Syngeneic Rat Liver Research Articles

Abstract 6136: Generation and characterization of colorectal cancer cell lines from the Buffalo rat

Abstract Introduction: Rat models of metastatic colorectal cancer (mCRC) to the liver are ideal for the development of transarterial therapies. To date, only one model of CRC cells grown in vitro and implanted into the liver of syngeneic rats is available. Yet, the small size of the WAG-Rij rat limits the use of the CC-531/WAG-Rij model for transarterial interventions. This work was aimed at generating an analogous model on the Buffalo rat, strain that has successfully been used for transarterial interventions in primary liver cancer. Materials and Methods: To induce CRC, 5 6-week old Buffalo rats were treated with Azoxymethane (AOM) and Dextran Sodium Sulfate (DSS). To assess tumor progression, the rats were regularly examined by colonoscopy as well as to T1- and T2-MR imaging from w20 up to w63. Standard procedures of tumor cell isolation and in vitro expansion were used. Once the polyclonal lines were established, expression of E-cadherin, Ceacam5, Mucin1, Vimentin1, and S100a4 was evaluated by flow cytometry (FACS) or qPCR. Additionally, the gene regions of APC (nt. 3049 to 3697) and β-catenin (nt. 371 to 1104) were analyzed by Sanger sequencing. Lastly, 23 adult Buffalo rats received implantations of 0.4-1 × 107 cells in the liver only or in the liver as well as subcutaneously. Results: Colonoscopies, MRIs, and histopathology demonstrated that all 5 rats treated with AOM/DSS developed colorectal neoplasms by w22. Tumors were harvested at various time points, at weeks 24, 32, 45, and 63. 2 polyclonal cell lines derived from invasive adenocarcinomas of a female (18L002) and a male (18L004) were successfully established and have proliferated in vitro >1year. FACS and qPCR results showed that both lines express the epithelial markers E-cadherin, Ceacam5, and Mucin1. Interestingly, despite high E-cadherin expression in both lines (>90% by FACS), qPCR data revealed that both lines also express the mesenchymal markers Vimentin and S100a4, suggestive of a trans-differentiated phenotype. DNA sequences of regions of the APC and β-catenin genes frequently mutated by AOM treatment appeared to be intact in both lines. MRIs of the 23 rats that received implants showed that 4 seem to have allowed for short-term growth (~14d) of the 18L004 cells in the liver before being re-absorbed. Histopathological analyses showed evidence of a serosal reaction at the site of implantation in those cases. Conclusions: Two polyclonal CRC lines with phenotypical characteristics of trans-differentiated cells were successfully generated from AOM/DSS-treated Buffalo rats. In spite of continued in vitro growth for over a year, neither line was tumorigenic in vivo, potentially due to the lack of mutations in key regions of the APC and β-catenin genes. Forced expression of mutant genes known to drive CRC progression such as β-catenin, Kras, or P53 could potentially increase in vivo tumorigenicity and allow for the establishment of a rat model of mCRC suitable for the study of transarterial therapies. Citation Format: Nina M. Muñoz, Crystal J. Dupuis, Katherine A. Dixon, Malea L. Williams, Kiersten L. Maldonado, Asif Rashid, Alda L. Tam. Generation and characterization of colorectal cancer cell lines from the Buffalo rat [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6136.

Fibrosis in small syngeneic rat liver grafts because of damaged bone marrow stem cells from chronic alcohol consumption.

A patient with liver failure due to chronic and acute alcohol abuse under consideration for an urgent liver transplant shortly after stopping alcohol may have residual abnormalities that threaten transplant success, particularly for a small graft. To address this, we studied a model in which reduced-size (50%) Lewis rat livers are transplanted into green fluorescence protein transgenic Lewis recipients after they are fed alcohol or a control diet for 5 weeks. Here we show that normal small Lewis grafts transplanted to alcohol-fed Lewis hosts developed fibrosis, whereas no fibrosis was observed in control-fed recipients. Host-derived CD133 + 8-hydroxy-2'-deoxyguanosine (8-OHdG) cells were significantly increased in livers recovered from both alcohol-fed and control recipients, but only alcohol-fed recipients demonstrated co-staining (a marker of oxidative DNA damage). α smooth muscle actin (α-SMA) staining, a marker for myofibroblasts, also co-localized with CD133 + cells only in the livers of alcohol-fed recipients. Immunostaining and polymerase chain reaction analysis confirmed that chronic alcohol consumption decreased the proportion of bone marrow stem cells (BMSCs) expressing CD133, c-Kit, and chemokine (C-X-C motif) receptor 4 markers and caused oxidative mitochondria DNA (mtDNA) damage. Culture of CD133 + cells from normal rats with medium containing 3% ethanol for 48 hours resulted in elevated mitochondrial 8-OHdG and mtDNA deletion, and ethanol exposure diminished CD133 expression but dramatically increased α-SMA expression. In conclusion, oxidative mtDNA damage and deletions occur in BMSCs of chronic alcohol-fed recipients, and these damaged cells mobilize to the small liver grafts and become myofibroblasts where they play a key role in the subsequent development of fibrosis. Liver Transplantation 23 1564-1576 2017 AASLD.

Erlotinib and sorafenib in an orthotopic rat model of hepatocellular carcinoma

The combination of erlotinib with sorafenib is currently being investigated in a phase III RCT. We studied the effect of erlotinib and sorafenib on HCC in a preclinical model. The Morris Hepatoma (MH) and HepG2 cells were treated in vitro with sorafenib (1-10 μM) and erlotinib (1-5 μM) and evaluated for tumor cell viability, apoptosis, and target regulation. Antiangiogenic effects were studied by measuring VEGF levels, endothelial cell viability, apoptosis, migration, and the aortic ring assay. In vivo, MH cells were implanted into the liver of syngeneic rats and treated with vehicle, sorafenib 5-10mg/kg, erlotinib 10mg/kg, and respective combinations. In vitro, erlotinib downregulated p-ERK but showed no significant effect on tumor cell viability in MH and HEPG2 cells. Despite a similar target regulation, sorafenib significantly reduced cell viability of HCC cells by induction of apoptosis, in a dose-dependent manner (11 ± 5%; 20 ± 10%; 51 ± 5% for sorafenib 1, 5, 10 μM). No additional effect was observed upon combination with erlotinib. Of note, erlotinib treatment resulted in endothelial cell migration and vascular sprouting of aortic rings through induction of VEGF mRNA and protein levels in endothelial and tumor cells, which was blocked by sorafenib. In vivo, erlotinib had no single agent antitumor activity, raised serum-VEGF levels, and lacked a synergistic effect in combination with sorafenib. Erlotinib had no antitumor effect on HCC in vitro nor in vivo, but induced VEGF, which may reflect a resistance mechanism to erlotinib monotherapy. No improvement of sorafenib efficacy was observed upon combination with erlotinib.

Novel organotypic culture model of cholangiocarcinoma progression

Recent studies have suggested that increased α-smooth muscle-actin positive myofibroblastic cells (α-SMA positive CAF) in the desmoplastic stroma may relate to a more aggressive cancer and worse survival outcomes for intrahepatic cholangiocarcinoma (ICC) patients. To facilitate investigating cellular and molecular interactions between α-SMA positive CAF and cholangiocarcinoma cells related to ICC progression, we developed a novel 3-D organotypic culture model of cholangiocarcinoma that more accurately mimics the stromal microenvironment, gene expression profile and select pathophysiological characteristics of desmoplastic ICC in vivo. This unique model was established by co-culturing within a type I collagen gel matrix, a strain of cholangiocarcinoma cells (derived from an ICC formed in syngeneic rat liver following bile duct inoculation of spontaneously-transformed rat cholangiocytes) with varying numbers of clonal α-SMA positive CAF established from the same tumor type. Cholangiocarcinoma cells and α-SMA positive CAF in monoculture each exhibited cell-specific biomarker gene expression profiles characteristic of stromal myofibroblastic cell versus malignant cholangiocyte cell types. In comparison, the gene expression profile and histopathological characteristics exhibited by the organotypic co-culture closely resembled those of whole tissue samples of the parent orthotopic ICC. We further showed α-SMA positive CAF to significantly enhance cholangiocarcinoma cell "ductal-like" growth and cancer cell migration/invasiveness in vitro, as well as to promote upregulated expression of select genes known to be associated with ICC invasion. This novel organotypic model provides an important new resource for studying the effects of microenvironment on cholangiocarcinoma progression in vitro and may have potential as a preclinical model for identifying molecularly targeted therapies.

Differential gene expression profiling of cultured neu-transformed versus spontaneously-transformed rat cholangiocytes and of corresponding cholangiocarcinomas

Previously, we described an orthotopic cholangiocarcinoma model based on bile duct inoculation of spontaneously-transformed low grade malignant rat BDE1 cholangiocytes (BDEsp cells) compared to high grade malignant erbB-2/neu- transformed BDE1 cholangiocytes (BDEneu cells) into the livers of syngeneic rats, which closely mimics clinical features of early versus advanced stages of the human cancer. We now used gene expression microarray together with quantitative real-time RT-PCR to profile genes differentially expressed in highly tumorigenic BDEneu cells and corresponding tumors compared to less aggressive tumorigenic BDEsp cells and tumors. Genes identified as being commonly overexpressed in parent BDEneu cells, tumors, and in a BDEneu tumor-derived cholangiocarcinoma cell line included Sox17, Krt20, Erbb2, and Sphk1 when respectively compared to BDEsp cells, tumors, and tumor-derived BDEsp cholangiocarcinoma cells. Muc1 was also prominently overexpressed in BDEneu cells and tumor-derived cholangiocarcinoma cells over that expressed in corresponding BDEsp cell lines. Periostin and tenascin-C, which were produced exclusively by cholangiocarcinoma-associated fibroblastic cells, were each significantly overexpressed in BDEneu tumors compared to BDEsp tumors. Interestingly, amphiregulin was representative of a gene found to be significantly underexpressed in vitro in BDEneu cells compared to BDEsp cells, but significantly overexpressed in BDEneu tumors compared to BDEsp tumors, and correlated with BDEneu cholangiocarcinoma progression in vivo. Our data support a unique animal model that recapitulates important molecular features of human cholangiocarcinoma progression, and may serve as a potentially powerful preclinical platform for identifying and rapidly testing novel molecular targeting strategies for cholangiocarcinoma therapy and/or prevention.

Abstract 2174: Differential gene overexpression in highly malignant erbB2/neu transformed rat BDE1 cholangiocytes versus less aggressive spontaneously transformed BDE1 cholangiocytes

Abstract Intrahepatic cholangiocarcinoma is a highly malignant primary hepatobiliary cancer often presenting as advanced disease with a dismal prognosis. Despite advances, the molecular pathogenesis of cholangiocarcinoma progression is still poorly understood. Previously, we described a unique “patient-like” animal model of intrahepatic cholangiocarcinoma based on bile duct inoculation of highly malignant erbB2/neu transformed rat BDE1 cholangiocytes (BDEneu cells) compared to less malignant spontaneously transformed BDE1 cholangiocytes (BDEsp cells) into the livers of syngeneic rats (Sirica, A.E., et al. 2008; 47:1178-1190). The aim of this study was to use gene expression microarray analysis, validated with quantitative real-time RT-PCR, to provide a profile of significantly overexpressed gene targets in highly tumorigenic BDEneu cells and resultant tumors compared to less malignant BDEsp cells and tumors. Our microarray analysis identified 4,711 probe sets that were significantly (q < 0.05) different between cultured ErbB2-overexpressing BDEneu cells compared to BDEsp cells and 3,090 probe sets that were significantly different between BDEneu tumors and BDEsp tumors. Among the genes identified as being most abundantly overexpressed in BDEneu cells compared to BDEsp cells included: radixin (57-fold), Sox17 (47-fold), metallothionein 2A (40-fold), insulin-like growth factor binding protein-7 (36-fold), keratin 20 (23-fold), fibronectin 1 (25-fold), transmembrane mucin1 (20-fold), cancer susceptibility candidate 4 (18-fold), tropomyosin 2 (17-fold), bone morphogenetic protein-7 (8-fold), ErbB2 (5-fold), and sphingosine kinase 1 (4-fold). Nine of these twelve abundantly overexpressed genes were also determined to be significantly overexpressed in orthotopic BDEneu liver tumors compared to BDEsp tumors, indicatative of being inherent indicators of transformed BDE cholangiocytes of increased malignant potential. In comparison, the extracellular matrix genes, periostin and tenascin, were found to be overexpressed by >600-fold and >100-fold, respectively, in the BDE liver tumors when compared to the cultured BDE cell lines, supporting their tumor stromal origin. Quantitative real-time RT-PCR analysis of BDEneu tumors further demonstrated a strong positive correlation between increased levels of mRNA transcripts for selected genes (i.e., Sox 17, keratin 20, sphingosine kinase 1, and amphiregulin) and progressively increased liver tumor size. Interestingly, amphiregulin mRNA, which was determined to be underexpressed in cultured BDEneu cells compared to BDEsp cells, was significantly increased in the BDEneu tumors over that expressed in the BDEsp tumors, and appeared to correlate with progression in BDEneu cholangiocarcinoma. (Supported by R01 CA 39225 and R01 CA 83650 to A.E.S). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2174.

Significance of Portosystemic Shunt on Graft Survival in Liver Transplantation: A Rat Model

Aims Distal splenorenal shunt effectively controls bleeding from esophageal and gastric varices but has a different effect on liver transplantation. This study sought to develop an animal model in rats to mimic the recipient with a portosystemic shunt and to investigate its hemodynamic consequences on liver transplantation. Methods We prepared 5 groups of allogeneic or syngeneic rat liver transplantation models with versus without portosystemic shunt, to investigate its effects on graft survival and portal flow. To explore the effects of excessive portal flow on graft survival in small-for-size liver transplantation, we transplanted partial liver grafts into syngeneic recipients. Results In allogeneic combinations, graft survival among the shunt group was shortened compared with their control counterparts. The graft survival of the large shunt group was significantly lower than that of a small shunt or without shunt group in a syngeneic liver transplantation model. Portal blood pressure of the large shunt group was significantly lower than that of the small shunt group. In contrast, excessive portal flow resulted in dysfunction of liver graft in small-for-size liver transplantation. Conclusions These results suggested that reduction in portal flow by portosystemic shunt lead to an acceleration of acute rejection and subsequent liver graft dysfunction, but it may be applicable to regulate the excessive portal flow in small-for-size transplantations. This study showed a valuable model mimicking the recipient with a portosystemic shunt.

Inhibition of mTOR in combination with doxorubicin in an experimental model of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is resistant to chemotherapy. We reported that sirolimus, an mTOR inhibitor, has antiangiogenic properties in HCC. Since antiangiogenic therapy may enhance chemotherapy effects, we tested the antitumorigenic properties of sirolimus combined with doxorubicin in experimental HCC. Morris Hepatoma (MH) cells were implanted into livers of syngeneic rats. Animals were assigned to sirolimus, pegylated liposomal doxorubicin, both combined or control groups. Tumoral growth was followed by MRI. Antiangiogenic effects were assessed by CD31 immunostaining and capillary tube formation assays. Cell proliferation was monitored in vitro by thymidine incorporation. Expression of p21 and phosphorylated MAPKAP kinase-2 was quantified by immunoblotting. Animals treated with the combination developed smaller tumors with decreased tumor microvessel density compared to animals that received monotherapies. In vitro, inhibition of mTOR further impaired capillary formation in the presence of doxorubicin. Doxorubicin reduced endothelial cell proliferation; inhibition of mTOR accentuated this effect. Doxorubicin stimulated p21 expression and the phosphorylation of MAPKAP kinase-2 in endothelial cells. Addition of mTOR inhibitor down-regulated p21, but did not decrease MAPKAP kinase-2 phosphorylation. Sirolimus has additive antitumoral and antiangiogenic effects when administered with doxorubicin. These findings offer a rationale for combining mTOR inhibitors with chemotherapy in HCC treatment.

Celecoxib inhibits growth of tumors in a syngeneic rat liver metastases model for colorectal cancer

IntroductionNonsteroidal anti-inflammatory drugs (NSAIDs) have been shown to reduce the risk of colorectal cancer in cyclooxygenase-2 (COX-2) overexpressing colorectal cancers. The present study was designed to evaluate the inhibitory effects of the COX-2 inhibitor celecoxib on the growth of colorectal cancer liver metastases in a syngeneic rat model, CC531.Materials and methodsThe effects of celecoxib on cell viability in vitro were evaluated by treatment of CC531 tumor cell cultures with celecoxib. In vivo, Wag/Rij rats were inoculated with CC531 tumor cells at two sites in the liver and treated with celecoxib starting one week before, or directly after tumor inoculation. Control rats were inoculated without treatment. Three weeks after tumor inoculation rats were sacrificed. Tumor size, immune cell infiltration, caspase-3 activity, PGE2 and celecoxib levels were determined.ResultsCC531 tumors did not show COX-2 expression. Tumor growth was significantly inhibited by celecoxib treatment in a dose dependent manner. Immune cell infiltration was decreased after celecoxib treatment, indicating that the immune system was not involved in preventing tumor growth. Tumor caspase-3 levels were only significantly increased if treatment was started before tumor inoculation. Celecoxib serum concentration starting at 0.84 μg/ml significantly inhibited the outgrowth of CC531 liver tumors. In contrast, in vitro concentrations of celecoxib of at least 12 μg/ml were needed to affect tumor cell viability.ConclusionThese results suggest that the inhibitory effects of celecoxib on tumor growth are not by direct cytotoxicity, but by creating an unfavorable environment for tumor growth.

Vascular remodeling and antitumoral effects of mTOR inhibition in a rat model of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is amenable to only few treatments. Inhibitors of the kinase mTOR are a new class of immunosuppressors already in use after liver transplantation. Their antiproliferative and antiangiogenic properties suggest that these drugs could be considered to treat HCC. We investigated the antitumoral effects of mTOR inhibition in a HCC model. Hepatoma cells were implanted into livers of syngeneic rats. Animals were treated with the mTOR inhibitor sirolimus for 4 weeks. Tumor growth was monitored by MR imaging. Antiangiogenic effects were assessed in vivo by microvessel density and corrosion casts and in vitro by cell proliferation, tube formation and aortic ring assays. Treated rats had significantly longer survival and developed smaller tumors, fewer extrahepatic metastases and less ascites than controls. Sirolimus decreased intratumoral microvessel density resulting in extensive necrosis. Endothelial cell proliferation was inhibited at lower drug concentrations than hepatoma cells. Tube formation and vascular sprouting of aortic rings were significantly impaired by mTOR inhibition. Casts revealed that in tumors treated with sirolimus vascular sprouting was absent, whereas intussusception was observed. mTOR inhibition significantly reduces HCC growth and improves survival primarily via antiangiogenic effects. Inhibitors of mTOR may have a role in HCC treatment.

Hydrodynamic Plasmid DNA Gene Therapy Model in Liver Transplantation

There is great interest in the field of transplantation to genetically modify grafts to decrease preservation injury or allograft rejection. Although adenoviral gene transfer has been effective in experimental liver transplantation, viral toxicity and safety concerns limit potential use in clinical trials. Therefore, the purpose of this study was to develop a model of nonviral gene transfer in the liver transplant setting, allowing for efficient transgene expression. Orthotopic syngeneic rat liver transplantation was performed with 3 h cold ischemia using University of Wisconsin (UW) preservation. A hydrodynamic gene transfer technique was developed where plasmid DNA was delivered to the liver graft by ex vivo rapid infusion of DNA in UW via the IVC with other vessels clamped. Expression plasmids for the marker genes luciferase and secreted human alpha1-antitrypsin (alpha1-AT) were used. Hepatic injury was assessed by graft histology and liver transaminases. Transgene expression was determined by hepatic luciferase relative light units activity (RLU) and serum alpha1-AT protein levels. Variables examined included the effect of (a) volume injected on the intravenous pressure in the liver graft; (b) injury to the liver, as measured by hepatic enzymes and histopathology; (c) variable expression between lobes; (d) volume of UW that the plasmid is administered in; (e) amount of DNA plasmid; (f) type of the promoter used; (g) clamp time; as well as (h) the time course of the marker gene expression. Control rats underwent standard orthotopic syngeneic rat liver transplantation and had no detectable hepatic luciferase activity or serum human alpha1-AT. The optimal DNA plasmid dose was found to be 400 mug/liver graft, as there was no increase in the luciferase expression by increasing the dose. Furthermore, cytomegalovirus promoter yielded greater expression than Rous sarcoma virus. A high injection pressure gradient allowed for more efficient transgene expression, but produced greater liver injury shown by elevated transaminases and centrilobular necrosis. Lowering injection volume from 75 to 50% of graft weight decreased liver injury by 4.5-fold. Although higher UW injection volumes were associated with increased expression, volumes of only 50% led to luciferase expression up to 10,000,000 RLU/mg; this expression was homogeneous between the different liver lobes. Human alpha1-AT was detected in recipient blood as early as 6 h, peaked at 24 h, and remained high for 5 days. We have developed a nonviral gene transfer technique where hydrostatic pressure across the cold-preserved liver vascular bed allows for efficient plasmid DNA delivery. This simple strategy should prove useful to genetically modify liver grafts in the transplantation setting.

Magnetic Resonance Imaging of Intrahepatically Transplanted Islets Using Paramagnetic Beads

Superparamagnetic agents can be reliably used for magnetic resonance imaging (MRI) of pancreatic islets located in the liver sinusoids. However, the main disadvantages seemed to be the rather long culture time necessary for islet labeling and the low specificity of these agents. In the present study we investigated a more specific approach with a shorter labeling time using immunomagnetic particles. Isolated rat islets were cultivated with immunomagnetic beads coated with antibody against rat MHC class I antigen. Labeled islets were transplanted into the livers of syngeneic rats. The animals were examined weekly by MRI or livers explanted 10 minutes after islet transplantation for in vitro experiments. In both in vitro and in vivo studies, labeled transplanted islets were imaged as hypointensive spots, diffusely distributed throughout the liver. This experiment represents an alternative way of islet imaging by magnetic resonance, which is as effective as the use of known superparamagnetic contrast agents and more specific owing to targeting to specific donor antigens.

Resistance to tumour challenge after tumour laser thermotherapy is associated with a cellular immune response.

Previous studies in our laboratory have shown that interstitial laser thermotherapy (ILT) of an experimental liver tumour is superior to surgical excision, at least partly due to a laser-induced immunological effect. The aim of the present study was to investigate the time–response relationship of the ILT-induced immunisation and the cellular response of macrophages and lymphocytes. A dimethylhydrazine-induced adenocarcinoma was transplanted into the liver of syngeneic rats. Rats with tumour were treated 6–8 days later (tumour size 0.25–0.40 cm3) with ILT of tumour or resection of the tumour-bearing lobe. Two groups of rats without tumour were treated with resection of a normal liver lobe or ILT of normal liver. A challenging tumour was implanted into the liver of each rat 2, 5 or 10 weeks after primary treatment. Rats were killed 6, 12 and 48 days (or earlier due to their condition) after challenge (n=8 in all groups). Immunohistochemical techniques were used to determine lymphocytes (CD8, CD4) and macrophages (ED1, ED2) in rats having had treatment of a primary tumour. Interstitial laser thermotherapy of the first tumour was followed by eradication of challenging tumour and absence of tumour spread. This contrasted with rapid growth and spread of challenging tumour in the other groups. In the challenging vital tumour tissue and in the interface between the tumour and surroundings, the number of ED1 macrophages and CD8 lymphocytes was higher in rats having been treated with the ILT of tumour than in those having undergone resection of the tumour-bearing lobe. The number of ED2 macrophages and CD4 lymphocytes was low and did not vary between these two groups. Interstitial laser thermotherapy elicited an immune response that eradicated a challenging tumour and was associated with increased numbers of tumour-infiltrating macrophages and CD8 lymphocytes.

Benefit of Kupffer cell modulation with glycine versus Kupffer cell depletion after liver transplantation in the rat: effects on postischemic reperfusion injury, apoptotic cell death graft regeneration and survival*

Inhibition or destruction of Kupffer cells (KC) may protect against ischemia-reperfusion (IR) induced primary graft nonfunction (PNF) in liver transplantation. Besides KC activation, PNF is characterized by microvascular perfusion failure, intrahepatic leukocyte accumulation, cell death and hepatocellular dysfunction. KCs can be inactivated by different agents including gadolinium chloride (GdCl3), methyl palmitate (MP) and glycine. The effects of three KC inactivators on IR-injury after rat liver transplantation were compared in the present study. Lewis liver donors were treated with GdCl3, MP, glycine or saline (control). Liver grafts were transplanted following 24 h storage (UW solution). KC populations and IR damage were assessed by histologic analysis, quantitative real-time polymerase chain reaction (RT-PCR) and intravital microscopy. The number of hepatic ED-1 positive macrophages was diminished after GdCl3 (114.8+/-4.4/mm2 liver tissue) and MP treatment (176.0+/-5.0), versus the glycine (263.9+/-5.5) and control (272.1+/-5.6) groups. All three treatment modalities downregulated phagocytic activity for latex particles, paralleled by reduced microvascular injury (acinar perfusion index, GdCl3: 0.75+/-0.03; MP: 0.83+/-.03; glycine: 0.84+/-0.03; 0.63+/-0.03). Quantitative RT-PCR revealed elevated myeloperoxidase mRNA after glycine versus GdCl3 and MP pretreatment (3.2- and 3.4-fold, P=0.011, respectively), without difference to controls (2.9-fold of glycine). TNFalpha-mRNA was reduced after glycine- (5.2-fold), GdCl3- (19.7-fold), MP-treatment (39.5-fold) compared with controls. However, profound prevention of intrahepatic cell death and liver graft failure was solely achieved with glycine preconditioning. Different than GdCl3 and MP, glycine modulates rather than destroys KCs. Glycine appears to preserve cell viability and to TNFalpha/leukocyte dependent organ regeneration capacity, which is related to increase graft survival following liver transplantation.

Nuclear factor-κB decoy oligodeoxynucleotides attenuates ischemia/reperfusion injury in rat liver graft

To evaluate the protective effect of NF-kappaB decoy oligodeoxynucleotides (ODNs) on ischemia/reperfusion (I/R) injury in rat liver graft. Orthotopic syngeneic rat liver transplantation was performed with 3 h of cold preservation of liver graft in University of Wisconsin solution containing phosphorothioated double-stranded NF-kappaB decoy ODNs or scrambled ODNs. NF-kappaB decoy ODNs or scrambled ODNs were injected intravenously into donor and recipient rats 6 and 1 h before operation, respectively. Recipients were killed 0 to 16 h after liver graft reperfusion. NF-kappaB activity in the liver graft was analyzed by electrophoretic mobility shift assay (EMSA). Hepatic mRNA expression of TNF-alpha, IFN-gamma and intercellular adhesion molecule-1 (ICAM-1) were determined by semiquantitative RT-PCR. Serum levels of TNF-alpha and IFN-gamma were measured by enzyme-linked immunosorbent assays (ELISA). Serum level of alanine transaminase (ALT) was measured using a diagnostic kit. Liver graft myeloperoxidase (MPO) content was assessed. NF-kappaB activation in liver graft was induced in a time-dependent manner, and NF-kappaB remained activated for 16 h after graft reperfusion. NF-kappaB activation in liver graft was significant at 2 to 8 h and slightly decreased at 16 h after graft reperfusion. Administration of NF-kappaB decoy ODNs significantly suppressed NF-kappaB activation as well as mRNA expression of TNF-alpha, IFN-gamma and ICAM-1 in the liver graft. The hepatic NF-kappaB DNA binding activity [presented as integral optical density (IOD) value] in the NF-kappaB decoy ODNs treatment group rat was significantly lower than that of the I/R group rat (2.16+/-0.78 vs 36.78+/-6.35 and 3.06+/-0.84 vs 47.62+/- 8.71 for IOD value after 4 and 8 h of reperfusion, respectively, P<0.001). The hepatic mRNA expression level of TNF-alpha, IFN-gamma and ICAM-1 [presented as percent of beta-actin mRNA (%)] in the NF-kappaB decoy ODNs treatment group rat was significantly lower than that of the I/R group rat (8.31+/-3.48 vs 46.37+/-10.65 and 7.46+/- 3.72 vs 74.82+/-12.25 for hepatic TNF-alpha mRNA, 5.58+/-2.16 vs 50.46+/-9.35 and 6.47+/-2.53 vs 69.72+/-13.41 for hepatic IFN-gamma mRNA, 6.79+/-2.83 vs 46.23+/-8.74 and 5.28+/-2.46 vs 67.44+/-10.12 for hepatic ICAM-1 mRNA expression after 4 and 8 h of reperfusion, respectively, P<0.001). Administration of NF-kappaB decoy ODNs almost completely abolished the increase of serum level of TNF-alpha and IFN-gamma induced by hepatic ischemia/reperfusion, the serum level (pg/mL) of TNF-alpha and IFN-gamma in the NF-kappaB decoy ODNs treatment group rat was significantly lower than that of the I/R group rat (42.7+/-13.6 vs 176.7+/-15.8 and 48.4+/-15.1 vs 216.8+/-17.6 for TNF-alpha level, 31.5+/-12.1 vs 102.1+/-14.5 and 40.2+/-13.5 vs 118.6+/-16.7 for IFN-gamma level after 4 and 8 h of reperfusion, respectively, P<0.001). Liver graft neutrophil recruitment indicated by MPO content and hepatocellular injury indicated by serum ALT level were significantly reduced by NF-kappaB decoy ODNs, the hepatic MPO content (A655) and serum ALT level (IU/L) in the NF-kappaB decoy ODNs treatment group rat was significantly lower than that of the I/R group rat (0.17+/-0.07 vs 1.12+/-0.25 and 0.46+/-0.17 vs 1.46+/-0.32 for hepatic MPO content, 71.7+/-33.2 vs 286.1+/-49.6 and 84.3+/-39.7 vs 467.8+/-62.3 for ALT level after 4 and 8 h of reperfusion, respectively, P<0.001). The data suggest that NF-kappaB decoy ODNs protects against I/R injury in liver graft by suppressing NF-kappaB activation and subsequent expression of proinflammatory mediators.

Activation of the lipopolysaccharide signaling pathway in hepatic transplantation preservation injury.

Endotoxin or lipopolysaccharide (LPS) initiates a cascade of complications of septic shock and multiple organ failure seen in Gram-negative bacterial infections. The first step of this pathway, which leads to activated nuclear factor (NF)-kappaB, activating protein (AP)-1, and other transcription factors, is the formation of the LPS receptor complex by LPS, LPS-binding protein (LBP), CD14, and toll-like receptor (TLR) 2 or 4. We examined whether the LPS signaling pathway is activated by hepatic ischemia/reperfusion injury in the transplant setting. Orthotopic syngeneic rat liver transplantation was performed with 0 to 18 hr of cold preservation in University of Wisconsin solution. Animals were killed 1 to 48 hr after reperfusion. Northern blot analysis for CD14, LBP, and TLR2 mRNA, immunohistochemistry for LBP, liver enzyme analysis, and gel shift assay for NF-kappaB and AP-1 were performed. LPS levels were elevated early after reperfusion. Aspartate aminotransferase and alanine aminotransferase maximally increased 12 hr after transplantation. LBP mRNA and protein and CD14 mRNA were significantly up-regulated peaking at 6 to 12 hr after reperfusion. TLR2 mRNA was also increased. NF-kappaB activity showed a biphasic peak at 1 to 3 hr and 12 hr after reperfusion, whereas AP-1 activity showed a peak at 3 to 6 hr. The induction of CD14 mRNA correlated with the length of cold ischemia time. These data indicate that multiple components of the LPS signaling pathway are activated during ischemia/reperfusion injury after liver transplantation.

Isolation, short-term culture, and transplantation of small hepatocyte-like progenitor cells from retrorsine-exposed rats.

Complete liver regeneration after partial hepatectomy (PH) in rats treated with the pyrrolizidine alkaloid retrorsine can be accomplished through the activation, expansion, and differentiation of a novel population of small hepatocyte-like progenitor cells (SHPCs). These cells have not been isolated in pure form, established in primary culture, or transplanted into syngeneic rats to examine their differentiation potential. Primary liver cells enriched for SHPCs were prepared by differential centrifugation of primary liver cell dispersions from retrorsine-exposed rats 6-8 days and 13-15 days after PH. Isolated SHPCs were characterized for cell size, morphology, and expression of cell type-specific markers (including hepatocyte and bile duct-oval cell markers), and established in short-term primary culture. Isolated SHPCs were transplanted into the livers of syngeneic rats to evaluate their ability to engraft and differentiate into mature hepatocytes. SHPCs obtained from retrorsine-exposed rats 6-8 days and 13-15 days after PH were small (10-12 microm in diameter), morphologically resembled hepatocytes, and were predominantly H.4 antigen-positive, alpha-fetoprotein-positive, and OV6-negative. SHPCs did not proliferate in culture and could not be passaged, but short-term cultures were established using protein substrates (collagen or laminin) and defined medium containing epidermal growth factor and nicotinamide. After transplantation into the livers of syngeneic hosts, SHPCs insert into hepatic plates and give rise to differentiated hepatocyte progeny. The SHPC-derived hepatocyte progeny express a differentiated phenotype (albumin-positive, transferrin-positive, alpha-fetoprotein-negative) and are able to proliferate in vivo in response to the growth stimulus provided by PH. The results demonstrate that enriched SHPC populations can be isolated from retrorsine-exposed rats and established in short-term culture, and they can engraft and differentiate after transplantation into the livers of syngeneic rats.

Cytological Examination of Rat Amniotic Epithelial Cells and Cell Transplantation to the Liver

It is hoped that amniotic epithelial cells can be useful in cell-mediated gene therapy. We report here an experimental cell transplantation model of amniotic cells in rats. There is an anatomical difference between human and rodent embryos. We established a method to isolate amniotic cells that are equivalent to human amniotic epithelial cells. An amniotic membrane distinct from the yolk sac was carefully collected and teased in saline containing deoxyribonuclease and hyaluronidase, followed by collagenase digestion. The cell yield was approximately 10(6) cells per pregnant female (10(5) cells per fetus), roughly in proportion to the age of fetus used, and 60% of the isolated cells were attached to the dish under culture conditions. Telomerase activity was higher in the cells isolated from fetuses in the middle stage (day 13.5 to 15.5) than in the late stage (day 17.5 to 21.5). Adherent cells exhibited two to three times more cell division, resulting in a ninefold increase in the number of cells. Immunohistochemical analysis revealed that approximately half of the adherent cells were albumin positive and formed clusters. The senescent cells survived for 2 months without apparent morphological changes. The adherent cells were able to be stored in liquid nitrogen and had a viability of 70% when thawed. Gene transduction with adenovirus vector was highly effective for rat amniotic cells. Transplantation of lacZ transfected amniotic cells into syngeneic rat liver resulted in the integration of the transplanted cells in the liver structure and the cells survived for at least 30 days.

Expression of GMP-140 (P-selectin) correlates with graft viability in cold-preserved rat livers.

Ischemia/reperfusion injury is an inflammatory process involving cytokine release, Kupffer cell activation, and sinusoidal endothelial cell activation. GMP-140 is synthesized by endothelial cells. We analyzed by Western blotting the expression of GMP-140 in a syngeneic rat liver transplantation model using grafts preserved for different periods of time. Compared with prereperfusion samples, expression did not change significantly in freshly harvested and 4-hr preserved livers. In grafts preserved for 24 hr (100% survival), GMP-140 levels increased dramatically at 1 hr, then returned to baseline at 24 hr after transplantation. Forty-eight hour preserved grafts (0% survival) showed a decreasing expression. To identify possible mediators, the effects of tumor necrosis factor-alpha and interleukin-1beta on GMP-140 expression in primary sinusoidal endothelial cells were analyzed. These cytokines increased both the percentage of stained cells as well as their mean staining fluorescence. The absence of increase in 48-hr grafts suggests that GMP-140 may distinguish viable from nonviable livers.

Endogenous natural killer cells do not play a role in antitumor effects induced by interleukin-2 in a syngeneic rat colon tumor model.

Previous experiments in a syngeneic rat liver tumor model using the colon adenocarcinoma CC531 demonstrated that injection of interleukin-2 (IL-2) induced significant antitumor responses. Furthermore, it was found that this treatment strategy was accompanied by an increase in the number of natural killer (NK) cells in and around the tumor. In the present study, the role of endogenous NK cells in IL-2-mediated antitumor responses was further elucidated by depleting tumor-bearing rats of NK cells, using the anti-CD161A mouse IgG1 antibody 3.2.3. Rats were depleted either after or prior to tumor induction and subsequently treated with IL-2. The results demonstrated that depletion of NK cells in tumor-bearing rats did not influence IL-2-induced antitumor effects. In addition, injection of IL-2 in NK-cell-depleted rats induced repopulation of NK cells in the peripheral blood from 3 days on and further after the last injection with IL-2. Therefore, the possibility cannot be excluded that de novo recruited NK cells play a role in attaining IL-2 mediated antitumor effects, but NK cells, which were present before or during the start of IL-2 therapy, were not relevant.