Limited therapeutic efficacy of thrombopoietin on the regeneration of steatotic livers

Liver regeneration after partial hepatectomy is impaired in steatotic livers of leptin-deficient ob/ob mice. Previous studies have shown that thrombopoietin (TPO) promotes liver regeneration and improves liver cirrhosis by an increase of platelet counts and the expansion of hepatic progenitor cells. Herein we studied whether TPO exerts pro-proliferative and hepatoprotective effects and thereby improves the regenerative capacity of steatotic livers. For this purpose, we studied hepatic regeneration at day 2, 3, 7 and 10 in a model of 55% hepatectomy in obese (ob/ob) and non-obese (C57BL/6J) mice. Liver function and injury, platelet counts, weight of the regenerated liver, proliferating liver cells as well as the number of hepatic (CK19-positive) oval cells were quantified by biochemical and immunohistochemical analysis. As expected, obese mice had a markedly decreased regenerative capacity of livers compared with lean animals. Pretreatment of mice with recombinant TPO (12.5 μg/kg) had no evident effect on regeneration of fatty livers, but ameliorated acute liver damage in obese mice, as indicated by decreased liver enzyme release early after resection. TPO was unable to enhance hepatocyte proliferation, but increased proliferation of non-parenchymal cells, including CK19-positive oval cells, at later observation time points after resection. Interestingly, TPO completely inhibited the resection-induced increase of plasma triglycerides immediately after resection in non-obese mice. In conclusion, TPO slightly prevents acute liver damage after resection in obese mice, but fails to significantly enhance regeneration of fatty livers.     

STAT-3 overexpression and p21 up-regulation accompany impaired regeneration of fatty livers

Fatty liver is an important cause of morbidity in humans and is linked to impaired liver regeneration after liver injury, but the mechanisms for impaired liver regeneration remain unknown. In the normal liver, the interleukin (IL)-6/STAT-3 pathway is thought to play a central role in regeneration because this pathway is disrupted in IL-6-deficient mice that exhibit impaired liver regeneration after 70% partial hepatectomy (PH). To determine whether inhibition of STAT-3 is involved in fatty liver-related mitoinhibition, regenerative induction of STAT-3 was compared in normal mice and leptin-deficient ob/ob mice that have fatty livers and markedly impaired liver regeneration after PH. In both groups, two waves of STAT-3 activation were observed, the first in endothelia and the second in hepatocytes. Before PH, a significantly higher percentage of ob/ob endothelial and hepatocyte nuclei expressed phosphorylated (activated) STAT-3. After PH, phospho-STAT-3 accumulated in liver nuclei of lean mice and this response was markedly exaggerated in ob/ob mice. Moreover, a striking inverse correlation was noted between hepatocyte nuclear accumulation of phospho-STAT-3 and DNA synthesis (as assessed by bromodeoxyuridine labeling), as well as cyclin D1 mRNA induction and protein expression. In contrast, STAT-3 activation was positively correlated with p21 protein expression in both groups of mice. Because these results link exaggerated STAT-3 activation with impaired hepatocyte proliferation, STAT-3 inhibition cannot be a growth-arrest mechanism in ob/ob fatty livers. Rather, hyperinduction of this factor may promote mitoinhibition by up-regulating mechanisms that impede cell cycle progression.     

Atypical ductular proliferation and its inhibition by transforming growth factor beta1 in the 3,5-diethoxycarbonyl-1,4-dihydrocollidine mouse model for chronic alcoholic liver disease

Many acute and chronic liver diseases are often associated with atypical ductular proliferation (ADP). These ADPs have gained increasing interest since a number of recent observations suggest that ADPs may represent progenies of the putative liver stem cell compartment. In this study, we show that feeding mice with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) results in persistent proliferation of primitive ductules with poorly defined lumens. Similar to oval cell proliferation in other rodent models as well as in various human liver diseases, DDC-induced ADP originated from the portal tract, spread into the hepatic lobule, and was associated closely with appearance of hepatocytes harboring an antigen (A6), which normally is expressed in biliary epithelium. Furthermore, DDC treatment severely inhibited the regenerative capacity of mice after partial hepatectomy. The development of ADP was selectively blocked in DDC-fed TGF-beta1 transgenic mice producing active TGF-beta1 in the liver and no accumulation of new hepatocytes expressing the A6 antigen was observed. Moreover, the transforming growth factor beta1 (TGF-beta1) transgenic mice did not survive beyond 3 weeks from starting the DDC-containing diet. The results suggest that persistent activation of the hepatic stem cell compartment is essential for liver regeneration in the DDC model and that active TGF-beta1 may negatively control activation of stem cells in the liver. These data further emphasize the relevance of the DDC model as an experimental tool for studying chronic liver diseases.     

IL-12 induces specific cytotoxicity against regenerating hepatocytes in vivo.

Although impaired liver regeneration is thought to be a major cause of death in patients with fulminant hepatitis, the mechanisms are not well defined. Since IL-12 synthesis has been reported to be up-regulated in murine hepatitis virus infection, we studied the influence of continuous IL-12 stimulation on murine liver regeneration using flow cytometric and functional analyses. In non-hepatectomized mice, interestingly, the number of hepatic NK cells was significantly decreased on day 7, after six IL-12 injections, and day 14, after 13 IL-12 injections. The number of hepatic NKT cells was markedly increased on day 7 and day 14 of daily IL-12 treatment. The cytotoxic activity of hepatic lymphocytes against both YAC-1 and p815 cells was enhanced on day 2, after single IL-12 injection, and day 7, after six IL-12 injections. In contrast, hepatic lymphocytes isolated 24 h after partial hepatectomy with IL-12 pretreatment did not show any cytolytic activity against either YAC-1 cells or p815 cells. However, continuous IL-12 stimulation resulted in a significantly higher serum alanine aminotransferase (sALT) level 24 h after the partial hepatectomy as compared with sALT levels in mice subjected to either partial hepatectomy or IL-12 pretreatment alone. On the other hand, the expression of hepatic TNF-alpha mRNA was markedly enhanced by continuous IL-12 stimulation even 24 h after partial hepatectomy, as compared with that in non-treated mice and hepatectomy alone. Simultaneous administration of anti-tumor necrosis factor (TNF)-alpha mAb completely inhibited IL-12-induced in vivo enhancement of liver damage after partial hepatectomy. In conclusion, IL-12 induces the specific cytolytic activity against regenerating hepatocytes in vivo mainly through the enhancement of TNF-alpha synthesis.     

Lateral hypothalamic lesions facilitate hepatic regeneration after partial hepatectomy in rats

It has been reported that ventromedial hypothalamic lesions facilitate hepatic regeneration through the hepatic vagal nerve after partial hepatectomy. However, whether the lateral area of the hypothalamus is involved in liver regeneration after partial hepatectomy is unknown. To determine the role of the lateral hypothalamic area in this phenomenon, we studied hepatic DNA synthesis during liver regeneration after partial hepatectomy with bilateral lesions of the area. Lesioning of the lateral hypothalamus accelerated the increase in hepatic DNA synthesis and raised the peak level of [methyl-³H]thymidine incorporation after partial hepatectomy. These effects of hypothalamic lesioning were inhibited by combined hepatic vagotomy and sympathectomy. Our results demonstrate that lesioning of the lateral hypothalamus promotes hepatic regeneration through the autonomic nervous system after partial hepatectomy and suggest that the lateral hypothalamic area is involved in liver regeneration through neural mediation.     

In-vitro immune response of splenic lymphocytes to portal serum agents from rats undergoing hepatic regeneration or hepatic carcinogenesis.

To clarify the physiologic response of splenic lymphocytes to liver damage and the role of this response in regeneration versus malignant transformation, we cultured rat spleen lymphocytes with portal sera from rats subjected either to partial (70%) hepatectomy or to long-term oral administration of the hepatic carcinogen 3'-methyl-4-dimethylaminoazobenzene. Sera taken within 24h after partial hepatectomy contained a previously described signal protein which serves as a marker of liver damage. The MW 5,000-10,000 serum fraction also contained a factor that promoted cell growth, DNA synthesis, glucose utilization, and the production of anti-sheep erythrocyte plaque-forming cells in cultures of rat splenic lymphocytes. In contrast, the sera of rats subjected to liver damage by the carcinogen had no more effect on the cultured lymphocytes than sera from sham-operated or untreated controls. The signal protein was present initially in portal sera from carcinogen-treated rats, but decreased as hepatitis gave way to cirrhosis. Subsequent malignant transformation was marked by the appearance of serum alpha-fetoprotein. Our results suggest that activation of splenic lymphocytes by serum factor(s) is involved in hepatic regeneration and that this process is deranged in carcinogenesis.     

Enhanced liver regeneration in IL-10-deficient mice after partial hepatectomy via stimulating inflammatory response and activating hepatocyte STAT3

Emerging evidence suggests that proinflammatory cytokines, including tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), play a critical role in the initiation and progression of liver regeneration; however, relatively little is known about the role of anti-inflammatory cytokine IL-10 in liver regeneration after partial hepatectomy (PHx). Here, we examined the role of IL-10 in liver regeneration using a model of PHx in several strains of genetically modified mice. After PHx, expression of IL-10 mRNA in the liver and spleen was significantly elevated. Such elevation was diminished in TLR4 mutant mice. Compared with wild-type mice, IL-10(-/-) mice had higher levels of expression of proinflammatory cytokines (IL-6, TNF-α, and IFN-γ) and inflammatory markers (CCR2 and F4/80) in the liver, as well as higher serum levels of proinflammatory cytokines after PHx. The number of neutrophils and macrophages was also higher in the livers of IL-10(-/-) mice than in wild-type mice after PHx. Liver regeneration as determined by BrdU incorporation after PHx was higher in IL-10(-/-) mice than in wild-type mice, which was associated with higher levels of activation of IL-6 downstream signal STAT3 in the liver. An additional deletion of STAT3 in hepatocytes significantly reduced liver regeneration in IL-10(-/-) mice after PHx. Collectively, IL-10 plays an important role in negatively regulating liver regeneration via limiting inflammatory response and subsequently tempering hepatic STAT3 activation.     

A novel inhibitor of the alternative complement pathway prevents antiphospholipid antibody-induced pregnancy loss in mice

Studies in gene-targeted mice have demonstrated that factor B of the alternative complement pathway plays an important role in several disease models, but an exogenous inhibitor of factor B has not previously been available. We have developed an inhibitory monoclonal antibody directed against a critical epitope on mouse factor B and have tested it in a model of antiphospholipid (aPL) antibody (Ab)-induced fetal loss. Gene-targeted factor B-deficient mice (fB-/-) were injected with a fusion protein comprised of the second and third short consensus repeat (SCR) domains of mouse factor B linked to a mouse IgG1 Fc domain. Hybridomas were made from splenocytes of the immunized mouse. One mAb, designated 1379, produced an IgG1 antibody that inhibited alternative pathway activation in vitro and in vivo by preventing formation of the C3bBb complex. Strikingly, this mAb inhibited alternative pathway activation in serum from mice, rats, humans, monkeys, pigs and horses. Fab fragments made from this mAb also inhibited alternative pathway activation. Epitope mapping demonstrated that this antibody binds to factor B within the third SCR domain. When mAb 1379 was administered to mice that also received human IgG containing antiphospholipid antibodies, it provided significant protection from antiphospholipid antibody-induced complement activation and fetal loss. Thus, this mAb to factor B has broad species reactivity and effectively inhibits alternative pathway activation. The mAb protects mice in an in vivo model of antiphospholipid antibody syndrome, demonstrating the therapeutic potential for the inhibition of factor B in this disease.     

Increase in serum alpha foetoprotein level in hepatic regeneration of the rat. Effects of age and of magnitude of regenerative activity.

The effects of variable degrees of liver regeneration induced by two-thirds partial hepatectomy or intoxication with different doses of carbon tetrachloride (CCl4) on increased production of alpha foetoprotein (AFP) have been studied in very young (5-6 weeks) and older (15 weeks and more) rats by counter-immunoelectrophoresis (sensitivity down to 250 ng/ml). In the young animal adequate regeneration following two-thirds hepatectomy as well as 100 myl CCl4 successfully induced large increases in serum AFP levels. Smaller doses as well as a large fatal dose of the toxin, all of which resulted in inadequate regenerative activity, failed to excite AFP synthesis and secretion up to detectable levels. The adult animal did not show detectable AFP with any of these procedures. However, necrosis induced in the regenerating adult liver 24 h following partial hepatectomy did result in the detection of small amounts of protein in the serum. It is concluded that in hepatic regeneration of this species, the age of the animal as well as the magnitude of regenerating activity in the liver are critical in the occurrence of high levels of serum AFP.     

Experimental models of hepatectomy and liver regeneration using newborn and weaning rats

OBJECTIVES: Liver regeneration is a complex process that has not been completely elucidated. The model most frequently used to study this phenomenon is 70% hepatectomy in adult rats; however, no papers have examined this effect in developing animals. The aims of the present study were: 1) to standardize two models of partial hepatectomy and liver regeneration in newborn suckling and weaning rats, and 2) to study the evolution of remnant liver weight and histological changes of hepatic parenchyma on the days that follow partial hepatectomy. METHODS: Fifty newborn and forty-four weaning rats underwent 70% hepatectomy. After a midline incision, compression on both sides of the upper abdomen was performed to exteriorize the right medial, left medial and left lateral hepatic lobes, which were tied inferiorly and resected en bloc. The animals were sacrificed on days 0 (just after hepatectomy), 1, 2, 3, 4 and 7 after the operation. Body and liver weight were determined, and hepatic parenchyma was submitted to histological analysis. RESULTS: Mortality rates of the newborn and weaning groups were 30% and 0%, respectively. There was a significant decrease in liver mass soon after partial hepatectomy, which completely recovered on the seventh day in both groups. Newborn rat regenerating liver showed marked steatosis on the second day. In the weaning rat liver, mitotic figures were observed earlier, and their amount was greater than in the newborn. CONCLUSIONS: Suckling and weaning rat models of partial hepatectomy are feasible and can be used for studies of liver regeneration. Although similar, the process of hepatic regeneration in developing animals is different from adults.     

Facilitation of liver regeneration after partial hepatectomy by ventromedial hypothalamic lesions in rats

Whether or not the hypothalamus is involved in initiating hepatic DNA synthesis after partial hepatectomy is unclear. To determine the role of the ventromedial hypothalamic nuclei in liver regeneration after partial hepatectomy, we studied hepatic DNA synthesis during liver regeneration in rats with bilateral lesions of these nuclei. Lesions of the ventromedial hypothalamus accelerated the increase in hepatic DNA synthesis and raised the peak level of thymidine incorporation after partial hepatectomy. These effects of hypothalamic lesions were completely inhibited by hepatic vagotomy. Thus, lesions of the ventromedial hypothalamus appear to promote hepatic regeneration by increasing vagal stimulation of the liver.     

Reduced liver cell death using an alginate scaffold bandage: A novel approach for liver reconstruction after extended partial hepatectomy

Extended partial hepatectomy may be needed in cases of large hepatic mass, and can lead to fulminant hepatic failure. Macroporous alginate scaffold is a biocompatible matrix which promotes the growth, differentiation and long-term hepatocellular function of primary hepatocytes in vitro. Our aim was to explore the ability of implanted macroporous alginate scaffolds to protect liver remnants from acute hepatic failure after extended partial hepatectomy. An 87% partial hepatectomy (PH) was performed on C57BL/6 mice to compare non-treated mice to mice in which alginate or collagen scaffolds were implanted after PH. Mice were scarified 3, 6, 24 and 48 h and 6 days following scaffold implantation and the extent of liver injury and repair was examined. Alginate scaffolds significantly increased animal survival to 60% vs. 10% in non-treated and collagen-treated mice (log rank = 0.001). Mice with implanted alginate scaffolds manifested normal and prolonged aspartate aminotransferases and alanine aminotransferases serum levels as compared with the 2- to 20-fold increase in control groups (P < 0.0001) accompanied with improved liver histology. Sustained normal serum albumin levels were observed in alginate-scaffold-treated mice 48 h after hepatectomy. Incorporation of BrdU-positive cells was 30% higher in the alginate-scaffold-treated group, compared with non-treated mice. Serum IL-6 levels were significantly decreased 3 h post PH. Biotin-alginate scaffolds were quickly well integrated within the liver tissue. Collectively, implanted alginate scaffolds support liver remnants after extended partial hepatectomy, thus eliminating liver injury and leading to enhanced animal survival after extended partial hepatectomy.     

A study of endotoxin-associated hepatotoxicity on proliferating hepatocytes.

It is thought that regeneration of the liver provides a state of preparedness for the Shwartzman reaction and contributes to the development of endotoxin-associated massive hepatic necrosis following partial hepatectomy. Therefore we examined endotoxin hepatotoxicity in rats with hepatic regeneration after 35% hepatectomy and in rats with liver cell proliferation induced by lead nitrate. Biochemical and histopathological studies showed no enhanced endotoxin hepatotoxicity in either partially hepatectomized rats or in rats with lead nitrate-induced liver cell proliferation. These results indicate that the development of endotoxin-associated hepatic damage after partial hepatectomy may not relate to regeneration and proliferation of the liver.     

肝细胞生长因子和C-met在肝纤维化肝部分切除后残余肝组织中的表达

目的 探讨肝细胞生长因子（HGF）和C-met在纤维化肝部分切除后残余肝组织中的表达变化。方法 雄性SD大鼠130只,随机分为正常组（n=7）、正常肝部分切除组（n=50）、肝纤维化组（n=7）和纤维化肝部分切除组（n=66）。正常肝部分切除组和纤维化肝部分切除组分别在术后12 h、1 d、3 d、5 d、7 d和14 d 取材,运用免疫组织化学和Western blotting方法,检测残肝组织中HGF、C-met的表达。结果 免疫组织化学显示,正常肝部分切除组,HGF表达于术后12 h达高峰,并维持高峰平台,7 d后逐渐降低,于14 d接近术前水平;C-met术后迅速上升,3 d达到高峰,而后逐步下降,14 d降回术前水平。纤维化肝部分切除组,HGF表达于术后迅速下降,12 h又迅速上升,于1 d时达高峰,然后逐步下降,14 d降至最低点;C-met术后迅速下降,12 h开始缓慢下降,于术后3 d达最低点,后缓慢上升,7 d又迅速上升,14 d达最高点。Western blotting显示,HGF、C-met蛋白条带变化规律与免疫组织化学结果吻合。结论 HGF和C-met同时高水平表达有利于肝细胞的分裂增殖,提示HGF和C-met表达不同步,可能是纤维化肝术后再生困难的重要原因。     

Expression of epidermal growth factor receptor protein in the liver of db/db mice after partial hepatectomy

Liver regeneration was impaired after partial hepatectomy (PH) in leptin receptor-deficient db/db mice with severe liver steatosis. In the present study, we analyzed the mode of epidermal growth factor receptor (EGFR) protein expression in the liver of 5- and 10-week-old db/db and age-matched control mice. In 5-week-old db/db mice, neither the expression of EGFR protein in the intact liver nor the rate of liver regeneration after PH was significantly different from that in age-matched control mice. However, in 10-week-old db/db mice, the level of EGFR protein expression was very low and liver regeneration was prominently suppressed. Histopathologically, much severer fatty change was observed in the liver of 10-week-old db/db mice than 5-week-old db/db mice. These results suggest that the down-regulation of EGFR protein expression is associated with an impairment of liver regeneration in db/db mice and that the severity of hepatic steatosis plays an indirect role in the impairment of liver regeneration by modifying EGFR expression.     

Effects of the liver homogenate obtained following partial hepatectomy on cirrhosis of the liver.

The authors studied the influence of liver homogenates as a whole and collected at various intervals after partial hepatectomy on cirrhosis of the liver. The lyophilized homogenates were administered over a period of 6 weeks to various groups of albino rats pretreated with CCl4 for a period of 6 months. The normal liver homogenate did not influence the histological and biochemical picture of the hepatocirrhosis. The material collected 48 hours after partial hepatectomy causes a moderate stimulation of the mechanisms of parenchymatous regeneration. 7 days after partial hepatectomy (in the postmitotic period) the hepatic regenerate shows a biological effect with lysis of collagen fibres and protection of parenchymatous cells.     

Liver damage using suicide genes. A model for oval cell activation

Liver regeneration from the facultative hepatic stem cells, the oval cells, takes place in situations in which liver regeneration from pre-existing hepatocytes is prevented. Different models have been used to stimulate oval cell response. Many of them involve the use of carcinogenic agents with or without partial hepatectomy. In this study we show that adenovirus-mediated gene transfer of the suicide gene thymidine kinase followed by ganciclovir administration caused hepatotoxicity of variable intensity. Rats with moderate elevation in serum transaminases recovered normal liver architecture few weeks after adenovirus injection. In contrast, rats with severe liver damage exhibited a marked and persisting activation of oval cells accompanied by ductular hyperplasia. In some rats, such lesion eventually evolved to cholangiofibrosis and in one rat to cholangiocarcinoma. Deposition of fibronectin and increased number of hepatic stellate cells were found in association with oval cells and cholangiofibrotic lesions. Hepatocyte growth factor was hyperexpressed in the livers with intense oval cell response or ductular proliferation, suggesting a participation of this factor in those lesions. In summary, our data demonstrate activation of oval cell response after gene transfer of thymidine kinase followed by ganciclovir administration. These findings indicate that high doses of this therapy causes liver damage together with an impairment in hepatocellular regeneration.     

Activation of extrathymic T cells in the liver during liver regeneration following partial hepatectomy.

Partial hepatectomy was performed in C57BL/6 mice to investigate whether extrathymic T cells in the liver are activated during liver regeneration. This study is based on the finding that in mice with malignant tumours, extrathymic T cells in the liver are activated and yet the intrathymic pathway is suppressed (i.e. thymic atrophy). Attention was therefore focused on whether a similar phenomenon is induced during benign cell regeneration. Extrathymic T cells were identified using the two-colour immunofluorescence test for CD3 and interleukin-2 receptor beta-chain (IL-2R beta) [or lymphocyte function-associated antigen-1 (LFA-1)] antigens. They were estimated to be intermediate CD3+ [or T-cell receptor (TcR)] cells with high expressions of IL-2R beta and LFA-1. It was demonstrated that the proportion and number of intermediate CD3+ cells increased in the early phase (days 2-4 after partial hepatectomy), and that the thymus was inversely atrophic at the same time. This raised the possibility that extrathymic T cells may also be responsible for regulation of normal cell regeneration.     

Defective hepatic regeneration after partial hepatectomy in leptin-deficient mice is not rescued by exogenous leptin

Liver regeneration after partial hepatectomy (PH) is impaired in leptin-deficient ob/ob mice. Here, we tested whether exogenous leptin and/or correction of the obese phenotype (by food restriction or long-term leptin administration) would rescue hepatocyte proliferation and whether the hepatic progenitor cell compartment was activated in leptin-deficient ob/ob livers after PH. Because of the high mortality following 70% PH to ob/ob mice, we performed a less extensive (55%) resection. Compared to lean mice, liver regeneration after 55% PH was deeply impaired and delayed in ob/ob mice. Administration of exogenous leptin to ob/ob mice at doses that restored circulating leptin levels during the surgery and postsurgery period or for 3 weeks prior to the surgical procedure did not rescue defective liver regeneration. Moreover, correction of obesity, metabolic syndrome and hepatic steatosis by prolonged administration of leptin or food restriction (with or without leptin replacement at the time of PH) did not improve liver regeneration in ob/ob mice. The hepatic progenitor cell compartment was increased in ob/ob mice. However, after PH, the number of progenitor cells decreased and signs of proliferation were absent from this cell compartment. In this study, we have conclusively shown that neither leptin replacement nor amelioration of the metabolic syndrome, obese phenotype and hepatic steatosis, with or without restitution of normal circulating levels of leptin, was able to restore replicative competence to ob/ob livers after PH. Thus, leptin does not directly signal to liver cells to promote hepatocyte proliferation, and the obese phenotype is not solely responsible for impaired regeneration.