Expression of matrilin-2 in liver cirrhosis and hepatocellular carcinoma.

The recently described matrilin protein family is part of the extracellular matrix, their pathophysiological role as well as distribution in liver diseases, however, have not yet been studied. Considering that matrilins have been found to play role in cell growth and tissue remodeling, their possible involvement in carcinogenesis has been raised. The main objective of this study was to investigate the changes in matrilin-2 expression which is one of the main components of basement membranes. Thirty-five cases of surgically resected hepatocellular carcinomas, 35 corresponding surrounding liver tissues and 10 normal liver samples were used for the study. In 15 of 35 cases the tumor developed on the basis of cirrhosis. Matrilin-2 protein expression was detected in normal liver around bile ducts, portal blood vessels, while sinusoids were negative by immunohistochemistry. Cirrhotic surrounding tissue showed intensive matrilin-2 staining along the sinusoids. Tumorous neovasculature was found strongly positive by immunohistochemistry. No differences, however, were detected by morphometry regarding the amount of protein expression based on the grade of hepatocellular carcinomas. Real-time RT-PCR did not show significant differences in matrilin-2 mRNA expression between normal, cirrhotic and tumor samples. This suggests posttranslational modification of matrilin-2 manifesting in altered distribution in liver fibrosis. Our data indicate that matrilin-2 is a novel basement membrane component in the liver, which is synthetised during sinusoidal "capillarization" in cirrhosis and in hepatocellular carcinoma. This is the first report to describe the expression and distribution of matrilin-2 in human normal and cirrhotic liver as well as in hepatocellular carcinoma.

Expression and localisation of claudin-1, -2, -3, -4, -5, -7 and -10 proteins in the normal canine mammary gland.

The recently identified claudins are dominant components of tight junctions, responsible for cell adhesion, polarity and paracellular permeability. Certain claudins have been shown to have relevance in tumour development. The aim of the present study was to analyse the expression of claudin-1, -2, -3, -4, -5, -7 and -10 in normal canine mammary glands. Samples from the inguinal mammary regions of 20 non-castrated, 1-13 years old female dogs were studied. Immunohistochemical analysis was performed on conventional specimens and tissue microarrays. The results of the immunohistochemical reactions detecting claudins in tissue sections were photodocumented. The immunoreactivity of claudins was quantitatively analysed on digital images using Leica QWin morphometry software. Intense membranous immunolabelling was found for claudin-1, -3 and -7, intense membranous with non-granular cytoplasmic immunolabelling for claudin-2, moderate membranous immunolabelling for claudin-4 and -5, and weak membranous immunolabelling for claudin-10. The occurrence of tight junctions was confirmed by ultrathin section electron microscopy. The available data suggested that claudins might be proteins preserved throughout the evolution of mammals. The results of our study support the concept that they are indeed preserved, since the same type of claudins, in identical distribution, could be detected in our canine mammary tissue samples as could be found in human mammary tissue.

Expression of matrilin-2 in oval cells during rat liver regeneration.

The matrilins represent a new family of oligomeric proteins that are assumed to act as adapter molecules connecting other proteins and proteoglycans in the extracellular matrix. Matrilin-2, the largest member of the family, displays a broad tissue distribution. It incorporates into loose and dense connective tissue and becomes associated with some basement membranes. The aim of our study was to analyse the expression of matrilin-2 in two liver regeneration models and to identify its cellular origin. Liver regeneration was induced in rats by partial hepatectomy (PH) and by the 2-acetylaminofluorene (AAF)/partial hepatectomy (PH) experimental models. Formalin fixed, paraffin embedded tissue sections were used for immunohistochemistry applying a rabbit matrilin-2 polyclonal antibody. Matrilin-2 was detected in normal rat liver and partially hepatectomized liver in the portal area, but could not be demonstrated in the acini. Matrilin-2 mRNA expression was analysed by RT-PCR and in situ hybridization. In the AAF/PH model the oval cells but not the hepatocytes produced matrilin-2 mRNA. Increase in protein level in the AAF/PH regenerating liver model was demonstrated by Western blotting. The protein was present in the basement membrane zone around the tubules formed by oval cells. Our data show that hepatic oval cells produce matrilin-2, a novel ECM protein, suggesting that matrilin-2 is an important component of ECM during stem cell-driven liver regeneration.

Comparison of the expression of agrin, a basement membrane heparan sulfate proteoglycan, in cholangiocarcinoma and hepatocellular carcinoma.

Heparan sulfate proteoglycans mediate cell adhesion and control the activities of numerous growth and motility factors. They play a critical role in carcinogenesis and tumor progression. Agrin is a large multidomain heparan sulfate proteoglycan associated with basement membranes in several tissues. The expression of agrin in the liver has recently been described under physiologic and pathologic conditions. However, little is known about its role in malignancies. We aimed to study the mRNA and protein expression of agrin in cholangiocarcinoma (CC) and focused on the differences between CC and hepatocellular carcinoma (HCC). Eighty surgically removed liver specimens were studied by immunohistochemistry. Representative samples were used for immunoblotting. mRNA expression was measured in 32 samples by real-time polymerase chain reaction. By immunohistochemistry, agrin was seen around bile ducts and blood vessels within the portal areas in the normal liver. Although no expression was found within the hepatic lobules, agrin was deposited in the neovascular basement membrane in HCCs. Agrin was abundant in the tumor-specific basement membrane in well-differentiated areas of CCs, whereas with immunostaining, it was fragmented, decreased, or it even disappeared in less differentiated areas and sites of infiltration. By real-time polymerase chain reaction, up-regulation of agrin expression was measured in HCCs compared with that in the normal liver. CC samples showed an even higher expression of agrin. Immunoblotting confirmed these findings. Our results indicate that agrin might play an important role in neoangiogenesis in human HCC, being a part of the newly formed vasculature. In CC, however, agrin might be involved in tumor progression.

Short-term alanyl-glutamine dipeptide pretreatment in liver ischemia-reperfusion model: effects on microcirculation and antioxidant status in rats.

BACKGROUND & AIMS: Ischemia-reperfusion (I-R) injury is responsible for the morbidity associated with liver surgery. Production of toxic free radicals influences the microcirculation. The aim of our study was to examine the effect of glutamine (Gln) supplementation--adminstered in alanyl-glutamine dipeptide form--on liver function, immuno/histopathology and the oxidative state of the liver after injury. METHODS: Two-hundred and fifty grams male Wistar rats underwent normothermic, 60 min, segmental liver ischemia followed by 6 h of reperfusion. The animals (n = 45) were divided into three groups: sham operated, I-R and parenteral Gln pretreatment. Hepatic microcirculation was monitored by laser Doppler flowmetry. At the 6 h of reperfusion, histological alterations, TUNEL reaction, active caspase-3 reaction, serum and liver tissue antioxidant levels, serum ALAT, ASAT and TNF-alpha levels were measured. RESULTS: Upon reperfusion, the Gln group had significantly (p<0.05) higher flow rates than the I-R group and, at the end of the 6h of reperfusion, significantly (p<0.05) lower serum ALAT and ASAT levels. The liver chemiluminescent intensity was lower, free SH-groups were elevated, while the reducing power was decreased in the Gln-pretreated group. Positive staining for caspase-3 after Gln pretreatment was significantly increased in contrast to the control tissues. CONCLUSION: Glutamine pretreatment is beneficial in supporting hepatic microcirculation and can prevent hepatocellular necrosis in liver reperfusion injury.

Agrin and CD34 immunohistochemistry for the discrimination of benign versus malignant hepatocellular lesions.

Agrin is a recently identified proteoglycan component of vascular and bile duct basement membranes in the liver. The selective deposition of agrin in hepatocellular carcinoma (HCC) microvessels versus sinusoidal walls prompted us to investigate the utility of agrin immunohistochemistry (IHC) in detecting malignant hepatocellular lesions. We focused on the differential diagnostic problems often presented by hepatocellular adenomas (HCAs) and dysplastic nodules. IHC for agrin was performed on 138 formalin-fixed, paraffin-embedded surgical specimens from 93 patients, including cirrhotic liver tissues (25), focal nodular hyperplasia (10), large regenerative nodules (8), low-grade (23) and high-grade (7) dysplastic nodules, small HCC (8), HCC (27), and HCA (30). Agrin immunostaining was compared with that of CD34 and, in selected cases, to glypican-3. The combination of agrin and CD34 sensitively (0.94) and specifically (0.93) identified lesions judged previously as malignant by histology. The majority of benign lesions were clearly agrin-negative, whereas the strength and extent of agrin IHC faithfully reflected dysplasia in "atypical" HCAs and in high-grade dysplastic nodules. Malignant lesions were uniformly positive. In conclusion, as agrin is highly selective for tumor blood vessels, IHC for agrin facilitates the discrimination of benign and malignant hepatocellular lesions. Moreover, whereas glypican-3 in some HCCs may appear in few scattered cells only, agrin is diffusely deposited in virtually all malignant lesions, which may prove advantageous in the evaluation of small specimens such as core biopsies.

Effect of PJ-34 PARP-inhibitor on rat liver microcirculation and antioxidant status.

BACKGROUND: Ischemia-reperfusion (I-R) injury during liver resection leads to the production of toxic free radicals and oxidants that influence the microcirculation. DNA single-strand breaks can be induced by these reactive species. In response to excessive DNA damage, PARP [poly(ADP-ribose) polymerase] becomes overactivated, which can lead to cellular ATP depletion and cell death. The aim of our study was to evaluate whether PARP is expressed in post-ischemic liver, and to examine the effect of the administration of PJ-34 PARP inhibitor on liver function, histopathology, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) reaction, and the oxidative state of the liver after injury. METHODS: Male Wistar rats (weighing 250 g) underwent 60 min of normothermic, segmental liver ischemia followed by 30 min of reperfusion. The animals (n = 45) were divided into three groups: sham operated; I-R (control) treated with saline; and PJ-34 pre-treated (10 mg/kg i.v.). Hepatic microcirculation was monitored by a laser Doppler flowmeter. The reperfusion was characterized as the integral of the reperfusion area (RA) and the maximal plateau (PM). Histological alterations, TUNEL-reaction, serum, and liver tissue antioxidant levels, as well as serum ALT and AST levels were measured. RESULTS: Upon reperfusion, the PJ-34 group had significantly (P < 0.05) higher flow rates than control groups (PM(PJ-34): 58%, PM(control): 37%; RA(PJ-34.): 48%, RA(control): 25%). At the end of the 30 min reperfusion, PJ-34 resulted in significantly (P < 0.05) lower serum ALT and AST levels and chemiluminescent intensity (free radicals) of the liver. The liver's free SH-group concentration and H-donor ability of the plasma was elevated in the PARP-inhibitor treated group. Positive staining for TUNEL, after PJ-34 pre-treatment was significantly increased (P < 0.05); in contrast, the control tissues were less positively stained for TUNEL but necrotic tissue was abundant. CONCLUSION: PARP plays a pathogenetic role in the deterioration of the hepatic microcirculation and promotes hepatocellular necrosis in liver reperfusion injury.

Agrin, a novel basement membrane component in human and rat liver, accumulates in cirrhosis and hepatocellular carcinoma.

Agrin is a multifunctional heparan sulfate proteoglycan originally discovered in the neuromuscular junctions and later observed in numerous other localizations. The presence of agrin in the liver, either healthy or diseased, has formerly not been reported. We detected agrin in minor amounts in the basement membranes of blood vessels and bile ducts in the healthy liver. The proliferation of bile ductules and the formation of new septal blood vessels in liver cirrhosis, as well as neoangiogenesis in the hepatocellular carcinoma (HCC) result in a dramatic increase in the quantity of agrin. Vascular and peribiliary basement membranes were strongly immunopositive for agrin in 29/29 human liver specimens with cirrhosis and HCC. However, sinusoidal walls of regenerative nodules in the cirrhotic liver consistently remained negative. Given the selectivity of agrin for tumor microvessels, agrin immunohistochemistry may prove helpful in recognizing malignant transformation in cirrhotic livers. Similar immunohistochemical observations were made on the liver of rats exposed to a combined cirrhosis/HCC induction treatment. In both human and rats, agrin probably originates from activated myofibroblasts, vascular smooth muscle cells and biliary epithelial cells. Increased agrin expression in human specimens, in the liver of 4/4 treated rats, as well as in isolated rat liver mesenchymal cells was verified by quantitative RT-PCR. Considering that agrin binds various growth factors, and it directly interacts with cell membrane receptors such as alphav-integrins, we hypothesize a stimulatory role for agrin in neoangiogenic processes such as tumor vascularization, and a supportive role in bile ductule proliferation.

Claudin-4 differentiates biliary tract cancers from hepatocellular carcinomas.

The recently identified claudins are dominant components of tight junctions, responsible for cell adhesion, polarity and paracellular permeability. Certain claudins have been shown to have relevance in tumor development, with some of them, especially claudin-4, even suggested as future therapeutic target. The aim of the present study was to analyze the expression of claudin-4 in the biliary tree, biliary tract cancers and hepatocellular carcinomas. A total of 107 cases were studied: 53 biliary tract cancers, 50 hepatocellular carcinomas, 10 normal liver and 10 normal extrahepatic biliary duct samples. Immunohistochemical analysis was performed on conventional specimens and on tissue microarrays as well. Claudin-4 was further investigated by Western blot analysis and real-time RT-PCR. Intense membranous immunolabeling was found for claudin-4 in all biliary tract cancers unrelated to the primary site of origin, namely intrahepatic, extrahepatic or gallbladder cancers. Normal biliary epithelium showed weak positivity for claudin-4. In contrast, normal hepatocytes and tumor cells of hepatocellular carcinomas did not express claudin-4. The results of Western immunoblot analysis and real-time RT-PCR were in correlation with the immunohistochemical findings. Cytokeratins, as CK7 (92%) and CK19 (83%) were mostly positive in biliary tract cancers, however, one-third of hepatocellular carcinomas also expressed CK7 (34%). HSA antibody (HepPar1) reacted with the majority of hepatocellular carcinomas (86%), while being positive in a low percentage of the biliary tract cancers (8%). In conclusion, this is the first report of a significantly increased claudin-4 expression in biliary tract cancers, which represents a novel feature of tumors of biliary tract origin. Claudin-4 expression seems to be a useful marker in differentiating biliary tract cancers from hepatocellular carcinomas and could well become a potential diagnostic tool.