Liver-specific gene expression in mesenchymal stem cells is induced by liver cells

AIM： The origin of putative liver cells from distinct bone marrow stem cells, e.g. hematopoietic stem cells or multipotent adult progenitor cells was found in recent in vitro studies. Cell culture experiments revealed a key role of growth factors for the induction of liver-specific genes in stern cell cultures. We investigated the potential of rat mesenchymal stem cells （MSC） from bone marrow to differentiate into hepatocytic cells in vitro. Furthermore, we assessed the influence of cocultured liver cells on induction of liver-specific gene expression. METHODS： Mesenchymal stem cells were marked with green fluorescent protein （GFP） by retroviral gene transduction. Clonal marked MSC were either cultured under liver stimulating conditions using fibronectin-coated culture dishes and medium supplemented with SCF, HGF, EGF, and FGF-4 alone, or in presence of freshly isolated rat liver cells. Cells in cocultures were harvested and GFP＋ or GFP- cells were separated using fluorescence activated cell sorting. RT-PCR analysis for the stem cell marker Thyl and the hepatocytic markers CK-18, albumin, CK-19, and AFP was performed in the different cell populations. RESULTS： Under the specified culture conditions, rat MSC cocultured with liver cells expressed albumin-, CK-18, CK-19, and AFP-RNA over 3 weeks, whereas MSC cultured alone did not show liver specific gene expression, CONCLUSION： The results indicate that （1） rat MSC from bone marrow can differentiate towards hepatocytic lineage in vitro, and （2） that the microenvironment plays a decisive role for the induction of hepatic differentiation of rMSC.

Cloning and characterization of a mouse liver-specific gene mfrep-1,up-regulated in liver regeneration

Human fibrinogen-related protein-1/liver fibrinogen-related protein-1 (HFREP-l/LFIRE-1), a liver-specific protein, is a member of fibrinogen superfamily that exerts various biological activities. However, the function of HFREP-l/LFIRE-1 in liver remains unknown. Here we isolated its mouse ortholog gene-mouse fibrinogen-related protein-1 (mfrep-1), which encoded 314 amino acids, exhibiting 80.4% similarity to HFREP-l/LFIRE-1. Northern blot analysis revealed that 1.2-kb mfrep-1 mRNA was detected selectively in mouse liver. To explore the function of MFREP-1, we examined the levels of mfrep-1 mRNA during regeneration after 70% partial hepatectomy (PHx) in mice, mfrep-1 mRNA increased in the regenerating liver and reached the first shoulder peak at 2-4 h after PHx. Cycloheximide pretreatment could suppress the induction of mfrep-1, indicating the up-regulation of this gene need de novo protein synthesis. Its mRNA continued to elevate at 6 h thereafter and reached the second peak at 24 h. The enhanced expression of mfrep-1 maintained high until 72 h and then declined slowly to the basal level. Immunohistochemistry assessment confirmed the up-regulated expression of MFREP-1 protein in parenchymal cells during liver regeneration. These data suggested that MFREP-1 might play an important role in liver regeneration and be involved in the regulation of cell growth.

Liver-specific glucocorticoid action in alcoholic liver disease:Study of glucocorticoid receptor knockout and knockin mice

Background and aim:Glucocorticoids are the only first-line drugs for severe alcoholic hepatitis(AH),with limited efficacy and various side effects on extrahepatic tissues.Liver-targeting glucocorticoid therapy may have multiple advantages over systemic glucocorticoid for AH.The aim of this study was to determine the role of hepatocellular glucocorticoid receptor(GR)in alcohol-associated steatosis(AS)and AH.Materials and methods:AS was induced by a high-fat diet plus binge alcohol in adult male and female mice with liver-specific knockout(LKO)and heterozygote of GR.AH was induced by chronic-plus-binge in middle-aged male mice with liver-specific knockin of GR.Changes in hepatic mRNA and protein expression were determined by quantitative real-time polymerase chain reaction and Western blot.Results:GR-LKO aggravated steatosis and decreased hepatic expression and circulating levels of albumin in both genders of AS mice but only increased markers of liver injury in male AS mice.Marked steatosis in GR-LKO mice was associated with induction of lipogenic genes and down-regulation of bile acid synthetic genes.Hepatic protein levels of GR,hepatocyte nuclear factor 4 alpha,and phosphorylated signal transducer and activator of transcription 3 were gene-dosage-dependently decreased,whereas that of lipogenic ATP citrate lyase was increased in male GR heterozygote and LKO mice.Interestingly,hepatic expression of estrogen receptor alpha(ERα)was induced,and the essential estrogen-inactivating enzyme sulfotransferase 1e1 was gene-dosage-dependently down-regulated in GR heterozygote and knockout AS mice,which was associated with induction of ERα-target genes.Liver-specific knockin of GR protected against liver injury and steatohepatitis in middle-aged AH mice.Conclusions:Hepatic GR deficiency plays a crucial role in the pathogenesis of AS induced by high-fat diet plus binge,and liver-specific overexpression/activation of GR protects against chronic-plus-binge-induced AH in middle-aged mice.Hepatocellular GR is important for protection against AS and AH.

Establishment of a transgenic mouse model with liver-specific expression of secretory immunoglobulin D

Mutation of mevalonate kinase （MVK） is thought to account for most cases of hyperimmunoglobulinemia D syndrome （HIDS） with recurrent fever. However, its mechanism and the relationship between elevated serum immunoglobulin D （IgD） and the clinical features of HIDS are unclear. In this study, we generated by fusion PCR a vector to express high levels of chimeric secretory IgD （cslgD） specifically in the liver. We then generated seven founder lines of transgenic mice by co-microinjection, and verified them using genomic PCR and Southern blotting. We detected the expression of csIgD by reverse transcription PCR, quantitative PCR, western blotting, and enzyme-linked immunosorbent assays. We demonstrated that csIgD could be specifically and stably expressed in the liver. We used flow cytometry to show that overexpression of csIgD in the bone marrow and spleen cells had no effect on B cell development. Morphologic and anatomical observation of the transgenic mice revealed skin damage, hepatosplenomegaly, and nephromegaly in some transgenic mice; in these mice, pathological sections showed high levels of cell necrosis and protein-like sediments in the liver, spleen, and kidney. We demonstrated that the genomic insertion sites of the transgeues did not disrupt the MVK gene on mouse chromosome 5. This transgenic mouse will be useful to explore the pathogenesis of HIDS.

Novel liver-specific nitric oxide(NO) releasing drugs with bile acid as both NO carrier and targeting ligand

Novel liver-specific nitric oxide（NO） releasing drugs with bile acid as both the NO carrier and targeting ligand were designed and synthesized by direct nitration of the hydroxyl group in bile acids or the 3-Ohydroxyl alkyl derivatives,with the intact 24-COOH being preserved for hepatocyte specific recognition.Preliminary biological evaluation revealed that oral administrated targeted conjugates could protect mice against acute liver damage induced by acetaminophen or carbon tetrachloride.The nitrate level in the liver significantly increased after oral administration of 1e while nitrate level in the blood did not significantly change.Co-administration of ursodeoxycholic acid（UDCA） significantly antagonized the increase of nitrate in the liver resulted by administration of 1e.

Effect of magnetic resonance imaging in liver metastases

This letter to the editor is a commentary on a study titled"Liver metastases:The role of magnetic resonance imaging."Exploring a noninvasive imaging evaluation system for the biological behavior of hepatocellular carcinoma(HCC)is the key to achieving precise diagnosis and treatment and improving prognosis.This review summarizes the role of magnetic resonance imaging in the detection and evaluation of liver metastases,describes its main imaging features,and focuses on the added value of the latest imaging tools(such as T1 weighted in phase imaging,T1 weighted out of phase imaging;diffusion-weighted imaging,T2 weighted imaging).In this study,I investigated the necessity and benefits of gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid for HCC diagnostic testing and prognostic evaluation.

Generation of the regulatory protein rtTA transgenic mice

AIM: To translate Tet-on system into a conditional mouse model, in which hepatitis B or C virus (HBV or HCV) gene could be spatiotemporally expressed to overcome 'immune tolerance' formed during the embryonic development and 'immune escape' against hepatitis virus antigen(s), an effector mouse, carrying the reverse tetracycline-responsive transcriptional activator (rtTA) gene under the tight control of liver-specific human apoE promoter, is required to be generated. METHODS: To address this end, rtTA fragment amplified by PCR was effectively inserted into the vector of pLiv.7 containing apoE promoter to create the rtTA expressing vector, I.e., pApoE-rtTA. ApoE-rtTA transgenic fragment (-6.9 kb) released from pApoE-rtTA was transferred into mice by pronucleus injection, followed by obtaining one transgene (+) founder animal from microinjection through PCR and Southern blot analysis.RESULTS: rtTA transgene which could be transmitted to subsequent generation (F1) derived from founder was expressed in a liver-specific fashion. CONCLUSION: Taken together, these findings demonstrate that rtTA transgenic mice, in which rtTA expression is appropriately targeted to the murine liver, are successfully produced, which lays a solid foundation to 'off-on-off' regulate expression of target gene (s) (e.g., HBV and/or HCV) in transgenic mice mediated by Tet-on system.

Glucose-responsive artificial promoter-mediated insulin gene transfer improves glucose control in diabetic mice

AIM:To investigate the effect of insulin gene therapy using a glucose-responsive synthetic promoter in type 2 diabetic obese mice.METHODS:We employed a recently developed novel insulin gene therapy strategy using a synthetic promoter that regulates insulin gene expression in the liver in response to blood glucose level changes.We intravenously administered a recombinant adenovirus expressing furin-cleavable rat insulin under the control of the synthetic promoter(rAd-SP-rINSfur) into diabetic Lepr db/db mice.A recombinant adenovirus expressing β-galactosidase under the cytomegalovirus promoter was used as a control(rAd-CMV-βgal).Blood glucose levels and body weights were monitored for 50 d.Glucose and insulin tolerance tests were performed.Immunohistochemical staining was performed to investigate islet morphology and insulin content.RESULTS:Administration of rAd-SP-rINSfur lowered blood glucose levels and normoglycemia was maintained for 50 d,whereas the rAd-CMV-βgal control virus-injected mice remained hyperglycemic.Glucose tolerance tests showed that rAd-SP-rINSfur-treated mice cleared exogenous glucose from the blood more efficiently than control virus-injected mice at 4 wk [area under the curve(AUC):21 508.80 ± 2248.18 vs 62 640.00 ± 5014.28,P < 0.01] and at 6 wk(AUC:29 956.60 ± 1757.33 vs 60 016.60 ± 3794.47,P < 0.01).In addition,insulin sensitivity was also significantly improved in mice treated with rAd-SP-rINSfur compared with rAd-CMV-βgal-treated mice(AUC:9150.17 ± 1007.78 vs 11 994.20 ± 474.40,P < 0.05).The islets from rAd-SP-rINSfur-injected mice appeared to be smaller and to contain a higher concentration of insulin than those from rAd-CMV-βgal-injected mice.CONCLUSION:Based on these results,we suggest that insulin gene therapy might be one therapeutic option for remission of type 2 diabetes.

Expression liver-directed genes by employing synthetic transcriptional control units

AIM： To generate and characcerize the synthetic transoiptional control units for transcriptional targeting of the liver, thereby compensating for the lack of specificity of currently available gene therapeutic vector systems. METHODS： Synthetic transcriptional control unit constructs were generated and analyzed for transcriptional activities in different cell types by FACS quantification, semi-quantitative RT-PCR, and Western blotting. RESULTS： A new bifunctionally-enhanced green fluorescent protein （EGFP）/neor fusion gene cassette was generated, and could flexibly be used both for transcript quantification and for selection of stable cell clones. Then, numerous synthetic transcriptional control units consisting of a minimal promoter linked to “naturally” derived composite enhancer elements from liver-specific expressed genes or binding sites of liver-specific transcription factors were inserted upstream of this reporter cassette. Following liposome-mediated transfection, EGFP reporter protein quantification by FACS analysis identified constructs encoding multimerized composite elements of the apolipoprotein B100 （APOB） promoter or the ornithin transcarbamoylase （OTC） enhancer to exhibit maximum transcriptional activities in liver originating cell lines, but only background levels in non-liver originating cell lines. In contrast, constructs encoding only singular binding sites of liver-specific transcription factors, namely hepatocyte nuclear factor （HNF）, HNF3, HNF4, HNF5, or CAAT/enhancer binding protein （C/EBP） only achieved background levels of EGFP expression. Finally, both semi-quantitative RT-PCR and Western blotting analysis of Hep3B cells demonstrated maximum transcriptional activities for a multimeric 4xAPOB cassette construct, which fully complied with the dataobtained by initial FACS analysis CONCLUSION： Synthetic transcriptional control unit constructs not only exhibit a superb degree of structural compactness, but also provide new means for liver-directed expression of therapeutic genes.

Optimized human factor IX expression cassettes for hepaticdirected gene therapy of hemophilia B

Gene therapy provides a potential cure for hemophilia B, and significant progress has been achieved in liver-directed gene transfer mediated by adeno-associated viral vectors. Recent clinical trials involving the use of a self-complementary adeno-associated virus serotype 8-human codon-optimized factor IX （AAV8-hFIXco） vector demonstrated encouraging efficacy with hFIX expression stabilized at 1% to 6% of normal level in patients, but safety concerns related to high vector doses are still present. Thus, further improvement of AAV vectors and hFIX expression cassette may positively contribute to the ultimate success of hemophilia B gene therapy. In this study, to obtain a higher expression level of hFIX that potentiates the coagulant capacity of recipients, human FIX expression vector was optimized by upgrading the codon adaption index and adjusting the GC content, inserting a Kozak sequence （GCCACC）, and introducing a gain-of-function mutation, R338L （FIX Padua）. The efficiency of the published and the presently constructed cassettes was compared through in vivo screening. In addition, the regulatory elements that control the FIX gene expression in these cassettes were screened for liver-specific effectiveness. Among all the constructed cassettes, scAAV-Pre-hFIXco-SIH-R338L, which was the construct under the control of the prothrombin enhancer and prealbumin promoter, resulted in the highest level of coagulant activity, and the expression levels of two constructed cassettes （scAAV-Chi-hFIXco-SIH-R338L and scAAV-Pre- hFIXco-SIH-R338L） were also higher than that of the published cassette （scAAV-LPI-hFIXco-SJ）. In summary, our strategies led to a substantial increase in hFIX expression at the protein level or a remarkably elevated coagulant activity. Thus, these reconstructs of hFIX with AAV vector may potentially contribute to the creation of an efficacious gene therapy of hemophilia B.