Compensatory Liver Regeneration Research Articles

Clinical Factors Affecting the Rate of Liver Regeneration in Living Donors after Right Hepatectomy.

Sufficient liver regeneration after a right hepatectomy is important in living donors for preventing postoperative hepatic insufficiency; however, it differs for each living donor so we investigated the clinical factors affecting the rate of liver regeneration after hepatic resection. This retrospective case-control study investigated fifty-four living donors who underwent a right hepatectomy from July 2015 to March 2023. Patients were classified into 2 groups by the remnant/total volume ratio (RTVR): Group A (RTVR < 30%, n = 9) and Group B (RTVR ≥ 30%, n = 45). The peak postoperative level of total bilirubin was more elevated in Group A than in Group B (3.0 ± 1.1 mg/dL vs. 2.3 ± 0.8 mg/dL, p = 0.046); however, no patients had hepatic insufficiency or major complications. The rates of residual liver volume (RLV) growth at Postoperative Week 1 (89.1 ± 26.2% vs. 53.5 ± 23.7%, p < 0.001) were significantly greater in Group A, and its significant predictors were RTVR (β = -0.478, p < 0.001, variance inflation factor (VIF) = 1.188) and intraoperative blood loss (β = 0.247, p = 0.038, VIF = 1.182). In conclusion, as the RLV decreases, compensatory liver regeneration after hepatic resection becomes more prominent, resulting in comparable operative outcomes. Further studies are required to investigate the relationship between hematopoiesis and the rate of liver regeneration.

Role of Genetic and Epigenetic Modifications in the Progression of Hepatocellular Carcinoma in Chronic HCV Patients

Globally, hepatocellular carcinoma (HCC) is a significant cause of mortality and morbidity among chronically infected HCV patients. It is established that HCV is a primary risk factor for HCC progression. The treatment of HCV infection has been transformed by the introduction of DAAs with high rates of virological clearance. The reduction in cirrhosis-related consequences, particularly HCC, is the long-term objective of DAAs therapy for HCV. Although the risk of developing HCC is decreased in HCV patients who achieve a disease-sustaining virological response, these patients are nevertheless at risk, especially those with severe fibrosis and cirrhosis. Previous studies have shown that HCV induce several mechanisms of hepatocarcinogenesis in the host’s hepatic micro- and macro-environment, which leads to HCC progression. In an HCV-altered environment, compensatory liver regeneration favors chromosomal instability and irreversible alterations, which encourage hepatocyte neoplastic transformation and the development of malignant clones. These mechanisms involve a series of genetic and epigenetic modifications including host genetic factors, dysregulation of several signaling pathways, histone, and DNA modifications including methylation and acetylation. This review highlights the genetic and epigenetic factors that lead to the development of HCC in chronic HCV-infected individuals and can be targeted for earlier HCC diagnosis and prevention.

Loss of hepatic mTORC1 signaling affects liver homeostasis which is partially compensated by Wnt/β‐catenin pathway activation

BackgroundsLiver has an innate capacity to regenerate following partial hepatectomy (PH). Pathways including the mTORC1 and the Wnt/β‐catenin pathways have been shown to become activated after PH to enable liver regeneration (LR). However, how one pathway could compensate during deficiency of another is less well understood. Here, we report discovery and characterization of a compensatory interplay between mTOR and Wnt signaling that enables LR following PH when mTORC1 pathway is interrupted genetically.MethodsWe generated hepatocyte‐specific Raptor knockout (Raptor∆HC) mice by delivering AAV8‐TBG‐Cre to 6‐week‐old Raptorflox/flox mice. Two weeks after AAV8 injection, animals were either harvested at baseline, or subjected to partial hepatectomy (PH) for analysis to address changes and compensations during the process of LR.ResultsAcute deletion of hepatocyte Raptor abolished mTORC1 activity and led to compensatory activation of mTORC2 and its downstream target p‐AKT (S473). Raptor∆HC mice had baseline liver injury as shown by elevated serum levels of ALT, AST, and ALP. Raptor∆HC mice exhibited decreased levels of pericentral enzyme (Glul, Cyp2e1), periportal enzyme (G6pc), and main secretory proteins (Alb, Trf, Ttr), suggesting global impairment of metabolic, synthetic, and secretory functions. These mice were also under metabolic stress as seen by ER whorl formation, mitochondrial swelling, and increased autophagy, by transmission electron microscopy (TEM). There was an increase in cell death seen as enhanced apoptotic body formation by TUNEL and cleaved caspase‐3 staining and an associated increase in macrophage infiltration in the immediate proximity of dying hepatocytes. An attempt at compensatory liver regeneration was concurrently evident through activation of the Wnt/β‐catenin pathway leading to enhanced Cyclin D1, Ki67, Axin2 and c‐Myc. β‐Catenin protein increased 5‐fold in Raptor∆HC mice and appears to be due to both increased gene transcription and decreased phosphorylation‐mediated degradation. Further, stabilization of β‐catenin protein appears to be due to both cell‐extrinsic mechanism seen as increased p‐LPR6 (S1490) suggesting Wnt stimulation, and cell‐intrinsic mechanism seen as decreased GSK3β activity which was phosphorylated and inhibited by compensatory elevation of AKT activity in the Raptor∆HC mice. Despite activation of the Wnt pathways, hepatocytes failed to finish cell cycle seen as an overall decrease in BrdU incorporation after a 14‐day continuous pulse. Indeed, when Raptor∆HC mice were subjected to PH, they all died within 48h.ConclusionsmTORC1 activity is essential for homeostatic liver functions and for LR after PH. Stabilization of β‐catenin due to multiple mechanisms including Wnt secretion likely from macrophages, secondary to AKT mediated GSK3β inhibition and via de novo β‐catenin transcription, attempts to compensate to maintain homeostasis in the Raptor∆HC mice.

Diploid hepatocytes promote compensatory liver regeneration following acetaminophen induced acute liver injury

Background Acetaminophen (APAP) is a commonly used analgesic that can be safely used at doses under 4 g/day. When taken in excess, APAP causes acute liver injury, which leads to acute liver failure and death. APAP overdose leads to 56,000 emergency room visits and 26,000 hospitalizations annually and is the leading cause of acute liver failure in the United States. The only available pharmacological treatment, N-acetyl cysteine, is effective within hours of overdose. The only other treatment option is orthotopic liver transplantation, and this is limited by the availability of donor organs. Considering the paucity of therapeutic options, there is an urgent need to understand mechanisms of liver regeneration and repair. Most mammalian somatic cells are diploid and contain pairs of each chromosome, but there are also polyploid cells, such as hepatocytes that contain additional sets of chromosomes. Polyploid hepatocytes are among the best described, and in adult humans and mice comprise more than 25% and 90%, respectively, of the hepatocyte population. The cellular and molecular mechanisms that regulate polyploidy have been well-characterized; however, it is poorly understood if diploid and polyploid hepatocytes play specialized roles in liver injury and repair. Methods Our lab recently found that diploid hepatocytes are inherently more proliferative than polyploid hepatocytes. Additionally, we can specifically study diploid hepatocytes using mice lacking E2f7 and E2f8 in the liver (referred to as “LKO” mice), which are functionally normal, but livers are depleted of polyploid hepatocytes. Considering that liver regeneration affects the outcome of APAP-medicated injury, we asked if LKO mice enriched with highly proliferative diploid hepatocytes would respond differently than control livers. We fasted LKO and control mice for 16 hours, injected intraperitoneally with 300 mg/kg (the LD50 dose) APAP and harvested livers after 0.5, 3, 6, 12, 24, 48, 72 and 96 hours. We used these tissues to investigate differences in liver injury, necrosis, and proliferation. Results Liver injury enzymes, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), were higher in control mice compared to LKO mice beginning at 3 hours. Necrotic liver tissue was abundant in control mice and was markedly reduced in LKO mice. We also measured more apoptotic, TUNEL positive hepatocytes in control mice. We next assessed the proliferation response after APAP-induced liver injury by staining tissues for PCNA which was induced by 6 hours in LKO mice compared to 24 hours in control mice. We treated wild-type 19-day old hepatocytes with 5 mM APAP for 12, 24, and 36 hours in vitro and found that diploid hepatocytes proliferate more readily compared to polyploids as marked by BrdU incorporation. Finally, we found higher doses of APAP eliminate the advantages seen in LKO mice. Conclusion Based on these data, diploid hepatocytes appear to be the main contributors of early-stage compensatory regeneration following APAP-mediated liver injury. These data suggest that, compared to polyploid hepatocytes, diploids initiate and drive liver healing/regeneration after acute injury.

Persistent hepatocyte apoptosis promotes tumorigenesis from diethylnitrosamine-transformed hepatocytes through increased oxidative stress, independent of compensatory liver regeneration

Hepatocellular carcinoma highly occurs in chronic hepatitis livers, where hepatocyte apoptosis is frequently detected. Apoptosis is a mechanism that eliminates mutated cells. Hepatocyte apoptosis induces compensatory liver regeneration, which is believed to contribute to tumor formation. Hepatocyte-specific Mcl-1 knockout mice (Mcl-1Δhep mice) developed persistent hepatocyte apoptosis and compensatory liver regeneration with increased oxidative stress in adulthood but had not yet developed hepatocyte apoptosis at the age of 2 weeks. When diethylnitrosamine (DEN) was administered to 2-week-old Mcl-1Δhep mice, multiple liver tumors were formed at 4 months, while wild-type mice did not develop any tumors. These tumors contained the B-Raf V637E mutation, indicating that DEN-initiated tumorigenesis was promoted by persistent hepatocyte apoptosis. When N-acetyl-L-cysteine was given from 6 weeks of age, DEN-administered Mcl-1Δhep mice had reduced oxidative stress and suppressed tumorigenesis in the liver but showed no changes in hepatocyte apoptosis or proliferation. In conclusion, enhanced tumor formation from DEN-transformed hepatocytes by persistent hepatocyte apoptosis is mediated by increased oxidative stress, independent of compensatory liver regeneration. For patients with livers harboring transformed cells, the control of oxidative stress may suppress hepatocarcinogenesis based on chronic liver injury.

Depletion of essential isoprenoids and ER stress induction following acute liver-specific deletion of HMG-CoA reductase

HMG-CoA reductase (Hmgcr) is the rate-limiting enzyme in the mevalonate pathway and is inhibited by statins. In addition to cholesterol, Hmgcr activity is also required for synthesizing nonsterol isoprenoids, such as dolichol, ubiquinone, and farnesylated and geranylgeranylated proteins. Here, we investigated the effects of Hmgcr inhibition on nonsterol isoprenoids in the liver. We have generated new genetic models to acutely delete genes in the mevalonate pathway in the liver using AAV-mediated delivery of Cre-recombinase (AAV-Cre) or CRISPR/Cas9 (AAV-CRISPR). The genetic deletion of Hmgcr by AAV-Cre resulted in extensive hepatocyte apoptosis and compensatory liver regeneration. At the biochemical level, we observed decreased levels of sterols and depletion of the nonsterol isoprenoids, dolichol and ubiquinone. At the cellular level, Hmgcr-null hepatocytes showed ER stress and impaired N-glycosylation. We further hypothesized that the depletion of dolichol, essential for N-glycosylation, could be responsible for ER stress. Using AAV-CRISPR, we somatically disrupted dehydrodolichyl diphosphate synthase subunit (Dhdds), encoding a branch point enzyme required for dolichol biosynthesis. Dhdds-null livers showed ER stress and impaired N-glycosylation, along with apoptosis and regeneration. Finally, the combined deletion of Hmgcr and Dhdds synergistically exacerbated hepatocyte ER stress. Our data show a critical role for mevalonate-derived dolichol in the liver and suggest that dolichol depletion is at least partially responsible for ER stress and apoptosis upon potent Hmgcr inhibition.

AAV-CRISPR Gene Editing Is Negated by Pre-existing Immunity to Cas9.

Adeno-associated viral (AAV) vectors are a leading candidate for the delivery of CRISPR-Cas9 for therapeutic genome editing in vivo. However, AAV-based delivery involves persistent expression of the Cas9 nuclease, a bacterial protein. Recent studies indicate a high prevalence of neutralizing antibodies and T cells specific to the commonly used Cas9 orthologs from Streptococcus pyogenes (SpCas9) and Staphylococcus aureus (SaCas9) in humans. We tested in a mouse model whether pre-existing immunity to SaCas9 would pose a barrier to liver genome editing with AAV packaging CRISPR-Cas9. Although efficient genome editing occurred in mouse liver with pre-existing SaCas9 immunity, this was accompanied by an increased proportion of CD8+ T cells in the liver. This cytotoxic T cell response was characterized by hepatocyte apoptosis, loss of recombinant AAV genomes, and complete elimination of genome-edited cells, and was followed by compensatory liver regeneration. Our results raise important efficacy and safety concerns for CRISPR-Cas9-based in vivo genome editing in the liver.

Hepatic deletion of MET aggravates acetaminophen hepatotoxicity and impairs liver regeneration in mice

MET is a receptor for hepatocyte growth factor (HGF) and is considered critical for liver regeneration after partial hepatectomy (Phx). Combined disruption of MET along with epidermal growth factor receptor (EGFR) signaling results in compete elimination of regenerative response after Phx, highlighting importance of these growth factor receptors in liver regeneration. A few studies also indicate a role of MET in cell survival signaling in hepatocytes. However, the role of MET in liver injury and compensatory regeneration after acetaminophen (APAP) overdose, a clinically significant model of acute liver failure (ALF), is not known, which was investigated in the current study. APAP (300 and 600 mg/kg) treatment in mice caused remarkable activation of MET in a dose‐dependent manner. Interestingly, MET activation was observed even at a very early stage (3 hr post‐APAP), prior to any observable necrosis or compensatory regenerative response, and was sustained during peak liver injury. Similar dose‐dependent activation of ERK1/2 signaling (one of the downstream mediators of MET) was also observed after APAP treatment. Next, albumin‐driven CRE approach was utilized for liver‐specific deletion of MET in order to study a causative role of MET in APAP‐induced liver injury and regeneration. WT or MET‐KO mice were treated with 300 mg/kg APAP and investigated at 0, 1, 3, 6, 12 and 24 hr post‐APAP. Liver‐specific MET deletion aggravated APAP‐induced liver injury as investigated by measuring serum ALT and AST levels, which was further corroborated by quantifying necrotic areas. Although, liver injury was not significantly different during early initiation phase, it was remarkably aggravated in MET‐KO mice during the progression phase of APAP hepatotoxicity (12 and 24 hr). Further, hepatocyte proliferation and activation of cell cycle machinery were strikingly lower in MET‐KO mice, indicating compensatory liver regeneration was compromised in these mice. While spontaneous recovery and 100% survival was observed in WT mice, significant mortality (67%) was observed in MET‐KO mice. Lastly, macrophage‐specific deletion of MET utilizing LysM‐CRE did not alter liver injury or regeneration after APAP overdose in mice, indicating macrophage‐specific MET is not involved. In conclusion, our study revealed a novel protective role of MET in liver injury and in promoting compensatory liver regeneration during APAP‐induced ALF.Support or Funding InformationThis study was supported by the Menten Endowment Foundation, University of Pittsburgh

Diploid Hepatocytes Resist Acetaminophen Induced Acute Liver Injury and Drive Compensatory Liver Regeneration

BackgroundAcetaminophen (APAP) is a commonly used analgesic that can be safely used at doses under 4 g/day. When taken in excess, APAP causes acute liver injury, which leads to acute liver failure and death. APAP overdose leads to 56,000 emergency room visits and 26,000 hospitalizations annually and is the leading cause of acute liver failure in the United States. The only available pharmacological treatment, N‐acetyl cysteine, is effective within hours of overdose. The only other treatment option is orthotopic liver transplantation, and this is limited by the availability of donor organs. Considering the paucity of therapeutic options, there is an urgent need to understand mechanisms of liver regeneration and repair.Most mammalian somatic cells are diploid and contain pairs of each chromosome, but there are also polyploid cells, such as hepatocytes that contain additional sets of chromosomes. Polyploid hepatocytes are among the best described, and in adult humans and mice comprise more than 25% and 90%, respectively, of the hepatocyte population. The cellular and molecular mechanisms that regulate polyploidy have been well‐characterized; however, it is poorly understood if diploid and polyploid hepatocytes play specialized roles in liver injury and repair.MethodsOur lab recently found that diploid hepatocytes are inherently more proliferative than polyploid hepatocytes. Additionally, we can specifically study diploid hepatocytes using mice lacking E2f7 and E2f8 in the liver (referred to as “LKO” mice), which are functionally normal, but livers are depleted of polyploid hepatocytes. Considering that liver regeneration affects the outcome of APAP‐medicated injury, we asked if LKO mice enriched with highly proliferative diploid hepatocytes would respond differently than control livers. We fasted LKO and control mice for 16 hours, injected intraperitoneally 300 mg/kg (the LD50 dose) APAP and harvested livers after 0.5, 3, 6, 12, 24, and 48 hours. We used these tissues to investigate differences in liver injury, necrosis, and proliferation.ResultsLiver injury enzymes, alanine aminotransferase and aspartate aminotransferase, were higher in control mice compared to LKO mice beginning at 3 hours. Necrotic liver tissue was abundant in control mice and was markedly reduced in LKO mice. We also measured more apoptotic, TUNEL positive hepatocytes in control mice. We next assessed the proliferation response after APAP‐induced liver injury by staining for PCNA which was induced by 6 hours in LKO mice compared to 24 hours in control mice. Finally, we treated wild‐type 19‐day old hepatocytes with 5 mM APAP for 12, 24, and 36 hours in vitro and found that diploid hepatocytes proliferate more readily compared to polyploids as marked by BrdU incorporation.ConclusionBased on these data, diploid hepatocytes appear to be the main contributors of early‐stage compensatory regeneration following APAP‐mediated liver injury. These data suggest that, compared to polyploid hepatocytes, diploids initiate and drive liver healing/regeneration after acute injury.Support or Funding InformationThis work supported by the NIH to AWD (R01DK103645).

Sphingosine kinase inhibitor regulates pro‐inflammatory cytokines to subdue the neoplastic lesions in hepatocellular carcinoma

Hepatocellular carcinoma (HCC), is the most leading cause of cancer‐related death in all over the world (seems to more than 80% of death cases). It is frequently linked with continuous hepatocytes death, inflammatory cell infiltration, and compensatory liver regeneration with higher angiogenic rate. Considering the different signalling process during this tumorogenesis with the involvement of new ‘lead’ identification of different sources, desire to be less toxic and higher bioavailability. The use of live, attenuated or genetically compensated microbes or its cellular component(s) or metabolites has begun to emerge as a potential new approach in medicinal research to deliver bioactive entities. Thus, advancing our knowledge of such microbes‐mediated therapy may suggest new avenues for therapeutic intervention in many fatal diseases.Sphingosine kinase inhibitor (SKI) II has been reported as a dual inhibitor of sphingosine kinases (SKs) 1 and 2 and has been extensively used to prove the involvement of SKs and sphingosine‐1‐phosphate (S1P) in cellular processes and able to induce apoptosis in human HepG2 (Hepatocellular carcinoma cell, human) cells, as characterized by phosphatidylserine serine exposure, activation of series of caspases and cleaved PARP. SKI also modulates the various transcription factors like NF‐kB‐65, p53 through regulation of cell proliferation exerted the death cause. Moreover, this study also focused that SKI effectively suppressed diethylnitrosamine (DEN) induced liver inflammation and hyperplasia in murine, also regulated the lipid peroxidation and enzymic antioxidants (SOD, CAT, GPx, GR and GST) status with an improvement of their survival rate. It reduced the elevated level of cytokines including tumor necrosis factors, interleukins, interferon (alpha and gamma), and transforming growth factors in hepatocytes from liver fibrosis bearing mice. As evident from an immunochemical analysis, DEN‐induced neoplastic lesions may up‐regulate the various growth factors including EGF, HGF, endothelin‐1 regulation with promoting angiogenic activity via modulation of CD34, VEGF, HIF‐1α expression and we found that the alteration of these changes were balanced by SKI treatment in vivo system. So taken together, these finding indicated that, SKI may recover in hyperproliferating cancer cells with tumor growth regulation in carcinogen‐exposed rodent system. In future direction, we will check the p53−/− and cmyc−/− mice that hoe it could regulate cancer metastasis with this SKI. and the involvement of od tumor microenvironment.Support or Funding InformationCSIR and Department of Science and Technology, IndiaThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 3942: The fraction of garlic protein(s) affect HCC via regulation of angiogenesis

Abstract Cancers are leading cause of human mortality. Most of deaths are due cancer because of its high resistance to the chemotherapeutic drugs. Thus, there is an ongoing demand for generation of new potent and more effective therapies for cancer treatment. Hepatocellular carcinoma (HCC), is the most leading cause of cancer-related death in all over the world (seems to more than 80% of death cases in USA). It is frequently linked with continuous hepatocytes death, inflammatory cell infiltration, and compensatory liver regeneration with higher angiogenic rate. Considering the different signaling process during this tumorigenesis with the involvement of new ‘lead' identification of various sources, desire to be less toxic and higher bioavailability. The use of natural or its active metabolic component(s) has begun to emerge as a potential innovative approach in medicinal research to deliver bioactive entities. Thus, garlic has already proven as the therapeutic implication on immune system and cancer due to presence of sulfur compound. But the role of active protein fraction present in fresh garlic is still not revealed yet. In our study, we are isolated an active garlic protein from fresh garlic aqueous extract with molecular weight about 40-45kD. This Garlic protein (GP) having non-cytotoxic to human/murine normal cells, could induce apoptosis in human HepG2 cells, as characterized by phosphatydylserine serine exposure, DNA fragmentation, alteration with loss of mitochondrial membrane potential and cytochrome C release, activation of series of caspases, other physiochemical changes lead to induce cancer cell death. This study also focused that the GP effectively suppressed DEN induced liver inflammation and hyperplasia in murine also decline in lipid peroxidation and changes in enzymic antioxidants and biochemical parameter with improvement of their survival rate. It reduced the elevated level of cytokines and growth factors in hepatocytes from liver fibrosis bearing mice. Thus, finding indicated that, GP may induce apoptosis and in hyper proliferating cancer cells via altering cytokines-chemokines level with tumor growth regulation in carcinogen exposed rodent system. Future direction, we are proposing to execute the experiments on p53-/- and cMyc-/- deficient mice to find out the role of GP in these circumstances. Citation Format: Nabanita Chatterjee, Subhadip Das, Prajnamoy Pal, Krishna Das Saha. The fraction of garlic protein(s) affect HCC via regulation of angiogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3942.

The immunology of hepatocellular carcinoma.

In contrast to most other malignancies, hepatocellular carcinoma (HCC), which accounts for approximately 90% of primary liver cancers, arises almost exclusively in the setting of chronic inflammation. Irrespective of etiology, a typical sequence of chronic necroinflammation, compensatory liver regeneration, induction of liver fibrosis and subsequent cirrhosis often precedes hepatocarcinogenesis. The liver is a central immunomodulator that ensures organ and systemic protection while maintaining immunotolerance. Deregulation of this tightly controlled liver immunological network is a hallmark of chronic liver disease and HCC. Notably, immunotherapies have raised hope for the successful treatment of advanced HCC. Here we summarize the roles of specific immune cell subsets in chronic liver disease, with a focus on non-alcoholic steatohepatitis and HCC. We review new advances in immunotherapeutic approaches for the treatment of HCC and discuss the challenges posed by the immunotolerant hepatic environment and the dual roles of adaptive and innate immune cells in HCC.

Abstract 4906: Sphingolipid regulates pro-inflammatory cytokines to subdue the neoplastic lesions in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC), is the most leading cause of cancer-related death in all over the world (seems to more than 80% of death cases). It is frequently linked with continuous hepatocytes death, inflammatory cell infiltration, and compensatory liver regeneration with higher angiogenic rate. Considering the different signalling process during this tumorogenesis with the involvement of new ‘lead’ identification of different sources, desire to be less toxic and higher bioavailability. The use of live, attenuated or genetically compensated microbes or its cellular component(s) or metabolites has begun to emerge as a potential new approach in medicinal research to deliver bioactive entities. Thus, advancing our knowledge of such microbes-mediated therapy may suggest new avenues for therapeutic intervention in many fatal diseases. Lipid (LSPL-1) isolated from Leishmania donovani could induce apoptosis in human HepG2 (Hepatocellular carcinoma cell, human) cells, as characterized by phosphatydylserine serine exposure, DNA fragmentation, alteration of BaX/Bcl2 ratio with loss of mitochondrial membrane potential and cytochrome C release, activation of series of caspases and cleaved PARP. LSPL-1 also modulates the various transcription factors like NF-kb65, p53 through regulation of cell proliferation exerted the death cause. Moreover this study also focused that the LSPL-1 effectively suppressed diethylnitrosamine (DEN) induced liver inflammation and hyperplasia in murine also decline in lipid peroxidation and increase in enzymic antioxidants (SOD, CAT, GPx, GR and GST) status with improvement of their survival rate. It reduced the elevated level of cytokines including tumor necrosis factors, interleukins, interferon (alpha and gamma), and transforming growth factors in hepatocytes from liver fibrosis bearing mice. As evident from immunochemical analysis, inflamed hepatocytes and liver tissue may up regulated the various growth factors including EGF, HGF, endothline-1 regulation with promoting angiogenic activity via modulation of CD34, VEGF, HIF-1α expression and alteration of these changes were balanced by LSPL-1. So taken together, these finding indicated that, LSPL-1 may induce apoptosis and in hyper proliferating cancer cells via altering cytokines level with tumour growth regulation in carcinogen exposed rodent system. Note: This abstract was not presented at the meeting. Citation Format: Nabanita Chatterjee, Krishna Das Saha. Sphingolipid regulates pro-inflammatory cytokines to subdue the neoplastic lesions in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4906. doi:10.1158/1538-7445.AM2017-4906

Inhibition of Glycogen Synthase Kinase 3 Accelerated Liver Regeneration after Acetaminophen-Induced Hepatotoxicity in Mice

Overdose of acetaminophen (APAP) is the leading cause of acute liver failure (ALF) in the United States. Timely initiation of compensatory liver regeneration after APAP hepatotoxicity is critical for final recovery, but the mechanisms of liver regeneration after APAP-induced ALF have not been extensively explored yet. Previous studies from our laboratory have demonstrated that activation of β-catenin signaling after APAP overdose is associated with timely liver regeneration. Herein, weinvestigated the role of glycogen synthase kinase 3 (GSK3) in liver regeneration after APAP hepatotoxicity using a pharmacological inhibition strategy in mice. Treatment with specific GSK3 inhibitor (L803-mts), starting from 4 hours after 600 mg/kg dose of APAP, resulted in early initiation of liver regeneration in a dose-dependent manner, without modifying the peak regenerative response. Acceleration of liver regeneration was not secondary to alteration of APAP-induced hepatotoxicity, which remained unchanged after GSK3 inhibition. Early cell cycle initiation in hepatocytes after GSK3 inhibition was because of rapid induction of cyclin D1 and phosphorylation of retinoblastoma protein. This was associated with increased activation of β-catenin signaling after GSK3 inhibition. Taken together, our study has revealed a novel role of GSK3 in liver regeneration after APAP overdose and identified GSK3 as a potential therapeutic target to improve liver regeneration after APAP-induced ALF.

Hepatitis B virus inhibits insulin receptor signaling and impairs liver regeneration via intracellular retention of the insulin receptor.

Hepatitis B virus (HBV) causes severe liver disease but the underlying mechanisms are incompletely understood. During chronic HBV infection, the liver is recurrently injured by immune cells in the quest for viral elimination. To compensate tissue injury, liver regeneration represents a vital process which requires proliferative insulin receptor signaling. This study aims to investigate the impact of HBV on liver regeneration and hepatic insulin receptor signaling. After carbon tetrachloride-induced liver injury, liver regeneration is delayed in HBV transgenic mice. These mice show diminished hepatocyte proliferation and increased expression of fibrosis markers. This is in accordance with a reduced activation of the insulin receptor although HBV induces expression of the insulin receptor via activation of NF-E2-related factor 2. This leads to increased intracellular amounts of insulin receptor in HBV expressing hepatocytes. However, intracellular retention of the receptor simultaneously reduces the amount of functional insulin receptors on the cell surface and thereby attenuates insulin binding in vitro and in vivo. Intracellular retention of the insulin receptor is caused by elevated amounts of α-taxilin, a free syntaxin binding protein, in HBV expressing hepatocytes preventing proper targeting of the insulin receptor to the cell surface. Consequently, functional analyses of insulin responsiveness revealed that HBV expressing hepatocytes are less sensitive to insulin stimulation leading to delayed liver regeneration. This study describes a novel pathomechanism that uncouples HBV expressing hepatocytes from proliferative signals and thereby impedes compensatory liver regeneration after liver injury.

Western diet in ApoE-LDLR double-deficient mouse model of atherosclerosis leads to hepatic steatosis, fibrosis, and tumorigenesis

Nonalcoholic fatty liver disease has been linked to cardiovascular diseases and atherosclerosis. The aim of the current study was to characterize the hepatic pathology leading to fibrosis and tumors in a murine model of atherosclerosis. Male apolipoprotein E/low-density lipoprotein receptor double-knockout mice (AL) mice were fed with a high fat and high cholesterol western diet for 35 weeks (AL mice on WD). Protein and mRNA analysis as well as micro-computed tomography (micro-CT) were performed to assess oxidative stress, liver damage, inflammation, fibrosis, signaling pathways, vascularization, and tumorigenesis. Controls were chosen to distinguish between genetically and dietary effects in steatohepatitis and associated tumorigenesis. Hepatic inflammation and dyslipidemia were increased in AL mice on WD compared with wild-type mice on WD. Uniquely, AL mice on WD showed a spontaneous development of tumors (30% of cases) and thickening of intrahepatic vessel walls. Functionally relevant underlying signaling pathways such as NF-κB, Stat3, JNK, and AKT were differentially regulated between AL and wild-type mice on WD. Micro-CT was capable of visualizing and quantitatively distinguishing tumor neovascularization from vascularization in non-neoplastic liver tissue. AL mice on WD diet represent a novel model combining atherosclerosis and nonalcoholic fatty liver disease. Signaling pathways of liver cell damage and compensatory liver regeneration in combination with enhanced inflammation appear to be crucial for the spontaneous development of tumors in AL mice on WD. Micro-CT represents a new and powerful technique for the ultrastructural and three-dimensional assessment of the vascular architecture of liver tumors.

Dynamic and coordinated regulation of KEAP1-NRF2-ARE and p53/p21 signaling pathways is associated with acetaminophen injury responsive liver regeneration.

quinone oxidoreductase 1, glutamate-cysteine ligase modifier subunit, and heme oxygenase-1 was inhibited during the first 24 hours and then induced to limit oxidative damage. The content of p53 and its downstream target p21 were significantly increased upon APAP exposure and subsequently decreased to normal levels at 48 hours. Furthermore, levels of cyclin D1, cyclin D-dependent kinase 4, proliferating cell nuclear antigen, and augmenter of liver regeneration at 48 hours were enhanced, suggesting initiation of hepatocyte proliferation and tissue repair. These results demonstrated that dynamic and coordinated regulation of KEAP1-NRF2-ARE and p53/p21 signaling pathways was associated with compensatory liver regeneration after APAP-induced acute liver injury.

Abstract 5235: A model for TNFα-mediated NFκB signalling: A systems biology study on hepatocytes and liver cancer cells.

Abstract It is known that the development of hepatocellular carcinoma (HCC) is frequently associated to inflammation, hepatocytic inhibition of apoptosis, and compensatory liver regeneration. In this context, the classical κ-light-chain-enhancer of activated B cells (NFκB) signaling pathway is one central regulator of inflammatory responses and hepatocyte survival, which may promote HCC development. Upon stimulation, Kupffer cell-derived cytokine tumor necrosis factor (TNF)-α is one of the key factors during the priming phase of liver regeneration as well as in hepatocarcinogenesis mainly driving cell survival and proliferation by NFκB signalling pathway. In order to understand the impact of this pathway in the earliest stages of liver damage and tumorigenesis, mathematical modelling is necessary to describe the dynamic behaviour of this pathway in normal as well as in malignant-transformed cells. Several mathematical models have been developed for TNFα-induced NFκB signalling in immortalized fibroblasts and tumor cell lines; however, a model specifically analyzing and comparing hepatocytes and HCC cells is still missing. We here present a hepatocyte-specific ordinary differential equation (ODE) model for TNFα-induced NFκB signaling that considers experimental data of protein expression after TNFα stimulation (p65/pp65, IκBα/pIκBα), basal protein turnover (p65, IκBα), and IκBα mRNA. In order to sufficiently describe the pathway dynamics, an additional nuclear phosphorylation step of p65 was included in the model. Possible candidate kinases are the mitogen- and stress-activated protein kinase1 (MSK1) and the protein kinase C zeta (PKCζ). The established model was able to predict TNFα-induced p65/IκBα complex formation, which was experimentally confirmed using primary mouse hepatocytes cells. Finally, we compared the dynamics of TNFα-induced NF-kB pathway activation in different species and cell types (primary murine hepatocytes, human and mouse HCC lines, mouse liver tissue after partial hepatectomy). In conclusion, we here present a mathematical model for TNFα/NFκB signalling in primary hepatocytes cells, providing an important basis to quantitatively disentangle the complex processes factors in liver regeneration and tumorigenesis. Citation Format: Federico Pinna, Sven Sahle, Katharina Beuke, Michaela Bissinger, Selcan Tuncay, Lorenza D'Alessandro, Ralph Gauges, Andreas Raue, Jens Timmer, Ursula Klingmüller, Peter Schirmacher, Ursula Kummer, Kai Breuhahn. A model for TNFα-mediated NFκB signalling: A systems biology study on hepatocytes and liver cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5235. doi:10.1158/1538-7445.AM2013-5235

Bile acid depletion increases susceptibility to acetaminophen‐induced hepatotoxicity in mice

Bile acids are known to play significant role in liver homeostasis. However, the role of bile acids in pathogenesis of acetaminophen (APAP)‐induced acute liver failure (ALF) is not known. Here we report that depletion of bile acids using cholestyramine (CSA), a bile acid sequestering resin, increases susceptibility of C57BL6 mice to APAP‐induced hepatotoxicity. C57BL6 mice were fed either normal diet or 2% CSA containing diet for 1 week, treated with 400 mg/kg APAP (ip) and analyzed over a time course of 0 to 24 hr. The CSA‐fed mice developed rapid and higher liver injury after APAP treatment. Depletion of bile acids by CSA did not alter metabolism of APAP. However, CSA‐fed mice exhibited rapid and significantly higher activation of c‐Jun N‐terminal protein Kinase (JNK) following APAP treatment. Furthermore, a significant decrease in intestinal fibroblast growth factor 15 (FGF15) mRNA was observed in CSA fed mice before and after APAP treatment. Compensatory liver regeneration following APAP overdose was delayed in the CSA‐fed mice up to 12 hr but was not different than the normal diet fed mice at 24 hr after APAP treatment. Taken together, these data indicate that depletion of intestinal bile acid increases susceptibility to APAP by rapid and higher hepatic JNK activation. These data indicate that bile acids and FGF15‐mediated gut‐liver signaling axis play an important role in cytoprotection following APAP‐induced ALF.These studies were supported by NIH ‐ P20 RR021940 (Udayan Apte), and AASLD/ALF Liver Scholar Award (Udayan Apte)

Comparative Study of Compensatory Liver Regeneration in a Rat Model: Portal Vein Ligation Only versus Sequential Ligation of the Portal Vein and Hepatic Artery

Comparative Study of Compensatory Liver Regeneration in a Rat Model: Portal Vein Ligation Only versus Sequential Ligation of the Portal Vein and Hepatic Artery