Noninvasive Biomarkers for the Diagnosis of Liver Fibrosis and Cirrhosis

The clinical importance of monitoring liver fibrosis lies in the morbidity and mortality of the chronic liver diseases in relation to the stage and progression of fibrosis. Whether the fibrosis stabilizes or regresses depends on the specific treatment. Liver biopsy, the current standard for the diagnosis, has implicit limitations due to sampling heterogeneity. There are noninvasive imaging methods, such as transient elastography that measures the stiffness of the liver, but it has some limitations (feasibility and unreliability), particularly in obese patients. FibroTest is the most widely used noninvasive serological method worldwide which is efficacious in the extreme stages of fibrosis, but these methods cannot discern intermediate stages. Liver fibrosis is a dynamic response that involves multiple cellular and molecular events with an excessive deposit of extracellular matrix. Even though there is much information on the pathophysiology of fibrosis, that knowledge is still incomplete, greatly hindering the development of both an accurate treatment and a noninvasive diagnostic method with adequate sensitivity for all the stages of fibrosis. It is known that IGFBP participates in liver homeostasis, and thus these proteins can be used as serum biomarkers during the progression of liver fibrosis in chronic hepatitis C.


Introduction
The etiologic factors that mainly induce liver fibrosis are alcoholic liver disease (ALD), chronic hepatitis C (CHC), and nonalcoholic fatty liver disease (NAFLD). Fibrogenic response can be organized in four different phases according to damage evolution: activation, production of extracellular matrix proteins (ECM), and

Diagnosis of liver fibrosis
Noninvasive measures of liver fibrosis have streamlined the management of patients with CHC [6]. Imageology methods, such as transient elastography (FibroScan™), which measures the stiffness of the liver, have received great acceptance by clinicians and patients. Clinical trials showed that FibroScan results, which are expressed in kPa, reported similar results to that of METAVIR score during biopsy interpretation of liver fibrosis in patients with CHC [10]. Moreover, this procedure has also been validated in chronic hepatitis B, NAFLD, alcoholic liver disease, primary biliary cirrhosis, and primary sclerosing cholangitis [14,15]. However, this methodology also has some limitations such as feasibility and unreliability, particularly in obese patients or under the circumstances of limited operator experience [16]. Furthermore, it is important to consider that this strategy is contraindicated during pregnancy, ascites, and implanted cardiac pacemaker patients [10].
A variety of "direct" serum markers reflecting ECM turnover (fibrogenesis and fibrolysis) and/or fibrogenic cell changes have been developed and used clinically [17]. In this sense, the multiple analyses of proteins and clinical trials provide valuable information of liver stage. FibroTest index is within the most accepted worldwide noninvasive serological method in the diagnosis of liver fibrosis by CHC, CHB, and NAFLD [17]. For this analysis are computed five surrogate parameters: DOI: http://dx.doi.org /10.5772/intechopen.84351 total bilirubin, haptoglobin, gamma-glutamyltranspeptidase (GGT), alpha2macroglobulin, and apolipoprotein-A. It is important to mention that the validation of any surrogate parameter needs to be validated by the calculation of the area under the receiver operating characteristic curve (AUROC) using liver biopsy as reference [8]. Systematic analysis of several studies revealed that FibroTest displayed an excellent discrimination to identify cirrhosis with AUROC = 0.90 but showed a lesser ability to identify ≥F2 fibrosis stages (AUROC = 0.81). The authors conclude that this index is not ready to substitute liver biopsy in the intermediate stages [18]. Additionally, some limitations such as cost and external validation are documented.
Even though there is much information on the pathophysiology of fibrosis, that knowledge is still incomplete, greatly hindering the development of both an accurate treatment and a noninvasive diagnostic method with adequate sensitivity for all the stages of fibrosis. Such a method must also be able to be performed with the necessary frequency to establish disease progression or regression, as well as the changes that occur in the processes, such as chronic inflammation, fibrogenesis, and fibrolysis. Based on our knowledge of fibrosis pathogenesis, attention is now directed towards strategies for antifibrotic therapies and regulatory challenges for conducting clinical trials with these agents. New therapies are attempting to: (1) control or cure the primary disease or reduce tissue injury; (2) target receptorligand interactions and intracellular signaling; (3) inhibit fibrogenesis; and (4) promote resolution of fibrosis. Progress is urgently needed in validating noninvasive markers of fibrosis progression and regression that can supplant biopsy and shorten the duration of clinical trials. Both scientific and clinical challenges remain, however, in the past three decades of steady progress in knowledge liver fibrosis.
This entails analyzing the molecules involved in these processes along with the participation of proteins such as insulin-like growth factor binding proteins (IGFBPs). Recently, IGFBP-1 and insulin-like growth factor (IGF) have been proposed as markers for advanced fibrosis in NAFLD [19]. However, the completed evaluation of IGFBPs in liver diseases is not fully understood.

Insulin-like growth factor (IGF) complex
The IGF is a family of proteins with high sequence homology to insulin. The IGF system functions as an endocrine, paracrine, and autocrine regulatory axis for cell proliferation, survival, and apoptosis in different types of cells [20]. In general, the IGF system consists of two surface receptors (IGF1R and IGF2R), two ligands (IGF-1 and IGF-2), and a family of IGFBPs [20,21].
Liver is the main source of IGFs but its highest concentrations are found in the blood. Both IGF-1 and -2 forms can be detected in small amounts in the kidney and other tissues of different species, for example, in the rat, IGF-I has been detected in serum, milk, amniotic fluid, and bile; it has also been detected in human adult bile [22]. In some experiments, it has been found that IGF-1concentrations are higher in bile of neonatal rats than in the adult rats. Thus, it is believed that IGF-I in bile should have an important role in the development of gastrointestinal tract. However, the precise role and the presence or absence of IGFs and IGFBPs in bile have not yet been clearly defined. The biosynthesis of IGFs depends mainly on the levels of growth hormone (GH), insulin, prolactin, and an adequate nutrition stimulus. In contrast, estrogens and cortisol can antagonize their formation [23].
The production of IGF-1 is stimulated by GH, which is secreted by somatotropic cells in the adenohypophysis. The hepatocytes present GH receptors that are stimulated by this hormone and in a consequence an increase in the transcription of Liver Cirrhosis -Debates and Current Challenges the IGF-1 gene is triggered. IGF-1 inhibits GH secretion either directly acting on the pituitary or indirectly by stimulating the hypothalamic secretion of somatostatin which, in turn, inhibits the release of GH. In this way, a negative feedback loop GH-IGF-1 is established [21,23].
IGFBPs play important roles in the bioavailability of circulating IGF-I, and their synthesis is under metabolic and hormonal control. Their functions can be summarized as follows: (1) they act as protein carriers in serum and control the influx of IGF-I from the vascular space to the tissues. (2) They prolong the half-life of IGF-I and regulate its metabolism. (3) They provide temporal localization of IGF-1 with the aim to be available under specific requirements. (4) They modulate the interaction of IGF-I with its receptor, thus acting as indirect control of the biological actions of IGF-I.
At present, different types of IGFBPs have been described that are mainly produced by hepatocytes and secreted into the blood serum. They can be a high affinity for binding IGFs (IGFBP-1 to IGFBP-6) or a low affinity (IGFBP-7 among others). Normal serum IGF-I levels are approximately 40 nmol/L, and 99% of circulating IGF-Iis was estimated to be associated with the different IGFBPs, mainly IGFBP-3 [23][24][25].
In recent years, these proteins have gained great attention due the association as biomarkers in different pathologies [25].

IGFBPs and liver fibrosis
An increase in IGFBP-1 levels has been observed during nonalcoholic liver disease and cirrhosis of the liver. At the same time, serum IGFBP-3 concentrations are low, correlating with the severity of liver dysfunction, and signifying poor prognosis in hepatocellular carcinoma (HCC) [26]. In in vitro studies, IGFBP-7 expression has been found to hepatocyte apoptosis and HSC activation [27,28]. In relation to the participation of IGFBP-2, -5 and -6, there is little evidence of their concentrations in serum and the possible association with liver diseases. Nevertheless, those proteins can inhibit angiogenesis (IGFBP-4) [29], regulate the role of TNF-α, tumor growth, and an increased expression in pulmonary and liver fibrosis (IGFBP-5) [30,31] and promote prostate cancer cell migration (IGFBP-6) [22,32]. The available information about the role, tissue production, and dependent and independent functions of IGFBPS as well as their regulation in related liver pathologies is resumed in Table 1.
In the present work, we include recently obtained data in our laboratory of a prospective comparative multicenter study to evaluate the production of IGFBPs according to liver fibrosis grade in patients with CHC. We provide a valuable and innovative approach to the analysis of IGFBPs as a group of proteins with important potential for improving diagnosis and maybe soon can be used as novel noninvasive biomarkers for liver fibrosis.
Even advanced stages of liver fibrosis have been described as reversible, stimulating considerable research to identify molecules for the development of antifibrotic therapies [35,36].
IGFBPs are produced in the liver, but there is little evidence of their participation in the process of liver damage in humans. Their study can further improve the knowledge of liver fibrosis pathophysiology and enable the identification of therapeutic targets.
The aim of the present study was to measure the serum concentrations of the different IGFBPs in patients with CHC and analyze them according to the grade of fibrosis grade.

Methods and patients for the study of IGFBPs in chronic hepatitis C
A prospective, cross-sectional, observational study was conducted. It included patients seen at the General Hospital of Mexico, University Hospital of Autonomous University of Nuevo Leon, and the National Institute of Medical Sciences and Nutrition, within the time frame of January 2011 to December 2015. The patients were treatment-naïve or untreated, and their grades of fibrosis were evaluated through the FibroTest and FibroScan methods. According to fibrosis grade, the patients were divided into four groups: F0, F1-F2, F3, and F4. Given the small number of patients with stages F1 and F2, they were combined as a single group (n = 25). Patients with atrisk alcohol consumption (AUDIT < 8) and whose fibrosis grade was not determined using the same diagnostic methods stated above were excluded. The control group was made up of healthy subjects, defined as persons that were not at-risk drinkers (AUDIT < 8) and had negative serology for hepatitis A, B, and C viruses (n = 160). All participants signed statements of informed consent and the study protocol followed the ethics guidelines of the 1975 Declaration of Helsinki. Blood samples (10 mL) were taken from all participants. The serum was separated and stored at −80°C until its use. The anthropometric variables obtained for each study subject were: sex, age, height (measured in centimeters with a stadiometer), weight (measured in kilograms with a manual scale), and body mass index (BMI) (kilograms/ meters 2 ; weight/height 2 formula). The following biochemical tests were performed on all the study subjects: total bilirubin, direct bilirubin, aspartate aminotransferase (AST), and alanine aminotransferase (ALT). A detailed clinical history was carried out for each patient, through which the presence of clinical data of liver damage was intentionally evaluated. IGFBP determination was carried out through multiple suspension array technology (Millipore®). The use of antibodies made it possible to study the concentration of a variable number of proteins in a single serum sample. Thus, seven proteins were analyzed in a single assay, reducing intra-assay and inter-assay error, %CV of <10 and of <15 for intra-and inter-assay, respectively, without cross-reactivity. The HIGFBMAG-53 K07 kit was employed. The data were acquired utilizing Luminex200 MAGPX® Systems equipment (series number 10294005), following the supplier's specifications. The sensitivity of the minimum and maximum detection values for each protein was obtained using Luminex XPONENT software. The continuous variables were described as mean ± standard deviation and the qualitative variables as absolute and relative frequencies (percentages). The qualitative variables were analyzed using the chi-square test and ANOVA test, orthogonal analyses, and Spearman correlation was used for the quantitative variables. Statistical significance was set at p < 0.05. Logistic regression analysis with advanced fibrosis stage as the dependent variable was applied. Receiver operating curves (ROCs) were made. Statistical analysis was carried out using the SPSS version 22 program.

Clinical determinations
Demographic analysis showed the predominance of women in the group of CHC; furthermore, the age mean was 50 years in CHC in comparison with the mean of 37 years in CT. Nevertheless, body mass index (BMI) did not show differences in both groups of study. Liver function tests that include bilirubin and transaminases (AST and ALT) showed evident and significant increment of values that reflect the liver dysfunction in CHC patients ( Table 1).
A total of 120 patients diagnosed with chronic hepatitis C (CHC) were included in the study. Many of the patients were women. All the patients were untreated and did not drink alcohol. The control population was made up of 165 healthy subjects with negative viral panels and no at-risk alcohol consumption ( Table 2).
There was a 2-fold greater increase in IGFBP-7 concentrations in patients, compared with controls. Upon fibrosis grade evaluation, we found a gradual increase in the concentration of that protein (Figure 2B), obtaining significant differences between F0 vs. F3 (p < 0.001), F0 vs. F4 (p < 0.001), F1-F2 vs. F3 (p = 0.002), and F3 vs. F4 (p = 0.005) (Figure 2B). The correlation of IGFBP-7 with fibrosis grade  produced a statistically significant r of 0.384 (p = 0.001), indicating that IGFBP-7 was associated with the grade of fibrosis, signifying that it could be a serologic biomarker for liver fibrosis.
Moreover, our study provides for first time completed screening of seven IGFBPs and their serum concentration in the initial, middle, and severe fibrosis stages. For IGFBP-1, slight differences in middle and severe stages and CT were found, whereas nonsignificant differences were observed for de IGFBP-3, and -6 among F0, F1-F2, F3, F4, and CT classifications. However, evident variations were observed for IGFBP-2 and -4 according to the fibrosis degrees and CT group. ( Table 3). The most evident concentration changes were observed in IGFBP-7, where the statistical analysis revealed a correlation in the fibrosis degrees and their production ( Table 3).

Sensitivity and specificity of IGFBPs in severe fibrosis stage
Additionally, we performed calculation of the area under the receiver operating characteristic curve (AUROC) using the severe stage as a reference parameter with the aim to determine the sensitivity and specificity of IGFBP-2, -4, -5, and -7 with F4 stage (Figure 2 and Table 4). The results showed that the value of area under the curve for IGFBP-2 was 0.760 (p < 0.001; 95% CI, 0.672-0.847); whereas in IGFBP-4 was 0.744 (p < 0.001; 95% CI, 0.659-0.828), IGFBP-5 was 0.600 (p = 0.049; 95% CI, 0.496-0.703), and for IGFBP-7 was 0.674 (p = 0.001; 95% CI, 0.579-0.770) ( Table 4). Based on the AUROC interpretation, where the  values take more significance when they are close to the unit and with p value, we observed that the degree of predictive significance indicator of these proteins is: IGFBP2 < IGFBP-4 < IGFBP-7 < IGFBP-5.

Discussion
Our study is the first to quantify seven IGFBPs in patients with hepatitis C virus (HCV) and associate those protein levels with fibrosis grade. Different studies consider IGFBP-1 as an insulin-sensitive protein that participates in the development of metabolic diseases, such as insulin-resistance or metabolic syndrome, in patients with or without liver disease. Some authors have reported elevated levels of IGFBP-1 expression [37], whereas others have found low levels [38,39]. Because of findings of its increased expression, IGFBP-1 has been identified as a possible biomarker for alcoholic liver disease (ALD). Controversial results have been reported in relation to NAFLD. A decrease in serum concentrations due to interaction with insulin was described [40,41], but Hagström et al. reported elevated values, with higher concentrations in patients with advanced fibrosis [19]. High concentrations have also been associated with hepatocellular carcinoma, albeit the role of that protein is still contradictory [37,42]. In our study, IGFBP-1 concentrations were higher in subjects with CHC, but no differences were found in relation to fibrosis grade. However, studies on patients with liver cirrhosis of different etiologies (hepatitis C, hepatitis B, NAFLD, ALD, and autoimmune hepatitis) have shown that IGFBP-1 increases [19,43] and is higher in advanced fibrosis (F3 and F4) [19].
IGFBP-2 has been suggested as a biomarker for metabolic diseases, such as diabetes and insulin resistance [33]. In our study, IGFBP-2 concentrations were until 6-fold higher in patients, compared with controls. In accordance with fibrosis grade, there was also a gradual increase in IGFBP-2 concentration, but with no statistically significant differences between fibrosis stages, suggesting that IGFBP-2 participates in liver damage and the development of fibrosis caused by HCV. Our results concur with those reported for idiopathic pulmonary fibrosis, in which high levels of IGFBP-2 were determined [44].
Additionally, in this work, we can determine for first time the evaluation of the specificity and sensibility of the seven IGFBPs. Interestingly, IGFBP-2 showed higher area values (AUROC) in F4 and thus can be considered as the best predictive IGFBP protein indicator in severe fibrosis stage followed by -4, -7 and -5.
IGFBP-3 had higher concentration levels than those of the other six IGFBPs. Nevertheless, the concentrations showed a tendency to be lower in patients than in controls. IGFBP-3 has been the most widely studied protein because of its high affinity for IGF-I. It has been described as a biomarker for liver dysfunction classified with the Child-Pugh scale, with a lower concentration in patients with Child-Pugh class C [45]. Since 1995, many studies have been conducted on IGFBPs in patients with liver cirrhosis due to different causes. Results have shown a decrease in IGFBP-3 concentrations and an increase in IGF-I [46,47]. IGFBPs are also thought to be associated with a high risk for liver cancer [48] and with poor prognosis [26]. Aleem et al. concluded that IGFBP-3 is superior to IGF-I and IGF-II for predicting the development of hepatocellular carcinoma in patients with cirrhosis caused by HCV [49], due to viral protein interaction that alters the IGF axis and the subsequent progression to liver cancer [50].
Miller et al. described the serum proteome of NAFLD, reporting increased IGFBP-3, compared with control subjects. They then analyzed fibrosis grade and severity and found that IGFBP-3 was able to distinguish between different disease stages [51]. In 2017, Chishima et al. studied the GH/IGF-I/IGFBP-3 axis DOI: http://dx.doi.org /10.5772/intechopen.84351 in patients with NAFLD and CHC and the relation to the histologic severity of NAFLD. IGFBP-3 levels were lower in patients with cirrhosis caused by NAFLD, whereas the levels did not decrease according to fibrosis grade in patients with HCVinduced chronic liver disease [46]. Our results concurred with those of that study.
Cirrhosis alters IGF-I production and suppresses protein metabolism. In studies on children with end-stage liver disease (ESLD) who underwent liver transplantation, the authors concluded that their results partially explained the failure to growth and the reduced number of functioning hepatocytes in patients with ESLD [52].
IGFBP-4 has been associated with the progression of lung cancer, finding high expression of that protein in lung tissue, and showing a decrease in survival [53]. In our study, IGFBP-4 concentration was 2-fold higher in patients, compared with controls, and behavior fluctuated in relation to fibrosis grade, with no statistically significant differences. In a study on patients with ESLD, no differences in IGFBP-4 were found upon comparison before and after liver transplantation [52]. In another study on cirrhotic patients and controls, there were no differences in IGFBP-4 when measured by the Western ligand blot technique [54]. Experimental studies have shown a regulation of the increase of that protein, along with IGFBP-1 and IGF-I, by AMPc, IL-6, IL-1β, and TNF-α [55,56].
IGFBP-5 has been studied in animal models of progressive intrahepatic cholestasis, suggesting that it plays a possible role in the pathogenesis of chronic cholangiopathy. The same authors reported that IGFBP-5, in human stellate cells (LX-2), increased pro-fibrotic marker expression, and concluded that IGFBP-5 participates in liver fibrosis progression [30]. In our study, IGFBP-5 concentrations were similar between patients and controls. However, upon classifying them by fibrosis grade, we found differences in F0 vs. F4 and F1-F2 vs. F3. These findings concur with the results reported by Colak et al. who showed that IGFBP-5 played an important role in many pathophysiologic stages of liver fibrosis [57]. One of the functions of IGFBP-5 was the trans-differentiation of HSCs into myofibroblasts, improving the survival of those cells through anti-apoptotic effects on the activated HSCs, increasing collagen Iα1, TIMP-1, and MMP-1profibrotic gene expression.
It is known that IGFBP-6 can induce chemotaxis in T cells and monocytes, but not in B cells. It also increases oxidative stress and may be a late amplifier of neutrophil activation [58]. However, there have been few studies conducted on IGFBP-6 in liver diseases. In our study, IGFBP-6 concentrations were the same in patients and controls, and we found a tendency for concentrations to increase with the increase in fibrosis grade. IGFBP-6 has been described to be affected by HCV proteins and to participate in the progression to liver cancer [50,59].
Finally, different studies demonstrate that IGFBP-7 (IGFBPrP1) contributes to liver fibrogenesis [27,60,61]. In our study, we found a significant increase in patients with liver disease. Likewise, we observed a gradual increase according to fibrosis grade. It was higher in F3, indicating that it could be a serum biomarker for liver fibrosis. IGFBP-7 has been widely studied in experimental models of liver fibrosis for identifying the mechanisms involved in the activation of HSCs and the signaling pathways, the result of which induces fibrosis. IGFBP-7 was inhibited in rat HSCs, inducing apoptosis in activated HSCs, and as a result, ameliorating liver fibrogenesis [61]. IGFBP-7 has also been shown to attenuate liver fibrosis through the regulation of MMPs/TIMPs in mice [62]. IGFBPrP1 contributed to the development of liver fibrosis in fibrotic and cirrhotic tissue biopsy samples and may be a novel molecule involved in the progression of liver fibrogenesis [28]. Studies conducted in vitro found that IGFBPrP1 induced liver fibrosis by means of HSC activation and hepatocyte apoptosis through the Smad 2/3 signaling pathway [27]. In addition, it acted as an initiator of liver fibrosis by inducing inflammation, HSC activation, and ECM protein deposit through the ERK1/2 pathway [63]. IGFBPrP1 has also been shown to promote fibrosis, by enhancing the TGF-β1 expression that it triggered, and the Egr1, PTEN, Hhip, MAP2K2 (MEK2), and MAPK3 (ERK1) genes were identified as candidates for the hepatic fibrosis-related pathway induced by IGFBPrP1 [60]. Mutual IGFBPrP1 and TGF-β1 regulation has been found that probably accelerates liver fibrosis progression [64]. IGFBPrP1 inhibition attenuates fibrosis by reestablishing the MMP2/TIMP2 and MMP9/TIMP1 balance concomitantly with the inhibition of HSC activation, low TGF-β1 expression, and ECM degradation [62]. Previous results, together with ours, show that IGFBP-7 is a molecule that can be diagnostically useful and a possible therapeutic target for liver fibrosis.
Currently, there are available treatment regiments against hepatitis C; in general, these drugs have as specific targets viral proteins (e. g. NS3/4A, NS5A, and NS5B) inhibiting their replication. The ratio of efficacy of different combinations of drugs can reach until 95% of efficiency [65,66]. Regarding the action of these drugs and liver fibrosis, it has been reported that peginterferon and/or ribavirin, daclatasvir, sofosbuvir, and simeprevir can cause regression of liver fibrosis [67]. The specific molecular events induced by the administration of these drugs are not very well understood. However, it has been observed that the liver parenchymal injury and hepatocyte death are associated with the host's inflammatory response and reactive oxygen species promoted by virus proteins [68,69], whereby the elimination of viral load abrogates the subsequent liver damage. Nevertheless, the molecular mechanisms triggered by the pharmacological therapy in the IGF system are not evaluated until now; it is possible that tissue fibrosis reversion can be orchestrated by IGF elements such as the IGFBPs. Thus, the study of these proteins could have beneficial implication for diagnosis and as well as complementary target to improve the liver regeneration.

Conclusion
We found higher serum concentrations of IGFBP-2, IGFBP-4, IGFBP-5, and IGFBP-7 in patients with CHC, and in accordance with fibrosis grade, IGFBP-2, IGFBP-4, and IGFBP-7 are associated with severe fibrosis. Based on our study, we strongly suggest the possibility that IGFBPs participate in ECM protein modulation and reuptake and regulate the progression of chronic liver disease and development of liver fibrosis. Therefore, we believe that IGF binding proteins play an important role in chronic hepatitis C and can be serum marker candidates for liver fibrosis.