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Evolutionary medicine emerged in the late twentieth century, integrating principles of natural selection and adaptation with the health sciences. Today, with a rapidly widening gap between the biology of Homo sapiens and its environment, maladaptation or maladaptive disorders can be detected in almost all diseases, including liver dysfunction. However, in hepatology, as in most medical specialties, evolutionary considerations are neglected because the majority of the medical community is not familiar with evolutionary principles. The aim of this brief review is to highlight an evolutionary approach that may facilitate understanding various liver diseases., Competing Interests: The authors declare no conflicts of interest., (© 2024 The Author(s). Evolutionary Applications published by John Wiley & Sons Ltd.)

Lysophosphatidic acid (LPA) is a bioactive phospholipid mediator that has been found to ameliorate nonsteroidal anti-inflammatory drug (NSAID)-induced gastric injury by acting on lysophosphatidic acid type 2 receptor (LPAR2). In this study, we investigated whether LPAR2 signaling was implicated in the development of NSAID-induced small intestinal injury (enteropathy), another major complication of NSAID use. Wild-type (WT) and Lpar2 deficient (Lpar2 -/- ) mice were treated with a single, large dose (20 or 30 mg/kg, i.g.) of indomethacin (IND). The mice were euthanized at 6 or 24 h after IND treatment. We showed that IND-induced mucosal enteropathy and neutrophil recruitment occurred much earlier (at 6 h after IND treatment) in Lpar2 -/- mice compared to WT mice, but the tissue levels of inflammatory mediators (IL-1β, TNF-α, inducible COX-2, CAMP) remained at much lower levels. Administration of a selective LPAR2 agonist DBIBB (1, 10 mg/kg, i.g., twice at 24 h and 30 min before IND treatment) dose-dependently reduced mucosal injury and neutrophil activation in enteropathy, but it also enhanced IND-induced elevation of several proinflammatory chemokines and cytokines. By assessing caspase-3 activation, we found significantly increased intestinal apoptosis in IND-treated Lpar2 -/- mice, but it was attenuated after DBIBB administration, especially in non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. Finally, we showed that IND treatment reduced the plasma activity and expression of autotaxin (ATX), the main LPA-producing enzyme, and also reduced the intestinal expression of Lpar2 mRNA, which preceded the development of mucosal damage. We conclude that LPAR2 has a dual role in NSAID enteropathy, as it contributes to the maintenance of mucosal integrity after NSAID exposure, but also orchestrates the inflammatory responses associated with ulceration. Our study suggests that IND-induced inhibition of the ATX-LPAR2 axis is an early event in the pathogenesis of enteropathy., (© 2023. The Author(s).)

Background: Small-cell lung cancer (SCLC) molecular subtypes have been primarily characterized based on the expression pattern of the following key transcription regulators: ASCL1 (SCLC-A), NEUROD1 (SCLC-N), POU2F3 (SCLC-P) and YAP1 (SCLC-Y). Here, we investigated the proteomic landscape of these molecular subsets with the aim to identify novel subtype-specific proteins of diagnostic and therapeutic relevance., Methods: Pellets and cell media of 26 human SCLC cell lines were subjected to label-free shotgun proteomics for large-scale protein identification and quantitation, followed by in-depth bioinformatic analyses. Proteomic data were correlated with the cell lines' phenotypic characteristics and with public transcriptomic data of SCLC cell lines and tissues., Results: Our quantitative proteomic data highlighted that four molecular subtypes are clearly distinguishable at the protein level. The cell lines exhibited diverse neuroendocrine and epithelial-mesenchymal characteristics that varied by subtype. A total of 367 proteins were identified in the cell pellet and 34 in the culture media that showed significant up- or downregulation in one subtype, including known druggable proteins and potential blood-based markers. Pathway enrichment analysis and parallel investigation of transcriptomics from SCLC cell lines outlined unique signatures for each subtype, such as upregulated oxidative phosphorylation in SCLC-A, DNA replication in SCLC-N, neurotrophin signalling in SCLC-P and epithelial-mesenchymal transition in SCLC-Y. Importantly, we identified the YAP1-driven subtype as the most distinct SCLC subgroup. Using sparse partial least squares discriminant analysis, we identified proteins that clearly distinguish four SCLC subtypes based on their expression pattern, including potential diagnostic markers for SCLC-Y (e.g. GPX8, PKD2 and UFO)., Conclusions: We report for the first time, the protein expression differences among SCLC subtypes. By shedding light on potential subtype-specific therapeutic vulnerabilities and diagnostic biomarkers, our results may contribute to a better understanding of SCLC biology and the development of novel therapies., (© 2022 The Authors. Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.)

Several studies have shown that liver fibrosis, and even cirrhosis can be reversed, disproving the old "dogma" that cirrhosis is irreversible. In addition to scaring, vascular alterations appear to be critically important in the progression of chronic liver diseases. To overcome the "tipping-point" of cirrhosis, we need to understand in depth what might make it irreversible in some cases. Morphologically, the initial, as well as the advanced stages of cirrhosis are characterized by specific structural changes. The hallmark of the initial stage is the division of the original liver parenchyma by centro-central or porto-portal septa. No significant vascular changes are observed in this stage. The advanced stage is characterized by several morphological alterations: (i) The main feature is the parenchymal extinction, with intact portal vein branches, hepatic artery branches, and biliary ductules; (ii) In the extinct areas we observed numerous loops in the ductular network, indicating the disruption of the hepato-biliary junctions; (iii) Although the ductular progenitor cells are able to generate hepatocytes via the budding process, the newly formed hepatocyte nodules cannot re-establish the original lobular architecture due to their disorganized growth. In conclusion, this regenerative process characteristic for the advanced stage, contributes to circulatory disorders, perpetuates parenchymal injury and may lead to the irreversibility of cirrhosis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Dezső, Paku, Kóbori, Thorgeirsson and Nagy.)

There is an increasing demand for transdermal transport measurements to optimize topical drug formulations and to achieve proper penetration profile of cosmetic ingredients. Reflecting ethical concerns the use of both human and animal tissues is becoming more restricted. Therefore, the focus of dermal research is shifting towards in vitro assays. In the current proof-of-concept study a three-layer skin equivalent using human HaCaT keratinocytes, an electrospun polycaprolactone mesh and a collagen-I gel was compared to human excised skin samples. We measured the permeability of the samples for 2% caffeine cream using a miniaturized dynamic diffusion cell ("skin-on-a-chip" microfluidic device). Caffeine delivery exhibits similar transport kinetics through the artificial skin and the human tissue: after a rapid rise, a long-lasting high concentration steady state develops. This is markedly distinct from the kinetics measured when using cell-free constructs, where a shorter release was observable. These results imply that both the established skin equivalent and the microfluidic diffusion chamber can serve as a suitable base for further development of more complex tissue substitutes.

Apelin, a ligand of the APJ receptor, is overexpressed in several human cancers and plays an important role in tumor angiogenesis and growth in various experimental systems. We investigated the role of apelin signaling in the malignant behavior of cutaneous melanoma. Murine B16 and human A375 melanoma cell lines were stably transfected with apelin encoding or control vectors. Apelin overexpression significantly increased melanoma cell migration and invasion in vitro, but it had no impact on its proliferation. In our in vivo experiments, apelin significantly increased the number and size of lung metastases of murine melanoma cells. Melanoma cell proliferation rates and lymph and blood microvessel densities were significantly higher in the apelin-overexpressing pulmonary metastases. APJ inhibition by the competitive APJ antagonist MM54 significantly attenuated the in vivo pro-tumorigenic effects of apelin. Additionally, we detected significantly elevated circulating apelin and VEGF levels in patients with melanoma compared to healthy controls. Our results show that apelin promotes blood and lymphatic vascularization and the growth of pulmonary metastases of skin melanoma. Further studies are warranted to validate apelin signaling as a new potential therapeutic target in this malignancy.

Small cell lung cancer (SCLC; comprising approximately 14% of all lung cancer cases in Hungary) is an aggressive tumor type characterized by rapid growth and early metastasis. Although SCLC is a particularly malignant form of cancer, targeted therapies in its treatment have remained largely unsuccessful and thus there were no major therapeutic advances in the last three decades. SCLC was once considered a molecularly homogeneous malignancy. However, recent analyses led to the classification of neuroendocrine and molecular subtypes, based on the dominant expression of one of the following four transcriptional regulator genes: ASCL1, NEUROD1, YAP1 and POU2F3. Because these genetically and biologically distinct subtypes might contribute to therapeutic resistance, the better understanding of their biological and clinicopathological characteristics may help in the development of more effective SCLC therapies.

Syndecan-1 is a transmembrane heparan sulfate proteoglycan which is indispensable in the structural and functional integrity of epithelia. Normal hepatocytes display strong cell surface expression of syndecan-1; however, upon malignant transformation, they may lose it from their cell surfaces. In this study, we demonstrate that re-expression of full-length or ectodomain-deleted syndecan-1 in hepatocellular carcinoma cells downregulates phosphorylation of ERK1/2 and p38, with the truncated form exerting an even stronger effect than the full-length protein. Furthermore, overexpression of syndecan-1 in hepatoma cells is associated with a shift of heparan sulfate structure toward a highly sulfated type specific for normal liver. As a result, cell proliferation and proteolytic shedding of syndecan-1 from the cell surface are restrained, which facilitates redifferentiation of hepatoma cells to a more hepatocyte-like phenotype. Our results highlight the importance of syndecan-1 in the formation and maintenance of differentiated epithelial characteristics in hepatocytes partly via the HGF/ERK/Ets-1 signal transduction pathway. Downregulation of Ets-1 expression alone, however, was not sufficient to replicate the phenotype of syndecan-1 overexpressing cells, indicating the need for additional molecular mechanisms. Accordingly, a reporter gene assay revealed the inhibition of Ets-1 as well as AP-1 transcription factor-induced promoter activation, presumably an effect of the heparan sulfate switch.

Introduction: The liver is well known for its enormous regenerative capacity. If the hepatocytes are compromised the reserve stem cells can regrow the lost tissue by means of oval cells differentiating into hepatocytes. We were curious whether this standby system was able to compensate for ontogenic liver growth arrested by 2-acetylaminofluorene (AAF) treatment or if it can be influenced by cholic acid, known to promote liver growth in several reactions., Methods: (i) Four weeks-old (60-70g) male F344 rats were kept on standard chow and treated with solvent only, (ii) others were kept on 0,2% cholic acid (CA) enriched diet, (iii) treated with AAF, or (iiii) given a combination of CA diet and AAF treatment (AAF/CA). The proliferative response of epithelial cells was characterized by pulse bromodeoxyuridine labelling. The relative gene expression levels of senescence-related factors and bile acid receptors were determined by quantitative real-time polymerase chain reaction analysis., Results: AAF administration efficiently inhibited the physiological proliferation of hepatocytes in young, male F344 rats after weaning. The activation of stem cells was indicated by the increased proliferation of periportal biliary/oval cells (B/OC). If the rats were fed additionally by cholic acid enriched diet, typical oval cell reaction emerged, subsequently the oval cells differentiated into hepatocytes restituting liver growth. This reaction was mediated by increased production of HGF, IL-6 and SCF by the damaged liver. Moreover, upregulation of FXR expression on B/OC made them competent for bile acids. Our results indicate that endogenous, autocrine mechanisms involved in liver ontogeny are also able to activate the backup regenerative machinery of stem cells., Competing Interests: The authors have declared that no competing interests exist.

Background and Aim: Massive hepatic necrosis is a rare but often fatal complication of various liver injuries. Nevertheless, some patients can survive by spontaneous hepatic regeneration. It is known that surviving hepatocytes and/or progenitor cells can participate in this process but the mechanism of hepatic recovery is vague., Methods: We examined 13 explanted human livers removed for acute liver failure. Combined immunohistochemistry, digital image analysis, and three-dimensional reconstruction of serial sections were applied., Results: Two patterns of regeneration could be distinguished. In livers with centrilobular necrosis, the surviving injured periportal hepatocytes started to proliferate and arrange into acinar structures and expressed α-fetoprotein. If the injury wiped out almost all hepatocytes, large areas of parenchymal loss were invaded by an intense ductular reaction. The cells at the distal pole of the ductules differentiated into hepatocytes and formed foci organized by the branches of the portal vein. The expanding foci often containing complete portal triads were arranged around surviving central veins. Their fusion eventually could be an attempt to re-establish the hepatic lobules., Conclusions: Regeneration of human livers following massive hepatic necrosis can occur in two ways-either through proliferation of α-fetoprotein-positive acinary-arranged hepatocytes or through ductular progenitor cells, with the latter being less efficient. Further investigation of these regenerative pathways may help identify biomarkers for likelihood of complete regeneration and hence have therapeutic implications., (© 2019 The Authors. Journal of Gastroenterology and Hepatology published by Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.)

The impact of growth pattern on the distribution of connective tissue and on the vascularization of brain metastases (40 colon, lung and breast carcinoma samples) was analyzed. Most of the cases showed either a "pushing-type" (18/40, mostly colon and lung carcinomas) or a "papillary-type" (19/40, mostly breast carcinomas) growth pattern. There was a striking difference in the growth pattern and vascularization of colon/lung versus breast carcinoma metastases. Pushing-type brain metastases incorporated fewer vessels and accumulated more collagen in the adjacent brain parenchyma, whereas papillary-type brain metastases incorporated more vessels and accumulated collagen in the center of the tumor. We observed duplication of the PDGFRβ-positive pericyte layer accompanied by an increase in the amount of collagen within the vessel walls. The outer layer of pericytes and the collagen was removed from the vessel by invasive activity of the tumors, which occurred either peri- or intratumorally, depending on the growth pattern of the metastasis. Our findings suggest that pericytes are the main source of the connective tissue in brain metastases. Vascularization and connective tissue accumulation of the brain metastases largely depend on the growth pattern of the tumors., (© 2019 American Association of Neuropathologists, Inc. All rights reserved.)

Background: It remains unclear if the vascular and connective tissue structures of primary and metastatic tumors are intrinsically determined or whether these characteristics are defined by the host tissue. Therefore we examined the microanatomical steps of vasculature and connective tissue development of C38 colon carcinoma in different tissues., Methods: Tumors produced in mice at five different locations (the cecal wall, skin, liver, lung, and brain) were analyzed using fluorescent immunohistochemistry, electron microscopy and quantitative real-time polymerase chain reaction., Results: We found that in the cecal wall, skin, liver, and lung, resident fibroblasts differentiate into collagenous matrix-producing myofibroblasts at the tumor periphery. These activated fibroblasts together with the produced matrix were incorporated by the tumor. The connective tissue development culminated in the appearance of intratumoral tissue columns (centrally located single microvessels embedded in connective tissue and smooth muscle actin-expressing myofibroblasts surrounded by basement membrane). Conversely, in the brain (which lacks fibroblasts), C38 metastases only induced the development of vascularized desmoplastic tissue columns when the growing tumor reached the fibroblast-containing meninges., Conclusions: Our data suggest that the desmoplastic host tissue response is induced by tumor-derived fibrogenic molecules acting on host tissue fibroblasts. We concluded that not only the host tissue characteristics but also the tumor-derived fibrogenic signals determine the vascular and connective tissue structure of tumors.

Tumor hypoxia promotes neoangiogenesis and contributes to the radio- and chemotherapy resistant and aggressive phenotype of cancer cells. However, the migratory response of tumor cells and the role of small GTPases regulating the organization of cytoskeleton under hypoxic conditions have yet to be established. Accordingly, we measured the proliferation, migration, RhoA activation, the mRNA and protein levels of hypoxia inducible factor-1alpha (HIF-1α) and three small G-proteins, Rac1, cdc42 and RhoA in a panel of five human tumor cell lines under normoxic and hypoxic conditions. Importantly, HT168-M1 human melanoma cells with high baseline migration capacity showed increased HIF-1α and small GTPases expression, RhoA activation and migration under hypoxia. These activities were blocked by anti- HIF-1α shRNA. Moreover, the in vivo metastatic potential was promoted by hypoxia mimicking CoCl2 treatment and reduced upon inhibition of HIF-1α in a spleen to liver colonization experiment. In contrast, HT29 human colon cancer cells with low migration capacity showed limited response to in vitro hypoxia. The expression of the small G-proteins decreased both at mRNA and protein levels and the RhoA activation was reduced. Nevertheless, the number of lung or liver metastatic colonies disseminating from orthotopic HT29 grafts did not change upon CoCl2 or chetomin treatment. Our data demonstrates that the hypoxic environment induces cell-type dependent changes in the levels and activation of small GTPases and results in varying migratory and metastasis promoting responses in different human tumor cell lines.

Background: Multiple studies concluded that oncometabolites (e.g. D-2-hydroxyglutarate (2-HG) related to mutant isocitrate dehydrogenase 1/2 (IDH1/2) and lactate) have tumour promoting potential. Regulatory mechanisms implicated in the maintenance of oncometabolite production have great interest. mTOR (mammalian target of rapamycin) orchestrates different pathways, influences cellular growth and metabolism. Considering hyperactivation of mTOR in several malignancies, the question has been addressed whether mTOR operates through controlling of oncometabolite accumulation in metabolic reprogramming., Methods: HT-1080 cells - carrying originally endogenous IDH1 mutation - were used in vitro and in vivo. Anti-tumour effects of rapamycin were studied using different assays. The main sources and productions of the oncometabolites (2-HG and lactate) were analysed by 13 C-labeled substrates. Alterations at protein and metabolite levels were followed by Western blot, flow cytometry, immunohistochemistry and liquid chromatography mass spectrometry using rapamycin, PP242 and different glutaminase inhibitors, as well., Results: Rapamycin (mTORC1 inhibitor) inhibited proliferation, migration and altered the metabolic activity of IDH1 mutant HT-1080 cells. Rapamycin reduced the level of 2-HG sourced mainly from glutamine and glucose derived lactate which correlated to the decreased incorporation of 13 C atoms from 13 C-substrates. Additionally, decreased expressions of lactate dehydrogenase A and glutaminase were also observed both in vitro and in vivo., Conclusions: Considering the role of lactate and 2-HG in regulatory network and in metabolic symbiosis it could be assumed that mTOR inhibitors have additional effects besides their anti-proliferative effects in tumours with glycolytic phenotype, especially in case of IDH1 mutation (e.g. acute myeloid leukemias, gliomas, chondrosarcomas). Based on our new results, we suggest targeting mTOR activity depending on the metabolic and besides molecular genetic phenotype of tumours to increase the success of therapies.

Background and Aims: Ductular reaction is a standard component of fibrotic liver tissue but its function is largely unknown. It is supposed to interact with the matrix producing myofibroblasts and compensate the declining regenerative capacity of hepatocytes. The relationship between the extent of fibrosis-ductular reaction, proliferative activity of hepatocytes and ductular reaction were studied sequentially in experimental hepatic fibrosis models., Methods: Liver fibrosis/cirrhosis was induced in wild type and TGFβ overproducing transgenic mice by carbon tetrachloride and thioacetamide administration. The effect of thioacetamide was modulated by treatment with imatinib and erlotinib. The extent of ductular reaction and fibrosis was measured by morphometry following cytokeratin 19 immunofluorescent labeling and Picro Sirius staining respectively. The proliferative activity of hepatocytes and ductular reaction was evaluated by BrdU incorporation. The temporal distribution of the parameters was followed and compared within and between different experimental groups., Results: There was a strong significant correlation between the extent of fibrosis and ductular reaction in each experimental group. Although imatinib and erlotinib temporarily decreased fibrosis this effect later disappeared. We could not observe negative correlation between the proliferation of hepatocytes and ductular reaction in any of the investigated models., Conclusions: The stringent connection between ductular reaction and fibrosis, which cannot be influenced by any of our treatment regimens, suggests that there is a close mutual interaction between them instead of a unidirectional causal relationship. Our results confirm a close connection between DR and fibrogenesis. However, since the two parameters changed together we could not establish a causal relationship and were unable to reveal which was the primary event. The lack of inverse correlation between the proliferation of hepatocytes and ductular reaction questions that ductular reaction can compensate for the failing regenerative activity of hepatocytes. No evidences support the persistent antifibrotic property of imatinib or erlotinib.

Background & Aims: In advanced cirrhosis new hepatocytic nodules are generated by budding of ductules in areas of parenchymal extinction. However, the vascular alterations in the areas of parenchymal extinction, the blood supply and the structure of the new hepatocytic nodules have not been analyzed in detail., Methods: Explanted human cirrhotic livers of three different etiologies and two experimental rat models of cirrhosis were thoroughly examined. 3D reconstruction of the immunohistochemically stained serial sections and casting of human and experimental cirrhotic livers have been used to reveal the structural organization of the regenerative buds., Results: In areas of parenchymal extinction the skeleton of the liver, the portal tree is preserved. The developing regenerative nodules are positioned along the portal tree and are directly supplied by terminal portal venules. The expanding nodules grow along the trunks of the portal vein. Casting of human and experimental cirrhotic livers by colored resin confirms that nodules are supplied by portal blood. The two other members of the portal triads become separated from the portal veins., Conclusions: As the structure of the hepatocyte nodules (centrally located portal vein branches, bile ducts at the periphery, hepatic veins and arteries in the connective tissue) impedes the restoration of normal liver structure, the basic architecture of hepatic tissue suffers permanent damage. We suggest that "budding" may initiate the second, irreversible stage of cirrhosis., Lay Summary: Cirrhosis is the final common outcome of long lasting hepatic injury defined as the destruction of the normal liver architecture by scar tissue. In the late phase of cirrhosis stem cells-derived hepatocyte nodules appear along the branches of the portal vein suggesting an important role of this specially composed blood vessels (containing digestive end-products from the stomach and intestines) in liver regeneration. Our results contribute to a better understanding of this serious liver disease., (Copyright © 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

The development of lung metastasis is a significant negative prognostic factor for cancer patients. The extravasation phase of lung metastasis involves interactions of tumour cells with the pulmonary endothelium. These interactions may have broad biological and medical significance, with potential clinical implications ranging from the discovery of lung metastasis biomarkers to the identification of targets for intervention in preventing lung metastases. Because of the potential significance, the mechanisms of tumour cell extravasation require cautious, systematic studies. Here, we discuss the literature pertaining to the proposed mechanisms of extravasation and critically compare a recently proposed mechanism (tumour cell-induced endothelial necroptosis) with the already described extravasation mechanisms in the lung. We also provide novel data that may help to explain the underlying physiological basis for endothelialization as a mechanism of tumour cell extravasation in the lung. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd., (Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Severe chronic hepatic injury can induce complex reparative processes. Ductular reaction and the appearance of small hepatocytes are standard components of this response, which is thought to have both adverse (e.g. fibrosis, carcinogenesis) and beneficial (regeneration) consequences. This complex tissue reaction is regulated by orchestrated cytokine action. We have investigated the influence of the tyrosine kinase inhibitor imatinib on a regenerative process. Ductular reaction was induced in mice by the widely used choline-deficient ethionine-supplemented diet (CDE). Test animals were treated daily with imatinib. After 6 weeks of treatment, imatinib successfully reduced the extent of ductular reaction and fibrosis in the CDE model. Furthermore, the number of small hepatocytes increased, and these cells had high proliferative activity, were positive for hepatocyte nuclear factor 4 and expressed high levels of albumin and peroxisome proliferator-activated receptor alpha. The overall functional zonality of the hepatic parenchyma (cytochrome P450 2E1 and glucose 6 phosphatase activity; endogenous biotin content) was maintained. The expression of platelet-derived growth factor receptor beta, which is the major target of imatinib, was downregulated. The anti-fibrotic activity of imatinib has already been reported in several experimental models. Additionally, in the CDE model imatinib was able to enhance regeneration and preserve the functional arrangement of hepatic lobules. These results suggest that imatinib might promote the recovery of the liver following parenchymal injury through the inhibition of platelet-derived growth factor receptor beta., (© 2016 The Authors. International Journal of Experimental Pathology © 2016 International Journal of Experimental Pathology.)

Background: Reproducible structural analysis was made on cirrhotic human liver samples in order to reveal potential connections between morphological and laboratory parameters., Material and Methods: Large histological samples were taken from segment VII of 56 cirrhotic livers removed in connection with liver transplantation. Picro Sirius red and immunohistochemically (smooth muscle actin [SMA], cytokeratin 7 [CK7], Ki-67) stained sections were digitalized and morphometric evaluation was performed., Results: The Picro Sirius-stained fibrotic area correlated with the average thickness of the three broadest septa, extent of SMA positivity, alkaline phosphatase (ALP) values and it was lower in the viral hepatitis related cirrhoses than in samples with non-viral etiology. The extent of SMA staining increased with the CK7-positive ductular reaction. The proliferative activity of the hepatocytes correlated positively with the Ki-67 labeling of the ductular cells and inversely with the septum thickness. These data support the potential functional connection among different structural components, for example, myofibroblasts, ductular reaction and fibrogenesis but challenges the widely proposed role of ductular cells in regeneration., Conclusion: Unbiased morphological characterization of cirrhotic livers can provide valuable, clinically relevant information. Similar evaluation of routine core biopsies may increase the significance of this 'Gold Standard' examination.

Treatment of patients with lung metastases remains a major challenge. A possible target for therapies is the inhibition of vascularization of metastases. Our study aimed to determine the possible mechanisms of the experimental lung metastasis vascularisation for tumours of various origins. We created lung metastases by intravenous injection of five tumour cell lines (HT1080, HT25, B16, C26 and MATB). Each cell line showed the same vascularisation type. Tumours gained vasculature by advancing through the alveolar spaces thereby incorporating the pre-existing alveolar capillaries (i.e. vessel co-option). From the alveolar spaces tumours entered into the alveolar walls. The tumour cells during the invasion/migration separated the pneumocytes from the capillaries. During this process the basement membrane was split into an epithelial and an endothelial layer. The heavily compressed pneumocytes inside the tumour became fragmented but the incorporated and stripped vessels remained functional, so they were able to provide blood supply for the metastases.

Background: The immunohistochemical detection of Enhancer of zeste homologue 2 (EZH2) proved to be a useful tool to recognize the malignant nature of tumors in a wide variety of neoplasms. The histological diagnostics of salivary gland tumors is a challenging task, and a reliable marker of malignancy would be extremely helpful., Methods: EZH2 expression was investigated in 54 malignant and 40 benign salivary gland tumors of various histological types by standard immunohistochemistry., Results: The majority (n = 52) of the malignant tumors stained positively, while all the investigated benign tumors were negative for EZH2., Conclusions: EZH2 expression in salivary gland tumors, similarly to the tumors of other organs is not characteristic for any tumor type, but is a solid marker of the malignant nature of the tumors.

Background: Ligation of a branch of the portal vein redirects portal blood to nonligated lobes resulting in lobar hypertrophy. Although the effect of portal vein ligation on liver volume is well documented, the parallel alterations in liver function are still the subject of controversy. Our aim was to assess the time-dependent reactions of regional hepatic function to portal vein ligation by selective biliary drainage., Methods: Male Wistar rats (n = 44) underwent 80% portal vein ligation. Before the operation as well as 1, 2, 3, 5, and 7 d after circulation, morphology and function (laboratory blood test; hepatic bile flow; plasma disappearance rate of indocyanine green; and biliary indocyanine green excretion) of the liver were examined., Results: Although portal vein ligation affected liver circulation and morphology to a great extent, serum albumin levels, bilirubin levels, and total hepatic bile flow did not change significantly after the operation. Nevertheless, plasma disappearance rate and biliary indocyanine green excretion indicated a temporary impairment of total liver function with the lowest value on the second day and normalization by the fifth day. Bile production and biliary indocyanine green excretion of ligated lobes decreased rapidly after the operation and remained persistently suppressed, whereas the secretory function of nonligated lobes--after a temporary decline--showed a greater increase than the weight of the lobes., Conclusions: Portal vein ligation induced temporary impairment of total liver function, followed by rapid recovery mainly by reason of increase in the function of nonligated lobes. Functional increase in nonligated lobes was more pronounced than suggested by the degree of volume gain., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Comparison of tissue microarray results of 29 cervical cancer and 27 normal cervix tissue samples using immunohistochemistry revealed considerable reorganization of the fibrillar stroma of these tumors. Preliminary densitometry analysis of laminin-1, α-smooth muscle actin (SMA) and fibronectin immunostaining demonstrated 3.8-fold upregulation of laminin-1 and 5.2-fold increase of SMA in the interstitial stroma, indicating that these proteins and the activated fibroblasts play important role in the pathogenesis of cervical cancer. In the present work we investigated the role of normal and tumor-associated fibroblasts., Methods: In vitro models were used to throw light on the multifactorial process of tumor-stroma interaction, by means of studying the cooperation between tumor cells and fibroblasts. Fibroblasts from normal cervix and cervical cancers were grown either separately or in co-culture with CSCC7 cervical cancer cell line. Changes manifest in secreted glycoproteins, integrins and matrix metallo-proteases (MMPs) were explored., Results: While normal fibroblasts produced components of interstitial matrix and TGF-β1 that promoted cell proliferation, cancer-associated fibroblasts (CAFs) synthesized ample amounts of laminin-1. The following results support the significance of laminin-1 in the invasion of CSCC7 cells: 1.) Tumor-associated fibroblasts produced more laminin-1 and less components of fibrillar ECM than normal cells; 2.) The production of laminin chains was further increased when CSCC7 cells were grown in co-culture with fibroblasts; 3.) CSCC7 cells were capable of increasing their laminin production; 4.) Tumor cells predominantly expressed integrin α6β4 laminin receptors and migrated towards laminin. The integrin profile of both normal and tumor-associated fibroblasts was similar, expressing receptors for fibronectin, vitronectin and osteopontin. MMP-7 secreted by CSCC7 cells was upregulated by the presence of normal fibroblasts, whereas MMP-2 produced mainly by fibroblasts was activated in the presence of CSCC7 cells., Conclusions: Our results indicate that in addition to degradation of the basement membrane, invasion of cervical cancer is accomplished by the remodeling of the interstitial stroma, which process includes decrease and partial replacement of fibronectin and collagens by a laminin-rich matrix.

A 2D model was previously presented that describes the gliding motility of human fibrosarcoma cells. The model was based on the observation that adhesions are present only on the outer rim of the leading lamella of the semicircular cell. The present model describes the organization of adhesions and the cytoskeleton of migrating HT1080 fibrosarcoma and LX2 hepatic stellate cells in three dimensions. The migration assays were performed in a modified Boyden chamber using fibronectin, Matrigel, or collagen I as chemoattractants. The distribution of the adhesions was analyzed by confocal laser scanning microscope, and following decoration with heavy meromyosin, the organization of actin filaments was analyzed by electron microscopy. Double labeling was performed to study the relationship of the actin and vimentin filament network in the moving cells. Vinculin containing adhesions were observed only at the front of the cell in the form of a ring while passing through a filter pore of the Boyden chamber. Actin filaments were present only below the plasma membrane, except the very tip of the leading lamella. Vimentin intermediate filaments were localized around the cell nucleus behind the actin filament-rich lamella. This paper describes a model of the organization of adhesions and the cytoskeleton of migrating cells in the Boyden chamber. The model is based on the observation that adhesions are present only at the leading edge of the cell. The results extend the earlier 2D model of cell locomotion into 3D.

The hepatic stem cells reside periportally forming the canals of Hering in normal liver. They can be identified by their unique immunophenotype in rat. The oval cells, the progenies of stem cells invade deep the liver parenchyma after activation and differentiate into focally arranged small-and eventually trabecularly ordered regular hepatocytes. We have observed that upon the completion of intense oval cell reactions narrow ductular structures are present in the parenchyma, we propose to call them parenchymal ductules. These parenchymal ductules have the same immunophenotype [cytokeratin (CK)7-/CK19+/alpha-fetoprotein (AFP)-/delta-like protein (DLK)-] as the resting stem cells of the canals of Hering, but different from them reside scattered in the parenchyma. In our present experiments, we have investigated in an in vivo functional assay if the presence of these parenchymal ductules has any impact on a progenitor cell driven regeneration process. Parenchymal ductules were induced either by an established model of oval cell induction consisting of the administration of necrogenic dose of carbontetrachloride to 2-acetaminofluorene pretreated rats (AAF/CCl4) or a large necrogenic dose of diethylnitrosamine (DEN). The oval cells expanded faster and the foci evolved earlier after repeated injury in the livers with preexistent parenchymal ductules. When the animals were left to survive for one more year increased liver tumor formation was observed exclusively in the DEN treated rats. Thus, repeated oval cell reactions are not necessarily carcinogenic. We conclude that the expansion of hepatic stem cell compartment conceptually can be used to facilitate liver regeneration without an increased risk of tumorigenesis.

Rationale: ABCC6 plays a crucial role in ectopic calcification; mutations of the gene cause pseudoxanthoma elasticum and general arterial calcification of infancy. To elucidate the role of ABCC6 in cellular physiology and disease, it is crucial to establish the exact subcellular localization of the native ABCC6 protein., Objective: In a recent article in Circulation Research, ABCC6 was reported to localize to the mitochondria-associated membrane and not the plasma membrane. As the suggested mitochondrial localization is inconsistent with published data and the presumed role of ABCC6, we performed experiments to determine the cellular localization of ABCC6 in its physiological environment., Methods and Results: We performed immunofluorescent labeling of frozen mouse and human liver sections, as well as primary hepatocytes. We used several different antibodies recognizing human and mouse ABCC6. Our results unequivocally show that ABCC6 is in the basolateral membrane of hepatocytes and is not associated with the mitochondria, mitochondria-associated membrane, or the endoplasmic reticulum., Conclusions: Our findings support the model that ABCC6 is in the basolateral membrane, mediating the sinusoidal efflux of a metabolite from the hepatocytes to systemic circulation.

Unlabelled: We have analyzed the architectural aspects of progenitor-cell-driven regenerative growth in rat liver by applying the 2-acetaminofluorene/partial hepatectomy experimental model. The regeneration is initiated by the proliferation of so-called oval cells. The oval cells at the proximal tips of the ductules have a more differentiated phenotype and higher proliferative rate. This preferential growth results in the formation of a seemingly random collection of small hepatocytes, called foci. These foci have no clonal origin, but possess a highly organized structure, which shows similarities to normal hepatic parenchyma. Therefore, they can easily remodel into the lobular structure. Eventually, the regenerated liver is constructed by enlarged hepatic lobules; no new lobules are formed during this process. The foci of the Solt-Farber experimental hepatocarcinogenesis model have identical morphological features; accordingly, they also represent only regenerative, not neoplastic, growth., Conclusion: Progenitor-cell-driven liver regeneration is a well-designed, highly organized tissue reaction, and better comprehension of the architectural events may help us to recognize this process and understand its role in physiological and pathological reactions., (Copyright © 2012 American Association for the Study of Liver Diseases.)

Background: The immunohistochemical demonstration of Enhancer of zeste homologue 2 (EZH2) proved to be a useful marker in several tumor types. It has been described to distinguish reliably hepatocellular carcinomas from liver adenomas and other benign hepatocellular lesions. However, no other types of malignant liver tumors were studied so far., Methods: To evaluate the diagnostic value of this protein in hepatic tumors we have investigated the presence of EZH2 by immunohistochemistry in hepatocellular carcinomas and other common hepatic tumors.EZH2 expression was examined in 44 hepatocellular carcinomas, 23 cholangiocarcinomas, 31 hepatoblastomas, 16 other childhood tumor types (rhabdomyosarcoma, neuroblastoma, Wilms' tumor and rhabdoid tumor), 17 metastatic liver tumors 24 hepatocellular adenomas, 15 high grade dysplastic nodules, 3 biliary cystadenomas, 3 biliary hamartomas and 3 Caroli's diseases., Results: Most of the malignant liver tumors were positive for EZH2, but neither of the adenomas, cirrhotic/dysplastic nodules, reactive and hamartomatous biliary ductules stained positively., Conclusions: Our immunostainings confirm that EZH2 is a sensitive marker of hepatocellular carcinoma, but its specificity is very low, since almost all the investigated malignant liver tumors were positive regardless of their histogenesis. Based on these results EZH2 is a sensitive marker of malignancy in hepatic tumors. In routine surgical pathology EZH2 could be most helpful to diagnose cholangiocarcinomas, because as far as we know this is the first marker to distinguish transformed and reactive biliary structures. Although hepatoblastomas also express EZH2, the diagnostic significance of this observation seems to be quite limited whereas, the structurally similar, other blastic childhood tumors are also positive., Virtual Slides: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1173195902735693.

The proliferative response of hepatocytes in vivo can be induced by two mechanisms: severe damage to hepatic tissue results in regenerative growth and so-called primary hepatocyte mitogens can initiate liver cell proliferation without preceding loss of parenchyma. The regulation of the two responses is quite different. The decreased regenerative response of cirrhotic/fibrotic liver is well known, and is a severe obstacle to surgery of the diseased liver. In the present experiments we investigated the efficiency of a primary hepatocyte mitogen 1,4-Bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOB) on two different liver cirrhosis/fibrosis models in mice induced by chronic administration of CCl(4) and thioacetamide respectively. BrdU incorporation and cyclin A expression established clearly that there is a reduced but still powerful mitogenic response of the fibrotic livers. Therefore, primary hepatocyte mitogens appear to be suitable to be used to rescue the regenerative response of cirrhotic livers., (© 2012 The Authors. International Journal of Experimental Pathology © 2012 International Journal of Experimental Pathology.)

Syndecans are transmembrane heparan sulphate proteoglycans. Their role in the development of the malignant phenotype is ambiguous and depends upon the particular type of cancer. Nevertheless, syndecans are promising targets in cancer therapy, and it is important to elucidate the mechanisms controlling their various cellular effects. According to earlier studies, both syndecan-1 and syndecan-2 promote malignancy of HT-1080 human fibrosarcoma cells, by increasing the proliferation rate and the metastatic potential and migratory ability, respectively. To better understand their tumour promoter role in this cell line, syndecan expression levels were modulated in HT-1080 cells and the growth rate, chemotaxis and invasion capacity were studied. For in vivo testing, syndecan-1 overexpressing cells were also inoculated into mice. Overexpression of full length or truncated syndecan-1 lacking the entire ectodomain but containing the four juxtamembrane amino acids promoted proliferation and chemotaxis. These effects were accompanied by a marked increase in syndecan-2 protein expression. The pro-migratory and pro-proliferative effects of truncated syndecan-1 were not observable when syndecan-2 was silenced. Antisense silencing of syndecan-2, but not that of syndecan-1, inhibited cell migration. In vivo, both full length and truncated syndecan-1 increased tumour growth and metastatic rate. Based on our in vitro results, we conclude that the tumour promoter role of syndecan-1 observed in HT-1080 cells is independent of its ectodomain; however, in vivo the presence of the ectodomain further increases tumour proliferation. The enhanced migratory ability induced by syndecan-1 overexpression is mediated by syndecan-2. Overexpression of syndecan-1 also leads to activation of IGF1R and increased expression of Ets-1. These changes were not evident when syndecan-2 was overexpressed. These findings suggest the involvement of IGF1R and Ets-1 in the induction of syndecan-2 synthesis and stimulation of proliferation by syndecan-1. This is the first report demonstrating that syndecan-1 enhances malignancy of a mesenchymal tumour cell line, via induction of syndecan-2 expression.

Angiogenesis is believed to be essential for the growth of metastatic tumors in the brain. We analyzed the vascularization of tumors formed by 4 epithelial cell lines (C38, ZR75, HT25, and H1650) and a fibrosarcoma (HT1080) cell line injected into the brains of mice. No peritumoral angiogenesis was observed. Tumors apparently acquired their vasculature by incorporation of native vessels. Vessel density was lower, but vessel diameter and vascular cell proliferation were higher within all tumors versus those in the peritumoral tissue. There was an inverse correlation between the number of incorporated vessels and vascular cell proliferation. Epithelial tumors with pushing growth patterns had lower vessel density and elevated vascular cell proliferation compared with invasive tumors. The incorporated vessels retained their normal structure, with the exception of astrocyte foot processes that were replaced by tumor cells. Attachment to the vascular basement membrane led to the differentiation of the ZR75 breast cancer cells. In the HT1080 metastases, there was intussusceptive angiogenesis, that is, the fibrosarcoma cells that were attached to the vessel caused lumen splitting and filled the developing pillars. Branching angiogenesis was not observed either in the tumors or in control cerebral wounds. These data suggest that sprouting angiogenesis is not needed for the incipient growth of cerebral metastases and that tumor growth in this model is a result of incorporation of host vessels.

One of the hallmarks of intussusceptive angiogenesis is the development of intraluminal connective tissue pillars. The exact mechanism of pillar formation has not yet been elucidated. By using electron and confocal microscopy, we observed intraluminal nascent pillars that contain a collagen bundle covered by endothelial cells (ECs) in the vasculature of experimental tumors. We proposed a new mechanism for the development of these pillars. First, intraluminal endothelial bridges are formed. Second, localized dissolution of the basement membrane occurs and a bridging EC attaches to a collagen bundle in the underlying connective tissue. A pulling force is then exerted by the actin cytoskeleton of the ECs via specific attachment points, which contain vinculin, to the collagen bundle, resulting in suction and subsequent transport of the collagen bundle into and through the vessel lumen. Third, the pillar matures through the immigration of connective tissue cells and the deposition of new collagenous connective tissue. The proposed simple mechanism generates a connection between the processes of endothelial bridging and intussusceptive angiogenesis and identifies the source of the force behind pillar formation. Moreover, it ensures the rapid formation of pillars from pre-existing building blocks and the maintenance of EC polarity. To describe it, we coined the term inverse sprouting., (Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Accumulation of connective tissue is a typical feature of chronic liver diseases. Decorin, a small leucine-rich proteoglycan, regulates collagen fibrillogenesis during development, and by directly blocking the bioactivity of transforming growth factor-β1 (TGFβ1), it exerts a protective effect against fibrosis. However, no in vivo investigations on the role of decorin in liver have been performed before. In this study we used decorin-null (Dcn-/-) mice to establish the role of decorin in experimental liver fibrosis and repair. Not only the extent of experimentally induced liver fibrosis was more severe in Dcn-/- animals, but also the healing process was significantly delayed vis-à-vis wild-type mice. Collagen I, III, and IV mRNA levels in Dcn-/- livers were higher than those of wild-type livers only in the first 2 months, but no difference was observed after 4 months of fibrosis induction, suggesting that the elevation of these proteins reflects a specific impairment of their degradation. Gelatinase assays confirmed this hypothesis as we found decreased MMP-2 and MMP-9 activity and higher expression of TIMP-1 and PAI-1 mRNA in Dcn-/- livers. In contrast, at the end of the recovery phase increased production rather than impaired degradation was found to be responsible for the excessive connective tissue deposition in livers of Dcn-/- mice. Higher expression of TGFβ1-inducible early responsive gene in decorin-null livers indicated enhanced bioactivity of TGFβ1 known to upregulate TIMP-1 and PAI-1 as well. Moreover, two main axes of TGFβ1-evoked signaling pathways were affected by decorin deficiency, namely the Erk1/2 and Smad3 were activated in Dcn-/- samples, whereas no significant difference in phospho-Smad2 was observed between mice with different genotypes. Collectively, our results indicate that the lack of decorin favors the development of hepatic fibrosis and attenuates its subsequent healing process at least in part by affecting the bioactivity of TGFβ1., Competing Interests: DISCLOSURE/CONFLICT OF INTEREST The authors declare no conflict of interest.

Background: Male infertility is an increasing problem in all domestic species including man. Localization and identification of genes involved in defects causing male infertility provide valuable information of specific events in sperm development. Correct condensation of the sperm head and development of the acrosome are required for fertile sperm. In the Finnish Yorkshire pig population a knobbed acrosome defect (KAD) has been reported which appears to be of genetic origin. In previous studies we have shown that a large number of affected spermatozoa have a cystic swelling anterior to the apical part of the acrosome., Results: Characterization of the knobbed acrosome affected sperm revealed that both the acrosomal granules and chromatin are affected. This type of KAD appears to be a previously unknown and serious form of the defect. A genome wide scan with PorcineSNP60 Genotyping BeadChip defined the KAD associated region within 0.7 Mbp on porcine chromosome 15. Two genes, STK17b and HECW2, located within this region were sequenced. The expression of these genes appeared comparable in KA-affected and control boars. The known function of HECW2 in acrosome development highlighted this gene as a good candidate responsible for the KAD. One nonsynonymous SNP was identified within the HECW2 gene. However, as this mutation was found in homozygous state in individuals with normal sperm, this is not likely to be the causal mutation., Conclusions: In this study we identified two candidate genes for a severe defect affecting both the sperm acrosome and chromatin that causes infertility. One of these genes, HECW2, plays an important role in ubiquitination, a prerequisite for chromatin remodelling and acrosome formation, highlighting the involvement of this gene in the knobbed acrosome defect and male infertility.

Background: Transforming growth factor (TGF)-β-1 is a very efficient inhibitor of hepatocyte proliferation in various in vivo and in vitro experimental systems. However, there are no data on whether it can influence the mitogenic response induced by primary hepatocyte mitogens., Aims: In this study, we compared the proliferative response in the liver between wild-type and transgenic mice, overexpressing active TGF-β-1 in their liver following the treatment by a primary hepatocyte mitogen TCPOBOP (1,4-bis[2-(3,5-dichloropyridyloxy)]benzene)., Methods: The proliferative response was characterized by the immunohistochemical examination of pulse and cumulative bromodeoxyuridine labelling and by quantitative real-time polymerase chain reaction analysis of cell cycle-related genes., Results: Neither of the applied techniques revealed significant differences between the two groups of mice; furthermore, we observed the upregulation of TGF-β-1 expression following the mitogenic treatment., Conclusions: TGF-β-1 does not inhibit the primary mitogen-induced proliferative response of the hepatocytes. This observation may provide an explanation for the divergent consequences of hepatic proliferations induced by partial hepatectomy or primary mitogenic treatment., (© 2010 John Wiley & Sons A/S.)

Aims:   Ductular reactions occur in a wide variety of liver diseases. Their origin and function is still debated. Our understanding of these histological reactions is impaired by their great diversity; therefore rational classification should precede further detailed analysis. The aim was to achieve a reproducible classification of hepatic ductular reactions based on their immunophenotype., Methods and Results:   Sixty-nine liver specimens with ductular reactions were analysed by immunohistochemistry. The majority of the samples could be classified into three categories based on their immunophenotype. Type P(rimitive) reaction is characterized by CD56 immunoreactivity. Most primary biliary cirrhosis and focal nodular hyperplasia samples fall into this group; these ductules do not show any sign of differentiation. Type D(ifferentiating) ductules are positive for CD56, epithelial membrane antigen (EMA) and CD10. Cirrhotic samples and regenerating livers following fulminant hepatic failure contain such ductular reactions; this immunophenotype indicates hepatocytic differentiation. Biliary obstruction results in EMA-positive type O(bstructive) reactions; these ductules are similar to the normal interlobular bile ducts., Conclusion:   Ductular reactions can be classified based on their immunophenotype. Our results may initiate further, similar, studies resulting in a generally accepted rational classification. We believe that such categorization is necessary for elucidating their biological and clinical significance., (© 2010 Blackwell Publishing Limited.)

The canals of Hering or biliary ductules have been described to connect the bile canaliculi with the interlobular bile ducts, and thus forming the distal part of the biliary tree. Studies in the last two decades suggested that the cells constructing these ductules could behave as hepatic progenitor cells. The canals of Hering are confined to the periportal space in the rat, while they have been reported to spread beyond the limiting plate in human liver. The distribution of the distal biliary ductules in normal human hepatic tissue has been investigated in our recent experiments. We could demonstrate the presence of interlobular connective tissue septa in a rudimentary form in healthy livers. The canals of Hering run in these septa in line with the terminal branches of the portal vein and hepatic arteries. This arrangement develops in the postnatal period but regresses after early childhood. The canals of Hering can be identified by the unique epithelial membrane antigen (EMA)-/CD56+/CD133+ immunophenotype. The canals of Hering leave the periportal space and spread into the liver parenchyma along rudimentary interlobular septa outlining the hepatic lobules. Our observations refine the original architectural description of the intraparenchymal portion of the canals of Hering in the human liver. The distinct immunophenotype supports their unique biological function.

Delta-like protein (DLK) is expressed in fetal and adult adrenal glands. We have investigated if this expression is maintained in adrenal gland-derived tumors. All the studied 37 cortical tumors, including five carcinomas, stained positively as well as the 13 examined pheochromocytomas. Thus, DLK is a very sensitive marker for adrenal tumors of cortical and medullary origin. Renal cell carcinomas, presenting the major differential diagnostic problem for cortical tumors, were all negative, as well as melanomas, which are similar to high portion of adrenocortical tumors that react with melan-A. However, all paragangliomas, some carcinoids, and thyroid medullary carcinomas were also positive for DLK. Therefore, this novel immunohistochemical marker seems useful for the identification of adrenocortical tumors while it has limited value for the distinction of pheochromocytomas from diagnostically related neuroendocrine tumors.

In this study, we present a mechanism for the development of arterial blood supply in experimental liver metastases. To analyze the arterialization process of experimental liver metastases, we elucidated a few key questions regarding the blood supply of hepatic lobules in mice. The microvasculature of the mouse liver is characterized by numerous arterioportal anastomoses and arterial terminations at the base of the lobules. These terminations supply one hepatic microcirculatory subunit per lobule, which we call an arterial hepatic microcirculatory subunit (aHMS). The process of arterialization can be divided into the following steps: 1) distortion of the aHMS by metastasis; 2) initial fusion of the sinusoids of the aHMS at the tumor parenchyma interface; 3) fusion of the sinusoids located at the base of the aHMSs, which leads to the disruption of the vascular sphincter (burst pipe); 4) incorporation of the dilated artery and the fused sinusoids into the tumor; and 5) further development of the tumor vasculature (arterial tree) by proliferation, remodeling, and continuous incorporation of fused sinusoids at the tumor-parenchyma interface. This process leads to the inevitable arterialization of liver metastases above the 2000- to 2500-mum size, regardless of the origin and growth pattern of the tumor.

Although liver architecture has a major impact on function, morphological aspects of liver growth are relatively neglected. In our recent experiments, the architectural changes of the rat liver were compared during 2 basic processes: ontogeny and regenerative liver growth. The hepatic tissue is constructed as structural/functional units, and probably the most established and well-defined such unit is the classic lobule. The extent and orientation of the lobules are variable in the liver, and this renders their accurate size determination more difficult. The filling of the liver vasculature by a colored resin nicely outlined the surface lobules, enabling an analysis of the alterations of these structures during liver growth. There are 3 structural components of postnatal physiological liver development: enlargement of the hepatocytes and expansion and multiplication of the liver lobules. However, the enlargement of the lobules is exclusively responsible for the regenerative liver growth following partial hepatectomy. The number of hepatic lobules does not change during this latter reaction, but they gain a more complex, irregular structure., ((c) 2009 AASLD.)

The 2-acetaminofluorene/partial hepatectomy (AAF/Phx) model is widely used to induce oval/progenitor cell proliferation in the rat liver. We have used this model to study the impact of a primary hepatocyte mitogen, triiodothyronine (T3) on the liver regenerating by the recruitment of oval/progenitor cells. Administration of T3 transiently accelerates the proliferation of the oval cells, which is followed by rapid differentiation into small hepatocytes. The oval cell origin of the small hepatocytes has been proven by tracing retrovirally transduced and BrdU marked oval cells. The differentiating oval cells become positive for hepatocyte nuclear factor-4 and start to express hepatocyte specific connexin 32, alpha1 integrin, Prox1, cytochrom P450s, and form CD 26 positive bile canaliculi. At the same time oval cell specific OV-6 and alpha-fetoprotein expression is lost. The upregulation of hepatocyte specific mRNAs: albumin, tyrosine aminotransferase and tryptophan 2,3-dioxygenase detected by real-time PCR also proves hepatocytic maturation. The hepatocytic conversion of oval cells occurs on the seventh day after the Phx in this model while the first small hepatocytes appear 5 days later without T3 treatment. The administration of the primary hepatocyte mitogen T3 accelerates the differentiation of hepatic progenitor cells into hepatocytes in vivo, and that may have therapeutic potential.

A large, rapidly growing malignant peripheral nerve sheath tumor (MPNST) of the liver in a young female patient, not associated with von Recklinghausen's disease, is presented. Diagnosis was based on detailed immunohistochemical and electromicroscopic examination beside the characteristic H&E picture. As far as we know, this is the first reported, unambiguously proven "de novo" MPNST in the liver. Differential diagnostic problems are discussed and a review of the literature is given.

Delta-like protein (DLK) is a membrane protein with mostly unknown function. It is expressed by several embryonic tissues among others by the hepatoblasts of rodent and human fetal livers. We have investigated in the present study if this protein is expressed in human hepatoblastomas. The presence of DLK has been studied by standard immunohistochemistry in 31 hepatoblastomas and in several differential diagnostically related tumours: hepatocellular carcinomas and in undifferentiated childhood neoplasms. All the hepatoblastomas were positive for DLK; the surrounding liver tissue remained negative. The reaction was present in the epithelial component of the tumours. The staining pattern was mostly membranous, occasionally cytoplasmic. The other studied tumours were negative for DLK, except one hepatocellular carcinoma and the differentiating cells of two ganglioneuroblastomas. Therefore, DLK seems to be a highly sensitive and specific marker for hepatoblastomas.

Thy-1, a marker of hematopoietic stem cells, has been reported to be expressed by oval cells proliferating during stem cell-mediated regeneration in rat liver, suggesting a relationship between the two cell populations. Consequently, Thy-1 has become an accepted cell surface marker to sort hepatic oval cells. In the present study we used the well-characterized 2-acetylaminfluorene/partial hepatectomy model to induce transit-amplification of hepatic oval cells in the regenerating liver and characterized Thy-1 expression using Northern hybridization, quantitative reverse transcriptase-polymerase chain reaction analysis, immunofluorescence confocal microscopy, and immunoelectronmicroscopy. We found that Thy-1 expression was induced during transit-amplification of the oval cell population, but Thy-1 mRNA was not present in the alpha-fetoprotein-expressing oval cells. Thy-1 protein was consistently present outside the basement membrane surrounding the oval cells. It overlapped frequently with smooth muscle actin staining. A similar cellular localization of the Thy-1 protein was found on human liver specimens with ductular reactions obtained from patients with fulminant liver failure. Furthermore, Thy-1 was expressed by myofibroblasts in experimental liver fibrosis models without oval cell proliferation. We conclude that Thy-1 is not a marker of oval cells but is present on a subpopulation of myofibroblasts/stellate cells.

Mycophenolic acid (MPA)/mycophenolate mofetil (MMF), a powerful immunosuppressive agent was tested on human B-lymphoma cells (Epstein-Barr virus +/-) in vitro and in SCID mouse xenograft model. Proliferation, apoptotic activity and tumor volume were evaluated. MPA inhibited lymphoma cell proliferation and induced apoptosis (50-60% at 72 h). In vivo, oral administration significantly inhibited subcutaneous tumor growth. Immunohistochemistry showed significantly decreased proliferation rate and higher apoptotic activity in tumors treated with MMF. Xenografted lymphoma cells remained sensitive to MPA. Our results suggest that MPA may be recommended as an additional component of lymphoma chemotherapeutical regimens, with special considerations to post-transplant lymphomas.

Although cancer cells are not generally controlled by normal regulatory mechanisms, tumor growth is highly dependent on the supply of oxygen, nutrients, and host-derived regulators. It is now established that tumor vasculature is not necessarily derived from endothelial cell sprouting; instead, cancer tissue can acquire its vasculature by co-option of pre-existing vessels, intussusceptive microvascular growth, postnatal vasculogenesis, glomeruloid angiogenesis, or vasculogenic mimicry. The best-known molecular pathway driving tumor vascularization is the hypoxia-adaptation mechanism. However, a broad and diverse spectrum of genetic aberrations is associated with the development of the "angiogenic phenotype." Based on this knowledge, novel forms of antivascular modalities have been developed in the past decade. When applying these targeted therapies, the stage of tumor progression, the type of vascularization of the given cancer tissue, and the molecular machinery behind the vascularization process all need to be considered. A further challenge is finding the most appropriate combinations of antivascular therapies and standard radio- and chemotherapies. This review intends to integrate our recent knowledge in this field into a rational strategy that could be the basis for developing effective clinical modalities using antivascular therapy for cancer.

Human papillomavirus (HPV) is known as a strictly epitheliotropic pathogen. Our results raised the possibility that HPV 16 is present in neural cells and in the vascular endothelium. By in situ hybridization, we have detected HPV 16 E6 ORF sequence in small blood vessels and peripheral nerves adjacent to oral and cervical cancers. The same structures have clearly shown immunohistochemical reactivity for the E6 oncoprotein. These results were verified by PCR applied to E6 and L1 ORFs following microscopic laser dissection of the immunohistochemically positive nerves and vessels. These observations suggest that HPV 16 DNA and protein are present in neurons and endothelial cells in the vicinity of HPV-associated tumors. The HPV 16 genome presumably exists in a non-replicating form in the neurons and constitutively produces high levels of E6 and E7 proteins with an unknown neuropathological outcome.

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.

Vascularization of cancer is a complex and heterogenous process where neoangiogenesis by sprouting is only one of the possible mechanisms that also include postnatal vasculogenesis, vessel incorporation, intussusceptive microvascular growth, glomeruloid angiogenesis and vascular mimicry. Furthermore, the mechanism of vascularization may also depend on the cancer type and the host tissue as well. Antivascular agents can be divided into angiosuppressive ones (endothelial cell proliferation inhibitors), vascular-targeted agents (microvessel disrupting agents) and anti-hypoxia agents (targeting the molecular pathways responsible for the development of the angiogenic phenotype). Since antivascular therapy is a special form of targeted therapy, it is necessary to apply it in a rational manner to consider the type of vascularization, the molecular background of the angiogenic phenotype, the stage of the disease and the standard anticancer therapy. Whithout such a fine-tuning, antivascular therapies cannot be integrated more successfully into the management of cancer patients.