Monitoring of Liver Fibrogenesis and Biochemical Diagnosis of Fibrosis

Chapter 41

Monitoring of Liver Fibrogenesis and Biochemical Diagnosis of Fibrosis O.A. Gressner1 and A.M. Gressner2 1

Wisplinghoff Medical Laboratories, Cologne, Germany; 2Wisplinghoff Medical Laboratories, Berlin, Germany

INTRODUCTION The last 20 years of biomedical research brought a plenitude of new insights into the pathogenesis of liver fibrosis, e.g., the composition of the fibrotic extracellular matrix (ECM), cellular sources of ECM, the nature of the molecular mediators regulating ECM expression, ECM turnover and paracrine cellular interactions, resolution of ECM, apoptosis of contributing cells [hepatic stellate cells (HSCs), hepatocytes], and reversibility of fibrosis (Fig. 41.1 and 41.2). Goals are to find therapeutic options, at least experimentally, and to establish innovative noninvasive biomarkers indicating the activity (progression) of development (fibrogenesis) rather than the stage (extent) of fibrotic organ transition. Therapeutic trials need frequent, reliable, objective, and cost-effective diagnostic and follow-up procedures, which complement liver biopsies as “surrogate markers.” Besides invasiveness (mortality rate 1:103 to 1:104, severe complications in 0.57% of cases) and the problem of sampling error [1/50,000th (about 30 mg) of the liver mass (w1500 g) can hardly be representative for the whole organ], biopsy histological examination depends on sample quality, that is, on length and size of the tissue specimen (coefficient of variation between 45% and 55%, accuracy 65e75%) and on the subjective evaluation of morphological changes (“observer error”), including grading of necroinflammatory activity (the driving force of fibrogenesis) and staging (extent) of fibrotic organ transition (Bedossa et al., 2003). Thus the diagnostic value of the biopsy as “gold standard” in the detection of fibrosis/fibrogenesis must be taken with care. This situation emphasizes the need for harmless, alternative, or complementary serum biomarkers.

Liver Pathophysiology. http://dx.doi.org/10.1016/B978-0-12-804274-8.00041-2 Copyright © 2017 Elsevier Inc. All rights reserved.

CURRENT BIOMARKERS OF HEPATIC FIBROGENESIS Despite its known limitations, no serum-based biomarker is currently available to equally replace liver biopsy in the evaluation of liver fibrosis. One possible reason is an imprecise and confusing classification of potential biomarkers. Most commonly, fibrosis biomarkers are classified in one of two classes. Class I fibrosis biomarkers are pathophysiologically derived from ECM turnover and/or from changes of fibrogenic cell types, in particular, HSCs (Ito-Cells) and their transdifferentiated counterpart, i.e., myofibroblasts (MFB) (Gressner and Weiskirchen, 2006). They should reflect the activity of the fibrogenic and/or fibrolytic process and, thus remodeling of ECM. These biomarkers do not indicate the extent of connective tissue deposition, i.e., the stage of fibrotic transition of the organ. Frequently, they are costly laboratory tests and are the result of translation of fibrogenic mechanisms into clinical application. Thus their selection is driven by hypothesis. Class II fibrosis biomarkers mostly estimate the degree of fibrosis (extent of ECM deposition). In general, they comprise common clinicalechemical tests (e.g., enzymes, proteins, and coagulation factors), which do not necessarily reflect ECM metabolism or fibrogenic cell changes. Their pathobiochemical relation with fibrogenesis is indirect, if at all. Thus their selection is not driven by hypothesis, but empiric. The markers are standard laboratory tests and are integrated into multiparametric panels. In general, both types of serum biomarkers follow different pathophysiological concepts. Class I markers inform about “what is going on” (i.e., grade of fibrogenic

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activity); class II markers indicate “where fibrosis is” (i.e., stage of fibrosis).

CLASS I FIBROSIS BIOMARKERS Class I biomarkers are matrix components increasingly expressed by activated HSC and MFBs, have a delayed clearance by Kupffer cells or sinusoidal endothelial cells in the liver due to metabolic dysfunction and/or hemodynamic bypasses, or are increasingly expressed mediators of fibrogenesis such as transforming growth factor (TGF)-b. An overview of the currently available class I biomarkers of fibrosis is given in Table 41.1 and Fig. 41.2. Taken together, of the several procollagen and collagen fragments proposed, only the N-terminal propeptide of type III-procollagen (PIIINP) has reached a limited clinical application, but there is no widespread acceptance (Collazos and Diaz, 1994). Sensitivities of about 76e78% and specificities of 71e81% have been reported, which can be further increased if combined with additional collagen fragment markers. It should be emphasized that PIIINP is not a liver-specific biomarker. Similarly, structural glycoproteins (e.g., undulin and tenascin), biosynthetic (e.g., prolylhydroxylase), or catabolic enzymes (e.g., matrix metalloproteinases) of collagen and other ECM components have not been convincing in the detection, grading, and staging of fibrosis. In several studies, hyaluronan (formerly termed hyaluronic acid) currently proves to be the relative best class I biomarker of fibrosis having sensitivities and specificities of 86e100% and 88%, respectively, if cirrhosis in nonalcoholic fatty liver disease (NAFLD) and other etiologies are considered (Lydatakis et al., 2006). The diagnostic power of hyaluronan is based on the high negative predictive value (98e100%) at a cut-off concentration of 60 mg/L, which is significantly higher than the positive predictive value of 61%. As such, the main utility of serum hyaluronan is particularly focused on its ability to exclude advanced fibrosis and cirrhosis. Its stimulated synthesis in activated HSC, secretion into the sinusoidal blood stream, and a short half-life of 2e9 min in the circulation are good suppositions for a valid fibrosis biomarker. Laminin was reported to be a predictor of portal hypertension because significantly elevated concentrations were found under these conditions (Kropf et al., 1991). TGF-b concentrations in plasma are elevated and correlate with the severity of liver disease and were suggested as noninvasive biomarker of fibrosis. However, the significant correlation with aspartate-aminotransferase (AST) and alanine-aminotransferase (ALT) activity (Flisiak et al., 2002) and the pathobiochemical finding that substantial amounts of TGF-b are localized in hepatocytes and released into the medium if hepatocytes are permeabilized suggest the elevation of TGF-b as a marker of necrosis instead of

fibrogenesis. However, it is functionally and immunologically quite difficult to detect TGF-b in body fluids such as blood due to binding to latent TGF-bebinding proteins (Breitkopf et al., 2001; Hyytiainen et al., 2004), alpha 2-macroglobulin (Crookston et al., 1993), and other ligands. Thus the measurement of an easier-to-detect mediator of TGF-b activity would generate important analytical advantages over the measurement of TGF-b itself (Grainger et al., 2000) (Fig. 41.2).

CLASS II FIBROSIS BIOMARKERS This category comprises a rapidly increasing, great variety of biochemical scores, and multiparameter combinations (i.e., biomarker panels), which are selected by various statistical models and mathematical algorithms (e.g., multiple logistic regression analysis). A summary of present day class II biomarkers of fibrosis is given in Table 41.2. They fulfill the most appropriate diagnostic criteria for detection and staging of fibrosis and, to a lesser extent, for grading of fibrogenic activity. In general, the panels consist of rather simple (standard) laboratory tests, which are subject to changes in the serum or plasma of fibrotic and cirrhotic patients. Several of the parameters included in the more than 20 scores, which are currently available, have no pathophysiological relation to fibrogenesis and/or fibrosis. Some of them have an indirect relation and only few parameters can be regarded as being directly related to the fibrogenic process. The parameters to be measured comprise those of necrosis such as ALT and AST, coagulationdependent tests, transport proteins, bilirubin and some ECM parameters. Frequently, the reduction of platelet counts in cirrhotic patients is included. Most prevalent are the Fibrotest and, for necroinflammatory activity, the Actitest (Biopredictive, Paris, France). They are based on gglutamyl transferase, total bilirubin, haptoglobin, a2macroglobulin, apolipoprotein A1, and, for Actitest, additionally on ALT (Poynard et al., 2004). The data of Fibrotest and Actitest are calculated with a patented artificial intelligence algorithm to give the measures of fibrosis stage and necroinflammatory grade (activity), respectively. The WAI score based on AST, alkaline phosphatase, and platelet count (Wai et al., 2003), the enhanced liver function (ELF) test based on TIMP-1, PIIINP, hyaluronan (Rosenberg et al., 2004), and the Hepascore based on bilirubin, gamma-glutamyl transpeptidase, hyaluronan, a2macroglobulin, age, and gender (Adams et al., 2005) are further scores with limited clinical application. The ELF test is currently the first fibrosis score that is further developed for application in routine medical laboratories and is currently provided as ready-made application by Siemens Healthcare Diagnostics for the Centaur immunological systems (Siemens, 2014). Fibrotest was recommended to be a better predictor than biopsy staging for hepatitis C

Monitoring of Liver Fibrogenesis and Biochemical Diagnosis of Fibrosis Chapter | 41

TABLE 41.1 Class I Biomarkers of Liver Fibrogenesis Specimen Serum

Urine

Liver Biopsy

þ



þ

Radioenzymatic, RIA

Analytical Method

Extracellular MatrixeRelated Enzymes Prolyl hydroxylase Monoamine oxidase

þ



(þ)

Enzymatic

Lysyl oxidase

þ



þ

RIA

Lysyl hydroxylase

þ





RIA

Galactosyl hydroxylysyleglucosyl transferase

þ



þ

RIA

Collagen peptidase

þ



þ

Enzymatic

N-Acetyl-b-D-glucosaminidase

þ

þ

þ

Enzymatic

Collagen Fragments and Split Products Type I procollagen l

N-terminal propeptide

þ



þ

ELISA

l

C-terminal propeptide

þ



þ

RIA

þ





RIA

Type III procollagen l

Intact procollagen

l

N-terminal propeptide

l

Complete propeptide (Col 1e3)

þ





RIA

l

Globular domain of propeptide (Col-1)

þ





RIA

NC1-fragment [C-terminal] cross-linking domain

þ

þ



ELISA, RIA

7S domain (“7S collagen”)

þ

þ



RIA

Type VI collagen

þ

þ

þ

RIA

Laminin, P1-fragment

þ





RIA, ELISA

Undulin

þ





ELISA

Vitronectin

þ





ELISA

Tenascin

þ





ELISA

YKL-40

þ



þ

RIA/ELISA

(Pro)matrix metalloproteinase (MMP-2)

þ





ELISA

Tissue inhibitor of metalloproteinases (TIMP-1, TIMP-2)

þ





ELISA

sICAM-1 (soluble intercellular adhesion molecule, sCD54)

þ





ELISA

þ





Radioligand assay

TGF-b

þ



þ

ELISA

CCN2

þ

?

þ

ELISA

Type IV collagen

Glycoproteins and Matrix Metalloproteinase (Inhibitors)

Glycosaminoglycans Hyaluronic acid (hyaluronan) Molecular Mediators

CCN2, connective tissue growth factor; ELISA, enzyme-linked immunosorbent assay; RIA, radioimmunoassay; TGF-b, transforming growth factor beta.

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FIGURE 41.1 Components of the extracellular matrix (connective tissue) of the fibrotic liver and their major changes. The binding of glycosaminoglycans (GAG) to the respective core proteins (CP) of proteoglycans (PG) is shown. BM, basement membranes; FACIT, fibril-associated collagens with interrupted triple helices.

complications and death (Poynard et al., 2005). Recently, Fibrotest and Actitest were included into biomarkers for the prediction of liver steatosis (steatotest), alcoholic steatohepatitis, and nonalcoholic steatohepatitis (NASH-test) by supplementation with serum cholesterol, triglycerides, glucose (and AST for NASH-test) adjusted for age, gender, and body mass index (Poynard et al., 2005). The diagnostic criteria elaborated in a large number of patients suggest steatotest as a simple and noninvasive quantitative measure of liver steatosis and the NASH-test as a useful screening procedure for advanced fibrosis and NASH in patients with various metabolic syndromes (Poynard et al., 2005). FibroMax (Biopredictive) was developed as a method of combined calculation of these fibrosis-related tests in a single procedure. Comparative evaluation of class II serum biomarker panels, however, could not highlight their clinical superiority (Parkes et al., 2006). Because only about 40% of the results were assigned to be correct, a fraction of about 50e70% was inaccurate with regard to the staging of fibrosis severity and a small fraction of results was even incorrect (Parkes et al., 2006). Thus the presently suggested multiparameter approaches with class II fibrosis biomarker panels have to be taken with caution in clinical practice. A successful approach to improve the diagnostic accuracy of

the panel markers in chronic hepatitis C might be their stepwise combination (Parkes et al., 2006). By combining the sequential algorithms of AST-toplatelet ratio index (APRI), Forns’ index, and Fibrotest, the diagnostic performance could be significantly improved, resulting in a reduction of the need for liver biopsy by 50e70% (Sebastiani et al., 2006). However, it should be emphasized that the combination of individually assessed parameters necessarily creates relatively high variance due to analytical imprecision and globally nonstandardized methods for measurement of biochemical routine parameters (Gressner et al., 2009). Coefficients of variation range from series to series between 3% and 6% for common clinicalechemical analytes and from 4% to more than 12% for hyaluronan, PIIINP, and other matrix parameters. Thus both, the comparability and reproducibility of grading the activity and staging the extent of fibrotic tissue transition, might scatter considerably between the various investigators, which limits their large-scale application and comparison. Furthermore, and even more important is the lack of standardized assay procedures (methods) for many of these parameters, which excludes the general use of cut-offs and algorithms (Rosenthal-Allieri et al., 2005).

Monitoring of Liver Fibrogenesis and Biochemical Diagnosis of Fibrosis Chapter | 41

551

FIGURE 41.2 Synopsis of pathogenetic mechanisms of liver fibrosis (fibrogenesis). An excess of extracellular matrix is produced by activated hepatic stellate cells (HSCs)/expanded pool of myofibroblasts (MFB) leading to fibrosis and, eventually, cirrhosis. Newly recognized pathogenetic mechanisms point to the (1) influx of bone marrowederived cells (fibrocytes) to the liver, (2) to circulating monocytes and to their TGF-bedriven differentiation to fibroblasts, and (3) to the epithelialemesenchymal transition (EMT) of hepatocytes and bile duct epithelial cells to fibroblasts. All three complementary mechanisms enlarge the pool of matrix-synthesizing (myo-) fibroblasts. Most important fibrogenic mediators are transforming growth factor (TGF)-b, platelet derived growth factor (PDGF), insulin-like growth factor 1 (IGF-1), endothelin-1 (ET-1), and reactive oxygen metabolites (ROS). ASH, alcoholic steatohepatitis; NAFLD, nonalcoholic fatty liver disease. The inset shows an electron micrograph of HSC containing numerous lipid droplets.

DEVELOPMENT OF INNOVATIVE BIOMARKERS The growing understanding of the pathogenesis of hepatic fibrosis indicates potentially powerful, noninvasive (blood) biomarkers of hepatic fibrogenesis and fibrosis. A selection of newly developed or proposed biomarkers is presented in Table 41.3.

Contribution of Bone MarroweDerived Cells to HSCs, MFBs, and Fibroblasts in Fibrotic Liver Tissue Several studies have pointed to the bone marrow as a source of immature, multipotent cells in various organs. Bone marrow cells have the capacity to differentiate to hepatocytes, cholangiocytes, sinusoidal endothelial cells, and Kupffer cells if the adequate microenvironment of the liver is present (Gao et al., 2001; Fujii et al., 2002). This phenomenon is of great importance for regenerative medicine (e.g., bone marrow stem cell therapy). It was recently extended for HSC and MFB under experimental

and clinical conditions. By the transplantation of genetically tagged bone marrow or of male bone marrow (Y chromosome) to female mice, it was demonstrated that up to 30% of HSC in the liver originate from the bone marrow and acquire the MFB phenotype under injurious conditions (Baba et al., 2004). Another study indicates that up to 68% of HSC and 70% of MFB in experimental CCl4cirrhotic mice liver derive from the bone marrow (Russo et al., 2006). Even in human liver fibrosis, a significant contribution of bone marrow cells to the population of MFB was proven, but it is presently unclear which type of specific bone marrow cells or mesenchymal stem cells is relevant for the generation of hepatic MFBs (Forbes et al., 2004). Another experimental study shows that myelogenic fibrocytes are present in the liver, which can be differentiated by TGF-b to collagen-producing MFB (Kisseleva et al., 2006). They are a subpopulation of circulating leukocytes, which display a unique surface phenotype with CD45þ (hematopoietic origin), CD34þ (progenitor cell), and type I collagenþ (capability of matrix synthesis) (Quan et al., 2006), and exhibit potent immunostimulatory activities (Abe et al., 2001). Fibrocytes represent a systemic

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TABLE 41.2 Class II Biomarkers of Liver Fibrogenesis Index

Parameters

Chronic Liver Disease

Sensitivity (%)

Specificity (%)

Actitest

Fibrotest þ ALT

HCV

Bonacini index

ALT/AST-ratio, INR, platelet count

HCV

46

19

ELF score

PIIINP, hyaluronan, TIMP-1

Mixed

11

41

FIB-4

Platelet count, AST, ALT, age

HCV/HIV

70

74

FibroIndex

Platelet count, AST, g-globulin

HCV

38

18

Fibrometer test

Platelet count, prothrombin index, AST, a2-macroglobulin, hyaluronan, urea, age

Mixed

81

5

Fibrotest (fibro-score)

Haptoglobin, a2-macroglobulin, apolipoprotein A1, GGT, bilirubin

HCV, HBV

75

6

Forns-index

Age, platelet count, GGT, cholesterol

HCV

15

51

Fortunato-score

Fibronectin, prothrombin time, PCHE, ALT, Mn-SOD, b-NAG

HCV

Hepascore

Bilirubin, GGT, hyaluronan, a2-macroglobulin, age, gender

HCV

63

10

Leroy-score

PIIINP, MMP-1

HCV

60

13

NAFLD fibrosis score

Age, BMI, IFG, diabetes, AST/ALT, platelet count, albumin HCV

47

17

Park-index

15

Patel-index (FibroSpect)

Hyaluronan, TIMP-1, a2-macroglobulin

HCV

77

73

PGAA-index

Prothrombin time, GGT, apolipoprotein A1, a2-macroglobulin

Alcohol

79

10

PGA-index

Prothrombin time, GGT, apolipoprotein A1

Mixed

12

81

Pohl-score

AST/ALT-ratio, platelet count

HCV

41

20

Sheth-index

AST/ALT (De Ritis)

HCV

53

21

Sud-index (FPI)

Age, AST, cholesterol, insulin resistance (HOMA), past alcohol intake

HCV

17

44

Testa-index

Platelet count/spleen diameter ratio

HCV

78

79

WAI index (APRI)

AST, platelet count

HCV

10

75

ALT, alanine aminotransferase; APRI, AST-to-platelet ratio index; AST, aspartate-aminotransferase; BMI, body mass index; ELF, enhanced liver function test; FIB, fibrosis; FPI, fibrosis probability index; GGT, gamma-glutamyltransferase; HBV, hepatitis B virus; HCV, hepatic C virus; HOMA, homeostasis model assessment; IFG, impaired fasting glucose; INR, international normalized ratio; MMP, matrix metalloproteinases; Mn-SOD, manganese superoxide dismutase; NAFLD, nonalcoholic fatty liver disease; b-NAG, N-acetyl-b-glucosaminidase; PCHE, Plasma cholinesterase; pseudocholinesterase; butyrylcholinesterase; PIIINP, N-terminal propeptide of type III-procollagen; TIMP, tissue inhibitors of metalloproteinases.

source of contractile MFB in various fibrotic lesions, such as lung, keloids, scleroderma, and fibrotic changes of the kidney (Quan et al., 2004). The mobilization of bone marrow cells and their recruitment into the damaged tissue is a general mechanism of tissue fibrosis and wound healing (Ishii et al., 2005), which is most likely regulated by colony stimulating factors (CSF), such as granulocyte-CSF (GCSF) (Higashiyama et al., 2007). This mediator together with chemokines regulates the migration of bone marrow cells to sites of

tissue injury, but also the efflux from the bone marrow into the circulation (Abe et al., 2001). Activated HSC, probably play an important role because these cells secrete a broad spectrum of inflammatory mediators (chemokines, macrophage colony-stimulating factor (M-CSF), stem cell factor, platelet activating factor) and leukocyte adhesion molecules (intercellular adhesion molecule 1, vascular cell adhesion molecule 1, neural cell adhesion molecule) required for recruitment, activation, and maturation of blood-born cells at the site of injury (Marra, 1999). The homing of

Monitoring of Liver Fibrogenesis and Biochemical Diagnosis of Fibrosis Chapter | 41

553

TABLE 41.3 Future Candidate Biomarkers of Noninvasive Diagnosis and Follow-Up of Liver Fibrogenesis Biomarker

Specimen

Analytical Method

Pathobiochemical Bias

BMP-7

Serum

Immunoassay

Antagonist of TGF-b, inhibitor of EMT

Colony stimulating factors (CSF): G-CSF GM-CSF M-CSF

Whole blood

Immunoassays

Mobilization of bone marrowederived fibrocytes

CTGF

Serum

Immunoassay

TGF-b induced expression in and secretion by hepatocytes and HSCs

Glycomics

Serum

Adaptation of DNA sequencer/fragment analyzer technology to profiling of desialylated N-linked oligosaccharides

Fibrosis-specific profiles of desialylated serum protein linked oligo-saccharides (N-glycans)

NX-DCP

Serum

Immunoassay

Increased release as result of fibrosisinduced hypoxia of liver parenchyma

Proteomics

Serum

Mass spectrometry (MS)

Fibrosis-specific serum protein profiles

Xylosyl transferase

Serum

Liquid chromatography tandem MS

Key enzyme of the biosynthesis of glycosaminoglycan chains in proteoglycans, e.g., in HSCs and hepatocytes

BMP, bone morphogenetic protein; CTGF, connective tissue growth factor; EMT, epithelialemesenchymal transition; G-CSF, granulocyte-colony stimulating factor; GM-CSF, Granulocyte macrophage colony-stimulating factor; HSCs, hepatic stellate cells; NX-DCP, NX-des-g-carboxyprothrombin; TGF-b, transforming growth factor beta.

myelogenic cells in the damaged liver was claimed to have an additional positive effect on the resolution of liver fibrosis because these cells express matrix metalloproteinases, which augment the degradation of fibrotic ECM (Higashiyama et al., 2007).

Transformation of Peripheral Blood Cells to MFBs of the Liver Recent studies indicate a highly developed multidifferentiation potential of a subgroup of circulating blood monocytes, which can be recruited quickly for tissue repair processes (Romagnani et al., 2006). In addition, the content of circulating myelogenic stem cells in the blood is suggested to be important for regenerative mechanisms in consequence of ischemic and degenerative diseases (i.e., myocardial infarction). Investigations over the last years have proven that peripheral blood monocytes can be differentiated in vitro to hepatocyte-like cells if they are exposed with the M-CSF and specific interleukins (monocyte-derived neo-hepatocytes) (Ruhnke et al., 2005a, 2005b). Although for liver fibrogenesis not yet proven, subgroups of monocytes can differentiate into fibroblast-like cells (fibrocytes) after migration into the damaged tissue. There, they participate in fibrotic processes, e.g., of the lung and kidney. The differentiation is positively influenced by granulocyte colony-stimulating factor (G-CSF), M-CSF, monocyte chemotactic peptide 1 (MCP-1), and other chemokines and hematopoietic growth and differentiation factors, which are expressed and secreted by activated HSC

(Kisseleva et al., 2006; Pinzani et al., 1992; Marra et al., 1993; Sprenger et al., 1997) and other liver cell types (Marra et al., 1998). It is of interest that very recently an inhibitory effect of the acute-phase protein serum amyloid P (SAP) on the process of differentiation of monocytes to fibrocytes could be established (Pilling et al., 2003) and, consequently, a preventive effect of SAP-injections on the development of bleomycininduced lung fibrosis was found (Pilling et al., 2007). Creactive protein failed to show an inhibitory effect on the differentiation of monocytes to fibrocytes. Because SAP is synthesized in hepatocytes, severe liver injury might facilitate the monocyte fibrocyte differentiation process due to reduction of the inhibitory SAP. Although this mechanism is presently speculative for the liver, circulating monocytes might nonetheless be a pool for immediate repair processes of liver damage. Besides special monocytes as source of fibroblasts in the fibrotic liver, circulating stem cells have to be considered, which are CD34þ and C-X-C chemokine receptor type 4 (CXCR4þ) (a chemokine receptor) (Romagnani et al., 2006). G-CSF and the stromal derived factor are probably the most important regulators of stem cell mobilization from bone marrow and their integration into the damaged tissue followed by differentiation to fibroblasts and other cells.

Parameters of EpithelialeMesenchymal Transition Beside activation and transdifferentiation of HSC, an increasing number of experimental studies points to an

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additional mechanism for the enlargement of the resident (local) pool of fibroblasts during the fibrotic reaction of the damaged organs, e.g., in kidney and lung (Lee et al., 2006). This process, termed epithelialemesenchymal transition (EMT), is well known in the context of embryonic development, but is now discussed as an important mechanism in the generation of fibroblasts during fibrogenesis in adult tissues (Kalluri and Neilson, 2003, Fig. 41.3). It was proven that in fibrotic kidney disease, tubulus epithelial cells can transdifferentiate to fibroblasts expressing the fibroblastspecific protein 1 (FSP-1), also known as S21A4 calcium-binding protein, and are able to express collagens (Kalluri and Neilson, 2003). Similarly, alveolar epithelial cells of the lung are subject to EMT and cardial endothelial cells can also switch to fibroblasts under conditions of damage (mesenchymalemesenchymal transition). It is estimated that in the kidney, about 66% of fibroblasts are the result of EMT; in the heart, the number climbs to about 20% (R. Kalluri, personal communication). In vitro and in vivo observations made in blood vessels following sustained inflammation support a hypothesis that endothelial cell transformation to MFB-like cells may explain the increase of matrix proteins and of MFB pathognomonic of fibrotic diseases (Karasek, 2007). Very recent studies have also discussed EMT in liver fibrogenesis, after a transition of albumin-positive hepatocytes

to FSP-1 positive and albumin-negative fibroblasts was shown. Preliminary studies claim that about 40% of hepatic fibroblasts derive from hepatocytes, but these data need further confirmation (R. Kalluri, personal communication). A recent report provides evidence for EMT of mature mouse hepatocytes in vitro and of the mouse hepatocyte cell line alpha mouse liver 12 (Kaimori et al., 2007). The EMT state was indicated by strong upregulation of a1(I) collagen mRNA expression and type I collagen deposition. Thus hepatocytes are capable of EMT changes and type I collagen synthesis. A further source of EMT is cholangiocytes (bile duct epithelial cells). In primary biliary cirrhosis (PBC), it was proven that bile duct epithelial cells express FSP-1 (S21A4) and vimentin as early markers of fibroblasts (Robertson et al., 2007). The bidirectional consequence of EMT of cholangiocytes is ductopenia (reduction of bile ducts) and enlargement of the pool of portal fibroblasts, which significantly contributes to portal fibrosis. In vitro studies with cultured human cholangiocytes have confirmed the clinical observations described. Thus EMT proves to be a general pathogenetic principle of chronic cholestatic liver diseases (Rygiel et al., 2008). In addition, activation and proliferation of portal/ periportal mesenchymal cells to peribiliary MFB, which are stimulated in a paracrine manner by bile duct epithelial cells via TGF-b, platelet-derived growth factor BB, and

FIGURE 41.3 Role of connective tissue growth factor (CTGF) in hepatic fibrogenesis. The effect of CTGF on epithelial to mesenchymal transition (EMT). By modulating receptor binding of transforming factor beta (TGF-b) and bone morphogenetic protein (BMP), important opponents in the regulation of this key process of hepatic fibrogenesis, CTGF leads to a shift in the balance toward mesenchymal activity, thus acting as a profibrogenic regulator. FSP1, fibroblast specific protein 1.

Monitoring of Liver Fibrogenesis and Biochemical Diagnosis of Fibrosis Chapter | 41

endothelin-1 (Kinnman et al., 2003), turned out to be an important pathogenetic mechanism of portal fibrosis and septa formation in cholestatic liver diseases. Indeed, only a minority of ECM-producing MFB in obstructive cholestatic injuries are derived from HSC (Beaussier et al., 2007; Ramadori and Saile, 2004). This also underlines the heterogeneous origin of MFB in fibrogenesis and emphasizes the importance of the underlying fibrogenic liver disease (Torok et al., 2015). The molecular inducers of EMT are TGF-b (Kalluri and Neilson, 2003), epidermal growth factor (EGF), insulin-like growth factor (IGF)-II, and fibroblast growth factor (FGF)-2, which promote the genetic and phenotypic programming of epithelial cells to mesenchymal cells (fibroblasts). The prototype of the most powerful inducer of EMT is TGF-b. The inducing function of TGF-b for the mesenchymal transition of mouse hepatocytes (described earlier) was shown by activation of Smad2/3 phosphorylation, inhibition by Smad4 silencing using small interfering RNA (siRNA) and induction of the snail transcription factor (Karasek, 2007). Interestingly, TGF-b induces EMT of only those hepatocytes resistant to the proapoptotic effects of this cytokine (Yang et al., 2006; Del Castillo et al., 2006). The subpopulation of surviving hepatocytes exhibits an overexpression of Snail by TGF-b conferring resistance to programmed cell death (Vega et al., 2004). Several additional pathways are involved in the generation of apoptosis resistance, e.g., proteinkinase A (Yang et al., 2006) and EGF/TGF-a (Del Castillo et al., 2006). Thus EMT of hepatocytes is dependent on the balance between apoptotic and survival mechanisms. The process of EMT also requires the action of metalloproteinases and a TGF-bedependent downregulation of E-cadherin, both contributing to the release of epithelial cells from cellecell and cellebasement membrane binding. The most important molecular counterpart is the bone morphogenetic protein (BMP)-7, also belonging to the TGF-b superfamily. BMP-7 not only inhibits EMT, but can even induce a mesothelial to epithelial transition (reverse EMT ¼ MET) (Zeisberg et al., 2003, Fig. 41.3). It has antiapoptotic properties and antiinflammatory and proliferation-stimulating effects (Sugimoto et al., 2007). BMP-7 inhibits TGF-b signaling via Smads (Wang and Hirschberg, 2004), which transduce the effect of the latter cytokine from its receptor; a serine/threonine kinase to the Smad-binding element of respective target genes in the nucleus (ten Dijke and Hill, 2004). In addition, several trapping proteins such as the small proteoglycans decorin and biglycan, latencyassociated peptide, BMP and activin membraneebound inhibitor, kielin-chordinelike protein, gremlin, and a2macroglobulin change the balance between TGF-b and BMP-7 in favor of an anti-EMT effect due to binding a neutralization of TGF-b (Neilson, 2005).

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Connective Tissue Growth Factor Preliminary studies point to connective tissue growth factor (CTGF; CCN2) in serum as an innovative class I biomarker of fibrogenesis, which reflects TGF-b activity (Gressner et al., 2006). This 38-kDa protein, a member of the CCN superfamily of secreted, cysteine-rich glycoproteins, has been implicated in the pathogenesis of hepatic fibrosis and is currently suggested to be an important downstream amplifier of the effects of the profibrogenic master cytokine TGF-b (Leask and Abraham, 2006). Its molecular mechanism of action is still not known in detail, but this cytokine, which is expressed in hepatocytes, HSC, portal fibroblasts, and cholangiocytes (Abreu et al., 2002; Gressner et al., 2007), seems to change the functional TGF-b/BMP-7 ratio (Abreu et al., 2002). CTGF is overexpressed in experimental and human liver cirrhosis (Rachfal and Brigstock, 2003; Abou-Shady et al., 2000; Paradis et al., 1999), which is mediated mainly by TGF-b, but also by endothelin-1, tumor necrosis factor-a, vascular endothelial growth factor (VEGF), nitrogen oxide, prostaglandin E2, thrombin, high glucose, and hypoxia (Blom et al., 2002). CTGF inhibits BMP, but activates TGF-b signaling by modulation of the receptor-binding of these ligands (Abreu et al., 2002). Additionally, CCN2/CTGF expression is sensitively upregulated by TGF-b. The crucial role in fibrogenesis was documented by recent studies in which knock-down of CCN2/CTGF by siRNA leads to substantial attenuation of experimental liver fibrosis (Li et al., 2006; George and Tsutsumi, 2007). Thus depletion of CTGF greatly attenuates the development of experimental liver fibrosis. Significant increases of CTGF in serum/plasma of patients with fibrogenic CLD were previously shown in this study using an in-house immunoassay for CTGF (Gressner et al., 2013; Kovalenko et al., 2009). The area under the curve (AUC) for fibrosis versus control and cirrhosis versus control were calculated to be 0.165 and 0.9, respectively; the sensitivities 21% and 5%, respectively, and the specificities 10% and 6%, respectively (Gressner et al., 2006). Thus there is good evidence for CTGF as a diagnostic relevant fibrogenic master switch in fibrotic CLD (Fig. 41.4). CTGF levels show a biphasic course with ascending concentrations in fibrotic and cirrhotic patients followed by slightly descending levels in HCC patients. CTGF concentrations furthermore decreased with tumor progression and size, with lower levels in tumor, nodes, metastases (TNM) stage II and stage III compared to TNM stage I (Gressner et al., 2013). Next to its diagnostic value in reflecting the degree of fibrosis, the course of CTGF serum concentrations may therefore be additionally recommended as a “negative” tumor marker in patients with established cirrhosis (Gressner et al., 2013). Taken together, both EMT and MET, and, under special circumstances, even MMT (mesothelial to mesenchymal

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FIGURE 41.4 Potential of connective tissue growth factor (CTGF) in serum for monitoring hepatic fibrogenesis. CTGF in serum as a new singular candidate parameter for the noninvasive diagnosis and staging of active liver fibrosis. Shown are receiver operating characteristic (ROC) curves for fibrotic or cirrhotic patients versus controls (Gressner et al., 2006). AUC, area under the curve.

transition, e.g., vascular endothelial cells to fibroblasts), and the fine tuning of the bioactive TGF-b/BMP-7 ratio and of their adaptor and trapping proteins offer multiple regulatory possibilities of influencing fibrogenesis. These mechanisms are known in some detail for the kidney (Zeisberg, 2006), but need more experimental proof for the liver, in particular with regard to its quantitative contribution to fibrogenesis.

YKL-40 Recently, the glycoprotein YKL-40 (“chondrex,” molecular mass 40 kDa), likely to be a growth factor for fibroblasts and endothelial cells, was shown to be strongly expressed in human liver tissue. In particular, HSCs contain YKL-40 mRNA. Several studies have found elevated YKL-40 concentrations in the sera of patients with liver diseases. An 80% sensitivity and 77% specificity as well as an AUC of 0.81 for fibrosis are reported for hepatic C virus (HCV) patients (Saitou et al., 2005); for those with alcoholic liver disease, a specificity of 88.5%, but a sensitivity of only 50.8%, however, was determined. Only 11.5% of the patients without severe fibrosis displayed a Chondrex plasma level above this threshold (Tran et al., 2000). Serum concentrations of this protein correlate with other ECM products secreted by MFB (e.g., PIIINP, hyaluronan, MMP-2, and TIMP-1) (Tran et al., 2000). It is claimed that YKL-40 concentrations reflect the degree of liver fibrosis, but extensive clinical evaluation is required and other inflammatory and malignant diseases as potential conditions of YKL-40 elevations have to be excluded.

Xylosyl Transferase Xylosyl transferase (XT), a key enzyme responsible for the covalent linkage of the glycosaminoglycan side chain to the core protein of the proteoglycan moiety, was shown to have increased activities in the sera of patients with connective tissue diseases. With high-performance liquid chromatography tandem mass spectrometry, measurements in large numbers of liver fibrotic patients are possible (Kuhn et al., 2006). Because MFB in fibrotic liver have a greatly stimulated proteoglycan synthesis (Gressner, 1994), XT activity in serum might be a promising class I biomarker of fibrogenesis. However, the analytical performance of the test and its lacking standardization impair further evaluation of this parameter.

Glycomics Further successful developments might emerge from serum proteome profiling (Poon et al., 2005) and from total serum protein glycomics, which is the pattern of N-glycans (Callewaert et al., 2004). It was reported that a unique serum proteomic fingerprint is identified in the sera of patients with fibrosis, which enables differentiation between different stages of fibrosis and a prediction of fibrosis and cirrhosis in patients with a chronic hepatitis B infection (Poon et al., 2005). Specificities, sensitivities, and accuracy of prediction of cirrhosis are about 10%. Similarly, N-glycan profiling can distinguish between compensated cirrhosis from noncirrhotic chronic liver diseases with sensitivity and specificity of 79% and 7%, respectively (Callewaert et al., 2004).

Monitoring of Liver Fibrogenesis and Biochemical Diagnosis of Fibrosis Chapter | 41

Supplementation of all these laboratory tests by modern high-resolution or molecular imaging analyses would be extremely helpful in the consolidation of objective and valid noninvasive biomarkers of diagnosis and follow-up of fibrogenic (liver) diseases.

NX-DES-g-CARBOXYPROTHROMBIN Des-g-carboxyprothrombin (DCP) has several variants based on the number of glutamic acid (Glu) residues from 0 to 10. NX-des-g-carboxyprothrombin (NX-DCP) is a DCP variant that contains less Glu residues. Earlier studies indicated that NX-DCP is increasingly released during fibrosis induced parenchymal hypoxia (Toyoda et al., 2012). A recent study from Japan performed a prospective cohort study on a consecutive group of 22 patients who underwent liver biopsy for HCV-related liver disease. Detected serum concentrations of NX-DCP correlated positively with fibrosis stage ( p ¼ .006). Moreover, NX-DCP was a multivariate factor associated with the presence of significant fibrosis F 3e4 (median 21 of F0e2 group vs. median 22 of F3e4 group with p ¼ .002). The AUC of NX-DCP showed no significant differences compared with multiparametric type II fibrosis markers such as the APRI, the modified-APRI, and the Pohl score (p > .05). Moreover, NX-DCP had a similar predictive ability to the models (mentioned earlier), and thereby should be furthermore evaluated as a potential new noninvasive prediction tool for fibrosis (Saito et al., 2015).

CONCLUSION Currently available types I and II serum biomarkers should be used with caution because neither single nor panel markers fulfill the requirements of an ideal noninvasive biomarker of fibrosis. Those are, in particular, (1) analytical simplicity, (2) good standardization of the test system, (3) cost-effectiveness, and (4) a high degree of specificity and sensitivity in the detection of fibrogenic liver diseases. Even the currently best and most extensively evaluated type I (i.e., hyaluronan) and type II (i.e., Fibrotest and Actitest) serum biomarkers do not meet all these criteria. As a result, further detailed insights into the mechanism of liver fibrosis and improvements of analytical procedures are necessary to trigger new approaches for noninvasive assessment of fibrosis with biochemical or physical means. Newly recognized pathogenetic mechanisms of fibrosis described earlier provide several innovative options for therapy of liver fibrogenesis and noninvasive diagnostic strategies (Table 41.3; Mehal and Schuppan, 2015; Torok et al., 2015). The determination of the TGF-b/BMP-7 ratio in serum or plasma is potentially promising, because this

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ratio might reflect the process of EMT and, thus at least partially the rate of progression of fibrosis. A decrease of this ratio might indicate those patients with slow progression (slow fibroser); an increase, a fast progression (rapid fibroser). However, some precautions have to be considered: The cytokine ratio in the circulation might not be an accurate reflection of their activity at the immediate environment of epithelial cells and fibroblasts, respectively, and major fractions of these cytokines might be in a biologically latent form. Thus the protein ratio does not necessarily mimic the diagnostically important activity ratio of these mediators. The determination of CTGF in serum or plasma is suggested as an innovative parameter of fibrogenesis because this modulator protein is strongly upregulated in the fibrotic liver, synthesized and secreted by parenchymal and nonparenchymal cells (Gressner et al., 2007) and the action of the profibrogenic TGF-b is stimulated but that of the antifibrogenic BMP-7 is inhibited (Abreu et al., 2002). Preliminary studies point to significantly enhanced concentrations of CTGF in blood of patients with active liver fibrogenesis (Gressner et al., 2006) in contrast to advanced cirrhosis with low concentration of active fibrogenesis, which is reflected by a relative decrease of serum CTGF. The flow cytometric detection of circulating fibrocytes in blood or in buffy coat leukocytes by using CD34þ, CD45þ, and collagen I positivities as identifying markers might be a way for the evaluation of their diagnostic potential. Alternatively, these antigens might be detected by amplifying their mRNA using a quantitative PCR approach. In addition, a re-evaluation of the high concentrations of G-CSF, granulocyte macrophage colony-stimulating factor (GMCSF), and M-CSF in serum of cirrhotic patients published previously (Kubota et al., 1995) as mobilizers of bone marrow cells and fibrocytes and of their integration into the damaged liver tissue (Yannaki et al., 2005) might be a promising task. It should be analyzed whether a systemic elevation of the hematopoietic growth factors correlates with the activity of liver fibrogenesis. Numerous publications discuss antifibrotic therapeutic strategies by inhibition of TGF-b (Abe et al., 2001; AbouShady et al., 2000; Torok et al., 2015; Liu et al., 2006), but the systemic application of inhibitors and, consequently, an overall and ubiquitous reduction of TGF-b activity will most likely have severe side effects, e.g., on tumor development and progression, autoimmunopathy, and degenerative diseases (Gressner and Weiskirchen, 2003). Therefore, the therapeutic application of recombinant human BMP-7 or functionally active BMP-7 fragments might be advantageous because BMP-7 inhibits experimental fibrosis in rats (Kinoshita et al., 2007), stimulates liver regeneration (Sugimoto et al., 2007), and inhibits TGFbedriven parenchymal cell apoptosis due to its antagonism of TGF-b.

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Experimental trials with thioacetamide-induced rat liver fibrosis point to successful antifibrotic results (Kinoshita et al., 2007). Similarly, extensive studies with experimental kidney diseases prove that BMP-7 can induce MET and, thus has regenerative and antifibrotic effects (Zeisberg and Shah, 2005). Presently, it is not known whether the positive CTGF-inhibitory experiments for suppression of experimental fibrosis (Li et al., 2006; George and Tsutsumi, 2007) can be translated into clinical practice, but studies, in which CTGF activity is reduced by systemic application of a humanized, monoclonal, blocking antibody (F-3019), which neutralizes and accelerates the clearance of this protein (Liu, 2007), are in progress and point to successful preliminary results. Pathophysiologically, the inhibitors of CTGF should have fibrosuppressive effects because the TGF-b/BMP-7 ratio is switched in favor of BMP-7. This was recently shown by inhibition of CTGF expression (Li et al., 2006; George and Tsutsumi, 2007). In conclusion, further intensive studies are required to translate the positive results of cell culture studies and of animal experiments into clinical application. The new pathogenetic insights justify strong optimism because the spectrum of potential approaches to interfere with the fibrogenic pathway is greatly broadened. The changing view on the pathogenetic mechanisms of liver fibrosis (mentioned earlier) clearly suggests that an exclusive role of HSC in the development of fibrosis has to be reconsidered. Although some of the newly proposed fibrogenic mechanisms have to be consolidated by additional experimental evidence in vitro and in situ, they indicate the presence of distinct subpopulations of MFBs/ fibroblasts in fibrosing liver, of which HSC-derived fibrogenic cells are only one of several sources. Most important, the composition of MFBs may vary with the etiology of fibrosis, i.e., PBC might activate a pathogenetic pathway different from alcoholic fibrosis. These facts point to the important notion that results obtained from various models of experimental fibrogenesis cannot be generalized because different classes of MFBs are generated by diverse pathways. Furthermore, HSC activation in culture cannot be regarded any longer as the almost dogmatic paradigm of the liver fibrogenic mechanism as it was in the past. Because now detailed information on the molecular cascades of intracellular fibrogenic signaling is available, we have learned that several of them are specifically celltype modulated. Therefore, it is conceivable that distinct subpopulations of fibroblasts and their transient precursor cell types respond differently to major fibrogenic cytokines, e.g., TGF-b. If this is the case, the complexity of the fibrogenic mechanisms will increase strongly in the future. The detailed insight into the mechanism of liver fibrosis and improvement of analytical techniques will result in new approaches for noninvasive assessment of fibrosis with biochemical or physical means. Those biochemical serum-

based markers of liver fibrosis/fibrogenesis will be complementary to quantitative elastography techniques for liver stiffness measurement such as monodimensional ultrasound transient elastography (FibroScan), magnetic resonance elastography, and acoustic radiation force impulse, which have found widespread clinical application (Patel et al., 2015). In addition, the analysis of genetic predisposition markers or the determination of special single nucleotide polymorphism signatures that are associated with the severity of fibrosis will potentially be another complementary diagnostic tool.

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