Circulating liver cancer stem cells and their stemness-associated MicroRNAs as diagnostic and prognostic biomarkers for viral hepatitis-induced liver cirrhosis and hepatocellular carcinoma.

Background: Liver cancer stem cells (LCSCs) are a subpopulation of tumor cells that can drive cancer initiation and relapses. Because of their significance, researchers are looking for biomarkers that characterize or regulate LCSCs so that they can be used as targets for the diagnosis and treatment of chronic liver diseases and hepatocellular carcinoma (HCC). Methodology: Six groups of patients having hepatitis C virus (HCV), HCV + cirrhosis, HCV + HCC, hepatitis B virus (HBV), HBV + cirrhosis, or HBV + HCC, in addition to a control group, were subjected to the measurement of LCSCs levels and analysis of miR-1290 and miR-1825 expression. Results: The percentages of the CD133/EpCAM-expressing LCSCs were increased in viral hepatitis and cirrhosis groups, compared to the control group. HCC patients had the highest percentages of LCSCs. CD133/EpCAM-expressing cells showed significant correlations with stemness-associated miRNAs; miR-1290 and miR-1825. Also, the miR-1290 and miR-1825 were significantly up-regulated in viral hepatitis-associated cirrhosis and HCC groups. Moreover, in HCV + HCC, miR-1290 and miR-1825 expression was significantly positively correlated with tumor size and number. However, only miR-1825 could distinguish between HCV- and HBV-associated HCC groups. MiR-1290 exhibited the highest sensitivity and specificity for detecting HCC, followed by miR-1825 and CD133/EpCAM-expressing LCSCs. Conclusions: These findings indicate the relevance of CD133/EpCAM-expressing cells in the pathogenesis of liver cirrhosis and HCC developed as a consequence of either chronic HCV or HBV infection. Accordingly, CD133/EpCAM-expressing cells, miR-1290, and miR-1825, could serve as promising diagnostic and prognostic biomarkers as well as therapeutic targets in patients suffering from liver cirrhosis or HCC.

Innovative model of surface-enhanced Raman spectroscopy for exosomes identification: An approach for the diagnosis of hepatocellular carcinoma.

BACKGROUND: The current hepatocellular carcinoma (HCC) diagnostic approaches lack adequate sensitivity and specificity. So, this study was performed to develop an innovative model of surface-enhanced Raman spectroscopy (SERS) that can detect HCC patients by identifying the circulating tumor-derived exosomes. METHODOLOGY: Sixty participants, including normal controls, hepatitis C virus (HCV)-infected patients, and HCV-associated HCC patients, had their whole blood samples and exosomes separated from these samples analyzed using Raman spectroscopy (RS). A revolutionary model of SERS, based on an innovative glass and nano-gold, was designed to directly identify exosomes. Its measurements were simulated by Comsol Multiphysics (5.6). RESULTS: The RS examination of the whole blood samples revealed no Raman peaks. Yet, the isolated exosomes from these samples generated Raman peaks at 400 and 1000 cm-1 wavenumbers in the HCV group. A Raman shift was detected in HCC patients at 812, 852, and 878 cm-1 wavenumbers with intensity ratios of 120, 130, and 60, respectively. The RS had a sensitivity and specificity of 95 % and 100 %, respectively, for detecting HCC. However, the newly-designed SERS was able to identify the HCC-derived exosomes, at 812 and 878 cm-1 wavenumbers, with boosted intensity ratios of 9*106 and 4*106, respectively, in the whole blood samples. CONCLUSION: The newly-developed SERS model has the potential to detect HCC patients through recognizing the tumor-derived exosomes non-invasively, with high accuracy, and without the need for laborious exosomal separation. Nonetheless, bringing this technology into the clinic demands the establishment of spectral databases and their validation using the current gold standards.

Effects of free and nanoparticulate curcumin on chemically induced liver carcinoma in an animal model.

INTRODUCTION: Curcumin therapeutic applications are constrained by its prominent metabolic instability as well as inadequate absorption and bioavailability. The current study was designed to enhance the curcumin bioavailability by exploiting nanoparticles. MATERIAL AND METHODS: Eleven groups of mice were divided into: normal and nanoparticle control groups, a hepatocellular carcinoma (HCC) group induced by diethylnitrosamine (DEN), 2 groups treated with DEN plus a high dose/low dose of free curcumin, 2 groups treated with a high dose/low dose of free curcumin, 2 groups treated with DEN plus a high dose/low dose of nanoparticulate curcumin, and 2 groups treated with a high dose/low dose of nanoparticulate curcumin. RESULTS: DEN administration significantly increased liver enzymes, vascular endothelial growth factor, tumor necrosis factor-α, α-fetoprotein, malondialdehyde, and nucelar factor-κB. Also, it decreased serum albumin and tissue antioxidant activities and caused severe histological changes in hepatic tissue. Oral treatment of DEN-injected mice with either a high dose of free curcumin or the tested doses of nanoparticulate curcumin resulted in a significant improvement of all the tested parameters. CONCLUSIONS: Although the two tested doses of nanoparticulate curcumin were much lower than free curcumin, both doses were effective in preventing HCC development while the low dose of free curcumin was hardly effective. Hence, we conclude that nanoparticles enhance the bioavailability of curcumin.

Impact of E-cadherin and its transcription regulators on assessing epithelial-mesenchymal transition in chronic hepatitis C virus infection.

BACKGROUND: The mechanisms of chronic hepatitis C virus (HCV)-induced liver fibrosis and hepatocarcinogenesis are still poorly recognized. Therefore, this study aimed to determine the effect of chronic HCV infection on the expression of the major regulators of epithelial-mesenchymal transition (EMT) including E-cadherin, Snail, Slug, and Twist2, in the Egyptian population. This will help to design more efficient strategies to treat HCV-associated cirrhosis and carcinoma. METHODS: Fifty-nine liver biopsies from patients, that were serologically proven to be HCV positive, were included in the current study. Histopathological examination was done. Grading of hepatitis activity (A) and staging of fibrosis (F) were assessed using the METAVIR Scoring System. Additionally, an immunohistochemical examination of E-cadherin, Snail, Slug, and Twist2 expression was performed. RESULTS: E-cadherin showed a significant progressive decline of its expression with increased fibrosis staging and development of hepatocellular carcinoma (HCC). In contrast, Snail and Slug expression was positively associated with the stage of fibrosis and HCC. Meanwhile, Twist2 expression was not affected by the degree of hepatitis activity, the stage of fibrosis, or by the development of HCC. CONCLUSIONS: E-cadherin and its transcriptional regulators; Snail and Slug may serve as indicators for assessing the stage of fibrosis and the progression of HCC associated with HCV infection but not for assessing the degree of hepatitis activity. Therefore, the Snail family could be a promising target for designing effective preventive and therapeutic strategies for chronic HCV infection and its serious comorbidities.

MicroRNA-195 vector influence on the development of gradually induced hepatocellular carcinoma in murine model.

Background: Recent studies implicate the role of microRNAs in the pathogenesis of hepatocellular carcinoma (HCC). This study was designed to induce HCC, in an experimental model, with the prospect to study the molecular pathophysiologic changes accompanying the development of HCC and the effect of miRNA-195 vector on the process of hepatocarcinogenesis.Methodology: This study incorporated three groups of male albino mice; one control group and two other groups injected intraperitoneal with diethylnitrosamine (DEN) weekly for 12 weeks for the gradual induction of HCC. The third group was injected intra-hepatic with miR-195 vector 1 month after DEN injection. At the 8th and 12th weeks post-DEN treatment, the tumor-associated biomarkers alpha-fetoprotein (AFP), vascular endothelial growth factor (VEGF), and tumor necrosis factor-alpha (TNF-α) were assessed in the serum of all mice. Hepatic specimens were subjected to ultra-structural pathological examination as well as to caspase-3 and survivin genes expression analysis.Results: All the assessed serological and molecular parameters of HCC development, in the miRNA-195-treated group of mice, showed a significant increase, versus the DEN-treated group, whereas survivin was significantly down-regulated, in the miR-195-treated group (P < 0.001). Additionally, ultra-structural criteria of HCC were depicted, in the 12th week, in DEN-injected group, versus the 8th week, in the miRNA-195-treated group.Conclusions: Intra-hepatic injection of miRNA-195 vector induced apoptotic gene expression and suppressed anti-apoptotic gene but these favorable anti-cancer effects could not counteract the inflammatory, and subsequently, the oncogenic effect probably caused by vector administration. Therefore, further studies are required to investigate the effect of miRNA in combination with anti-inflammatory medications.

The interaction between microRNA-152 and DNA methyltransferase-1 as an epigenetic prognostic biomarker in HCV-induced liver cirrhosis and HCC patients.

The necessity for early detection and hence improving the outcome of treatment of hepatocellular carcinoma (HCC) is critical especially in Hepatitis C virus (HCV)-Genotype 4 induced cases. In our current work, we examined the miRNA-152 and DNMT-1 expression in chronic liver disease (CLD) due to HCV genotype 4 infection with/without cirrhosis and HCC patients as an attempt to evaluate the potential benefits of these new circulating, noninvasive, prognostic, epigenetic markers for liver cirrhosis and carcinogenesis of Egyptian patients. Eighty subjects were included in this study, divided into two groups; group I (40 patients) were classified into subgroup Ia (CLD without cirrhosis, n = 18) and subgroup Ib (CLD with cirrhosis, n = 22), group II (CLD patients with HCC, n = 20), and control (Healthy volunteer, n = 20). The expression of miRNA-152 and DNMT-1 genes were analyzed using Real-Time PCR. MiRNA-152 showed a persistent and significant downregulation in all diseased groups, which was in consistence with the progression of the disease toward the HCC stage. DNMT-1 showed upregulation in all diseased groups when compared to control and subgroup Ia. The miRNA-152 was shown to correlate inversely with DNMT-1 in subgroup Ia, Ib and group II (r = -0.557, p < 0.01), (r = -0.850, p < 0.001) and (r = -0.544, p < 0.02) respectively. In addition, miRNA-152 and DNMT-1 showed a diagnostic ability to discriminate between cases of cirrhosis and HCC against CLD without cirrhosis (p < 0.01), while DNMT-1 did not, except between HCC and cirrhotic cases. Furthermore, both genes can be considered as predictor and prognostic parameters for cirrhosis (OR = 1.041, p = 0.043) and (OR = 1.039, p = 0.04) respectively, while miRNA-152 alone is proved as a prognostic marker for HCC (OR = 1.003, p = 0.044). Finally, the persistent reverse correlation between miRNA-152 with DNMT-1 prompts their use as noninvasive prognostic biomarkers for HCV induced liver cirrhosis and HCC in HCV Genotype 4 patients.

MicroRNA-122a as a non-invasive biomarker for HCV genotype 4-related hepatocellular carcinoma in Egyptian patients.

INTRODUCTION: Hepatitis C virus (HCV) infection persists in most infected individuals and can lead to the development of chronic hepatitis, cirrhosis and hepatocellular carcinoma (HCC). MicroRNAs (miRNAs) have a crucial role in various liver diseases, especially HCC. The expression profiles of circulating microRNAs have been studied aiming at the identification of novel non-invasive biomarkers. This study aims to develop a non-invasive diagnostic tool based on measuring the serum levels of different miRNAs in order to detect HCV-induced HCC at the early stages of the disease. MATERIAL AND METHODS: Five main miRNAs (miRNA-122a, miRNA-125a, miRNA-139, miRNA-145, and miRNA-199a) were selected according to the literature that demonstrated their unique expression pattern during HCC development. Serum samples were collected from 42 cases of chronic hepatitis C (CHC) without cirrhosis, 45 cases of CHC with cirrhosis (LC), 38 cases of HCC with HCV, and 40 healthy individuals serving as a control. The five miRNAs were measured using real-time reverse transcription PCR. The conventional HCC markers α-fetoprotein (AFP) and des-γ-carboxyprothrombin (DCP) were measured with commercial kits. RESULTS: Serum levels of miRNA-122a, miRNA-125a, miRNA-139, miRNA-145, and miRNA-199a were significantly lower (p < 0.01) in HCC than in CHC and LC groups. As a single marker, miRNA-122a had the highest sensitivity for HCC, followed by miRNA-199a, miRNA-145, miRNA-139, and miRNA-125a. CONCLUSIONS: These findings indicate that measurement of serum levels of miRNA-122a, miRNA-125a, miRNA-139, miRNA-145, and miRNA-199a can differentiate HCC from CHC and LC. Our results suggest that serum miR-122 might serve as a novel and potential noninvasive biomarker for HCV-induced HCC.

Antihepatocarcinogenic activity of whey protein concentrate and lactoferrin in diethylnitrosamine-treated male albino mice.

Hepatocellular carcinoma is considered one of the most prevalent and lethal malignancies worldwide. Chemotherapy with cytotoxic agents showed a low response rate with possible toxic effects. Recently, some emphases have been placed on the anticancer properties of bovine whey protein and its components, especially lactoferrin. The present study aimed to evaluate and compare the antihepatocarcinogenic activity of bovine whey protein concentrate (WPC, 300 and 600 mg/kg body weight) and lactoferrin (30 and 60 mg/kg body weight), orally and daily for 14 weeks, in the mice model of diethylnitrosamine (DEN)-induced hepatocarcinogenesis. The results showed that both WPC and lactoferrin (in a dose-dependent manner) alleviated significantly (P < .001) the elevation in serum markers of liver carcinoma and inflammation in the DEN-treated mice. Also, they exhibited a great amelioration in the livers' histological structure of the DEN-treated mice by 37.0% to 66.7%. In addition, they decreased significantly (P < .001) the hepatic DNA fragmentation in the DEN-treated mice by 23.1% to 32.7%. Only, the high doses of WPC and lactoferrin completely modulated the decrease in the activity of liver enzymic antioxidant defense system (catalase, glutathione peroxidase, and superoxide dismutase) and improved significantly (P < .01-.001) the concentration of hepatic reduced glutathione of the DEN-treated mice. Moreover, the high doses of WPC and lactoferrin reduced significantly (P < .05-.001) the elevation in the concentrations of hepatic active caspases 3, 8, and 9 of the DEN-treated mice. In conclusion, both WPC and lactoferrin were effective in inhibiting the hepatocarcinogenic activity of DEN in mice model through their ability to alleviate the hepatic inflammation and apoptosis.

miRNA-221 and miRNA-222 are promising biomarkers for progression of liver fibrosis in HCV Egyptian patients.

BACKGROUND: Current methodologies used to determine the progression of hepatic fibrosis rely heavily on liver biopsy, a dangerous and invasive procedure, with semi-subjective analysis of the results of the biopsy. Thus, a new approach is immensely needed for monitoring the progression of liver fibrosis in Hepatitis C virus (HCV) patients. AIM OF WORK: The purpose of this study was to find highly specific and sensitive miRNA biomarkers that can be used to detect different stages of liver fibrosis. METHODOLOGY: The study consisted of 42 cases of chronic hepatitis C (CHC) with early-stage fibrosis, 45 cases of CHC with late-stage fibrosis, and 40 healthy subjects with no CHC or fibrosis as controls. Expression patterns of 5 miRNAs (miR-16, miR-146a, miR-214-5p, miR-221, and miR-222) were analyzed in each group using TaqMan real-time PCR. RESULTS: Serum levels of miRNA-16, miRNA-146a, miRNA-221, and miRNA-222 were all significantly up-regulated in early and late stages of liver fibrosis. miRNA-222 had the highest sensitivity and specificity values in early and late fibrosis. miRNA-221 had the second highest sensitivity and specificity with the late-stage fibrosis group. Furthermore, miRNA-221 showed significant positive correlations with both miRNA-16 and miRNA-146a in the early- and late-stage fibrosis groups, with the early stage having a stronger correlation. CONCLUSIONS: The results indicated that miRNA-16, miRNA-146a, miRNA-221, and miRNA-222 can be used to detect the presence of liver fibrosis. The high sensitivity and specificity of miRNA-222 and miRNA-221 in late-stage fibrosis indicate promising prognostic biomarkers for HCV-induced liver fibrosis.

Expression analysis of liver-specific circulating microRNAs in HCV-induced hepatocellular Carcinoma in Egyptian patients.

OBJECTIVES: Due to the absence of reliable and accurate biomarkers for the early detection of liver malignancy, circulating microRNAs have recently emerged as great candidates for prompt cancer identification. Therefore, the aim of this study was to investigate the potential of liver-specific circulating microRNAs as an accurate non-invasive diagnostic tool for early diagnosis of hepatitis C virus (HCV)-induced hepatocellular carcinoma (HCC). METHODOLOGY: A total of 165 patients were enrolled in this study and categorized into four main groups: 42 chronic hepatitis C (CHC) without cirrhosis, 45 CHC with cirrhosis (LC), 38 HCC with HCV patients, and 40 healthy controls. The expression profiles of seven miRNAs (miR-16, miR-34a, miR-125a, miR-139, miR-145, miR-199a, and miR-221) were analyzed using real-time PCR. RESULTS: Serum levels of miRNA-125a, miRNA-139, miRNA-145, and miRNA199a were significantly lower (p < 0.01) in HCC than in both CHC and LC groups. On the other hand, no significant difference was shown in the expression of miR-16, miR-34a, and miR-221 between the CHC, LC, and HCC groups. MiR-16, miR-34a, and miR-221 were significantly elevated in the HCC group compared to the control group. MiR-34a showed the highest specificity and sensitivity. CONCLUSIONS: The results indicated that the measurement of serum levels of miR-125a, miR-139, miR-145, and miR-199a can help to differentiate HCC from CHC and LC. Also, miR-16, miR-34a, and miR-221 serum levels would have a prognostic value. MiR-34a had the highest specificity and sensitivity, indicating that it might serve as a novel and potential non-invasive biomarker for HCV-induced HCC.

Potential ultrastructure predicting factors for hepatocellular carcinoma in HCV infected patients.

Hepatitis C virus represents one of the rising causes of hepatocellular carcinoma (HCC). Although the early diagnosis of HCC is vital for successful curative treatment, the majority of lesions are diagnosed in an irredeemable phase. This work deals with a comparative ultrastructural study of experimentally gradually induced HCC, surgically resected HCC, and potential premalignant lesions from HCV-infected patients, with the prospect to detect cellular criteria denoting premalignant transformation. Among the main detected pathological changes which are postulated to precede frank HCC: failure of normal hepatocyte regeneration with star shape clonal fragmentation, frequent elucidation of hepatic progenitor cells and Hering canals, hepatocytes of different electron density loaded with small sized rounded monotonous mitochondria, increase junctional complexes bordering bile canaliculi and in between hepatocyte membranes, abundant cellular proteinaceous material with hypertrophied or vesiculated rough endoplasmic reticulum (RER), sequestrated nucleus with proteinaceous granular material or hypertrophied RER, formation of lipolysosomes, large autophagosomes, and micro-vesicular fat deposition. In conclusion, the present work has visualized new hepatocytic division or regenerative process that mimic splitting or clonal fragmentation that occurs in primitive creature. Also, new observations that may be of value or assist in predicting HCC and identifying the appropriate patient for surveillance have been reported. Moreover, it has pointed to the possible malignant potentiality of liver stem/progenitor cells. For reliability, the results can be subjected to cohort longitudinal study.

Spotlight on the three main hepatic fibrogenic cells in HCV-infected patients: Multiple immunofluorescence and ultrastructure study.

The present work deals with the simultaneous ultrastructure and triple immunofluorescence study of the three main hepatic fibrogenic cells, hepatic stellate cell, myofibroblast (MF), and fibroblast, in a group of hepatitis C virus (HCV) RNA positive patients, as their exact interrelation behavior in vivo with the progress of hepatic fibrosis is still inadequate. In this study, for the first time, cells having the morphological characteristic of MF and not bone marrow fibrocytes were revealed in liver portal vessels. This necessitates the reevaluation of the available knowledge concerning bone marrow fibrocyte. Also, the distribution, cellular interrelations, and the fate of MF were highlighted.

The Role of MicroRNAs in Response to Interferon Treatment of Chronic Hepatitis C patients.

INTRODUCTION: Treatment of HCV using a combination of pegylated interferon (PEG-IFN) and ribavirin fails in about 40% of the patients with HCV genotype 4 infections, and it is physically and economically demanding. Thus, it is highly important to identify factors that can help to predict the likelihood that a patient will respond to this treatment. METHODS: In this study, five miRNAs, i.e., miRNA-122, miRNA-199, miRNA-192, miRNA-30, and miRNA-128, were selected according to previous studies that demonstrated their noticeable functions in viral replication, indicating that they potentially could be used by host cells to control viral infections. The five miRNAs were measured using real-time, reverse transcription-polymerase chain reactions. The data were analyzed using the t-test and chi-squared test. RESULTS: We found that the expression level of miRNA-122 was significantly increased in the responders' group (p < 0.01) over that in the non-responders' groups before and after treatment; both increased significantly (p < 0.01) compared with the normal control group. CONCLUSION: miR-122 might be a useful predictor for virological responses to treatment with PEG-interferon plus ribavirin therapy in patients with HCV.

Circulating miRNAs as Predictor Markers for Activation of Hepatic Stellate Cells and Progression of HCV-Induced Liver Fibrosis.

INTRODUCTION: Liver fibrosis is the excessive accumulation of extracellular matrix that occurs by activation of hepatic stellate cells (HSCs), which has been identified as the major driver of liver fibrosis. Several studies confirmed that miRNAs have regulatory effects on the activation of HSCs by affecting the signaling pathways. The aim of this study was to develop non-invasive diagnostic markers by measuring different circulating miRNAs in serum as predictor markers for early diagnosis of liver fibrosis and its progression. METHODS: In this case-control study, we enrolled 66 subjects with chronic hepatitis C (CHC) with early stage of fibrosis and 65 subjects with CHC with late-stage fibrosis. Also, 40 subjects were included as normal controls. The six main miRNAs, i.e., miR-138, miR-140, miR-143, miR-325, miR-328, and miR-349, were measured using the reverse transcription-polymerase chain reaction. RESULTS: In the cases of CHC both with early and late stage of fibrosis, the circulating levels of the six main miRNAs were significantly higher than the levels in the control group. ROC analysis indicated that the sensitivity and specificity of miR-138 were 89.3% and 71.43%, respectively, in the early stage of fibrosis. In the late stage, the sensitivity and specificity of miR-138 were 89.3 and 93.02%, respectively, whereas, for miR-143, they were 75.0 and 88.4%, respectively. CONCLUSIONS: Circulating miR-138 could serve as a non-invasive biomarker for the detection of early fibrosis. Also, miR-138 and miR-143 could be specific biomarkers for indicating the late stage of liver fibrosis.

ROLE OF T REGULATORY CELLS IN CHRONIC HCV INFECTED EGYPTIAN PATIENTS AND THEIR IMPACT ON THE RESPONSE TO PEGYLATED INTERFERON THERAPY.

Treatment of patients with chronic hepatitis C with the current standard pegylated interferon (PEG-IFN) and ribavirin achieves overall response (SVR) rates of ~55%. A role of CD4+ CD25+ regulatory T cells (Treg cells) has been proposed as they can suppress HCV-specific T cells in HCV-infected patients. Patients with chronic HCV legible for PEG-IFN plus ribavirin treatment, were classified according to their response to treatment into two groups (responders and non-responders, 32 and 27 patients respectively). Blood and plasma samples were collected at the start of treatment and at 12 and 24 weeks during treatment. Immunophenotyping by flow cytometry for Treg cells, the FOXP-3 expression using real-time PCR and measurement of IL-10, TGF-ß CXCL-9 and CXCL-10 were performed. Increased expression of Treg cells was detected in patients who didn't respond to treatment before and during treatment. Also, the levels of IL-10, TGF-ß, CXCL-9 and CXCL-10 revealed significant increase.in non-responders all through compared to responders group. Evaluation of Treg cells, cytokines (IL-10 & TGF-ß) and chemokines (CXCL-9 & CXCL-10) before starting the treatment could be a predictor of response to treatment with PEG-IFN plus ribavirin. The optimum levels which would differentiate between responders and non-responders are needed to be defined before-hand.

ROLE OF EPITHELIAL MESENCHYMAL TRANSITION IN HEPATIC FIBROGENESIS.

Liver fibrosis is a gradual process of increased secretion and decreased degradation of extra-cellular materials. Two cell types are now well recognized as being involved in liver fibrosis, i.e. hepatic stellate cells (HSCs) and portal mesenchymal cells. T iis process is initiated by the damage of hepatic cells, which leads to activation of hepatic stellate cells that differentiate into myofibroblasts leading to the formation of liver fibrosis. On the other hand, the epithelial-mesenchymal transition and mesenchymal-epithelial transition are crucial for the regulation of cellular plasticity during liver fibrosis. The EMT is a process in which molecular reprogramming leads epithelial cells to adopt a mesenchymal phenotype. During EMT, epithelial cells gain mesenchymal features which include changes in the expression of epithelial markers. The EMT process plays fundamental roles during embryogenesis, tissue fibrosis, and carcinogenesis. As multiple experimental studies of liver fibrosis have confirmed that established liver fibrosis is reversible upon cessation of the causative agent, modulation of the EMT markers could be promising as potential therapeutic agents. Better understanding of the molecular cascades of intracellular fibrogenic signaling and genetic factors that controlling the expression of EMT markers would be a powerful strategy for early diagnosis and treatment liver fibrosis at the genetic level. Activating or silencing of the responsible genes may be an efficient and more specific approach for treating liver fibrosis either through the arrest of EMT or the induction of MET.

Serum Markers of Epithelial Mesenchymal Transition as Predictors of HCV-induced Liver Fibrosis, Cirrhosis and Hepatocellular Carcinoma.

INTRODUCTION: Hepatitis C virus (HCV) is a major cause of chronic liver disease in Egypt, leading to hepatic fibrosis, liver cirrhosis (LC), and hepatocellular carcinoma (HCC). Liver fibrosis is characterized by excessive deposition of extracellular matrix (ECM). Newly-recognized pathogenic mechanisms point to the epithelial-mesenchymal transition (EMT) of hepatocytes to matrix synthesizing (myo-) fibroblasts. Transforming growth factor-beta (TGF-ß1), bone morphogenic protein (BMP)-7, and connective tissue growth factor (CTGF) are biomarkers reflecting the EMT process. YKL-40 is a glycoprotein member of ECM and plays a role in cancer cell proliferation. The purpose of this study was to determine the serum biomarkers of EMT and its impact on the fibrogenic process and tumorigenesis in HCV-genotype 4 patients. METHODS: In this case-control study that was conducted in 2013-2014, 97 HCV-infected patients were subjected to clinical examination, laboratory investigations, and liver biopsy. According to the histopathologic examination, they were classified to F0 (14 cases), F1 (17 cases), F2 (15 cases), F3 (18 cases), F4 (22 cases), and HCC (11 cases). Fifteen age- and gender-matched subjects were included as normal controls. Serum levels of TGF-ß1, BMP-7, CTGF, YKL-40 were assessed, and the TGF-ß1/BMP-7 ratios were calculated. The data were analyzed by plotting the receiver operating characteristic curve (ROC), Pearson product-moment correlation coefficient, and Spearman's rank correlation coefficient (Spearman's rho). RESULTS: Serum levels of TGF-ß1, BMP-7, CTGF, and YKL-40 were significantly increased in all patient groups compared to controls (p < 0.001). LC exhibited the highest CTGF level and YKL-40 was highest in HCC. The TGF-ß1/ BMP-7 ratios reflected the progression of EMT from CHC to LC, however, there was no significant difference between LC and HCC. TGF-ß1/ BMP-7 ratio is considered to reflect positive correlation with CTGF in LC group (r = 0.629; p < 0.03) and YKL-40 in HCC group (r = 0.504; p < 0.04). CONCLUSION: Increased TGF-ß1/BMP-7 ratio and CTGF levels reflect the rate of EMT and provide information about fibrogenic activity. Also, this ratio, in association with YKL-40, can be used to predict malignant transformation in HCV-genotype 4 Egyptian patients.

Value of reelin for assessing hepatic fibrogenesis in a group of Egyptian HCV infected patients.

BACKGROUND: Development of non-invasive markers that can predict the stages of hepatic fibrosis without resorting to repeated liver biopsies is still an important goal to evaluate the effectiveness of antifibrotic treatment. The present work investigates the value of the assessment of peripheral circulating reelin, in which the liver represents its prime source, as a marker for monitoring hepatic fibrogenesis. METHODS: Seventy-four cases with chronic hepatitis positive for serum HCV RNA and 15 healthy volunteers were enrolled in this study. Assessment of reelin in the harvested serum and in 64 corresponding liver biopsies using immunofluorescence technique was done. The results were evaluated in relation to the stages and quantitative morphometric analysis of hepatic fibrosis as well as the serum levels of the validated biomarker hyaluronic acid. RESULTS: Significant correlation was detected between the levels of serum reelin and the semiquantitative assessment of reelin immunoreactivity in liver tissue, the stages of hepatic fibrosis, the morphometrically determined collagen and serum hyaluronic acid with a correlation coefficient of 0.675, 0.623, 0.479, 0.772, respectively with p<0.001. The sensitivity and the specificity of reelin for the determination of advanced (F2+F3) and significant fibrosis (F2-F4) were nearly comparable to the result of hyaluronic acid. In addition the area under curve (AUC) were 0.859, 0.871 for the reelin versus 0.878, and 0.891 for the hyaluronic acid. CONCLUSIONS: In conclusion serum reelin may be considered an additional useful parameter for monitoring the progression of hepatic fibrosis in HCV-infected patients specially in those with active rheumatological conditions which result in an increase in serum hyaluronic acid.