Shashikiran Donthamsetty

Enhanced liver regeneration following changes induced by hepatocyte-specific genetic ablation of integrin-linked kinase

Following liver regeneration after partial hepatectomy, liver grows back precisely to its original mass and does not exceed it. The mechanism regulating this “hepatostat” is not clear and no exceptions have been found to date. Although pathways initiating liver regeneration have been well studied, mechanisms involved in the termination of liver regeneration are unclear. Here, we report that integrin‐linked kinase (ILK) (involved in transmission of the extracellular matrix [ECM] signaling by way of integrin receptors) and/or hepatic adaptations that ensue following ILK hepatocyte‐targeted removal are critical for proper termination of liver regeneration. Following partial hepatectomy (PHx), mice with a liver‐specific ILK ablation (ILK‐KO‐Liver) demonstrate a termination defect resulting in 58% larger liver than their original pre‐PHx mass. This increase in post‐PHx liver mass is due to sustained cell proliferation driven in part by increased signaling through hepatocyte growth factor (HGF), and the β‐catenin pathway and Hippo kinase pathways. Conclusion: The data indicate that ECM‐mediated signaling by way of ILK is essential in proper termination of liver regeneration. This is the first evidence of a defect leading to impaired termination of regeneration and excessive accumulation of liver weight following partial hepatectomy. (HEPATOLOGY 2009.)

Nonalcoholic fatty liver sensitizes rats to carbon tetrachloride hepatotoxicity

This study tested whether hepatic steatosis sensitizes liver to toxicant‐induced injury and investigated the potential mechanisms of hepatotoxic sensitivity. Male Sprague‐Dawley rats were fed a methionine‐ and choline‐deficient diet for 31 days to induce steatosis. On the 32nd day, administration of a nonlethal dose of CCl4 (2 mL/kg, intraperitoneally) yielded 70% mortality in steatotic rats 12‐72 hours after CCl4administration, whereas all nonsteatotic rats survived. Neither CYP2E1 levels nor covalent binding of [14C]CCl4‐derived radiolabel differed between the groups, suggesting that increased bioactivation is not the mechanism for this amplified toxicity. Cell division and tissue repair, assessed by [3H]thymidine incorporation and proliferative cell nuclear antigen assay, were inhibited in the steatotic livers after CCl4administration and led to progressive expansion of liver injury culminating in mortality. The hypothesis that fatty hepatocytes undergo cell cycle arrest due to (1) an inability to replenish ATP due to overexpressed uncoupling protein‐2 (UCP‐2) or (2) induction of growth inhibitor p21 leading to G1/S phase arrest was tested. Steatotic livers showed 10‐fold lower ATP levels due to upregulated UCP‐2 throughout the time course after CCl4 administration, leading to sustained inhibition of cell division. Western blot analysis revealed an up‐regulation of p21 due to overexpression of TGF β1 and p53 and down‐regulation of transcription factor Foxm1b in steatotic livers leading to lower phosphorylated retinoblastoma protein. Thus, fatty hepatocytes fail to undergo compensatory cell division, rendering the liver susceptible to progression of liver injury. Conclusion: Impaired tissue repair sensitizes the steatotic livers to hepatotoxicity. (HEPATOLOGY 2007;45:391–403.)

High fat diet-fed obese rats are highly sensitive to doxorubicin-induced cardiotoxicity

Often, chemotherapy by doxorubicin (Adriamycin) is limited due to life threatening cardiotoxicity in patients during and posttherapy. Recently, we have shown that moderate diet restriction remarkably protects against doxorubicin-induced cardiotoxicity. This cardioprotection is accompanied by decreased cardiac oxidative stress and triglycerides and increased cardiac fatty-acid oxidation, ATP synthesis, and upregulated JAK/STAT3 pathway. In the current study, we investigated whether a physiological intervention by feeding 40% high fat diet (HFD), which induces obesity in male Sprague-Dawley rats (250-275 g), sensitizes to doxorubicin-induced cardiotoxicity. A LD(10) dose (8 mg doxorubicin/kg, ip) administered on day 43 of the HFD feeding regimen led to higher cardiotoxicity, cardiac dysfunction, lipid peroxidation, and 80% mortality in the obese (OB) rats in the absence of any significant renal or hepatic toxicity. Doxorubicin toxicokinetics studies revealed no change in accumulation of doxorubicin and doxorubicinol (toxic metabolite) in the normal diet-fed (ND) and OB hearts. Mechanistic studies revealed that OB rats are sensitized due to: (1) higher oxyradical stress leading to upregulation of uncoupling proteins 2 and 3, (2) downregulation of cardiac peroxisome proliferators activated receptor-alpha, (3) decreased plasma adiponectin levels, (4) decreased cardiac fatty-acid oxidation (666.9+/-14.0 nmol/min/g heart in ND versus 400.2+/-11.8 nmol/min/g heart in OB), (5) decreased mitochondrial AMP-alpha2 protein kinase, and (6) 86% drop in cardiac ATP levels accompanied by decreased ATP/ADP ratio after doxorubicin administration. Decreased cardiac erythropoietin and increased SOCS3 further downregulated the cardioprotective JAK/STAT3 pathway. In conclusion, HFD-induced obese rats are highly sensitized to doxorubicin-induced cardiotoxicity by substantially downregulating cardiac mitochondrial ATP generation, increasing oxidative stress and downregulating the JAK/STAT3 pathway.

Inhibition of cyclooxygenase-2 aggravates secretory phospholipase A2-mediated progression of acute liver injury

Our previous study [Bhave, V. S., Donthamsetty, S., Latendresse, J. R., Muskhelishvili, L., and Mehendale, H. M. 2008-this issue. Secretory phospholipase A(2) mediates progression of acute liver injury in the absence of sufficient COX-2. Toxicol Appl Pharmacol] showed that in the absence of sufficient induction and co-presence of cyclooxygenase-2 (COX-2), secretory phospholipase A(2) (sPLA(2)) appearing in the intercellular spaces for cleanup of post-necrotic debris seems to contribute to the progression of toxicant-initiated liver injury, possibly by hydrolysis of membrane phospholipids of hepatocytes in the perinecrotic areas. To further test our hypothesis on the protective role of COX-2, male Fisher-344 rats were administered a selective COX-2 inhibitor, NS-398, and then challenged with a moderately toxic dose of CCl(4). This led to a 5-fold increase in the susceptibility of the COX-2 inhibited rats to CCl(4) hepatotoxicity and mortality. The CCl(4) bioactivating enzyme CYP2E1 protein, CYP2E1 enzyme activity, and the (14)CCl(4)-derived radiolabel covalently bound to the liver proteins were unaffected by the COX-2 inhibitor suggesting that the increased hepatotoxic sensitivity of the COX-2 inhibited rats was not due to higher bioactivation of CCl(4). Further investigation showed that this increased mortality was due to higher plasma and hepatic sPLA(2) activities, inhibited PGE(2) production, and progression of liver injury as compared to the non-intervened rats(.) In conclusion, inhibition of COX-2 mitigates the tissue protective mechanisms associated with COX-2 induction, which promotes sPLA(2)-mediated progression of liver injury in an acute liver toxicity model. Because increased sPLA(2) activity in the intercellular space is associated with increased progression of injury, and induced COX-2 is associated with hepatoprotection, ratios of hepatic COX-2 and sPLA(2) activities may turn out to be a useful tool in predicting the extent of hepatotoxicities.

Genes inducing iPS phenotype play a role in hepatocyte survival and proliferation in vitro and liver regeneration in vivo

Reprogramming factors have been used to induce pluripotent stem cells as an alternative to somatic cell nuclear transfer technology in studies targeting disease models and regenerative medicine. The neuronal repressor RE‐1 silencing transcription factor (REST) maintains self‐renewal and pluripotency in mouse embryonic stem cells by maintaining the expression of Oct3/4, Nanog, and cMyc. We report that primary hepatocytes express REST and most of the reprogramming factors in culture. Their expression is up‐regulated by hepatocyte growth factor (HGF) and epidermal growth factor (EGF). REST inhibition results in down‐regulation of reprogramming factor expression, increased apoptosis, decreased proliferation, and cell death. The reprogramming factors are also up‐regulated after 70% partial hepatectomy in vivo. Conclusion: These findings show that genes inducing the iPS phenotype, even though expressed at lower levels than embryonic stem cells, nonetheless are associated with control of apoptosis and cell proliferation in hepatocytes in culture and may play a role in such processes during liver regeneration. (HEPATOLOGY 2011)

Secretory phospholipase A2 mediates progression of acute liver injury in the absence of sufficient cyclooxygenase-2

Previous studies have shown that injury initiated by toxicants progresses even after most of the toxicant is eliminated from the body. One mechanism of progression of injury is the extracellular appearance of hydrolytic enzymes following leakage or upon cell lyses. Under normal conditions, after exposure to low to moderate doses of toxicants, secretory phospholipase A(2) (sPLA(2)) and other hydrolytic enzymes are known to appear in the extracellular spaces in order to cleanup the post-necrotic debris in tissues. We tested the hypothesis that sPLA(2) contributes to progression of toxicant-initiated liver injury because of hydrolysis of membrane phospholipids of hepatocytes in the perinecrotic areas in the absence of sufficient cyclooxygenase-2 (COX-2). Male Sprague-Dawley rats were administered either a moderately hepatotoxic dose (MD, 2 ml CCl(4)/kg, ip) or a highly hepatotoxic dose (HD, 3 ml CCl(4)/kg, ip) of CCl(4). After MD, liver sPLA(2) and COX-2 were co-localized in the necrotic and perinecrotic areas and their activities in plasma and liver increased before decreasing in tandem with liver injury (ALT and histopathology) leading to 100% survival. In contrast, after the HD, high extracellular and hepatic sPLA(2) activities were accompanied by minimal COX-2 activity and localization in the liver throughout the time course. This led to progression of liver injury and 70% mortality. These data suggested a destructive role of sPLA(2) in the absence of sufficient COX-2. Time- and dose-dependent destruction of hepatocytes by sPLA(2) in isolated hepatocyte incubations confirmed the destructive ability of sPLA(2) when present extracellularly, suggesting its ability to spread injury in vivo. These findings suggest that sPLA(2), secreted for cleanup of necrotic debris upon initiation of hepatic necrosis, requires the co-presence of sufficiently induced COX-2 activity to prevent the run-away destructive action of sPLA(2) in the absence of the tissue protective mechanisms afforded by COX-2 induction.

Hepatocyte proliferation and hepatomegaly induced by phenobarbital and 1,4‐bis [2‐(3,5‐dichloropyridyloxy)] benzene is suppressed in hepatocyte‐targeted glypican 3 transgenic mice

Glypican 3 (GPC3) is a family of glycosylphosphatidylinositol‐anchored, cell‐surface heparan sulfate proteoglycans. Loss‐of‐function mutations of GPC3 cause Simpson‐Golabi‐Behmel syndrome characterized by overgrowth of multiple organs, including liver. Our previous study showed that in GPC3 transgenic (TG) mice, hepatocyte‐targeted overexpression of GPC3 suppresses hepatocyte proliferation and liver regeneration after partial hepatectomy and alters gene expression profiles and potential cell cycle‐related proteins. This study investigates the role of GPC3 in hepatocyte proliferation and hepatomegaly induced by the xenobiotic mitogens phenobarbital (PB) and TCPOBOP (1, 4‐bis [2‐(3, 5‐dichloropyridyloxy)] benzene). Wildtype (WT) and GPC3 TG mice were given 0.1% PB in drinking water for 10 days or a single dose of TCPOBOP (3 mg/kg) by oral gavage. At day 5 the WT mice showed a 2.2‐ and 3.0‐fold increase in liver weight, whereas the GPC3 TG mice showed a 1.3‐ and 1.6‐fold increase in liver weight after PB and TCPOBOP administration, respectively. There was a significant suppression of proliferative response in the GPC3 TG mice, as assessed by percent of Ki67‐positive hepatocyte nuclei. Moreover, gene array analysis showed a panel of changes in the gene expression profile of TG mice, both before and after administration of the xenobiotic mitogens. Expression of cell cycle‐related genes in the TG mice was also decreased compared to the WT mice. Conclusion: Our results indicate that in GPC3 TG mice, hepatocyte‐targeted overexpression of GPC3 plays an important role for regulation of liver size and termination of hepatocyte proliferation induced by the xenobiotic mitogens PB and TCPOBOP, comparable to the effects seen in the GPC3 TG mice during liver regeneration after partial hepatectomy. (HEPATOLOGY 2011;)

Nonalcoholic steatohepatitic (NASH) mice are protected from higher hepatotoxicity of acetaminophen upon induction of PPARa with clofibrate

The objective was to investigate if the hepatotoxic sensitivity in nonalcoholic steatohepatitic mice to acetaminophen (APAP) is due to downregulation of nuclear receptor PPARalpha via lower cell division and tissue repair. Male Swiss Webster mice fed methionine and choline deficient diet for 31 days exhibited NASH. On the 32nd day, a marginally toxic dose of APAP (360 mg/kg, ip) yielded 70% mortality in steatohepatitic mice, while all non steatohepatitic mice receiving the same dose survived. (14)C-APAP covalent binding, CYP2E1 protein, and enzyme activity did not differ from the controls, obviating increased APAP bioactivation as the cause of amplified APAP hepatotoxicity. Liver injury progressed only in steatohepatitic livers between 6 and 24 h. Cell division and tissue repair assessed by (3)H-thymidine incorporation and PCNA were inhibited only in the steatohepatitic mice given APAP suggesting that higher sensitivity of NASH liver to APAP-induced hepatotoxicity was due to lower tissue repair. The hypothesis that impeded liver tissue repair in steatohepatitic mice was due to downregulation of PPARalpha was tested. PPARalpha was downregulated in NASH. To investigate whether downregulation of PPARalpha in NASH is the critical mechanism of compromised liver tissue repair, PPARalpha was induced in steatohepatitic mice with clofibrate (250 mg/kg for 3 days, ip) before injecting APAP. All clofibrate pretreated steatohepatitic mice receiving APAP exhibited lower liver injury, which did not progress and the mice survived. The protection was not due to lower bioactivation of APAP but due to higher liver tissue repair. These findings suggest that inadequate PPARalpha expression in steatohepatitic mice sensitizes them to APAP hepatotoxicity.

Excessive hepatomegaly of mice with hepatocyte‐targeted elimination of integrin linked kinase following treatment with 1,4‐bis [2‐(3,5‐dichaloropyridyloxy)] benzene

TCBOPOP (1,4‐bis [2‐(3,5‐dichaloropyridyloxy)] benzene) an agonist of the constitutive androstane receptor (CAR), produces rapid hepatocyte hyperplasia and hepatomegaly in the absence of hepatic injury. In this study we demonstrate that integrin‐linked kinase (ILK), which is involved in transmission of the extracellular matrix (ECM) signaling by way of integrin receptors, plays an important role in regulating TCPOBOP‐induced proliferation of hepatocytes and hepatomegaly. Hepatocyte‐specific ILK knockout mice (ILK/liver−/− mice) and wildtype mice (WT) were given a single dose of TCPOBOP (3 mg/kg) by oral gavage. Mice were sacrificed at days 1, 2, 5, and 7 after TCPOBOP administration. WT mice showed maximum proliferation on days 1 and 2, which came back to baseline levels by days 5 and 7 after TCPOBOP administration. The ILK/liver−/− mice, on the other hand, showed a prolonged and a sustained proliferative response as evident by an increased number of proliferative cell nuclear antigen assay (PCNA)‐positive cells even at days 5 and 7 after TCPOBOP administration. At day 7 the WT mice showed close to a 2.5‐fold increase in liver weight, whereas the ILK/liver−/− mice showed a 3.7‐fold increase in liver weight. The prolonged proliferative response in the ILK/liver−/− mice seems to be due to sustained induction of CAR leading to sustained induction of c‐Myc, which is known to be a key mediator of TCPOPOP‐CAR induced direct liver hyperplasia. Conclusion: The data indicate that ECM‐mediated signaling by way of ILK is essential for adjustment of final liver size and proper termination of TCPOBOP‐induced proliferation of hepatocytes. (HEPATOLOGY 2011;53:587‐595)

Liver-Specific Ablation of Integrin-Linked Kinase in Mice Results in Enhanced and Prolonged Cell Proliferation and Hepatomegaly after Phenobarbital Administration

We have recently demonstrated that disruption of extracellular matrix (ECM)/integrin signaling via elimination of integrin-linked kinase (ILK) in hepatocytes interferes with signals leading to termination of liver regeneration. This study investigates the role of ILK in liver enlargement induced by phenobarbital (PB). Wild-type (WT) and ILK:liver-/- mice were given PB (0.1% in drinking water) for 10 days. Livers were harvested on 2, 5, and 10 days during PB administration. In the hepatocyte-specific ILK/liver-/- mice, the liver:body weight ratio was more than double as compared to 0 h at day 2 (2.5 times), while at days 5 and 10, it was enlarged three times. In the WT mice, the increase was as expected from previous literature (1.8 times) and seems to have leveled off after day 2. There were slightly increased proliferating cell nuclear antigen-positive cells in the ILK/liver-/- animals at day 2 as compared to WT after PB administration. In the WT animals, the proliferative response had come back to normal by days 5 and 10. Hepatocytes of the ILK/liver-/- mice continued to proliferate up until day 10. ILK/liver-/- mice also showed increased expression of key genes involved in hepatocyte proliferation at different time points during PB administration. In summary, ECM proteins communicate with the signaling machinery of dividing cells via ILK to regulate hepatocyte proliferation and termination of the proliferative response. Lack of ILK in the hepatocytes imparts prolonged proliferative response not only to stimuli related to liver regeneration but also to xenobiotic chemical mitogens, such as PB.