Oval Cell Response Research Articles

Long-Term Expansion of Functional Mouse and Human Hepatocytes as 3D Organoids

The mammalian liver possesses a remarkable regenerative ability. Two modes of damage response have been described: (1) The "oval cell" response emanates from the biliary tree when all hepatocytes are affected by chronic liver disease. (2) A massive, proliferative response of mature hepatocytes occurs upon acute liver damage such as partial hepatectomy (PHx). While the oval cell response has been captured invitro by growing organoids from cholangiocytes, the hepatocyte proliferative response has not been recapitulated in culture. Here, we describe the establishment of a long-term 3D organoid culture system for mouse and human primary hepatocytes. Organoids can be established from single hepatocytes and grown for multiple months, while retaining key morphological, functional and gene expression features. Transcriptional profiles of the organoids resemble those of proliferating hepatocytes after PHx. Human hepatocyte organoids proliferate extensively after engraftment into mice and thus recapitulate the proliferative damage-response of hepatocytes.

Effect of human umbilical cord blood stem cell transplantation on oval cell response in 2-AAF/CCL4 liver injury model: experimental immunohistochemical study

BackgroundOval cells, specific liver progenitors, are activated in response to injury. The human umbilical cord blood (hUCB) is a possible source of transplantable hepatic progenitors and can be used in cases of severe liver injury. We detected the effect of hUCB stem cell transplantation on natural response of oval cells to injury.MethodsTwenty-four female albino rats were randomly divided into three groups: (A) control, (B) liver injury with hepatocyte block, and (C) hUCB transplanted group. Hepatocyte block was performed by administration of 2-acetylaminofluorene (2-AAF) for 12 days. CCL4 was administrated at day 5 from experiment start. Animals were sacrificed at 9 days post CCL4 administration, and samples were collected for biochemical and histopathological analysis. Oval cell response to injury was evaluated by the percentage of oval cells in the liver tissue and frequency of cells incorporated into new ducts.ResultsImmunohistochemical analysis of oval cell response to injury was performed. There was significant deviation in the hUCB-transplanted (4.9 ± 1.4) and liver injury groups (2.4 ± 0.9) as compared to control (0.89 ± 0.4) 9 days post injury. Detection of oval cell response was dependant on OV-6 immunoreactivity. For mere localization of cells with human origin, CD34 antihuman immunoreactivity was performed. There was no significant difference in endogenous OV-6 immunoreactivity following stem cell transplantation as compared to the liver injury group.ConclusionsIn vivo transplantation of cord blood stem cells (hUCB) does not interfere with natural oval cell response to liver injury.

MiR-133b Regulation of Connective Tissue Growth Factor: A Novel Mechanism in Liver Pathology

miRNAs are involved in liver regeneration, and their expression is dysregulated in hepatocellular carcinoma (HCC). Connective tissue growth factor (CTGF), a direct target of miR-133b, is crucial in the ductular reaction (DR)/oval cell (OC) response for generating new hepatocyte lineages during liver injury in the context of hepatotoxin-inhibited hepatocyte proliferation. Herein, we investigate whether miR-133b regulation of CTGF influences HCC cell proliferation and migration, and DR/OC response. We analyzed miR-133b expression and found it to be down-regulated in HCC patient samples and induced in the rat DR/OC activation model of 2-acetylaminofluorene with partial hepatectomy. Furthermore, overexpression of miR-133b via adenoviral system invitro led to decreased CTGF expression and reduced proliferation and Transwell migration of both HepG2 HCC cells and WBF-344 rat OCs. Invivo, overexpression of miR-133b in DR/OC activation models of 2-acetylaminofluorene with partial hepatectomy in rats, and 3,5-diethoxycarbonyl-1,4-dihydrocollidine in mice, led to down-regulation of CTGF expression and OC proliferation. Collectively, these results show that miR-133b regulation of CTGF is a novel mechanism critical forthe proliferation and migration of HCC cells and OC response.

Lessons from hepatocyte-specific Cyp51 knockout mice: impaired cholesterol synthesis leads to oval cell-driven liver injury.

We demonstrate unequivocally that defective cholesterol synthesis is an independent determinant of liver inflammation and fibrosis. We prepared a mouse hepatocyte-specific knockout (LKO) of lanosterol 14α-demethylase (CYP51) from the part of cholesterol synthesis that is already committed to cholesterol. LKO mice developed hepatomegaly with oval cell proliferation, fibrosis and inflammation, but without steatosis. The key trigger was reduced cholesterol esters that provoked cell cycle arrest, senescence-associated secretory phenotype and ultimately the oval cell response, while elevated CYP51 substrates promoted the integrated stress response. In spite of the oval cell-driven fibrosis being histologically similar in both sexes, data indicates a female-biased down-regulation of primary metabolism pathways and a stronger immune response in males. Liver injury was ameliorated by dietary fats predominantly in females, whereas dietary cholesterol rectified fibrosis in both sexes. Our data place defective cholesterol synthesis as a focus of sex-dependent liver pathologies.

The plastic liver: differentiated cells, stem cells, every cell?

The liver is capable of full regeneration following several types and rounds of injury, ranging from hepatectomy to toxin-mediated damage. The source of this regenerative capacity has long been a hotly debated topic. The damage response that occurs when hepatocyte proliferation is impaired is thought to be mediated by oval/dedifferentiated progenitor cells, which replenish the hepatocyte and ductal compartments of the liver. Recently, reports have questioned whether these oval/progenitor cells truly serve as the facultative stem cell of the liver following toxin-mediated damage. In this issue of the JCI, Kordes and colleagues use lineage tracing to follow transplanted rat hepatic stellate cells, a resident liver mesenchymal cell population, in hosts that have suffered liver damage. Transplanted stellate cells repopulated the damaged rat liver by contributing to the oval cell response. These data establish yet another cell type of mesenchymal origin as the progenitor for the oval/ductular response in the rat. The lack of uniformity between different damage models, the extent of the injury to the liver parenchyma, and potential species-specific differences might be at the core of the discrepancy between different studies. Taken together, these data imply a considerable degree of plasticity in the liver, whereby several cell types can contribute to regeneration.

Hepatic loss of survivin impairs postnatal liver development and promotes expansion of hepatic progenitor cells in mice

Hepatocytes possess a remarkable capacity to regenerate and reconstitute the parenchyma after liver damage. However, in the case of chronic injury, their proliferative potential is impaired and hepatic progenitor cells (HPCs) are activated, resulting in a ductular reaction known as oval cell response. Proapoptotic and survival signals maintain a precise balance to spare hepatocytes and progenitors from hyperplasia and cell death during regeneration. Survivin, a member of the family of inhibitor of apoptosis proteins (IAPs), plays key roles in the proliferation and apoptosis of various cell types. Here, we characterized the in vivo function of Survivin in regulating postnatal liver development and homeostasis using mice carrying conditional Survivin alleles. Hepatic perinatal loss of Survivin causes impaired mitosis, increased genome ploidy, and enlarged cell size in postnatal livers, which eventually leads to hepatocyte apoptosis and triggers tissue damage and inflammation. Subsequently, HPCs that retain genomic Survivin alleles are activated, which finally differentiate into hepatocytes and reconstitute the whole liver. By contrast, inducible ablation of Survivin in adult hepatocytes does not affect HPC activation and liver homeostasis during a long-life period. Perinatal Survivin deletion impairs hepatic mitosis in postnatal liver development, which induces HPC activation and reconstitution in the liver, therefore providing a novel HPC induction model.

Failure of Fibrotic Liver Regeneration in Mice Is Linked to a Severe Fibrogenic Response Driven by Hepatic Progenitor Cell Activation

Failure of fibrotic liver to regenerate after resection limits therapeutic options and increases demand for liver transplantation, representing a significant clinical problem. The mechanism underlying regenerative failure in fibrosis is poorly understood. Seventy percent partial hepatectomy (PHx) was performed in C57Bl/6 mice with or without carbon tetrachloride (CCl4)-induced liver fibrosis. Liver function and regeneration was monitored at 1 to 14 days thereafter by assessing liver mass, alanine aminotransferase (ALT), mRNA expression, and histology. Progenitor (oval) cell mitogen tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and TWEAK-neutralizing antibody were used to manipulate progenitor cell proliferation in vivo. In fibrotic liver, hepatocytes failed to replicate efficiently after PHx. Fibrotic livers showed late (day 5) peak of serum ALT (3542 ± 355 IU/L compared to 93 ± 65 IU/L in nonfibrotic livers), which coincided with progenitor cell expansion, increase in profibrogenic gene expression and de novo collagen deposition. In fibrotic mice, inhibition of progenitor activation using TWEAK-neutralizing antibody after PHx resulted in strongly down-regulated profibrogenic mRNA, reduced serum ALT levels and improved regeneration. Failure of hepatocyte-mediated regeneration in fibrotic liver triggers activation of the progenitor (oval) cell compartment and a severe fibrogenic response. Inhibition of progenitor cell proliferation using anti-TWEAK antibody prevents fibrogenic response and augments fibrotic liver regeneration. Targeting the fibrogenic progenitor response represents a promising strategy to improve hepatectomy outcomes in patients with liver fibrosis.

Contribution of mature hepatocytes to small hepatocyte-like progenitor cells in retrorsine-exposed rats with chimeric livers

The potential lineage relationship between hepatic oval cells, small hepatocyte-like progenitor cells (SHPCs), and hepatocytes in liver regeneration is debated. To test whether mature hepatocytes can give rise to SHPCs, rats with dipeptidyl peptidase IV (DPPIV) chimeric livers, which harbored endogenous DPPIV-deficient hepatocytes and transplanted DPPIV-positive hepatocytes, were subjected to retrorsine treatment followed by partial hepatectomy (PH). DPPIV-positive hepatocytes comprised about half of the DPPIV chimeric liver mass. Tissues from DPPIV chimeric livers after retrorsine/PH treatment showed large numbers of SHPC clusters. None of the SHPC clusters were stained positive for DPPIV in any analyzed samples. Furthermore, serial sections stained for gamma-glutamyl-transpeptidase (GGT, a marker of fetal hepatoblasts) and glucose-6-phosphatase (G6Pase, a marker of mature hepatocytes) showed inverse expression of the two enzymes and a staining pattern consistent with a lineage that begins with GGT(+)/G6Pase(-) to GGT(-)/G6Pase(+) within a single SHPC cluster. Using double immunofluorescence staining for markers specific for hepatic oval cells and hepatocytes in serial sections, oval cell proliferations with CK-19(+)/laminin(+) and OV-6(+)/C/EBP-α(-) were shown to extend from periportal areas into the SPHC clusters, differentiating into hepatic lineage by progressive loss of CK-19/laminin expression and appearance of C/EBP-α expression towards the cluster side. Cells in the epithelial cell adhesion molecule (EpCAM(+)) SHPC clusters showed membranous EpCAM(+)/HNF-4α(+) (hepatocyte nuclear factor-4α) staining and were contiguous to the surrounding cytoplasmic EpCAM(+)/HNF-4α(-) ductular oval cells. Extensive elimination of oval cell response by repeated administration of 4,4'-methylenedianiline (DAPM) to retrorsine-exposed rats impaired the emergence of SHPC clusters. These findings highly suggest the hepatic oval cells but not mature hepatocytes as the origin of SHPC clusters in retrorsine-exposed rats.

Adult HNF1beta+ cells give rise to oval cells but do not significantly contribute to normal homeostasis or injury repair of hepatocytes in mice

In response to liver injury or loss of liver mass, hepatocytes and cholangiocytes proliferate to replace the injured tissue. However, in case of chronic liver and biliary disease, when hepatocyte and cholangiocyte proliferation is impaired, an expansion of small putative progenitors is observed (oval cell response). Oval cells reside in a niche close to the terminal bile ducts, called canals of Hering, and are believed to give rise to both, hepatocytes and bile ducts. Though controversially debated, these progenitors were recently reported to not only give rise to hepatocytes after liver injury but even provide a continuous supply of hepatocytes of the entire liver in normal homeostasis (“streaming hypothesis”).

NF-κB Essential Modifier Is Required for Hepatocyte Proliferation and the Oval Cell Reaction After Partial Hepatectomy in Mice

The transcription factor nuclear factor κB (NF-κB) is activated by the IκB kinase complex. The regulatory subunit of this complex, NF-κB essential modifier (NEMO or IKBKG), is a tumor suppressor. Hepatocyte-specific deletion of NEMO induces chronic liver inflammation that leads to apoptosis, oxidative stress, development of nonalcoholic steatohepatitis, and hepatocarcinogenesis. We performed partial hepatectomies in mice with hepatocyte-specific disruption of NEMO (Nemo(Δhepa)). Some mice were fed a diet that contained the antioxidant butylated hydroxyanisole (BHA), and others were given daily intraperitoneal injections of the oxidant phenetyl isothiocyanate (PEITC). Nemo(Δhepa) mice had impaired liver regeneration after partial hepatectomy and 50% mortality, indicating that NEMO is required for the regenerative response. Liver cells of the mice had a strong oxidative stress response; these cells down-regulated the NF-κB-dependent antioxidant response and reduced levels of proteins that repair DNA double-strand breaks. However, the impairments to hepatocyte proliferation were compensated by a response of oval cells in Nemo(Δhepa) mice. Oval cells expressed low levels of albumin and thereby expressed normal levels of NEMO. Repopulation of the liver with oval cells that expressed NEMO reversed liver damage in Nemo(Δhepa) mice. Interestingly, these mice still developed hepatocellular carcinomas 6 months after partial hepatectomy, whereas Nemo(Δhepa) mice fed the BHA diet were protected from carcinogenesis. In livers of mice, expression of NEMO and activation of NF-κB are required for hepatocyte proliferation and liver regeneration. These mechanisms require control of oxidative stress and DNA integrity.

The inhibitory effect of rapamycin on the oval cell response and development of preneoplastic foci in the rat

Oval cell activation occurs under conditions of severe liver injury when normal hepatocyte proliferation is blocked. Recent studies have shown that a subset of hepatocellular carcinomas expresses oval cell markers, suggesting that these cells are targets of hepatocarcinogens. However, the signaling pathways that control oval cell activation and proliferation are not well characterized. Based on the role of the nutrient signaling kinase complex, mTORC1, in liver development, we investigated the role of this pathway in oval cell activation. Oval cell proliferation was induced in male Fisher rats by a modification of the traditional choline deficient plus ethionine model (CDE) or by 2-acetylaminoflourene treatment followed by 2/3 partial hepatectomy with or without initiation by diethylnitrosamine. To assess the role of mTOR in the oval cell response and development of preneoplastic foci, the effect of the mTORC1 inhibitor, rapamycin, was studied in all models. Rapamycin induced a significant suppression of the oval cell response in both models, an effect that coincided with a decrease in oval cell proliferation. Rapamycin administration did not affect the abundance of neutrophils or natural killer cells in CDE-treated liver or the expression of key cytokines. Gene expression studies revealed the fetal hepatocyte marker MKP-4 to be expressed in oval cells. In an experimental model of hepatic carcinogenesis, rapamycin decreased the size of preneoplastic foci and the rate of cell proliferation within the foci. mTORC1 signaling plays a key role in the oval cell response and in the development of preneoplastic foci. This pathway may be a target for the chemoprevention of hepatocellular carcinoma.

Oval Cell Response Is Attenuated by Depletion of Liver Resident Macrophages in the 2-AAF/Partial Hepatectomy Rat

Background/AimsMacrophages are known to play an important role in hepatocyte mediated liver regeneration by secreting inflammatory mediators. However, there is little information available on the role of resident macrophages in oval cell mediated liver regeneration. In the present study we aimed to investigate the role of macrophages in oval cell expansion induced by 2-acetylaminofluorene/partial hepatectomy (2-AAF/PH) in rats.Methodology/Principal FindingsWe depleted macrophages in the liver of 2-AAF/PH treated rats by injecting liposome encapsulated clodronate 48 hours before PH. Regeneration of remnant liver mass, as well as proliferation and differentiation of oval cells were measured. We found that macrophage-depleted rats suffered higher mortality and liver transaminase levels. We also showed that depletion of macrophages yielded a significant decrease of EPCAM and PCK positive oval cells in immunohistochemical stained liver sections 9 days after PH. Meanwhile, oval cell differentiation was also attenuated as a result of macrophage depletion, as large foci of small basophilic hepatocytes were observed by day 9 following hepatectomy in control rats whereas they were almost absent in macrophage depleted rats. Accordingly, real-time polymerase chain reaction analysis showed lower expression of albumin mRNA in macrophage depleted livers. Then we assessed whether macrophage depletion may affect hepatic production of stimulating cytokines for liver regeneration. We showed that macrophage-depletion significantly inhibited hepatic expression of tumor necrosis factor-α and interleukin-6, along with a lack of signal transducer and activator of transcription 3 phosphorylation during the early period following hepatectomy.ConclusionsThese data indicate that macrophages play an important role in oval cell mediated liver regeneration in the 2-AAF/PH model.

1234 PRO-APOPTOTIC MIR-34A/SIRT1/P53 PATHWAY IN NAFLD PATIENTS AND TARGETING BY URSODEOXYCHOLIC ACID IN RAT LIVER

histology was graded and staged according to the Kleiner system. Hepatic triglycerides were quantified, and qPCR/Western blotting used to assess inflammation, fibrosis and lipid metabolism. Hepatic (oval) cell response was assessed by pan-cytokeratin (panCK) and Sox9 immunohistochemistry with morphometric analysis. Results: ALIOS fed mice developed lobular inflammation/ballooning by 6m and F3 fibrosis by 12m. By 6m, 5aR1 mice fed ALIOS diet demonstrated greater steatosis histologically (median Kleiner steatosis score; 3 vs 1), and biochemically (mean hepatic triglyceride; 93.1 vs 62.4 nmol/mg, p < 0.01) than WT mice fed ALIOS diet. There was no difference in inflammation or fibrosis. 5aR1 mice had reduced carnitine palmitoyl transferase-1a mRNA expression (p < 0.05), and increased acetyl-CoA carboxylase protein expression, consistent with decreased fatty acid b-oxidation and increased de novo lipogenesis. Focal hepatocellular lesions consistent with dysplasia/hepatocellular carcinoma developed after 12m ALIOS diet in 5/10 WT mice, with none developing in 5aR1 mice. Dysplastic/hepatocellular carcinoma lesions expressed the oval cell marker Sox9. Hepatic oval cell proliferation increased after ALIOS diet, but was attenuated in 5aR1 compared to WT mice (mean surface area panCK positive; 0.62% vs. 0.94%; Sox9 positive cells per view; 60 vs. 76/field of view respectively). Conclusions: 5aR1 deficiency promotes hepatic steatosis, but protects against the development of NASH-related dysplasia/hepatocellular carcinoma and reduces hepatic oval cell proliferation. Prolonged ALIOS diet is a unique murine model for studying NASH-related oval cell proliferation and hepatocarcinogenesis in the absence of cirrhosis. These data support a role of 5aR1 in hepatocarcinogenesis associated with NASH.

P86 5α-reductase-1 knockout promotes steatosis but protects against hepatocarcinogenesis in a murine model of NAFLD

IntroductionGlucocorticoids have been implicated in the pathogenesis of NAFLD, but as most patients with NAFLD have normal circulating cortisol levels it has been suggested that it is hepatic levels which...

Inhibition of Notch signaling affects hepatic oval cell response in rat model of 2AAF-PH

Background and aimsActivation of the oval cell compartment occurs in the liver when hepatocytes are functionally compromised and/or unable to divide. Our goal was to investigate the systemic signals responsible for determining the efficiency of oval cell-mediated liver regeneration, focusing on the Notch signaling cascade.MethodsThe established oval cell induction protocol of 2-acetylaminofluorine (2-AAF) implantation followed by 70% surgical resection of the liver (partial hepatectomy, PH) was employed in a rat model. This oval cell induction model was further combined with injections of a γ-secretase inhibitor (GSI XX) to examine the effects of Notch inhibition on oval cell-aided regeneration of the liver.ResultsNotch signaling was found to be upregulated at the peak of oval cell induction during 2AAF-PH alone. Treatment with GSI XX led to interruption of the Notch signal, as shown by a decrease in expression of Hes1. While there was a robust oval cell response seen at day 11 post-PH, there was a measurable delay in differentiation when Notch was inhibited. This was confirmed morphologically as well as by immunohistochemistry for the oval cell markers, α-fetoprotein, OV-6, and CK19. The hepatocytes seen at day 22 demonstrated an enhanced hepatocellular mitoinhibition index (p21Waf1/Ki67), suggestive of dysregulated proliferation and cell cycle progression. Moreover, these hepatocytes exhibited decreased expression of hepatocyte functional markers, such as cytochrome P450 and glucose-6-phosphatase-α.ConclusionTaken together, these results identify the Notch signaling pathway as a potent regulator of differentiation and proliferation in oval cells, which is necessary for functional repair of the liver by oval cells.

Prospective isolation of a bipotential clonogenic liver progenitor cell in adult mice

The molecular identification of adult hepatic stem/progenitor cells has been hampered by the lack of truly specific markers. To isolate putative adult liver progenitor cells, we used cell surface-marking antibodies, including MIC1-1C3, to isolate subpopulations of liver cells from normal adult mice or those undergoing an oval cell response and tested their capacity to form bilineage colonies in vitro. Robust clonogenic activity was found to be restricted to a subset of biliary duct cells antigenically defined as CD45(-)/CD11b(-)/CD31(-)/MIC1-1C3(+)/CD133(+)/CD26(-), at a frequency of one of 34 or one of 25 in normal or oval cell injury livers, respectively. Gene expression analyses revealed that Sox9 was expressed exclusively in this subpopulation of normal liver cells and was highly enriched relative to other cell fractions in injured livers. In vivo lineage tracing using Sox9creER(T2)-R26R(YFP) mice revealed that the cells that proliferate during progenitor-driven liver regeneration are progeny of Sox9-expressing precursors. A comprehensive array-based comparison of gene expression in progenitor-enriched and progenitor-depleted cells from both normal and DDC (3,5-diethoxycarbonyl-1,4-dihydrocollidine or diethyl1,4-dihydro-2,4,6-trimethyl-3,5-pyridinedicarboxylate)-treated livers revealed new potential regulators of liver progenitors.

Response of sinusoidal mouse liver cells to choline-deficient ethionine-supplemented diet

BackgroundProliferation of oval cells, the bipotent precursor cells of the liver, requires impeded proliferation and loss of hepatocytes as well as a specific micro-environment, provided by adjacent sinusoidal cells of liver. Despite their immense importance for triggering the oval cell response, cells of hepatic sinusoids are rarely investigated. To elucidate the response of sinusoidal liver cells we have employed a choline-deficient, ethionine-supplemented (CDE) diet, a common method for inducing an oval cell response in rodent liver. We have utilised selected expression markers commonly used in the past for phenotypic discrimination of oval cells and sinusoidal cells: cytokeratin, E-cadherin and M2-pyruvate kinase for oval cells; and glial fibrillary acidic protein (GFAP) was used for hepatic stellate cells (HSCs).ResultsCDE diet leads to an activation of all cells of the hepatic sinusoid in the mouse liver. Beside oval cells, also HSCs and Kupffer cells proliferate. The entire fraction of proliferating cells in mouse liver as well as endothelial cells and cholangiocytes express M2-pyruvate kinase. Concomitantly, GFAP, long considered a unique marker of quiescent HSCs was upregulated in activated HSCs and expressed also in cholangiocytes and oval cells.ConclusionsOur results point to an important role of all types of sinusoidal cells in regeneration from CDE induced liver damage and call for utmost caution in using traditional marker for identifying specific cell types. Thus, M2-pyruvate kinase should no longer be used for estimating the oval cell response in mouse liver. CDE diet leads to activation of GFAP positive HSCs in the pericentral zone of liver lobulus. In the periportal zone the detection of GFAP in biliary cells and oval cells, calls other cell types as progenitors of hepatocytes into question under CDE diet conditions.

Hepatic stellate cells’ involvement in progenitor-mediated liver regeneration

Earlier studies conducted by our laboratory have shown that suppression of transforming growth factor-β (TGFβ)-mediated upregulation of connective tissue growth factor (CTGF) by iloprost resulted in a greatly diminished oval cell response to 2-acetylaminofluorene/partial hepatectomy (2AAF/PH) in rats. We hypothesized that this effect is due to decreased activation of hepatic stellate cells. To test this hypothesis, we maintained rats on a diet supplemented with 2% L-cysteine as a means of inhibiting stellate cell activation during the oval cell response to 2AAF/PH. In vitro experiments show that L-cysteine did, indeed, prevent the activation of stellate cells while exerting no direct effect on oval cells. Desmin immunostaining of liver sections from 2AAF/PH animals indicated that maintenance on the L-cysteine diet resulted in an 11.1-fold decrease in the number of activated stellate cells within the periportal zones. The total number of cells proliferating in the periportal zones of livers from animals treated with L-cysteine was drastically reduced. Further analyses showed a greater than fourfold decrease in the magnitude of the oval cell response in animals maintained on the L-cysteine diet as determined by immunostaining for both OV6 and α-fetoprotein (AFP). Global liver expression of AFP as measured by real-time PCR was shown to be decreased 4.7-fold in the L-cysteine-treated animals. These data indicate that the activation of hepatic stellate cells is required for an appropriate oval cell response to 2AAF/PH.

Hepatic Fibrosis Is Enhanced and Accompanied by Robust Oval Cell Activation after Chronic Carbon Tetrachloride Administration to Egr-1-Deficient Mice

The transcription factor early growth response (Egr)-1 regulates the expression of genes required for execution of the wound healing response. Multiple cycles of injury, coupled to incomplete wound healing, lead to fibrosis. Therefore, we hypothesized that Egr-1 is required for the development of hepatic fibrosis. To test this hypothesis, we exposed wild-type and egr-1(-/-) mice to acute or chronic carbon tetrachloride (CCl(4)). Acute CCl(4) exposure established a profibrotic milieu in the liver, including activation of hepatic stellate cells as well as expression of type 1 collagen genes and tissue inhibitor of matrix metalloproteinase 1 in both wild-type and egr-1(-/-) mice. This response was exacerbated in egr-1(-/-) mice. After chronic CCl(4) exposure, hepatic fibrosis was established in both genotypes; however, the fibrotic response was profoundly worsened in Egr-1-deficient mice. Importantly, enhanced fibrosis in egr-1(-/-) mice was accompanied by a robust activation of the oval cell response, suggesting more severe liver injury and/or reduced hepatocyte proliferation when compared with wild-type mice. Hepatic expression of genes indicative of oval cell activation, as well as the number of cells expressing A6, a mouse oval cell marker, was greater in egr-1(-/-) mice. Taken together, these data reveal novel roles for Egr-1 as a negative regulator of both CCl(4)-induced hepatic fibrosis and the oval cell response.

A study of oval cell proliferation kinetics in the rat 2-acetylaminofluorene/partial hepatectomy model

To investigate the changes of oval cell proliferation rate in the rat 2-acetylaminofluorene/partial hepatectomy (2-AAF/PH) model. Livers were collected from 2-AAF/PH rats at different time points after hepatectomy. Paraffin sections were investigated by double immunofluorescent staining with confocal microscopy for oval cell marker epithelial cell adhesion molecule and proliferative index proliferating cell nuclear antigen, or epithelial cell adhesion molecule and alpha-smooth muscle actin. Deposition of matrix in liver tissue was detected by sirius red staining. Response of ductular oval cells could be observed in portal area at 2 days after PH, and the number of oval cells reached its peak at 9 days and then gradually declined. Oval cell proliferation rate decreased from (91.3 +/- 1.6)% at 2 days after PH to (53.6 +/- 4.4)% at 12 days (P < 0.01). In addition, oval cells infiltrating into liver parenchyma were closely associated with activated hepatic stellate cells and extracellular matrix. Oval cell proliferation rate starts decreasing before its number reaches a peak in 2-AAF/PH model. Hepatic stellate cells probably tightly regulate oval cell number through secreting several factors and producing extracellular matrix.