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George K Michalopoulos

Publications and source records attributed to George K Michalopoulos.

At least 19 recordsLinked to original sources
PubMed2006

Integrin-linked kinase is involved in matrix-induced hepatocyte differentiation.

Hepatocytes have restricted proliferative capacity in culture and when cultured without matrix, lose the hepatocyte-specific gene expression and characteristic cellular micro-architecture. Overlay of matrix-preparations on de-differentiated hepatocytes restores differentiation. Integrin-linked kinase (ILK) is a cell-matrix-adhesion protein crucial in fundamental processes such as differentiation and survival. In this study, we investigated the role of ILK, and its binding partners PINCH, alpha-parvin, and Mig-2 in matrix-induced hepatocyte differentiation. We report here that ILK is present in the liver and localizes at cell-matrix adhesions of cultured hepatocytes. We also show that ILK, PINCH, alpha-parvin, and Mig-2 expression level is dramatically reduced in the re-differentiated hepatocytes. Interestingly, hepatocytes lacking ILK undergo matrix-induced differentiation but their differentiation is incomplete, as judged by monitoring cell morphology and production of albumin. Our results show that ILK and cell-matrix adhesion proteins play an important role in the process of matrix-induced hepatocyte differentiation.

Animals↗
PubMed2006

Phenobarbital regulates nuclear expression of HNF-4alpha in mouse and rat hepatocytes independent of CAR and PXR.

Phenobarbital is a lipophilic molecule used as a sedative and antiepileptic drug that elicits a multitude of effects in the liver, including gross liver enlargement, hepatocyte hypertrophy, and induced expression of drug-metabolizing enzymes and other liver-specific genes. The constitutive androstane receptor (CAR; NR1I3) and to a lesser extent the pregnane X receptor (PXR; NR1I2) are responsible for mediating induction of many phenobarbital-responsive genes. However, CAR-mediated transcriptional control of some genes is critically dependent on hepatocyte nuclear factor 4 alpha (HNF-4alpha; NR2A1), which itself regulates multiple liver-specific genes involved in hepatic growth, metabolism, and differentiation. We studied the effects of phenobarbital on HNF-4alpha expression in hepatocytes and provide evidence that HNF-4alpha nuclear expression is regulated in response to phenobarbital. Real-time polymerase chain reaction analyses revealed that HNF-4alpha mRNA is modestly up-regulated by phenobarbital. In addition, nuclear expression of HNF-4alpha protein is significantly elevated 3 hours after the administration of phenobarbital in wild-type, CAR-/-, and CAR-/-/PXR-/- mice. In vitro analysis revealed that phenobarbital-induced HNF-4alpha expression is both time- and dose dependent. In addition, the phosphatase inhibitor okadaic acid and the Ca2+/calmodulin-dependent protein kinase II inhibitor KN62 block nuclear induction of HNF-4alpha by phenobarbital. Furthermore, HNF-4alpha nuclear expression is enhanced by inhibition of cyclic AMP-dependent protein kinase A. In conclusion, induced nuclear expression of HNF-4alpha and CAR is an integral part of the phenobarbital response, aimed at coordinated regulation of genes involved in drug metabolism and detoxification as well as maintenance of liver function.

Animals↗
PubMed2006

Transcriptomic and genomic analysis of human hepatocellular carcinomas and hepatoblastomas.

This study analyzed gene expression patterns and global genomic alterations in hepatocellular carcinomas (HCC), hepatoblastomas (HPBL), tissue adjacent to HCC and normal liver tissue derived from normal livers and hepatic resections. We found that HCC and adjacent non-neoplastic cirrhotic tissue have considerable overlap in gene expression patterns compared to normal liver. Several genes including Glypican 3, spondin-2, PEG10, EDIL3 and Osteopontin are over-expressed in HCC vs. adjacent tissue whereas Ficolin 3 is the most consistently under-expressed gene. HCC can be subdivided into three clusters based on gene expression patterns. HCC and HPBL have clearly different patterns of gene expression, with genes IGF2, Fibronectin, DLK1, TGFb1, MALAT1 and MIG6 being over-expressed in HPBL versus HCC. In addition, specific areas of the genome appear unstable in HCC, with the same regions undergoing either deletion or increased gene dosage in all HCC. In conclusion, a set of specific genes and areas of genomic instability are found across the board in liver neoplasia.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Prostaglandin E2 receptor EP1 transactivates EGFR/MET receptor tyrosine kinases and enhances invasiveness in human hepatocellular carcinoma cells.

Recent evidence indicates that cyclooxygenase-2 (COX-2) and epidermal growth factor receptor (EGFR) are involved in hepatocarcinogenesis. This study was designed to evaluate the possible interaction between the COX-2 and EGFR signaling pathways in human hepatocellular carcinoma (HCC) cells. Immunohistochemical analysis using serial sections of human HCC tissues revealed positive correlation between COX-2 and EGFR in HCC cells (P < 0.01). Overexpression of COX-2 in cultured HCC cells (Hep3B) or treatment with PGE(2) or the selective EP(1) receptor agonist, ONO-DI-004, increased EGFR phosphorylation and tumor cell invasion. The PGE(2)-induced EGFR phosphorylation and cell invasiveness were blocked by the EP(1) receptor siRNA or antagonist ONO-8711 and by two EGFR tyrosine kinase inhibitors, AG1478 and PD153035. The EP(1)-induced EGFR transactivation and cell invasion involves c-Src, in light of the presence of native binding complex of EP(1)/Src/EGFR and the inhibition of PGE(2)-induced EGFR phosphorylation and cell invasion by the Src siRNA and the Src inhibitor, PP2. Further, overexpression of COX-2 or treatment with PGE(2) also induced phosphorylation of c-Met, another receptor tyrosine kinase critical for HCC cell invasion. Moreover, activation of EGFR by EGF increased COX-2 promoter activity and protein expression in Hep3B and Huh-7 cells, whereas blocking PGE(2) synthesis or EP(1) attenuated EGFR phosphorylation induced by EGF, suggesting that the COX-2/PGE(2)/EP(1) pathway also modulate the activation of EGFR by its cognate ligand. These findings disclose a cross-talk between the COX-2/PGE(2)/EP(1) and EGFR/c-Met signaling pathways that coordinately regulate human HCC cell invasion.

Alprostadil↗
PubMed2006

Conditional deletion of beta-catenin reveals its role in liver growth and regeneration.

BACKGROUND & AIMS: The Wnt/beta-catenin pathway plays a role in liver growth and development. To address this conclusively, we used a conditional knockout approach to delete beta-catenin in the liver. METHODS: Floxed beta-catenin (exons 2-6) mice were intercrossed with Albumin-Cre recombinase transgenic mice; considerable beta-catenin deletion was evident 15 days after birth by Western blot and immunohistochemistry analyses. RESULTS: Although these mice were viable, there was a significant decrease in their liver weight/body weight ratio by 14% at 1 month and 28%-35% by 2-6 months of age, which was sustained throughout their normal life span. There was an accompanying decrease in basal hepatocyte proliferation showed by Ki-67 staining. Additional analysis revealed several known and novel genes to be down-regulated in these mice that play a role in normal liver homeostasis. When subjected to two-thirds partial hepatectomy, the Ctnnb1(loxp/loxp); Alb-Cre(+/-) mice were sick and lethargic, especially during the first 2-3 days only. These mice display a 2-fold decrease in the number of Ki-67- or PCNA-positive cells at the time of peak hepatocyte proliferation at 40 hours, which coincided with decreased cyclin A, D, and E expression. However, a rebound increase in hepatocyte proliferation was evident in the knockout mice at 3 days. Also, increased apoptosis was observed in the knockout livers during regeneration at all stages. CONCLUSIONS: Thus, beta-catenin is essential for normal liver growth and development. Also, although regeneration is delayed in the absence of beta-catenin, it does occur suboptimally, showing its redundancy in the liver.

Animals↗
PubMed2006

Hepatocyte growth factor attenuates liver fibrosis induced by bile duct ligation.

Hepatic fibrosis is a common outcome of a variety of chronic liver diseases. Here we evaluated the therapeutic efficacy of hepatocyte growth factor (HGF) on liver fibrosis induced by bile duct ligation (BDL) and investigated potential mechanisms. Mice underwent BDL, followed by intravenous injections of naked HGF expression plasmid or control vector. HGF gene therapy markedly ameliorated hepatic fibrotic lesions, as demonstrated by reduced alpha-smooth muscle actin (alphaSMA) expression, attenuated deposition of type I and type III collagen, and normalized total hydroxyproline content. HGF also suppressed transforming growth factor-beta1 (TGF-beta1) expression. Interestingly, colocalization of alphaSMA and cytokeratin-19 in bile duct epithelium was observed, suggesting the possibility of biliary epithelial to myofibroblast transition after BDL. Cells that were still positive for cytokeratin-19 but actively producing type I collagen were found in the biliary epithelia and periductal region. Laminin staining revealed an impaired basement membrane of the bile duct epithelium in diseased liver. These lesions were largely prevented by HGF administration. In vitro, treatment of human biliary epithelial cells with TGF-beta1 induced alphaSMA and fibronectin expression and suppressed cytokeratin-19. HGF abolished the phenotypic conversion of biliary epithelial cells induced by TGF-beta1. These results suggest that HGF ameliorates hepatic biliary fibrosis in part by blocking bile duct epithelial to mesenchymal transition.

Animals↗
PubMed2006

Peroxisomal localization of hypoxia-inducible factors and hypoxia-inducible factor regulatory hydroxylases in primary rat hepatocytes exposed to hypoxia-reoxygenation.

Many signals involved in pathophysiology are controlled by hypoxia-inducible factors (HIFs), transcription factors that induce expression of hypoxia-responsive genes. HIFs are post-translationally regulated by a family of O2-dependent HIF hydroxylases: four prolyl 4-hydroxylases and an asparaginyl hydroxylase. Most of these enzymes are abundant in resting liver, which is itself unique because of its physiological O2 gradient, and they can exist in both nuclear and cytoplasmic pools. In this study, we analyzed the cellular localization of endogenous HIFs and their regulatory hydroxylases in primary rat hepatocytes cultured under hypoxia-reoxygenation conditions. In hepatocytes, hypoxia targeted HIF-1alpha to the peroxisome, rather than the nucleus, where it co-localized with von Hippel-Lindau tumor suppressor protein and the HIF hydroxylases. Confocal immunofluorescence microscopy demonstrated that the HIF hydroxylases translocated from the nucleus to the cytoplasm in response to hypoxia, with increased accumulation in peroxisomes on reoxygenation. These results were confirmed via immunotransmission electron microscopy and Western blotting. Surprisingly, in resting liver tissue, perivenous localization of the HIF hydroxylases was observed, consistent with areas of low pO2. In conclusion, these studies establish the peroxisome as a highly relevant site of subcellular localization and function for the endogenous HIF pathway in hepatocytes.

Animals↗
PubMed2005

Distinctive role of Stat3 and Erk-1/2 activation in mediating interferon-gamma inhibition of TGF-beta1 action.

Interferon-gamma (IFN-gamma) is a multifunctional cytokine that elicits antifibrotic activity in a variety of organs. In this study, we investigated the potential role and mechanism of IFN-gamma in modulating the fibrogenic action of transforming growth factor (TGF)-beta(1) in tubular epithelial cells. Incubation of human proximal tubular epithelial (HKC) cells with IFN-gamma inhibited TGF-beta(1)-mediated alpha-smooth muscle actin (alpha-SMA) expression. IFN-gamma also abolished TGF-beta(1)-induced fibronectin and plasminogen activator inhibitor-1 (PAI-1) expression. To explore the mechanisms by which INF-gamma inhibits TGF-beta(1) action, the signaling pathways that are critical for mediating the antifibrotic activity of IFN-gamma were studied. Stimulation of HKC cells with IFN-gamma triggered a sustained activation of Erk-1/2 and signal transducer and activator of transcription-3 (Stat3). Blockade of Erk-1/2 activation with an Mek1 inhibitor abolished the inhibitory effect of IFN-gamma on alpha-SMA expression, whereas inhibition of Stat3 activation had no influence. Constitutive activation of Erk-1/2 by ectopic expression of activated Mek1 mimicked IFN-gamma and suppressed TGF-beta(1)-mediated alpha-SMA expression. Interestingly, inhibition of Stat3 activation abolished the ability of IFN-gamma to attenuate TGF-beta(1)-mediated PAI-1 and fibronectin expression in HKC cells. These findings indicate that IFN-gamma is capable of antagonizing the fibrogenic actions of TGF-beta(1) in renal tubular epithelial cells. The antifibrotic action of IFN-gamma appears to be mediated through a coordinated activation of both Erk-1/2 and Stat3 signal pathways in a mutually independent fashion.

Fibrosis↗
PubMed2005

Transdifferentiation of rat hepatocytes into biliary cells after bile duct ligation and toxic biliary injury.

Rats with chimeric livers were generated by using the protocol of injecting hepatocytes from dipeptidyl peptidase IV (DPPIV)-positive donors into retrorsine-treated DPPIV-negative recipients subjected to partial hepatectomy. Rats with established chimeric livers were subjected to bile duct ligation, with or without pretreatment with the biliary toxin methylene diamiline (DAPM). Ductules bearing the donor hepatocyte marker DPPIV were seen at 30 days after bile duct ligation. The frequency of the ductules derived from the donor hepatocytes was dramatically enhanced (36-fold) by the pretreatment with DAPM. In conclusion, our results show that hepatocytes can function as facultative stem cells and rescue the biliary epithelium during repair from injury when its proliferative capacity is being compromised.

Aniline Compounds↗
PubMed2005

Liver regeneration.

This review summarizes the functional aspects, cellular kinetics and molecular mechanisms related to liver regeneration. Liver regeneration is a model of regenerative growth of tissues in adult animals. Rapid biochemical and gene expression changes following initiation of regeneration are mediated by specific stimuli, including growth factors and cytokines. The whole process involves multiple feedback loops between same or different types of cells. The end result is restoration of hepatic mass and preservation of the normal histology of the liver. All of this complex phenomenology occurs while the liver continues to provide all the metabolic support required to sustain life of the organism.

Adaptation, Physiological↗
PubMed2005

Epidermal growth factor receptor: a novel target of the Wnt/beta-catenin pathway in liver.

BACKGROUND & AIMS: Wnt/beta-catenin activation is observed in normal liver development, regeneration, and liver cancer. Our aim was to elucidate the regulation and mechanism of this pathway in liver. METHODS: We report the generation and characterization of liver-specific nonmutated beta-catenin-overexpressing transgenic mice. Transgenic livers were examined for their morphology and phenotype by histology, proliferation, apoptosis, and microarray analysis. RESULTS: Transgenic livers displayed a significant increase in cytoplasmic, membranous, and nuclear beta-catenin in hepatocytes as compared with their wild-type littermates, which display a predominant membranous localization only. A 15%-20% increase in the liver weight-body weight ratio was evident in transgenic mice secondary to increased hepatocyte proliferation. Microarray analysis showed differential expression of approximately 400 genes in the transgenic livers. Epidermal growth factor receptor RNA and protein and increased levels of activated epidermal growth factor receptor and Stat3 were observed in the transgenic livers. Epidermal growth factor receptor promoter analysis showed a T-cell factor-binding site, and subsequent reporter assay confirmed epidermal growth factor receptor activation in response to Wnt-3A treatment that was abrogated by frizzled related protein 1, a known Wnt antagonist. Epidermal growth factor receptor inhibition successfully decreased liver size in transgenic mice. Next, 7 of 10 hepatoblastomas displayed simultaneous beta-catenin and epidermal growth factor receptor up-regulation, thus suggesting a strong relationship between these 2 proteins in tumors. CONCLUSIONS: beta-Catenin transgenic mice show an in vivo hepatotrophic effect secondary to increased basal hepatocyte proliferation. Epidermal growth factor receptor seems to be a direct target of the pathway, and epidermal growth factor receptor activation might contribute toward some mitogenic effects of increased beta-catenin in liver: epidermal growth factor receptor inhibition might be useful in such states.

Animals↗
PubMed2004

Wnt impacts growth and differentiation in ex vivo liver development.

The Wnt-beta-catenin pathway plays a role in liver growth and development. Here, we investigate the direct effect of Wnt-3A on ex vivo liver development. Livers from mouse embryos at day 10 were cultured in serum-free Wnt-3A-conditioned media alone or with HGF and insulin for 72 h and analyzed for histology, proliferation, apoptosis and lineage. Control cultures grown in serum-free conditions or Wnt-3A and sFRP-1 combination display loss of architecture and proliferation and increased apoptosis. In the presence of Wnt-3A, embryonic liver cultures show CK-19-positive cells (biliary phenotype) displaying proliferation, minimal apoptosis and duct-like histological arrangement. HGF and Wnt combination exhibited normal histology as seen in the presence of 10% serum displaying stem cells, hepatocytes and primitive bile ducts. HGF, insulin and Wnt combination provided no additional benefits rather had an overall deleterious effect. Thus, Wnt supports biliary differentiation by enhancing stem cell specification, hepatocyte trans-differentiation and promoting biliary survival. HGF and Wnt combination supports stem cells, hepatocytes and bile ducts. The addition of insulin to the combination of HGF and Wnt provided no growth or differentiation advantage. Our results indicate usefulness of Wnt and HGF in hepatocyte cultures and suggest their balance during normal liver development.

Animals↗
PubMed2004

Expression of Notch-1 and its ligand Jagged-1 in rat liver during liver regeneration.

The Notch/Jagged signaling pathway is important for cellular differentiation and proliferation. Its dysfunction is associated with human pathologies in several tissues including liver. Point mutations in Jagged-1 gene are the cause for Alagille syndrome, associated with paucity of intrahepatic bile ducts. To determine the putative role of the trans-membrane receptor Notch and its ligand Jagged-1 in liver regeneration, we investigated the expression of Notch and Jagged-1 in rat liver following 2/3 partial hepatectomy. Immunohistochemical staining of normal rat liver showed that Notch was expressed in hepatocytes, bile duct cells and endothelial cells, whereas Jagged-1 was expressed in bile duct cells and hepatocytes. Both Notch-1 and Jagged-1 proteins were upregulated in hepatocytes after partial hepatectomy up to day 4. After partial hepatectomy, nuclear translocation of the intracellular cytoplasmic domain of Notch (NICD) increased and peaked within 15 minutes, indicating the activation of Notch. Expression of the Notch-dependent target gene (HES-1) expression increased within 30-60 minutes. Addition of recombinant Jagged-1 protein to primary cultures of hepatocytes stimulated hepatocyte DNA synthesis. Furthermore, injection of silencing RNA for Notch and Jagged-1 to livers 2 days before partial hepatectomy significantly suppressed proliferation of hepatocytes at days 2 to 4 of the regenerative response. In conclusion, Notch/Jagged signaling pathway is activated during liver regeneration and is potentially contributing to signals affecting cell growth and differentiation.

Animals↗
PubMed2004

Webcasting pathology department conferences in a geographically distributed medical center.

As academic pathology departments have become increasingly based in large, regional medical systems spread across hundreds of square miles, new methods are needed to tie these increasingly distributed departments together as integrated units. An important part of that integration is the ability to share academic and teaching conferences across long distances. In this article we present an effective, low-cost webcasting system that has evolved at the University of Pittsburgh Medical Center Department of Pathology over the past several years based on inexpensive, widely available software. To date, the system has broadcast and archived more than 400 conferences and currently serves approximately 80 to 100 requests each week. Important factors in the success of the program include the creation of a faculty steering committee to control resources and manage growth, the availability of informatics faculty and support for technical staff, and the decision to operate the service as part of the core information technology infrastructure of the department. Webcasting will likely become an even more important academic and operational tool in the future as more of the department's conferences, seminars, and even working meetings are communicated through the webcasting infrastructure.

Academic Medical Centers↗
PubMed2004

Beta-catenin is temporally regulated during normal liver development.

BACKGROUND & AIMS: beta-Catenin, a key component of the Wnt pathway, plays an important role in unregulated liver growth in liver tumors, in regulated growth during liver regeneration, and in ex vivo embryonic liver cultures. METHODS: We used developing livers from several stages of gestational development to examine beta-catenin expression, protein-protein interactions, localization, and regulation in prenatal and postnatal livers. RESULTS: Microarray, Northern, and protein analyses showed peak expression of beta-catenin during early liver development at Embryonic day 10 (E10)-E12, followed by a decrease and a complete loss of normal beta-catenin (97-kilodalton species) after E16 through the remaining prenatal period. At the early stages, beta-catenin localized to the cytoplasm and nuclei of resident cells in addition to its normal membranous localization, which was seen at all later stages and in adult liver. Decreases in beta-catenin levels at E14 onward coincided with its decreased gene expression and increased degradation, as seen by an increase in serine 45/threonine 41-phosphorylated beta-catenin and its other negative regulators, such as axin, adenomatous polyposis coli gene product (APC), and glycogen synthase kinase-3 beta. Finally, we showed an intact association of E-cadherin and beta-catenin despite the loss of beta-catenin at E16-E18, owing to the presence of membrane-associated smaller-molecular-weight beta-catenin species. CONCLUSIONS: We also identified a stage-specific expression and regulation of beta-catenin during liver development that might be crucial for physiological liver development. Nuclear and cytoplasmic beta-catenin corresponded to cell proliferation in liver development. Finally, a smaller-molecular-weight species of beta-catenin might be maintaining normal interactions at the membrane.

Animals↗
PubMed2003

Gene expression analysis of human soft tissue leiomyosarcomas.

Leiomyosarcoma of the somatic soft tissue is a rare malignant mesenchymal neoplasm that metastasizes to other organs in a subset of cases. Much remains to be learned about the mechanisms underlying the development of aggressive behavior of this tumor. It has been difficult to predict the clinical behavior of leiomyosarcomas using the morphology-based grading system, even though tumor size and histological grade have correlated with biologic behavior in some studies. In this study we analyzed the gene expression patterns of 35 samples of mesenchymal origin, including 11 cases of leiomyosarcomas of different histological grades arising in soft tissue and the retroperitoneum, using the Affymetrix U133a chips, which contain more than 22,000 genes and expression sequence tags (ESTs). We identified a set of genes whose expression was commonly altered in all leiomyosarcoma samples. In addition, we identified specific gene expression patterns in several subsets of the tumor. We used these alterations of gene expression to subclassify the leiomyosarcomas into 3 groups. Interestingly, the grouping of these samples correlated well with tumor differentiation and clinical aggressiveness. The analysis identified 92 genes that distinguish low-grade, well-differentiated leiomyosarcomas from less well-differentiated, high-grade, and metastatic leiomyosarcoma. Thesse alterations of gene expression appear to be correlated with the clinical behavior and histological grade of the tumor. The striking differences in terms of gene expression pattern among leiomyosarcomas of different differentiation status and clinical aggressiveness imply that several genetic abnormalities are responsible for the genesis and progression of this tumor.

Adolescent↗