Early Phase Of Liver Regeneration Research Articles

Calcium signalling from the type I inositol 1,4,5-trisphosphate receptor is required at early phase of liver regeneration.

Liver regeneration is a multistage process that unfolds gradually, with different mediators acting at different stages of regeneration. Calcium (Ca(2+) ) signalling is essential for liver regeneration. In hepatocytes, Ca(2+) signalling results from the activation of inositol 1,4,5-trisphosphate receptors (InsP3 R) of which two of the three known isoforms are expressed (InsP3 R-I and InsP3 R-II). Here, we investigated the role of the InsP3 R-I-dependent Ca(2+) signals in hepatic proliferation during liver regeneration. Partial hepatectomy (HX) in combination with knockdown of InsP3 R-I (AdsiRNA-I) was used to evaluate the role of InsP3 R-I on liver regeneration and hepatocyte proliferation, as assessed by liver to body mass ratio, PCNA expression, immunoblots and measurements of intracellular Ca(2+) signalling. AdsiRNA-I efficiently infected the liver as demonstrated by the expression of β-galactosidase throughout the liver lobules. Moreover, this construct selectively and efficiently reduced the expression of InsP3 R-I, as evaluated by immunoblots. Expression of AdsiRNA-I in liver decreased peak Ca(2+) amplitude induced by vasopressin in isolated hepatocytes 2 days after HX. Reduced InsP3 R-I expression prior to HX also delayed liver regeneration, as measured by liver to body weight ratio, and reduced hepatocyte proliferation, as evaluated by PCNA staining, at the same time point. At later stages of regeneration, control hepatocytes showed a decreased expression of InsP3 R, as well as reduced InsP3 R-mediated Ca(2+) signalling, events that did not affect liver growth. Together, these results show that InsP3 R-I-dependent Ca(2+) signalling is an early triggering pathway required for liver regeneration.

An integrated approach for the identification of USF1-centered transcriptional regulatory networks during liver regeneration

Liver regeneration after partial hepatectomy (PH) is a synchronized process that is precisely controlled by system-wide transcriptional regulatory networks. To clarify the transcriptional changes and regulatory networks that involve transcription factors (TFs) and their target genes during the priming phase, an advanced mouse oligonucleotide array-based transcription factor assay (MOUSE OATFA), mRNA microarray analysis, bioinformatic analysis and ChIP-on-chip experiments were used. A total of 774 genes were upregulated or downregulated in PH liver samples compared with the sham operation (SH) group. Seventeen TFs showed significant changes in activity in the regenerating livers, some of which have not been extensively studied in previous reports, including upstream stimulatory transcription factor 1 (USF1). The TF signatures from MOUSE OATFA were combined with mRNA expression profiles and ChIP-on-chip analyses to construct experimental transcriptional regulatory networks in regenerating livers. USF1-centered regulatory networks were further confirmed by ChIP assays, revealing some of its target genes and novel coregulatory networks. The combination of MOUSE OATFA with transcriptome profiling and bioinformatic analysis represents a novel paradigm for the comprehensive prediction of transcriptional coregulatory networks during the early phase of liver regeneration.

Proteomic analysis of the regenerating liver following 2/3 partial hepatectomy in rats.

BackgroundLiver regeneration (LR) after 2/3 partial hepatectomy (PH) is one of the most studied models of cell, organ, and tissue regeneration. Although the transcriptional profile analysis of regenerating liver has been carried out by many reserachers, the dynamic protein expression profile during LR has been rarely reported up to date. Therefore, this study aims to detect the global proteomic profile of the regenerating rat liver following 2/3 hepatectomy, thereby gaining some insights into hepatic regeneration mechanism.ResultsProtein samples extracted from the sham-operated and the regenerating rat livers at 6, 12, 24, 72, 120 and 168 h after PH were separated by IEF/SDS-PAGE and then analyzed by MALDI-TOF/TOF mass spectrometry. Compared to sham-operated groups, there were totally 220 differentially expressed proteins (including 156 up-regulated, 62 down-regulated, and 2 up/down-regulated ones) identified in the regenerating rat livers, and most of them have not been previously related to liver regeneration. According to the expression pattern analysis combined with gene functional analysis, it showed that lipid and carbohydrate metabolism were enhanced at the early phase of LR and continue throughout the regeneration process. Ingenuity Pathway Analysis indicated that YWHAE protein (one of members of the 14-3-3 protein family) was located at the center of pathway networks at all the timepoints after 2/3 hepatectomy under our experimental conditions, maybe suggesting a central role of this protein in regulating liver regeneration. Additionally, we also revealed the role of Cdc42 (cell division cycle 42) in the termination of LR.ConclusionsFor the first time, our proteomic analysis suggested an important role of YWHAE and pathway mediated by this protein in liver regeneration, which might be helpful in expanding our understanding of LR amd unraveling the mechanisms of LR.Electronic supplementary materialThe online version of this article (doi:10.1186/0717-6287-47-59) contains supplementary material, which is available to authorized users.

Epigallocatechin Gallate Does Not Accelerate the Early Phase of Liver Regeneration After Partial Hepatectomy in Rats

BackgroundTwo-thirds partial hepatectomy (PHx) is an established model for the study of liver regeneration after resection. This process is accompanied by oxidative stress.AimsIn our study, we tested the effect of epigallocatechin gallate (EGCG), a green tea antioxidant, on the early phase of liver regeneration after PHx.MethodsMale Wistar rats were divided into five groups: (I) laparotomy + water for intraperitoneal injections, (II) laparotomy + EGCG 50 mg/kg body weight, (III) PHx + water for injections, (IV) PHx + EGCG 20 mg/kg and (V) PHx + EGCG 50 mg/kg, for 3 consecutive days. The rats were killed 24 h after surgery. Biochemical analysis of rat sera was performed. Histological samples were stained with hematoxylin & eosin and bromodeoxyuridine (BrdU). In hepatectomized rats, we also measured plasma malondialdehyde, tissue malondialdehyde, glutathione and cytokines levels, the activity of caspases 3/7, expression of Nqo-1 and HO-1 genes at the mRNA level, and expression of p21, p-p27 and p-p53 genes at the protein level.ResultsWe observed lower accumulation of BrdU in group V when compared to groups III and IV. The activity of caspases 3/7 and expression of p-p53 were lower in group V than in groups III and IV. Tissue levels of IL-6 were lower in group V when compared to group III. Significant differences were not noted in other parameters.ConclusionsAdministration of EGCG did not stimulate early phase liver regeneration in rats after PHx. There was even lower DNA synthesis in the group treated with a high dose of EGCG.

Downregulation of hepatic stimulator substance during the early phase of liver regeneration inhibits E-cadherin expression in mice

Hepatic stimulatory substance (HSS), which encodes a sulfhydryl oxidase enzyme, promotes liver regeneration (LR) and maintains the viability of hepatocytes. Surprisingly, we found that the levels of the HSS mRNA and expressed protein were both strongly repressed at 12h after a 70% partial hepatectomy (PH) in mice. Understanding the mechanism and effect of this extraordinary suppression can provide a novel path for exploring the molecular function of HSS during LR. We observed that the EGF levels in the serum were negatively correlated with HSS expression in regenerating livers. Treating primary mouse hepatocytes or Hepa1-6 cells with EGF suppressed HSS mRNA expression. This suppression was transcriptional and was mediated by the effect of EGF on the phosphorylation of CCAAT/enhancer-binding protein β (C/EBPβ), which regulates HSS expression. We further showed that the enhanced phosphorylation of C/EBPβ after PH promoted its interaction with the HSS promoter and repressed HSS expression at early time-points after PH. Interestingly, the knockdown of HSS caused a dramatic decrease in E-cadherin expression in hepatocytes. E-cadherin expression was also significantly suppressed at 12h after PH. Moreover, the pre-injection of HSS-expressing adenovirus vectors prevented E-cadherin suppression after PH. Treatment with C/EBPβ siRNA reversed the EGF-mediated inhibition of HSS expression and led to enhanced E-cadherin expression and reduced cell migration. Our findings suggest that C/EBPβ directly inhibits the HSS promoter after PH and that this inhibition can downregulate E-cadherin expression. These data provide novel insight into the potential role of HSS in hepatic structural reconstruction during LR.

Expression of microRNA-150 targeting vascular endothelial growth factor-A is downregulated under hypoxia during liver regeneration

Vascular endothelial growth factor (VEGF) is a factor that stimulates the proliferation of sinusoidal endothelial cells and hepatocytes during liver regeneration(LR). The present study aimed to screen and validate a microRNA(miRNA) that targets VEGF-A with relative specificity and to elucidate the potential association between hypoxia-inducible factor-1α (HIF‑1α) and miRNA expression in the early phase of LR. Changes in the expression of miRNAs, which were predicted to target VEGF-A using online databases, were detected at 12, 24 and 48h following a 70%partial hepatectomy(PHx) using quantitative PCR(qPCR). An inhibitor of the most downregulated miRNA was transfected into the primary hepatocytes in order to observe changes in the expression of the VEGF-A gene. The expression of HIF-1α protein in the regenerating liver was investigated using western blot analysis. The expression levels of HIF-1α mRNA (messenger RNA), the selected miRNA and VEGF-A mRNA in an anoxic model of hepatocytes was examined with qPCR. Of seven putative miRNAs, the expression of miR-150 exhibited the sharpest downregulation from 12-48h. The micrOFF™ miR-150 inhibitor significantly elevated the expression levels of VEGF-A mRNA and protein 48h after transfection. Thus, VEGF-A may be a downstream target of miR-150 during LR. Furthermore, HIF-1α protein expression increased to its highest level 24h following PHx. miR-150 expression was inhibited and the expression of VEGF-A mRNA increased accordingly in the hypoxia-induced hepatocytes. Our results suggest that miR-150 expression is subject to negative regulation by HIF-1α.

Impaired liver regeneration in Ldlr−/− mice is associated with an altered hepatic profile of cytokines, growth factors, and lipids

It is widely recognized that in the early stages of liver regeneration after partial hepatectomy, the hepatocytes accumulate a significant amount of lipids. The functional meaning of this transient steatosis and its effect on hepatocellular proliferation are not well defined. In addition, the basic mechanisms of this lipid accumulation are not well understood although some studies suggest the participation of the Low Density Lipoprotein Receptor (Ldlr). To address these questions, we studied the process of liver regeneration in Ldlr null mice and wild type mice following partial hepatectomy. Ldlr deficiency was associated with a significant decrease in serum albumin concentration, during early stages of liver regeneration, and a delayed hepatic regeneration. Remnant livers of Ldlr(-)(/)(-) showed a time-shifted expression of interleukin-6 (IL6) and a defective activation of tumor necrosis factor-α (TNFα) and hepatocyte growth factor (HGF) expression in early phases of liver regeneration. Unexpectedly, Ldlr(-)(/)(-) showed no significant differences in the content of lipid droplets after partial hepatectomy compared to wild type mice. However, lipidomic analysis of the regenerating liver from Ldlr(-)(/)(-) revealed a lipid profile compatible with liver quiescence: high content of cholesterol esters and ceramide, and low levels of phosphatidylcholine. Ldlr deficiency is associated with significant changes in the hepatic lipidome that affect cytokine-growth factor signaling and impair liver regeneration. These results suggest that the analysis of the hepatic lipidome may help predict the success of liver regeneration in the clinical environment, specifically in the context of pre-existing liver steatosis.

Significance of alterations in the metabolomics of sulfur-containing amino acids during liver regeneration

It has been known that liver regeneration is accompanied with a profound change in the metabolomics of sulfur-containing substances in liver. However, its physiological significance in the liver regenerative process is still unclear. Our previous work showed that buthioninesulfoximine and phorone, both widely used to deplete intracellular glutathione (GSH) in biological experiments, induced contrasting changes in the sulfur-containing amino acid metabolism in liver. In this study we employed these GSH-depleting agents to evaluate the role of sulfur-containing substances in the early phase of liver regeneration. Male rats treated with buthioninesulfoximine or phorone were subjected to two-thirds partial hepatectomy (PHx). At the doses used, the magnitude of GSH depletion after PHx was comparable, but buthioninesulfoximine administration inhibited the progression of liver regeneration as determined by liver weight increase, elevation of serum alanine aminotransferase activity, and cyclin D1 and proliferating cell nuclear antigen (PCNA) protein expressions, whereas liver recovery was significantly accelerated in the phorone-treated rats, suggesting that the role of GSH in this process is minimal. Hepatic concentrations of methionine, S-adenosylmethionine, cysteine, taurine and GSH were all elevated by PHx. Methionine adenosyltransferase activity was also induced in the remnant liver. Buthioninesulfoximine administration depressed the elevation of S-adenosylmethionine, but increased the catabolism of cysteine to taurine. In contrast, S-adenosylmethionine elevation was augmented whereas cysteine, hypotaurine and taurine were decreased in the phorone-treated rats. PHx elevated hepatic putrescine and spermidine, but lowered spermine concentrations. Buthioninesulfoximine administration increased putrescine further, but decreased spermidine and spermine concentrations. On the contrary, both spermidine and spermine concentrations were elevated in the rats treated with phorone. The results suggest that the availability of S-adenosylmethionine plays a critical role in the progression of liver regeneration via enhancement of polyamine synthesis. These findings raise the possibility that regulating hepatic transsulfuration reactions may be capable of modifying the recovery process after liver injury.

Genome‐wide combinatorial transcriptional regulatory dynamics during early onset of liver regeneration and chronic alcohol intake

The objective of this study is to identify genome‐scale transcriptional regulatory network dynamics affected by chronic alcohol intake during the early phase of liver regeneration. Rats were fed liquid diet containing 36% of total calories derived from ethanol for 5 weeks, and controls were pair‐fed an isocaloric liquid diet. A 70% partial hepatectomy (PHx) was performed. Genome‐wide binding targets for NF‐kB, C/EBP‐beta, and STAT3 were detected either by Roche NimbleGen ChIP‐chip or ABI SOLiD sequencing approach. Our findings indicate significant differences in the binding profiles of all the three regulators in chronic ethanol compared to isocaloric controls, at baseline as well as in response to PHx. Pathway analysis revealed that potential target genes specific to the chronic ethanol group participate in key processes such as mitochondria processing, DNA methylation, cell death, proteolysis, histone modification, lipid biosynthetic process, and regulation of cell cycle etc. Our results revealed combinatorial binding dynamics of multiple TFs from ChIP‐seq and DNA binding motif. Our results provide a first system‐wide view of the regulatory network dynamics in liver regeneration as it is affected by alcohol, and implicate combinatorial regulatory modules as participating in the adverse liver regeneration response. Research Support: NIH R01 AA018873, K05 AA017261, and T32 AA007463.

Calcitonin gene–related peptide regulates the early phase of liver regeneration

ObjectiveTo investigate the expression of calcitonin gene–related peptide (CGRP) and its role in the liver regeneration process after 70% hepatectomy (Hx). Materials and methodsWistar rats were divided into eight groups based on time after Hx. Remnant liver samples were collected serially 0 h, 1 h, 6 h, 12 h, 1 d, 2 d, 7 d, and 14 d after Hx (n = 6 for each time point). The expression level of the calcitonin/CGRP gene in the remnant liver was measured. Western bolts and immunohistochemistry were performed to determine the levels of CGRP in the regenerating liver. Furthermore, CGRP8-37 (a CGRP receptor antagonist) was used to examine the role of CGRP during liver regeneration. ResultsA marked upregulation of the calcitonin/CGRP gene was observed immediately after Hx, and the protein levels of CGRP in the liver, which were measured by western blot and immunohistochemistry, also rapidly increased after Hx. The liver regeneration rate was significantly attenuated by an administration of CGRP8-37 2 d after Hx. The mitotic index was evaluated by histologic examination 1 and 2 d after Hx and was also significantly lower in the CGRP8-37 group. In addition, CGRP8-37 treatment inhibited the phosphorylation of extracellular-signal regulated kinase 1/2. The levels of early response genes, such as c-fos, c-jun, and c-myc, were also downregulated by CGRP8-37. ConclusionThe calcitonin/CGRP gene may have an important role in the early phase of liver regeneration after Hx.

The Role of TWEAK/Fn14 Signaling in Liver Regeneration after Mouse Partial Hepatectomy

Introduction: Liver regeneration is important after liver resection and transplantation, though the mechanisms surrounding this process are complex and incompletely understood. Tumor necrosis factor-like weak inducer of apoptosis (TWEAK), acts via its specific receptor fibroblast growth factor-inducible 14 (Fn14), and Fn14 receptors have been shown to be highly expressed in chronically damaged livers, such as in hepatitis C, and in regenerating livers after partial hepatectomy (PH), suggesting a strong role for the TWEAK/Fn14 pathway in the proliferation of hepatic epithelial cells and/or their transformation. This is the first attempt to study the effect of stimulating the TWEAK/Fn14 pathway via administration of an agonistic anti-Fn14 monoclonal antibody (mAb), on liver regeneration after mouse PH. Methods: Mice were divided into 4 groups: sham (control), PH alone, PH + anti-Fn14 mAb, and sham + anti-Fn14 mAb, and sacrificed at 24, 48, 72 hrs (n=5 per group at each time point), or 7 days (n=2 per group). Anti-Fn14 mAb (ITEM-4: 200 μg/mouse) was administered at the time of initial surgery via intraperitoneal injection. Liver regeneration at point of sacrifice was estimated by remnant liver weight to body weight ratio (LBWr). Serum was taken for liver function assessment, and mice livers were resected for western blot assessment of the Fn14 receptor. Fn14 to actin ratios were calculated to standardize the results. Results: Resected LBWr, a marker for consistency of resection, was similar in both PH and PH + anti-Fn14 mAb groups (0.027 vs. 0.028, p=0.2). Remnant LBWr, a marker of regeneration, was significantly higher at 24 and 48 hrs after PH + anti-Fn14 mAb vs. PH alone, although it became similar at 72 hrs after PH. Western blot data indicates a trend toward higher expression of Fn14 at 24 hrs in the PH + anti-Fn14 mAb than in the sham group (Fn14:actin: 1.21 vs. 0.49, p=0.1) or PH alone group (Fn14:actin: 1.21 vs. 0.75, p=0.3). Values of LFTs (ALP, ALT, AST and total Bili) were similar between PH alone and PH + anti-Fn14 mAb groups at 24 and 48 hrs after PH, but all values were significantly higher in the PH + anti-Fn14 mAb group at 72 hrs compared with PH alone. At 7 days, all LFTs in both PH and PH + anti-Fn14 mAb groups returned to baseline. Conclusion: The TWEAK/Fn14 signaling pathway contributes to the early phase of liver regeneration by stimulating it after PH with upregulation of Fn14 in the liver. Agonistic anti-Fn14 mAb might be useful to stimulate liver regeneration after liver resection or liver transplantation with cautious monitoring of LFTs.

HSCs play a distinct role in different phases of oval cell‐mediated liver regeneration

Hepatic stem cell niche plays an important role in hepatic oval cell-mediated liver regeneration. As a component of hepatic stem cell niche, the role of hepatic stellate cells (HSCs) in oval cell proliferation needs further studies. In the present study, we isolated HSCs from rats at indicated time point after partial hepatectomy (PH) in 2-acetylaminofluorene/PH oval cell proliferation model. Conditional medium (CM) from HSCs were collected to detect their effects on proliferation and the mitogen-activated protein kinase pathway activation of two oval cell lines. We found that CM collected from HSCs at early phase of liver regeneration (4 and 9 days group) contained high levels of hepatocyte growth factor (HGF) and stimulated oval cell proliferation via extracellular signal-regulated kinase and p38 pathway. CM collected from HSCs at terminal phase of liver regeneration (12 and 15 days group) contained high levels of transforming growth factor (TGF)-β1, which suppressed DNA synthesis of oval cells. The shift between these two distinct effects depended on the balance between HGF and TGF-β1 secreted by HSCs. Our study demonstrated that HSCs acted as a positive regulator at the early phase and a negative regulator at the terminal phase of the oval cell-mediated liver regeneration.

A microRNA-21 surge facilitates rapid cyclin D1 translation and cell cycle progression in mouse liver regeneration

MicroRNA-21 (miR-21) is thought to be an oncomir because it promotes cancer cell proliferation, migration, and survival. miR-21 is also expressed in normal cells, but its physiological role is poorly understood. Recently, it has been found that miR-21 expression is rapidly induced in rodent hepatocytes during liver regeneration after two-thirds partial hepatectomy (2/3 PH). Here, we investigated the function of miR-21 in regenerating mouse hepatocytes by inhibiting it with an antisense oligonucleotide. To maintain normal hepatocyte viability and function, we antagonized the miR-21 surge induced by 2/3 PH while preserving baseline expression. We found that knockdown of miR-21 impaired progression of hepatocytes into S phase of the cell cycle, mainly through a decrease in levels of cyclin D1 protein, but not Ccnd1 mRNA. Mechanistically, we discovered that increased miR-21 expression facilitated cyclin D1 translation in the early phase of liver regeneration by relieving Akt1/mTOR complex 1 signaling (and thus eIF-4F-mediated translation initiation) from suppression by Rhob. Our findings reveal that miR-21 enables rapid hepatocyte proliferation during liver regeneration by accelerating cyclin D1 translation.

Effect of laser on the remnant liver after the first 24 hours following 70% hepatectomy in rats

To evaluate the mitochondrial function of the remnant liver (RL) in the early phase of liver regeneration in rats after 70% partial hepatectomy (PH). Sixty male Wistar rats (200-250g) submitted to 70% PH were divided into five groups according to the time of euthanasia and application or not of laser light: C = Control, time zero; 2 minutes, 4, 6 and 24 hours after PH. The dose of laser radiation was 22.5 J/cm(2), wavelength of 660 nm (visible/red), in the remnant liver. We studied the respiration activated by ADP (state 3), basal mitochondrial respiration (state 4), respiratory control ratio (RCR) and mitochondrial membrane potential (MMP). The mitochondrial function of RL changed at 4 and 6 hours after PH, with a significant increase in state 3 and a concomitant increase in state 4 and with maintenance of RCR. MMP differed significantly between the groups biostimulated with laser radiation and the control group 4 hours after HP, with a substantial reduction in the non-laser groups. The laser light at the dose used in this study did not induce additional damage to the RL and seems to have delayed the hepatocellular metabolic overload of the remnant liver.

Interleukin-1 Receptor Antagonist Modulates the Early Phase of Liver Regeneration after Partial Hepatectomy in Mice

BackgroundCytokine administration is a potential therapy for acute liver failure by reducing inflammatory responses and favour hepatocyte regeneration. The aim of this study was to evaluate the role of interleukin-1 receptor antagonist (IL-1ra) during liver regeneration and to study the effect of a recombinant human IL-1ra on liver regeneration.MethodsWe performed 70%-hepatectomy in wild type (WT) mice, IL-1ra knock-out (KO) mice and in WT mice treated by anakinra. We analyzed liver regeneration at regular intervals by measuring the blood levels of cytokines, the hepatocyte proliferation by bromodeoxyuridin (BrdU) incorporation, proliferating cell nuclear antigen (PCNA) and Cyclin D1 expression. The effect of anakinra on hepatocyte proliferation was also tested in vitro using human hepatocytes.ResultsAt 24h and at 48h after hepatectomy, IL-1ra KO mice had significantly higher levels of pro-inflammatory cytokines (IL-6, IL-1β and MCP-1) and a reduced and delayed hepatocyte proliferation measured by BrdU incorporation, PCNA and Cyclin D1 protein levels, when compared to WT mice. IGFBP-1 and C/EBPβ expression was significantly decreased in IL-1ra KO compared to WT mice. WT mice treated with anakinra showed significantly decreased levels of IL-6 and significantly higher hepatocyte proliferation at 24h compared to untreated WT mice. In vitro, primary human hepatocytes treated with anakinra showed significantly higher proliferation at 24h compared to hepatocytes without treatment.ConclusionIL1ra modulates the early phase of liver regeneration by decreasing the inflammatory stress and accelerating the entry of hepatocytes in proliferation. IL1ra might be a therapeutic target to improve hepatocyte proliferation.

Genome-wide expression profiling of hepatic oval cells after partial hepatectomy in rats

To examine the changes of biological activities in hepatic oval cells (HOCs) elicited by 70% partial hepatectomy (PH) and understand the relationship between this cell and liver regeneration (LR), this study isolated and obtained the high purity HOCs (≥95%) from rat regenerating livers, and then monitored gene expression profiling of rat hepatic oval cells following surgical operation. Results showed that there were LR-related 1059 genes. These genes were grossly classified into three groups using a fold change cut-off threshold of three-fold: up-regulation, down-regulation and up/down regulation. Analyses of gene expression patterns combined with gene functional categorization suggested that genes in the categories “nucleic acid metabolism” and “cell cycle” were dominated by up-regulated expression. Genes in the functional groups “cell metabolism” and “oxidation reduction” were significantly enriched in expression pattern characterized by down-regulation. According to above mentioned results, the synchronized induction of DNA replication and cell proliferation-involved genes suggested that the peak of oval cell proliferation might occur between 30 and 36 h post-PH. The amino acid transformation-involved genes were down-regulated at the early phase of LR, which perhaps trigger the storage of those amino acids essential for protein synthesis. Reduced oxidative-reduction activity at early phase might be related to negative influence of surgical operation on its detoxification capacity. Conclusively, the genome-wide transcriptional analysis of oval cells would contribute to our understanding of the nature of LR at cell level.

Genomewide microRNA down-regulation as a negative feedback mechanism in the early phases of liver regeneration

The liver is one of the few organs that have the capacity to regenerate in response to injury. We carried out genomewide microRNA (miRNA) microarray studies during liver regeneration in rats after 70% partial hepatectomy (PH) at early and mid time points to more thoroughly understand their role. At 3, 12, and 18 hours post-PH ∼40% of the miRNAs tested were up-regulated. Conversely, at 24 hours post-PH, ∼70% of miRNAs were down-regulated. Furthermore, we established that the genomewide down-regulation of miRNA expression at 24 hours was also correlated with decreased expression of genes, such as Rnasen, Dgcr8, Dicer, Tarbp2, and Prkra, associated with miRNA biogenesis. To determine whether a potential negative feedback loop between miRNAs and their regulatory genes exists, 11 candidate miRNAs predicted to target the above-mentioned genes were examined and found to be up-regulated at 3 hours post-PH. Using reporter and functional assays, we determined that expression of these miRNA-processing genes could be regulated by a subset of miRNAs and that some miRNAs could target multiple miRNA biogenesis genes simultaneously. We also demonstrated that overexpression of these miRNAs inhibited cell proliferation and modulated cell cycle in both Huh-7 human hepatoma cells and primary rat hepatocytes. From these observations, we postulated that selective up-regulation of miRNAs in the early phase after PH was involved in the priming and commitment to liver regeneration, whereas the subsequent genomewide down-regulation of miRNAs was required for efficient recovery of liver cell mass. Our data suggest that miRNA changes are regulated by negative feedback loops between miRNAs and their regulatory genes that may play an important role in the steady-state regulation of liver regeneration.

Unique pathway for anandamide synthesis and liver regeneration

The endocannabinoid system (ECS) includes cannabinoid (CB1 and CB2) receptors and their endogenous ligands (i.e., the endocannabinoids anandamide and 2-arachydonylglycerol) as well as proteins involved in endocannabinoids biosynthesis and degradation (1). The ECS is present in the liver and undergoes adaptive changes in response to noxious stimuli. Endocannabinoids as well as CB1 and CB2 receptors (which are, respectively, either faintly or not expressed in normal livers) are up-regulated in experimental liver injury and liver cirrhosis of various etiologies. In vivo, CB1 receptor activation promotes fat accumulation, triggers inflammation in nonalcoholic and alcoholic fatty liver diseases, contributes to the progression of chronic hepatitis to cirrhosis by stimulating fibrogenesis, and is also implicated in hemodynamic and neurological consequences associated with liver cirrhosis, including portal hypertension, encephalopathy, and cardiomyopathy (2–4). Conversely, activation of CB2 receptors exerts antifibrogenic and antiinflammatory effects in experimental models of liver disease (5). These pharmacological effects make CB2 agonists and CB1 antagonists promising candidates for the treatment of fibrosis in chronic liver pathologies. By disclosing a prominent CB1-mediated role of anandamide in the early phase of liver regeneration, Mukhopadhyay et al. (6) provide additional and significant support to the prominent role of the ECS in liver biology in an article in PNAS.

Decorin gene expression in normal and tumor liver tissue

Decorin, a small leucine ‐ rich proteoglycan is involved in a number of cellular processes. Decorin has been shown to suppress proliferation of cancer cells in vitro. Decorin interacts with a number of receptor tyrosine kinases like Met, and Epidermal Growth Factor Receptor. In view of the role of Decorin as a negative regulator of Met and EGFR, we decided to investigate whether Decorin played a role in hepatocyte proliferation, both in vitro and in vivo. Initially we investigated the effects of Decorin on induction of DNA synthesis by HGF and EGF in primary cultures of rat hepatocytes. Addition of Decorin in the culture medium resulted in more than 50% inhibition of DNA synthesis. Decorin gene expression was also found to be regulated during the early stages of liver regeneration in rats following a PHx. Decorin gene expression was found to be suppressed in Liver tissue samples obtained from HCC, Adjacent tissue, Normal and hepatoma cases by microarray analysis. We also investigated Decorin expression by RT‐PCR and western blot analysis in a number of transformed cell lines like HepG3, Hep3b and HuH7, JM1 and JM2. Decorin expression could not be detected by RT‐PCR and western blot analysis in these cell lines. In conclusion, preliminary data indicated that Decorin expression, following a PHx, is significantly suppressed during early phases of liver regeneration and Decorin has a modulating role in hepatocyte proliferation.

Elevation and characteristics of Rab30 and S100a8/S100a9 expression in an early phase of liver regeneration in the mouse

Recent studies have revealed that cytokines, including TNFα and IL-6 play key roles in the priming phase of liver regeneration. However, further knowledge of molecular events in the priming phase is needed for more comprehensively understanding the initiation of liver regeneration. In the present study, we attempted to identify additional genes involved in an early phase (2-6h post partial hepatectomy, PH). The expression of 71 genes was shown to be up-regulated more than 3-fold in the liver at 2h and 6h post PH, as compared to 0h (normal livers) using microarray analysis. Among them, Rab30 and S100a8/S100a9, were identified as novel genes up-regulated over 20-fold at 2h post PH as compared to normal liver, and were further examined by RT-qPCR to confirm microarray results. Rab30 showed no significant up-regulation in organs other than the liver, whereas S100a8/S100a9 showed significant up-regulation in other organs, such as the lung and spleen at 6h post PH as compared to those of sham-operated mice, indicating the existence of a different up-regulation machinery between Rab30 and S100a8/S100a9. Their expression was further investigated in the liver at various developmental stages. Rab30 was shown to be expressed only in newborn liver, whereas S100a8/S100a9 was highly expressed in embryo stages, and exhibited the highest levels in newborn liver. These findings imply that Rab30 and S100a8/S100a9 are possibly involved in the functional switch from hematopoiesis support to metabolism in the newborn stage, but might play different roles in liver development. In conclusion, Rab30 and S100a8/S100a9 were indicated to play roles in the initiation of liver regeneration as well as possibly in the functional switch of the liver in the newborn stage.