver cirrhosis and hepatic failure.Although even acute injury will activate mechanisms of fibrogenesis, the sustained signals of inflammation associated with chronic liver disease caused by infection, drugs, metabolic disorders, or immune attacks are required for significant fibrosis to accumulate.Cirrhosis is the result of many liver diseases and consists of fibrosis and regenerating nodules.Clinical presentations vary from asymptomatic to advanced end stage liver diseases with complications.In addition, a significant need exists for developing safe and accurate noninvasive technique for detecting progression or regression of hepatic fibrosis in patients with chronic liver disease (Fallowfield JA et al., 2006).The past few years have seen remarkable progress in the field of liver fibrosis, including understanding the crosstalk between innate immunity and inflammatory cells & pathways regulating fibrosis regression.This article will review recent advances in this field as well as imaging and diagnostic test of liver fibrosis.

Advances in the mechanisms of fibrogenesis
Liver fibrogenesis is characterized by cellular activation of hepatic stellate cells (HSCs) and its mediators.HSCs have dominated studies exploring mechanisms of liver fibrosis over the last two decades.HSCs are resident vitamin A-storing cells in the perisinusoidal space of Disse between the sinusoidal endothelium and hepatocytes.Following hepatic injury, HSCs become activated into a myofibroblast-like phenotype that is contractile, proliferative and fibrogenic.Collagen and other ECM components are deposited which result in a fibrous scar eventually leading to cirrhosis and liver failure (Lee & Friedman, 2011).Liver fibrosis is a dynamic process, resulting from the equilibrium between fibrogenesis and altered matrix degradation, and may be reversible prior to the establishment of advanced architectural epithelial-mesenchymal transition (EMT) may be involved in the fibrotic process in the liver as well as in the kidney and lung (Zeisberg et al., 2007).Perpetuation of the hepatic fibrotic process is supported by the activation of HSC and the presence of MFB, which are continuously stimulated by growth factors, cytokines and oxidative stress derived from nearby cells.Damaged hepatocytes are a source of lipid peroxides and ROS generated from hepatocytic mitochondria (Jiang et al., 2010).Macrophage chemotactic protein-1 (MCP-1) and osteopontin derived from activated Kupffer cells are involved in the infiltration of inflammatory cells into the liver (Syn et al., 2011).Liver fibrosis is the final common pathway of the wound-healing response of the liver which can progress to liver fibrosis and eventually cirrhosis.At the cellular and molecular level, this progressive process is characterized by cellular activation of HSCs and aberrant activity of TGF-with its downstream cellular mediators.

Reversibility of liver fibrosis and cirrhosis
Regression of fibrosis and cirrhosis in humans is not a novel concept.Anecdotal reports published more than forty years ago recorded an improvement in patients with cirrhosis treated for hemochromatosis and Wilson disease (Powell & Ker, 1970;Falkmer et al., 1970).Advanced fibrosis and cirrhosis has recently been described with respect to matrix turnover in trials of antiviral therapy for chronic viral hepatitis (Poynard et al., 2002).The issue of regression/reversibility of cirrhosis originates from evidence obtained in animal models upon the discontinuation of the cause of liver damage or following treatment with antifibrotic agent.In chronic liver injury, activated HSC are major source of fibrillar ECM as well as of the TIMPs which inhibit collagen degradation (Bataller & Brenner, 2005).HSC survival and apoptosis are regulated by growth factors expressed during fibrotic liver injury.Thus,HSC apoptosis plays a critical role in the recovery from biliary as well as toxicinduced liver fibrosis (Iredale et al., 1998).Moreover, the very striking improvement in the histological appearance in these reports suggests that the number of activated HSCs is reduced as well.By definition, the resolution of an injury with a return to normal histology must involve the loss or phenotypic reversal to quiescence of activated HSCs (Iredale, 2001).On the other hand, activated HSC produce the fibrolytic MMPs resulting in extracellular degradation and scar remodelling.Similarly, kuffer cells/macrophages appear instrumental in the reversal of established fibrosis when the fibrogenic stimulus is absent; while they can fuel fibrogenesis when the trigger is present (Duffield et al., 2005).Clearly, at some point (probably coinciding with the onset of the clinical symptoms of cirrhosis), fibrosis becomes irreversible.Animal models suggest that this event coincides with a significant collagen cross-linking by tissue transglutaminase, leading to the production of an insoluble matrix (Issa et al., 2004).These observations have enhanced our understanding of the pathogenesis of liver fibrosis and defined our approach to its treatment.When hepatocyte necrosis occurs, the remaining hepatocytes undergo proliferation, leading to repair of the local environment.These processes are stimulated by growth factors derived from HSC, such as HGF, epidermal growth factor, epimorphin and pleiotrophin (Sawitza et al., 2009).It is difficult to say when cirrhosis becomes irreversible.Fibrotic deposition related to recent disease and characterized by the presence of thin reticulin fibers, often in the presence of a diffuse inflammatory infiltrate, is likely fully reversible.However, longstanding fibrosis, branded by extensive collagen cross-linking, dense acellular/paucicellular ECM and decreased expression and/or activity of specific metalloproteinases, is likely irreversible (Issa et al., 2004;Pinzani & Rombouts, 2004).For example, cirrhosis following withdrawal of an injurious stimulus can undergo remodeling of dense micronodular cirrhosis to a more attenuated, macronodular pattern.However, some septa will persist, likely representing those laid down early in the injury, and are therefore the most "mature" (i.e., cross-linked) (Friedman, 2003).Evidence of fibrotic regression has now been documented in chronic liver diseases, including autoimmune hepatitis, biliary obstruction, iron overload, Non-alcoholic steatohepatitis, and viral hepatitis B and C (Ismail & Pinzani, 2009).Poynard et al., 2002 examined liver biopsy specimens taken before and after therapy from 153 patients with HCV-related cirrhosis treated with different pegylated interferon and ribavirin regimens.Using the METAVIR scoring system, they found that the extent of liver fibrosis had improved in 75 (49%) Stage-4 patients: to Stage 3 in 23 patients, to Stage 2 in 26 patients, to Stage 1 in 23 patients, and to a virtually normal histological appearance in three patients.No such improvements were recorded in the control group of patients treated with interferon monotherapy.Reversal of cirrhosis was more common among younger patients.

Evaluation of liver fibrosis
The accurate and early diagnosis of liver fibrosis is crucial for long-term prognosis (Castera & Pinzani, 2010).The complete evaluation of a patient with diffuse liver diseases requires clinical evaluation, laboratory tests and pathological examination.Standard liver tests (ALT, AST, Bilirubin…etc) are of limited value in assessing the degree of fibrosis.Currently, histological examination of a liver biopsy specimen is the reference standard for the diagnosis, staging, and monitoring of liver fibrosis (Bravo et al., 2001;Campbell & Reddy, 2004).Three staging systems are commonly used to classify liver fibrosis: the Knodell, METAVIR and Ishak scores (Knodell et al., 1981;Desmet et al., 1994;Anthony et al., 1978).Knodell and METAVIR score fibrosis from Stages 0 to 4, with Stage 4 as cirrhosis, whereas Ishak scores fibrosis from Stages 0 to 6, with 5 as incomplete or early cirrhosis and 6 as established cirrhosis.There are several situations in which the role of liver biopsy (LB) is being challenged.These methods are semi-quantitative which make it a poor choice when considering assessment of liver fibrosis progression or regression.Furthermore, there is the issue of sampling error, defined as variable levels of fibrosis throughout the liver, with biopsy only examining a small portion of the liver (around 1/50000th of liver mass is obtained) (Bedossa et al., 2003).In addition, histological examination is prone to intraobserver and interobserver variation, which may occur even when widely validated systems are used to assess liver damage (Regev et al., 2002).Finally, liver biopsy is an invasive procedure with associated morbidity: pain occurs in 20% of patients and major complications (such as bleeding or hemobilia) in 0.5% of patients (Huang et al., 2007;Rockey et al., 2009).

Noninvasive markers of fibrosis
Over the past years, several non-invasive tests have become available for clinicians to use to assess liver fibrosis and determine the best course of management for their patients, especially those with chronic hepatitis C (Afdhal, 2003;Kotlyar et al., 2008).Initial studies of noninvasive markers largely consisted of single components, but the field has evolved into combining these single components into panel markers.Several laboratory tests, scores, and indices have been proposed for noninvasive prediction of hepatic fibrosis in patients with chronic hepatitis C.Among these is the aspartate aminotransferase (AST)/alanine aminotransferase (ALT) ratio (AAR) (Saadeh et al., 2001;Giannini et al., 2003;Williams & Hoofnagle, 1988).A ratio >1 has been proposed as a test for cirrhosis.However, the test is disadvantaged by both poor sensitivity (53.2%) and a negative predictive value (80.7%).Forns fibrosis index (FFI) (Forns, 2002) was developed as a model to predict fibrosis in patients with HCV based on age, gamma glutamyl transferase (gGT), cholesterol and platelet count.The score derived from this was validated against liver histology using the METAVIR scoring system for hepatic fibrosis, and it was found to have a sensitivity of 94% & specificity of 51% for a cut-off value of >4.2 (for absence of fibrosis) and a sensitivity of 30% & specificity of 95% for a cut-off value of > 6.9 (for presence of fibrosis).Low platelet count per se (Ono et al., 1999) can predict advanced fibrosis.The AST-to-platelet ratio index (APRI) (Wai et al., 2003) is formulated by dividing the AST by the upper limit of normal AST divided by the platelet count and multiplied by 100.In the initial study in patients with HCV, the APRI was derived from a training set of 192 patients undergoing liver biopsy.The area under the curve (AUC) for predicting significant fibrosis (Ishak fibrosis score ≥ 3) and cirrhosis were 0.82 and 0.92, respectively, in the training set, and 0.80 and 0.89, respectively, in the validation set for 78 patients.Using optimized cut-off values, this test predicted significant fibrosis in 51% and cirrhosis in 81% of patients.Other similar scoring systems that have been applied include the cirrhosis discriminant score (Bonacini et al., 1997), ageplatelet index ( Poynard et al., 1997) and Pohl score (Pohl et al., 2007).These tests were compared in a study comparing AAR, CDS, AP index, Pohl score and APRI (Lackner et al., 2005).In this study of 194 patients, AUC analysis revealed similar diagnostic accuracy for CDS, AP index, APRI and platelet count for the prediction of significant fibrosis (Ishak score ≥3) (AUCs of 0.71, 0.74, 0.80 and 0.71, respectively) and cirrhosis (AUCs of 0.91, 0.91, 0.90 and 0.89, respectively).FIB-4 ( Vallet-Pichard et al., 2007) is a recently described marker of fibrosis derived from a formula utilizing AST, ALT, age and platelets.In a series of 847 biopsies from HCV monoinfected patients comparing FIB-4 values to liver biopsy and FibroTest values, FIB-4 values <1.45 or >3.25 (64.6% cases) were concordant with FibroTest results in 92.1% and 76% of cases, respectively.Using the METAVIR scoring system to record fibrosis, this test had an AUC of 0.85 (95% CI 0.82-0.89)for prediction of severe fibrosis (F3-4) and 0.91 (95% CI 0.86-0.93)for prediction of cirrhosis.The FibroIndex was derived from an estimation set of 240 patients and validated in 120 subsequent patients.The test was derived from platelet count, AST and gamma globulin.Using this method, the AUC for the detection of significant fibrosis using the METAVIR histological classification of fibrosis (≥F2) was 0.83 in the estimation set and 0.82 in the validation set.Although it was calculated using best cut-offs for PPV, only 35% of patients avoided LB.FibroTest-ActiTest (FT-AT), from BioPredictive, Paris, France, is a noninvasive blood test that combines the quantitative results of six serum biochemical markers [alfa2macroglobulin, haptoglobin, gamma glutamyl transpeptidase, total bilirubin, apolipoprotein A1 and ALT] with patients' age and gender in a patented algorithm to generate a measure of fibrosis and necroinflammatory activity in the liver (Imbert-Bismut et al., 2001;Poynard et Abbreviations used: GGT: g-glutamyl-transpeptidase; AST: aspartate transaminase; chronic hepatitis C: HCV; Se: sensitivity; Sp: specificity; AUC: area under the receiver operator characteristic curve; t: Training group; v: Validation group.An AUC of 1.0 is characteristic of an ideal test, where as an AUC of 0.5 or less indicates a test of no diagnostic value.al., 2002).FT-AT provides an accurate measurement of bridging fibrosis and/or moderate necroinflammatory activity with the area under the receiver operating curve (AUROC) predictive value, between 0.70 and 0.80, when compared to liver biopsy ( Sporea et al.,  2008) (Table 1 summarized the diagnostic performance of noninvasive tests of liver fibrosis).

Limitations of serum biomarkers
One limitation of biomarkers is that none is liver-specific and they may be influenced by changes in their clearance and excretion.The inter-laboratory reproducibility of scores such as FibroTest has been shown to be satisfactory for use in clinical practice (Cales et al., 2008).Careful interpretation of each test is required e.g., when using FibroTest, the existence of haemolysis or Gilbert syndrome can lead to false-positive results (by a decrease haptoglobin or an increase in bilirubin, respectively) (Castera et al., 2011).

Imaging methods
Imaging techniques are an attractive way of evaluating fibrosis because they are noninvasive and have the ability to detect structural changes, which serological-based tests of fibrosis and inflammation are unable to do.Using the modalities of ultrasound, computed tomography or magnetic resonance imaging (MRI), it is possible to diagnose features of advanced chronic liver disease by recognizing surrogate markers of portal hypertension (e.g.splenomegaly, ascites..etc) with a high degree of sensitivity and specificity.However, these techniques do not reliably detect lesser degrees of fibrosis.Even diagnosis of cirrhosis is often based only on signs of advanced liver cirrhosis, e.g., signs of portal hypertension, a shrunken right liver lobe with enlargement of the caudate lobe, resulting in a high specificity but lower sensitivity of the methods (Honda et al., 1990).Ultrasound studies combining several ultrasound parameters and Doppler measurements achieved accuracies for the diagnosis of cirrhosis up to a maximum of 88% (Aubé et al., 1999).Commonly used imaging methods are discussed in the next section.

Transient elastography or fibroscan
Transient elastography (TE), or FibroScan (Echosens, Paris, France), is a novel technology for measuring liver stiffness (Ziol et al., 2005).The scan was developed on the principle that livers with increasing degrees of scarring or fibrosis have decreasing elasticity and that a shear wave propagating through stiffer material would progress faster than in a more elastic material.Thus, the stiffer the liver, the faster the sheer waves propagate.The ultrasound transducer probe is mounted on the axis of a vibrator.Pulse-echo ultrasound waves then measure the velocity of the shear wave in the liver tissue at a distance of 2.5-6.5 cm under the skin level, which corresponds to a measured distance of 4 cm in the liver tissue.TE is rapid (less than 5 minutes), highly reproducible and can easily be performed bedside or in the outpatient clinic with immediate results.Liver stiffness corresponds to the median value of ten validated measurements that range from 2.5 to 75 kPa, with normal values around 5.5 kPa (Castera et al., 2008).TE provides clinicians with a noninvasive, accurate, and reproducible tool to estimate liver fibrosis.Numerous studies have shown that this technique is an excellent tool for the detection of advanced fibrosis or cirrhosis, but the results for the prediction of different stages of moderate fibrosis are less conclusive.Due to its noninvasive nature, simple training and ease of use, TE can be used repeatedly on patients and is optimal for large-scale studies, in which healthy patients with no indication for liver biopsy can also be included.This technique has the advantage of being safe, reproducible, and rapid.However, falsely high liver stiffness measurements might also occur during acute hepatitis, extrahepatic cholestasis, congestive heart failure, and amyloidosis (Castera et al., 2010).Failed acquisition was commonly due to obesity, particularly increased waist circumference, and limited operator experience.However, development of S and XL probes might overcome this limitation.A meta-analysis of nine studies (Talwalkar et al., 2007) showed that TE has a sensitivity of 87% (95% CI 84%-90%) and a specificity of 91% (95% CI 89%-92%) for diagnosing cirrhosis.In seven of the nine studies, Stage 2 to 4 fibrosis was diagnosed with 70% sensitivity (95% CI 67%-73%) and 84% specificity (95% CI 80%-88%  et al., 2010).

Magnetic resonance elastography
Few studies that focus on MRI detection and quantification of liver fibrosis currently exist.
More recently, a liver stiffness evaluation (LSE) by MR elastography (MRE) has been demonstrated to provide high accuracy for the noninvasive diagnosis of liver fibrosis (Talwalkar et al., 2008;Huwart et al., 2008).The technique used is similar to that used in ultrasound elastography in that it uses a vibration device to induce a shear wave in the liver.Liver elasticity is evaluated using an external probe at the back of the patient and sending low frequency vibrations (60 Hz) through the liver and measuring the MRI spin-echo sequence.With this technique, shear elasticity and viscosity maps are obtained, and a colorcoded image is generated that depicts the wave velocity, and thus the stiffness, throughout the organ.A study comparing the MRE of thirty healthy volunteers and fifty patients with chronic liver disease with liver histology showed a sensitivity of 86% and a specificity of 85% for discrimination between patients with moderate and severe fibrosis (Metavir F2-F4) and those with mild fibrosis (Yin et al., 2007).However, MRE is expensive, and cost may limit its use.Thus, it may not be readily available at all hospitals.

Acoustic radiation force impulse
Acoustic radiation force impulse (ARFI) is a new technology for LSE which uses an add-on module to the standard ultrasound imaging device.It involves targeting an anatomic region for interrogation of the elastic properties with the use of a region of interest cursor while performing real-time B-mode imaging.An initial ultrasonic pulse is transmitted at diagnostic intensity levels to obtain a baseline signal for later comparison.A short-duration (approximately 0.3 s), high-intensity acoustic 'pushing pulse' is then transmitted by the same transducer and is followed by a series of diagnostic intensity pulses, which are used to track the displacement of the tissue caused by the pushing pulse (Palmeri et al., 2005).By measuring the time-to-peak displacement at each lateral location, one can reproduce the shear-wave speed of the tissue.

Combination of serum markers and imaging methods
The use of sequential or combined serum tests and imaging to provide better prediction for significant fibrosis (METAVIR stages F2-F4) or cirrhosis, and thus a reduction in the need for biopsy in patients with chronic hepatitis C (CHC), is gaining support.Certainly, the stepwise concept of using a highly sensitive test to first rule out significant disease and then using a more specific test to confirm the diagnosis if the test results are positive is advantageous.One recent approach integrated a biopsy into a clinical decision algorithm by targeting patients with indeterminate or misclassified values with marker panels.The Sequential Algorithms for Fibrosis Evaluation (SAFE) biopsy study (Sebastiani et al., 2009) evaluated the APRI followed by FT-AT in 2035 CHC patients from nine clinical centers in the United States and Europe.Using this sequential approach, the number of biopsies saved at baseline was 47 and 82% with an overall accuracy of 90% for Stages F2-F4 and cirrhosis, respectively.Another study (Paggi et al., 2008) noted that the combination of APRI and a simple ultrasound assessment of the presence of liver surface nodularity could predict stages F3-F4 in 54% of CHC patients.These techniques certainly appear to be valid approaches for reducing the need for biopsy in CHC patients.The combined use of TE and biochemical markers seems to be the most promising noninvasive techniques which can help the clinician decide whether a liver biopsy is necessary in some patients, and accordingly decide who to treat.

Conclusions
Our understanding of the mechanism of liver fibrosis has changed dramatically over the last decade and is no longer viewed as permanent but rather as a dynamic process.The HSC play a critical role in fibrogenesis.Reversal of fibrosis is accompanied by clearance of HSC and treatment of the primary cause of injury can allow complete resolution of fibrosis.Liver biopsy is the current reference test for the assessment of hepatic fibrosis, but because of its limitations, noninvasive markers of liver fibrosis have been developed.Although none of the currently available noninvasive marker of fibrosis are an ideal test to accurately differentiate between disease stages, the combination of serum markers, and imaging appear to have good predictive values in excluding patients with cirrhosis.

Table 1 .
Diagnostic performance of common non-invasive tests of liver fibrosis.
). Foucher et al. reported that, in 144 chronic hepatitis C patients with fibrosis at stage 3 or 4, the cut-off values of liver stiffness measured by TE were 27.5, 49.1, 53.7 and 62.7 kPa for the appearance of esophageal varices (Stage 2/3), ascites, HCC and rupture of esophageal varices (Castéra Friedrich-Rust et al., 2009velocity is proportional to the square root of tissue elasticity.Results are expressed in meters per second (m/s) (range, 0.5-4.4m/sec;±20%accuracy over the range).In a study performed byFriedrich-Rust et al., 2009; ARFI was compared to LB and blood markers of liver fibrosis in 86 patients with chronic hepatitis (B or C).The Spearman correlation coefficients between the histological fibrosis stage and ARFI, TE, FibroTest and APRI scores indicated significant correlations: 0.71, 0.73, 0.66 and 0.45, respectively (p<0.001).Newer imaging estimation of hepatic fibrosis appears promising.TE & ARFI appear to be excellent tools for early detection of cirrhosis with likely prognostic value in this setting.