Genomic instability causes HGF gene activation in colon cancer cells, promoting their resistance to necroptosis.

BACKGROUND & AIMS: Genomic instability promotes colon carcinogenesis by inducing genetic mutations, but not all genes affected by this process have been identified. We investigated whether genomic instability in human colorectal cancer (CRC) cells produces mutations in the hepatocyte growth factor (HGF) gene. METHODS: We genotyped human colon tumor tissues and adjacent nontumor tissues collected from 78 patients University of Pittsburgh Health Sciences and Veterans Hospital, along with 40 human CRC and adjacent nontumor tissues in a commercial microarray. We used cellular, biochemical, and molecular biological techniques to investigate the factors that alter HGF signaling in colon cancer cells and its effects on cell proliferation and survival. RESULTS: All tested human CRC tissues and cell lines that had microsatellite instability contained truncations in the regulatory deoxyadenosine tract element (DATE) of the HGF gene promoter. The DATE was unstable in 14% (11 of 78) of CRC samples; DATE truncation was also polymorphic and detected in 18% (13 of 78) of CRC tissues without microsatellite instability. In CRC cell lines, truncation of DATE activated expression of HGF, resulting in its autocrine signaling via MET. This promoted cell proliferation and resistance to necroptosis. HGF signaling via MET reduced levels of the receptor-interacting serine-threonine kinase 1, a mediator of necroptosis, in CRC cells. High levels of HGF protein in tumor tissues correlated with lower levels of receptor-interacting serine-threonine kinase 1 and shorter survival times of patients. CONCLUSIONS: Thirty-one percent of CRC samples contain alterations in the DATE of the HGF promoter. Disruption of the DATE increased HGF signaling via MET and reduced levels of receptor-interacting serine-threonine kinase 1 and CRC cell necroptosis. DATE alteration might be used as a prognostic factor or to select patients for therapies that target HGF-MET signaling.

Novel Death Defying Domain in Met entraps the active site of caspase-3 and blocks apoptosis in hepatocytes.

UNLABELLED: Met, the transmembrane tyrosine kinase receptor for hepatocyte growth factor (HGF), is known to function as a potent antiapoptotic mediator in normal and neoplastic cells. Herein we report that the intracellular cytoplasmic tail of Met has evolved to harbor a tandem pair of caspase-3 cleavage sites, which bait, trap, and disable the active site of caspase-3, thereby blocking the execution of apoptosis. We call this caspase-3 cleavage motif the Death Defying Domain (DDD). This site consists of the following sequence: DNAD-DEVD-T (where the hyphens denote caspase cleavage sites). Through functional and mechanistic studies, we show that upon DDD cleavage by caspase-3 the resulting DEVD-T peptide acts as a competitive inhibitor and entraps the active site of caspase-3 akin to DEVD-CHO, which is a potent, synthetic inhibitor of caspase-3 activity. By gain- and loss-of-function studies using restoration of DDD expression in DDD-deficient hepatocytic cells, we found that both caspase-3 sites in DDD are necessary for inhibition of caspase-3 and promotion of cell survival. Employing mutagenesis studies, we show that DDD could operate independently of Met's enzymatic activity as determined by using kinase-dead human Met mutant constructs. Studies of both human liver cancer tissues and cell lines uncovered that DDD cleavage and entrapment of caspase-3 by DDD occur in vivo, further proving that this site has physiological and pathophysiological relevance. CONCLUSION: Met can directly inhibit caspase-3 by way of a novel mechanism and promote hepatocyte survival. The results presented here will further our understanding of the mechanisms that control not only normal tissue homeostasis but also abnormal tissue growth such as cancer and degenerative diseases in which apoptotic caspases are at play.

Mesenchymal stem cells enhance survival and bacterial clearance in murine Escherichia coli pneumonia.

RATIONALE: Bacterial pneumonia is the most common infectious cause of death worldwide and treatment is increasingly hampered by antibiotic resistance. Mesenchymal stem cells (MSCs) have been demonstrated to provide protection against acute inflammatory lung injury; however, their potential therapeutic role in the setting of bacterial pneumonia has not been well studied. OBJECTIVE: This study focused on testing the therapeutic and mechanistic effects of MSCs in a mouse model of Gram-negative pneumonia. METHODS AND RESULTS: Syngeneic MSCs from wild-type mice were isolated and administered via the intratracheal route to mice 4 h after the mice were infected with Escherichia coli. 3T3 fibroblasts and phosphate-buffered saline (PBS) were used as controls for all in vivo experiments. Survival, lung injury, bacterial counts and indices of inflammation were measured in each treatment group. Treatment with wild-type MSCs improved 48 h survival (MSC, 55%; 3T3, 8%; PBS, 0%; p<0.05 for MSC vs 3T3 and PBS groups) and lung injury compared with control mice. In addition, wild-type MSCs enhanced bacterial clearance from the alveolar space as early as 4 h after administration, an effect that was not observed with the other treatment groups. The antibacterial effect with MSCs was due, in part, to their upregulation of the antibacterial protein lipocalin 2. CONCLUSIONS: Treatment with MSCs enhanced survival and bacterial clearance in a mouse model of Gram-negative pneumonia. The bacterial clearance effect was due, in part, to the upregulation of lipocalin 2 production by MSCs.

A Novel Humanized Model of NASH and Its Treatment With META4, A Potent Agonist of MET.

BACKGROUND & AIMS: Nonalcoholic fatty liver disease is a frequent cause of hepatic dysfunction and is now a global epidemic. This ailment can progress to an advanced form called nonalcoholic steatohepatitis (NASH) and end-stage liver disease. Currently, the molecular basis of NASH pathogenesis is poorly understood, and no effective therapies exist to treat NASH. These shortcomings are due to the paucity of experimental NASH models directly relevant to humans. METHODS: We used chimeric mice with humanized liver to investigate nonalcoholic fatty liver disease in a relevant model. We carried out histologic, biochemical, and molecular approaches including RNA-Seq. For comparison, we used side-by-side human NASH samples. RESULTS: Herein, we describe a "humanized" model of NASH using transplantation of human hepatocytes into fumarylacetoacetate hydrolase-deficient mice. Once fed a high-fat diet, these mice develop NAFLD faithfully, recapitulating human NASH at the histologic, cellular, biochemical, and molecular levels. Our RNA-Seq analyses uncovered that a variety of important signaling pathways that govern liver homeostasis are profoundly deregulated in both humanized and human NASH livers. Notably, we made the novel discovery that hepatocyte growth factor (HGF) function is compromised in human and humanized NASH at several levels including a significant increase in the expression of the HGF antagonists known as NK1/NK2 and marked decrease in HGF activator. Based on these observations, we generated a potent, human-specific, and stable agonist of human MET that we have named META4 (Metaphor) and used it in the humanized NASH model to restore HGF function. CONCLUSIONS: Our studies revealed that the humanized NASH model recapitulates human NASH and uncovered that HGF-MET function is impaired in this disease. We show that restoring HGF-MET function by META4 therapy ameliorates NASH and reinstates normal liver function in the humanized NASH model. Our results show that the HGF-MET signaling pathway is a dominant regulator of hepatic homeostasis.

Conditional beta-catenin loss in mice promotes chemical hepatocarcinogenesis: role of oxidative stress and platelet-derived growth factor receptor alpha/phosphoinositide 3-kinase signaling.

UNLABELLED: Activation of beta-catenin, the central effector of the canonical Wnt pathway and a recognized oncogene, has been implicated in hepatocellular carcinoma. We examined N-nitrosodiethylamine (DEN)-induced tumorigenesis in hepatic beta-catenin conditional knockout mice (beta-cat KO). Male beta-cat KO and age- and sex-matched littermate controls were given a single intraperitoneal DEN injection and followed for 6-12 months for hepatic tumors. Hepatic tumors were characterized for histology, proliferation, apoptosis, oxidative stress, and specific proteins by way of western blot, immunohistochemistry, and coprecipitation studies. For in vivo tumor intervention studies, specific inhibitors were administered intraperitoneally or through drinking water. Intriguingly, beta-cat KO mice showed a paradoxical increase in susceptibility to DEN-induced tumorigenesis. This accelerated tumorigenesis is due to increased injury and inflammation, unrestricted oxidative stress, fibrosis, and compensatory increase in hepatocyte proliferation secondary to platelet-derived growth factor receptor alpha (PDGFRalpha)/phosphoinositide 3-kinase (PIK3CA)/Akt activation and c-Myc overexpression. In vitro suppression of beta-catenin expression in hepatoma cells led to enhanced PDGFRalpha expression, which was abrogated in the presence of nuclear factor kappaB (NF-kappaB) inhibitor. Daily treatment of 6-month-old DEN-exposed beta-cat KO with PDGFRalpha inhibitor dramatically reduced tumor numbers and size. Inclusion of N-acetyl-L-cysteine, a known antioxidant and NF-kappaB inhibitor, in the drinking water led to complete abolition of tumorigenesis in DEN-exposed beta-cat KO. CONCLUSION: Loss of beta-catenin impairs the liver's ability to counteract DEN-induced oxidative stress and enhances tumorigenesis through PDGFRalpha/PIK3CA/Akt signaling. Blockade of PDGFRalpha or oxidative stress dramatically affects beta-catenin-deficient tumorigenesis. Also, hepatoma cells use PDGFRalpha/PIK3CA signaling as an escape mechanism following beta-catenin suppression, and their sequential suppression profoundly impedes tumor proliferation.

Beta-catenin regulates vitamin C biosynthesis and cell survival in murine liver.

Because the Wnt/beta-catenin pathway plays multiple roles in liver pathobiology, it is critical to identify gene targets that mediate such diverse effects. Here we report a novel role of beta-catenin in controlling ascorbic acid biosynthesis in murine liver through regulation of expression of regucalcin or senescence marker protein 30 and L-gulonolactone oxidase. Reverse transcription-PCR, Western blotting, and immunohistochemistry demonstrate decreased regucalcin expression in beta-catenin-null livers and greater expression in beta-catenin overexpressing transgenic livers, HepG2 hepatoma cells (contain constitutively active beta-catenin), regenerating livers, and in hepatocellular cancer tissues that exhibit beta-catenin activation. Interestingly, coprecipitation and immunofluorescence studies also demonstrate an association of beta-catenin and regucalcin. Luciferase reporter and chromatin immunoprecipitation assays verified a functional TCF-4-binding site located between -163 and -157 (CTTTGCA) on the regucalcin promoter to be critical for regulation by beta-catenin. Significantly lower serum ascorbate levels were observed in beta-catenin knock-out mice secondary to decreased expression of regucalcin and also of L-gulonolactone oxidase, the penultimate and last (also rate-limiting) steps in the synthesis of ascorbic acid, respectively. These mice also show enhanced basal hepatocyte apoptosis. To test if ascorbate deficiency secondary to beta-catenin loss and regucalcin decrease was contributing to apoptosis, beta-catenin-null hepatocytes or regucalcin small interfering RNA-transfected HepG2 cells were cultured, which exhibited significant apoptosis that was alleviated by the addition of ascorbic acid. Thus, through regucalcin and L-gulonolactone oxidase expression, beta-catenin regulates vitamin C biosynthesis in murine liver, which in turn may be one of the mechanisms contributing to the role of beta-catenin in cell survival.

Beta-catenin deletion in hepatoblasts disrupts hepatic morphogenesis and survival during mouse development.

UNLABELLED: Beta-catenin, the central component of the canonical Wnt pathway, plays important roles in the processes of liver regeneration, growth, and cancer. Previously, we identified temporal expression of beta-catenin during liver development. Here, we characterize the hepatic phenotype, resulting from the successful deletion of beta-catenin in the developing hepatoblasts utilizing Foxa3-cyclization recombination and floxed-beta-catenin (exons 2 through 6) transgenic mice. Beta-catenin loss in developing livers resulted in significantly underdeveloped livers after embryonic day 12 (E12) with lethality occurring at around E17 stages. Histology revealed an overall deficient hepatocyte compartment due to (1) increased cell death due to oxidative stress and apoptosis, and (2) diminished expansion secondary to decreased cyclin-D1 and impaired proliferation. Also, the remnant hepatocytes demonstrated an immature phenotype as indicated by high nuclear to cytoplasmic ratio, poor cell polarity, absent glycogen, and decreased expression of key liver-enriched transcription factors: CCAAT-enhancer binding protein-alpha and hepatocyte nuclear factor-4alpha. A paucity of primitive bile ducts was also observed. While the stem cell assays demonstrated no intrinsic defect in hematopoiesis, distorted hepatic architecture and deficient hepatocyte compartments resulted in defective endothelial cell organization leading to overall fetal pallor. CONCLUSION: Beta-catenin regulates multiple, critical events during the process of hepatic morphogenesis, including hepatoblast maturation, expansion, and survival, making it indispensable to survival.

Platelet-derived growth factor receptor-alpha: a novel therapeutic target in human hepatocellular cancer.

Hepatocellular cancer (HCC) is a disease of poor prognosis. Identifying novel molecular aberrations might present opportunities to identify new therapeutic targets. Due to the similarities between the processes of development and cancer, we used early developing livers to identify genes that might play a primary role in HCC. Platelet-derived growth factor receptor-alpha (PDGFRalpha) was identified from microarray using early developing mouse livers. Expression of PDGFRalpha and its upstream effectors, PDGF-AA and PDGF-CC, were examined in HCC tissues (n = 43) by Western blot, real-time PCR, and immunohistochemistry. Finally, effect of anti-PDGFRalpha antibody (mAb 3G3, ImClone Systems, Inc.) was examined on human hepatoma cells. A high expression of PDGFRalpha was observed during early liver development. HCCs (17 of 21) revealed cytoplasmic PDGFRalpha and activated PDGFRalpha (phospho-Tyr(754)) by immunohistochemistry. Additional HCCs (14 of 22) showed elevated PDGFRalpha levels when compared with the adjacent normal livers by Western blots. Of these 14 patients, 3 showed increased PDGFRalpha gene expression, 3 showed elevated PDGF-AA, and 4 had higher PDGF-CC levels in the tumors compared with adjacent livers. Multiple hepatoma cell lines, when treated with mAb 3G3, showed significant decreases in cell proliferation and survival (P < 0.05). In conclusion, approximately 70% of HCC tissues had elevated PDGFRalpha levels due to diverse mechanisms. PDGFRalpha inhibition in hepatoma cells led to diminution of tumor cell survival and proliferation and thus might be of therapeutic significance.

A hepatocyte growth factor receptor (Met)-insulin receptor hybrid governs hepatic glucose metabolism.

Met is the transmembrane tyrosine kinase cell surface receptor for hepatocyte growth factor (HGF) and is structurally related to the insulin receptor (INSR) tyrosine kinase. Here we report that the HGF-Met axis regulates metabolism by stimulating hepatic glucose uptake and suppressing hepatic glucose output. We show that Met is essential for an optimal hepatic insulin response by directly engaging INSR to form a Met-INSR hybrid complex, which culminates in a robust signal output. We also found that the HGF-Met system restores insulin responsiveness in a mouse model of insulin refractoriness. These results provide new insights into the molecular basis of hepatic insulin resistance and suggest that HGF may have therapeutic potential for type 2 diabetes in the clinical setting.

Wnt'er in liver: expression of Wnt and frizzled genes in mouse.

UNLABELLED: The Wnt signaling pathway is essential for a wide array of developmental and physiological processes. Wnts are extracellular ligands that bind to frizzled (Fz) receptors at the membrane, canonically inducing beta-catenin nuclear translocation and activation. Although beta-catenin has been shown to be critical in liver biology, the expression of the 19 Wnt and 10 Fz genes in liver remains undetermined. We report comprehensive analysis of Wnt and Fz expression in whole liver as well as individual cell types: freshly isolated and plated hepatocytes, biliary epithelial cells, normal and activated stellate and Kupffer cells, and sinusoidal endothelial cells (SECs). Oligonucleotides for the 19 Wnt, 10 frizzled receptors genes, and secreted Frizzled-related protein-1 (sFRP or Fzb) were synthesized based on the available sequences. A total of 11 Wnts and 8 Fz genes and Fzb were expressed in normal liver. Although only 6 Wnt and 5 Fz genes were expressed in freshly isolated hepatocytes, 8 Wnt genes, 7 Fz genes, and Fzb were expressed in plated hepatocytes. Although 12 Wnt and 7 Fz genes were expressed in biliary tree, additional Fz9 and Fzb were only expressed in cultured biliary epithelial cells. The same 14 Wnt and 7 Fz genes were expressed in both activated and normal stellate and Kupffer cells; only Fzb was expressed in their activated state. Also, 11 Wnt, seven Fz, and Fzb genes were expressed in SECs. CONCLUSION: These data indicate that most Wnt and frizzled genes are expressed in the liver and might be playing important roles in liver pathobiology via canonical and noncanonical pathways.

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.

Activation of Wnt/beta-catenin pathway during hepatocyte growth factor-induced hepatomegaly in mice.

Hepatocyte growth factor (HGF) and beta-catenin both play a crucial role in stimulating hepatocyte proliferation, but whether these 2 pathways cooperate in inducing hepatocyte proliferation is unclear. We have previously reported that beta-catenin forms a complex with c-Met (HGF receptor) that undergoes dissociation because of beta-catenin tyrosine phosphorylation on stimulation by HGF. It is also known that delivery of the human HGF gene cloned in a plasmid under a CMV promoter results in hepatomegaly in mice. In addition, recently characterized beta-catenin transgenic mice also showed hepatomegaly. The present study was based on the hypothesis that HGF-induced hepatomegaly is mediated, at least in part, by activation of the Wnt/beta-catenin pathway. Here we report that delivery of the human HGF gene delivery in mice led to hepatomegaly via beta-catenin activation in the liver in 1- and 4-week studies. The mechanisms of beta-catenin activation in the 1-week study included loss of c-Met-beta-catenin association as well as canonical beta-catenin activation, leading to its nuclear translocation. In the 4-week study, beta-catenin activation was observed via canonical mechanisms, whereas the c-Met-beta-catenin complex remained unchanged. In both studies there was an associated increase in the E-cadherin-beta-catenin association at the membrane. In addition, we generated liver-specific beta-catenin knockout mice, which demonstrated significantly smaller livers. HGF gene delivery failed to induce hepatomegaly in these beta-catenin conditionally null mice. In conclusion, beta-catenin- and HGF-mediated signaling pathways cooperate in hepatocyte proliferation, which may be crucial in liver development, regeneration following partial hepatectomy, and pathogenesis of hepatocellular carcinoma.

beta-Catenin and met deregulation in childhood Hepatoblastomas.

Activation of the Wnt/beta-catenin and hepatocyte growth factor/Met signaling has been implicated in various tumors. Owing to the cross-talk between these pathways and aberrant redistribution of beta-catenin in hepatoblastomas, we examined their status in this tumor. This study examined changes in beta-catenin and Met in paired pretreatment and post-treatment hepatoblastoma tissues in relation to their effects on proliferation and target genes such as c-myc and cyclin-D1. In this study we compared proliferation indices, beta-catenin staining and its known molecular targets, c-myc and cyclin-D1, and Met, a tyrosine kinase receptor for hepatocyte growth factor in pretreatment and post-treatment specimens. Pretreatment and post-treatment sections from 13 children, ages 11 weeks to 9 years, were analyzed for these markers by immunohistochemistry. All tumors (13 of 13) displayed increased proliferation and beta-catenin (cytoplasmic and nuclear) staining in pretreatment biopsies that remained relatively unaffected after treatment. Aberrant Met staining (cytoplasmic) was observed in all pretreatment samples that decreased considerably after treatment in 11 of 13 patients. A significant subset of these tumors showed increased c-myc and cyclin-D1 staining in pretreatment biopsies that decreased after chemotherapy in most cases. beta-Catenin redistribution in tumor cells corresponds to proliferation in hepatoblastomas. However, beta-catenin nuclear localization remains unaffected in viable hepatoblastoma tissue after chemotherapy. In contrast, Met undergoes a prominent decrease after treatment and thus might be important in pathogenesis of hepatoblastoma.

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.

Fibroblast growth factor enriches the embryonic liver cultures for hepatic progenitors.

Fibroblast growth factors (FGFs) play an important role in hepatic induction during development. The aim of our study was to investigate the effect of exogenous FGFs on ex vivo liver development. We begin our analysis by examining FGF signaling during early mouse liver development. Phospho-FGF receptor (Tyr653/654) was detected in embryonic day 10 (E10) to E12 livers only. Next, E10 livers were cultured in the presence of FGF1, FGF4, or FGF8 for 72 hours and examined for histology, proliferation, apoptosis, and differentiation. FGFs especially FGF8 promoted sheet-like architecture, cell proliferation, and survival as compared to the control. All FGFs induced a striking increase in the number of c-kit and alpha-fetoprotein-positive progenitors, without altering albumin staining. However these progenitors were CK-19-positive (biliary and bipotential progenitor marker) only in the presence of FGF1 or FGF4 and not FGF8. FGFs also induced beta-catenin, a stem cell renewal factor in these cultures. In conclusion, the presence of activated FGFR indicates a physiological role of FGF during early liver development. FGF1 and FGF4 enrich the embryonic liver cultures for bipotential hepatic progenitors. FGF8 promotes such enrichment and induces a one-step differentiation toward a unipotential hepatocyte progenitor. Thus, FGFs might be useful for enrichment and propagation of developmental hepatic progenitors.

[Clinical analysis of binocular anisei konia after laser in situ keratomileusis on myopic patients].

PURPOSE: To explore the effect produced by laser in situ keratomileusis (LASIK) on binocular aniseikonia(BA) and steropsis of myopic patients. METHODS: Sixty-four cases who received LASIK were divided into 4 groups by different binoeular diopter with the binocular aniserkonia (BA) designed by Liugeping and BA. The patients were tested on 6 months before and after the operation respectively for studing the relationship between the post-operative BA and steropsis. RESULTS: When the binocular diopter difference was < or = 2.5 D, there was no significant difference between the preoperative and postoperative BA. When the diopter difference was > 2.50 D, the incongruons images of simultaneous perception and stereoscopic vision after operation had significant divergence compared with those before the operation. The postoperative stereopsis was closely related to binocular vision. CONCLUSION: LASIK can not only reduce the BA of high myopic anisome tropia patients to a range that can be endured, but also be helpful to restore the stereopsis. Moreover, the better the postoperative binocular vision, the patients have the finer the stereopsis will be.

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.

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.

Beta-catenin antisense studies in embryonic liver cultures: role in proliferation, apoptosis, and lineage specification.

BACKGROUND & AIMS: Wnt/beta-catenin pathway activation occurs during liver growth in hepatoblastomas, hepatocellular cancers, and liver regeneration. The aim of this study was to investigate the role of beta-catenin, a key component of the Wnt pathway, in liver development as well as its normal distribution in developing liver. METHODS: Embryonic liver cultures and beta-catenin antisense phosphorodiamidate morpholino oligomer (PMO) were used to elucidate the role of beta-catenin in liver development. Livers from embryos at 10 days of gestational development were cultured in the presence of antisense or control PMO for 72 hours and analyzed. RESULTS: Beta-catenin shows stage-specific localization and distinct distribution compared with known markers in developing liver. A substantial decrease in beta-catenin protein was evident in the organs cultured in the presence of antisense. Beta-catenin inhibition decreased cell proliferation and increased apoptosis in these organ cultures. Presence of antisense resulted in loss of CK19 immunoreactivity of the bipotential stem cells. Beta-catenin inhibition also promoted c-kit immunoreactivity of the hepatocytes. CONCLUSIONS: We conclude that the PMO antisense to beta-catenin effectively inhibits synthesis of its protein. Beta-catenin modulates cell proliferation and apoptosis in developing liver. It may play a significant role in early biliary lineage commitment of the bipotential stem cells and also seems to be important in hepatocyte maturation.