The clinical course of chronic liver diseases is significantly dependent on the progression rate and the extent of fibrosis, i.e. the non-structured replacement of necrotic parenchyma by extracellular matrix. Fibrogenesis, i.e. the development of fibrosis can be regarded as an unlimited wound healing process, which is based on matrix (connective tissue) synthesis in activated hepatic stellate cells, fibroblasts (fibrocytes), hepatocytes and biliary epithelial cells, which are converted to matrix-producing (myo-)fibroblasts by a process defined as epithelial-mesenchymal transition. Blood (non-invasive) biomarkers of fibrogenesis and fibrosis can be divided into class I and class II analytes. Class I biomarkers are those single tests, which are based on the pathophysiology of fibrosis, whereas class II biomarkers are mostly multiparametric algorithms, which have been statistically evaluated with regard to the detection and activity of ongoing fibrosis. Currently available markers fulfil the criteria of ideal clinical-chemical tests only partially, but increased understanding of the complex pathogenesis of fibrosis offers additional ways for pathophysiologically well based serum (plasma) biomarkers. They include TGF-β-driven marker proteins, bone marrow-derived cells (fibrocytes), and cytokines, which govern pro- and anti-fibrotic activities. Proteomic and glycomic approaches of serum are under investigation to set up specific protein or carbohydrate profiles in patients with liver fibrosis. These and other novel parameters will supplement or eventually replace liver biopsy/histology, high resolution imaging analysis, and elastography for the detection and monitoring of patients at risk of developing liver fibrosis.

Tissue fibrosis is characterized by the excess deposition of extracellular matrix (ECM) involving molecular and histological re-arrangement of various types of collagens, proteoglycans, structural glycoproteins and hyaluronan (Figure 1). It is a hallmark of liver cirrhosis and contributes significantly to the deleterious outcome of chronic liver diseases[1]. The deposition of ECM in the space of Disse (perisinusoidal fibrosis) between the sinusoidal surface of hepatocytes and the endothelial cell layer of liver sinusoids, the generation of (incomplete) subendothelial basement membranes, and the strangulation of hepatocytes by surrounding matrix impair not only the blood flow through the organ, but also the biosynthetic function of hepatocytes and the clearance capability of these and other cell types[2].

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Figure 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) are shown. BM: Basement membranes; FACIT: Fibril-associated collagens with interrupted triple-helices.

The molecular pathogenesis of the fibrotic transition of liver parenchyma turns out to be a muti-faceted process largely due to the activation of resting, vitamin A-storing stellate cells to matrix-producing myofibroblasts[34] in the immediate neighbourhood of hepatocytes, to the phenotypic switch of hepatocytes and bile duct epithelial cells to fibroblasts termed epithelial-mesenchymal transition (EMT)[5–7], and to the influx of bone marrow-derived cells (fibrocytes) reaching the liver via the systemic circulation[89] (Figure 2). The fractional contribution of these pathways to fibrosis depends on the underlying disease and probably on the stage of the fibrotic transition[2]. The activation of stellate cells results from interaction with damaged hepatocytes, activated Kupffer cells, disintegrated platelets and various subfractions of leucocytes. Among the cytokines involved in the pathogenetic processes, TGF-β plays a dominant role, but PDGF, endothelin-1, VEGF, and others also contribute significantly. Antagonistic (antifibrotic) mediators might also exist among which bone morphogenetic protein (BMP)-7 plays an important role, e.g. in the inhibition of EMT-derived fibroblasts[10].

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Figure 2 Synopsis of pathogenetic mechanisms of liver fibrosis (fibrogenesis). The cells produce an increase in extracellular matrix derived from activated hepatic stellate cells (HSC)/expended pool of myofibroblasts (MFB) produce various components of the extracellular matrix (fibrosis) leading to cirrhosis. Newly recognized pathogenetic mechanisms point to the (i) influx of bone marrow-derived cells (fibrocytes) to the liver, (ii) to circulating monocytes and to their TGF-β-driven differentiation to fibroblasts and (iii) to the epithelial-mesenchymal transition (EMT) of hepatocytes and bile duct epithelial cells to fibroblasts. All three complementary mechanisms enlarge the pool of matrix-synthesizing (myo-)fibroblasts. Some important fibrogenic mediators are transforming growth factor (TGF)-β, platelet-derived growth factor (PDGF), insulin-like growth factor I (IGF-I), endothelin-1 (ET-1), and reactive oxygen metabolites (ROS). Abbreviations: ASH: Alcoholic steatohepatitis; NAFLD: Non-alcoholic fatty liver disease. The insert shows an electron micrograph of hepatic stellate cells containing numerous lipid droplets.

The pathogenetic complexity is mirrored by multiple approaches of a clinical diagnosis and a follow-up of ongoing liver fibrosis.

The widely used diagnostic “gold standard” of liver biopsy has many draw-backs besides its invasiveness such as sampling error (around 1/50 000th of liver mass is obtained), irreproducible sample quality depending on length and size of the tissue specimen (coefficient of variation 45%-35%) and a histological evaluation strictly dependent on the experience of the pathologist (observer error)[11]. Therefore, the development of non-invasive, objective and quantitative serum- or plasma-based biomarkers of fibrogenesis is an important goal, which can be approached by the assessment of two, principally different lines of blood-borne (non-invasive) analytes: Class I and class II serum fibrosis markers.

Class I fibrosis biomarkers are pathophysiologically derived from ECM turnover and/or from changes of the fibrogenic cell types, in particular hepatic stellate cells (HSC) and (myo-)fibroblasts[3]. They should reflect the activity of the fibrogenic and/or fibrolytic process and, thus, remodelling 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 involve costly laboratory tests and are the result of translation of fibrogenic mechanisms into clinical application. Thus, their selection is hypothesis-driven.

Class II fibrosis biomarkers mostly estimate the degree of fibrosis (extent of ECM deposition). In general, they comprise common clinical-chemical tests (enzymes, proteins, coagulation factors), which do not necessarily reflect ECM metabolism or fibrogenic cell changes. Their pathobiochemical relationship with fibrogenesis is indirect if at all. Thus, their selection is not hypothesis-driven, 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” (grade of fibrogenic activity), class II markers indicate “where fibrosis is” (stage of fibrosis).

A growing understanding of the pathogenesis of hepatic fibrosis has indicated potentially powerful non-invasive (blood) biomarkers of hepatic fibrogenesis and fibrosis (Table 3). CTGF/CCN2 was already mentioned as a pluripotent downstream modulator of TGF-β, and was found to be up-regulated by TGF-β in hepatocytes. Although most CTGF will only have a defined paracrine function in fibrogenic tissue, a certain fraction spills over into the circulation, resulting in elevated serum concentrations during active fibrogenesis[18]. The circulating level of CTGF might be an objective and sensitive measure of ongoing fibrogenesis in necro-inflammatory liver tissue.

Bone-marrow-derived fibrocytes might offer new approaches not only for understanding the pathogenesis, but also for the diagnosis of liver fibrosis. Fibrocytes are circulating progenitor cells (CD34 positive) of hematopoietic origin (CD45 positive) capable of differentiating into diverse mesenchymal cell types[35]. The additional markers of fibrocytes, i.e. positivity of type I collagen and the CXCR4 chemokine expression can be used to quantitate this special sub-population of circulating leucocytes applying quantitative PCR and/or flow cytometry. The determination of the colony stimulating factors M-CSF, G-CSF, and GM-CSF, which are increasingly expressed in fibrotic liver tissue and elevated in serum[36], are possibly involved in the mobilisation of fibrocytes from the bone marrow and their homing in the liver during fibrogenesis. These factors may be further candidates for diagnostic evaluation.

A new, but currently still controversial aspect of fibrogenesis is epithelial-mesenchymal transition (EMT) of hepatocytes and biliary epithelial cells, respectively, to (myo-)fibroblasts[2]. EMT is governed by the balance of TGF-β (pro-EMT) and its antagonist, i.e. BMP-7 (anti-EMT). In addition to its anti-EMT effect, BMP-7 was shown to have anti-apoptotic and anti-inflammatory activities. Thus, the measurement of BMP-7 alone or even in relation to TGF-β in serum might reflect the activity of fibrogenesis and, hence, the velocity of fibrotic organ transition[37].

Xylosyltransferase (XT), a key enzyme in the biosynthesis of glycosaminoglycans in proteoglycans, was shown to have increased activities in the serum of patients with connective tissue diseases. With HPLC-tandem mass spectrometry, measurements in large cohorts of liver fibrotic patients may to be possible[38]. Since HSC in fibrotic liver tissue (myofibroblasts) have a greatly stimulated proteoglycan synthesis[39], XT activity in serum might be a promising class I biomarker of fibrogenesis.

Further successful developments could emerge from serum proteome profiling[40] and from total serum protein glycomics, i.e. the pattern of N-glycans[41]. It was reported that a unique serum proteomic fingerprint is powerful enough (accuracy > 90%) to differentiate between various stages of fibrosis and to allow prediction of fibrosis and cirrhosis in patients with a chronic hepatitis B infection[40]. Specificities, sensitivities and accuracy of prediction of cirrhosis are around 89%. Similarly, N-glycan profiling can distinguish between compensated cirrhosis from non-cirrhotic chronic liver diseases with a sensitivity and specificity of 79% and 86%, respectively[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 non-invasive biomarkers of diagnosis and follow-up of fibrogenic (liver) diseases. In conclusion, currently available type I and II serum biomarkers should be used with caution, because neither single nor panel markers fulfil the requirements of an ideal non-invasive biomarker of fibrosis[42], i.e. analytical simplicity allowing performance in any laboratory, standardization of the test system and calibrators allowing comparison between laboratories over a long period, cost effectiveness, specificity for the liver and the disease, clear association with the stage of fibrosis or grade of fibrogenesis and independency of the aetiology of fibrosis. Even the best and most extensively evaluated type I (i.e. hyaluronan) and type II (i.e. Fibrotest, Actitest) serum biomarkers do not meet the criteria of an ideal marker. Further detailed insight into the mechanism of liver fibrosis and improvements in analytical techniques will result in new approaches for the non-invasive assessment of fibrosis with biochemical or physical means.

In addition, genetic markers linked with the progression rate of fibrosis will become important diagnostic and prognostic tools for patients with liver fibrosis.

Non-invasive evaluation of the fibrogenic response of the chronically injured liver has made considerable progress over the past few years, in particular over the last three years multiple algorithms based on a combination of more or less routine parameters have been suggested frequently. A rigorous, independent and widespread evaluation of the utility of these panels in the diagnosis and follow-up of chronic liver diseases is still needed for a final decision and the recommendation for use in routine clinical practice. Novel single biochemical markers have been suggested, but there putative diagnostic value in clinical practice is far from defined. The fundamental problem in the evaluation of existing and novel non-invasive parameters lies in the limited validity of the present diagnostic “gold standard”, i.e. histology of liver biopsy specimens. Perhaps new developments in highly sensitive and tissue-specific scanning techniques of the liver will solve this problem. These procedures will then be suitable for the correct validation of effective antifibrotic treatments.