Observation of the effect of bone marrow mesenchymal stem cell transplantation by different interventions on cirrhotic rats.

Bone marrow mesenchymal stem cells (BMSCs) transplantation has attracted attention for the treatment of liver cirrhosis and end-stage liver diseases. Therefore, in this study, we evaluated the effect of different methods of BMSCs transplantation in the treatment of liver cirrhosis in rats. Seventy-two male Sprague-Dawley rats were divided into 7 groups: 10 were used to extract BMSCs, 10 were used as normal group, and the remaining 52 rats were randomly divided into five groups for testing: control group, BMSCs group, BMSCs+granulocyte colony-stimulating factor (G-CSF) group, and BMSCs+Jisheng Shenqi decoction (JSSQ) group. After the end of the intervention course, liver tissue sections of rats were subjected to hematoxylin and eosin (H&E) and Masson staining, and pathological grades were scored. Liver function [aminotransferase (ALT), aspartate aminotransferase (AST), albumin (ALB)] and hepatic fibrosis markers [hyaluronidase (HA), laminin (LN), type III procollagen (PCIII), type IV collagen (CIV)] were measured. BMSCs+JSSQ group had the best effect of reducing ALT and increasing ALB after intervention therapy (P<0.05). The reducing pathological scores and LN, PCIII, CIV of BMSCs+G-CSF group and BMSCs+JSSQ group after intervention therapy were significant, but there was no significant difference between the two groups (P>0.05). The effect of JSSQ on improving stem cell transplantation in rats with liver cirrhosis was confirmed. JSSQ combined with BMSCs could significantly improve liver function and liver pathology scores of rats with liver cirrhosis.

Potentials of Differentiated Human Cord Blood-Derived Unrestricted Somatic Stem Cells in Treatment of Liver Cirrhosis.

OBJECTIVES: Liver transplantation is the well-known treatment for chronic liver diseases; however, postoperative complications and lack of donors continue to be limitations with this treatment. Investigating new modalities for treatment of chronic liver illness is a must. In the present study, we aimed to clarify the effects of an in vitro hepatocyte-differentiated human unrestricted somatic stem cell transplant as a new cell-based therapy in an experimental model of chronic liver failure. MATERIALS AND METHODS: Human umbilical cord blood-derived unrestricted somatic stem cells were isolated, cultured, propagated, and characterized. Cells were directed to differentiate into hepatocyte-like cells. An animal model of carbon tetrachloride cirrhotic liver failure was prepared, and the human in vitro differentiated unrestricted somatic stem cells were transplanted into the experimental model. Animals that did not receive transplant served as the pathologic control group. Animals were euthanized 12 weeks after transplant, and liver functions and histopathology were assessed. RESULTS: Compared with the pathologic control group, the transplant group showed improvements in levels of alanine aminotransferase, aspartate aminotransferase, albumin, and bilirubin. Histopathologic examination of the transplant group also showed improvements in hydropic degeneration and fibrosis. CONCLUSIONS: The use of unrestricted somatic stem cells, isolated and propagated from cord blood and then differentiated into hepatocyte-like cells, improved both fibrosis and normal function of cirrhotic livers. These cells could be considered as a line of cell-based therapy in cases of chronic liver disease.

Observation of the effect of bone marrow mesenchymal stem cell transplantation by different interventions on cirrhotic rats

Bone marrow mesenchymal stem cells (BMSCs) transplantation has attracted attention for the treatment of liver cirrhosis and end-stage liver diseases. Therefore, in this study, we evaluated the effect of different methods of BMSCs transplantation in the treatment of liver cirrhosis in rats. Seventy-two male Sprague-Dawley rats were divided into 7 groups: 10 were used to extract BMSCs, 10 were used as normal group, and the remaining 52 rats were randomly divided into five groups for testing: control group, BMSCs group, BMSCs+granulocyte colony-stimulating factor (G-CSF) group, and BMSCs+Jisheng Shenqi decoction (JSSQ) group. After the end of the intervention course, liver tissue sections of rats were subjected to hematoxylin and eosin (H&E) and Masson staining, and pathological grades were scored. Liver function [aminotransferase (ALT), aspartate aminotransferase (AST), albumin (ALB)] and hepatic fibrosis markers [hyaluronidase (HA), laminin (LN), type III procollagen (PCIII), type IV collagen (CIV)] were measured. BMSCs+JSSQ group had the best effect of reducing ALT and increasing ALB after intervention therapy (P<0.05). The reducing pathological scores and LN, PCIII, CIV of BMSCs+G-CSF group and BMSCs+JSSQ group after intervention therapy were significant, but there was no significant difference between the two groups (P>0.05). The effect of JSSQ on improving stem cell transplantation in rats with liver cirrhosis was confirmed. JSSQ combined with BMSCs could significantly improve liver function and liver pathology scores of rats with liver cirrhosis.

Preoperative erythropoietin treatment improves survival following major hepatic resection in a cirrhotic rat model.

AIM: Major hepatic resection of a cirrhotic liver may result in a fatal clinical course. Preoperative erythropoietin (EPO) treatment has been shown to have protective properties and to stimulate liver regeneration. This study aims to investigate the effect of preoperative EPO on survival following major hepatic resection in a cirrhotic rat model. METHODS: Cirrhotic liver was induced by intraperitoneal injection of thioacetamide (200mg/kg/mL) in 72 Lewis rats. Each 36 rats received EPO (1IU/g, every second day, 5 times preoperatively) or saline (control) and major hepatectomy (removal of the left and half of the median lobe) was performed. Biochemical and immunohistochemical parameters, cytokines and overall survival were compared following surgery. RESULTS: Rats that received preoperative EPO had decreased hepatic aspartate aminotransferase, alanine aminotransferase and interleukin (IL)-1ß expression, 48hours following surgery. They had increased hepatocyte growth factor and vascular endothelial growth factor expression at 1hour, increased IL-6 expression at 24, 48 and 120hours and increased Ki-67, 120hours following surgery. Overall, survival was significantly improved among EPO-treated rats (P=0.034). CONCLUSION: Preoperative EPO treatment has a protective effect and stimulates liver regeneration, leading to improved overall survival following major hepatectomy in a cirrhotic rat model.

Transplant of Hepatocytes, Undifferentiated Mesenchymal Stem Cells, and In Vitro Hepatocyte-Differentiated Mesenchymal Stem Cells in a Chronic Liver Failure Experimental Model: A Comparative Study.

OBJECTIVES: Liver transplant is the cornerstone line of treatment for chronic liver diseases; however, the long list of complications and obstacles stand against this operation. Searching for new modalities for treatment of chronic liver illness is a must. In the present research, we aimed to compare the effects of transplant of undifferentiated human mesenchymal stem cells, in vitro differentiated mesenchymal stem cells, and adult hepatocytes in an experimental model of chronic liver failure. MATERIALS AND METHODS: Undifferentiated human cord blood mesenchymal stem cells were isolated, pro-pagated, and characterized by morphology, gene expression analysis, and flow cytometry of surface markers and in vitro differentiated into hepatocyte-like cells. Rat hepatocytes were isolated by double perfusion technique. An animal model of chronic liver failure was developed, and undifferentiated human cord blood mesenchymal stem cells, in vitro hepato-genically differentiated mesenchymal stem cells, or freshly isolated rat hepatocytes were transplanted into a CCL4 cirrhotic experimental model. Animals were killed 3 months after transplant, and liver functions and histopathology were assessed. RESULTS: Compared with the cirrhotic control group, the 3 cell-treated groups showed improved alanine aminotransferase, aspartate aminotransferase, albumin, and bilirubin levels, with best results shown in the hepatocyte-treated group. Histopathologic examination of the treated groups showed improved fibrosis, with best results obtained in the undifferentiated mesenchymal stem cell-treated group. CONCLUSIONS: Both adult hepatocytes and cord blood mesenchymal stem cells proved to be promising candidates for cell-based therapy in liver regeneration on an experimental level. Improved liver function was evident in the hepatocyte-treated group, and fibrosis control was more evident in the undifferentiated mesenchymal stem cell-treated group.

Fetal Liver Stem/Progenitor Cell Transplantation: A Model to Study Tissue Mass Replacement and Cell-Based Therapies.

Liver transplantation is the only therapeutic treatment for patients with end-stage liver diseases. However, donor organ scarcity is the major limitation, and therefore, alternative strategies are urgently needed. The ultimate goal for successful cell-based therapies is the ability of transplanted cells to efficiently engraft and reconstitute injured liver mass. To evaluate the repopulation capacity of transplanted cells, it is essential to identify their specific characteristics, as well as to study the mechanism(s) Through which transplanted donor cells replace tissue mass in hepatic microenvironments, using well-established cell transplantation models. To date, rat fetal liver stem/progenitor cells represent the most efficient cell population to reconstitute the near-normal liver and the liver microenvironment with advanced fibrosis/cirrhosis, and therefore, can be used for developing strategies in engineering potential donor cells in the future that will be useful for clinical application in hepatic cell therapy.The present protocol describes the isolation of epithelial stem/progenitor cells derived from ED14/15 fetal livers of DPPIV+ F344 or F344-Tg(EGFP) F455/Rrrc rats, the immunohistochemical staining method to detect E-cadherin-positive epithelial cells within unfractionated cell isolates, their transplantation into different DPPIV- liver microenvironments (near-normal, retrorsine-treated, and TAA-induced fibrotic/cirrhotic liver), as well as detection methods to follow the fate of transplanted cells in the recipient liver (see Fig. 1).

Human urokinase-type plasminogen activator gene-modified bone marrow-derived mesenchymal stem cells attenuate liver fibrosis in rats by down-regulating the Wnt signaling pathway.

AIM: To evaluate the therapeutic effects of bone marrow-derived mesenchymal stem cells (BMSCs) with human urokinase-type plasminogen activator (uPA) on liver fibrosis, and to investigate the mechanism of gene therapy. METHODS: BMSCs transfected with adenovirus-mediated human urokinase plasminogen activator (Ad-uPA) were transplanted into rats with CCl4-induced liver fibrosis. All rats were sacrificed after 8 wk, and their serum and liver tissue were collected for biochemical, histopathologic, and molecular analyzes. The degree of liver fibrosis was assessed by hematoxylin and eosin or Masson's staining. Western blot and quantitative reverse transcription-polymerase chain reaction were used to determine protein and mRNA expression levels. RESULTS: Serum levels of alanine aminotransferase, aminotransferase, total bilirubin, hyaluronic acid, laminin, and procollagen type III were markedly decreased, whereas the levels of serum albumin were increased by uPA gene modified BMSCs treatment. Histopathology revealed that chronic CCl4-treatment resulted in significant fibrosis while uPA gene modified BMSCs treatment significantly reversed fibrosis. By quantitatively analysing the fibrosis area of liver tissue using Masson staining in different groups of animals, we found that model animals with CCl4-induced liver fibrosis had the largest fibrotic area (16.69% ± 1.30%), while fibrotic area was significantly decreased by BMSCs treatment (12.38% ± 2.27%) and was further reduced by uPA-BMSCs treatment (8.31% ± 1.21%). Both protein and mRNA expression of ß-catenin, Wnt4 and Wnt5a was down-regulated in liver tissues following uPA gene modified BMSCs treatment when compared with the model animals. CONCLUSION: Transplantation of uPA gene modified BMSCs suppressed liver fibrosis and ameliorated liver function and may be a new approach to treating liver fibrosis. Furthermore, treatment with uPA gene modified BMSCs also resulted in a decrease in expression of molecules of the Wnt signaling pathway.

Splenectomy enhances the therapeutic effect of adipose tissue-derived mesenchymal stem cell infusion on cirrhosis rats.

BACKGROUND & AIMS: Clinical studies suggest that splenectomy improves liver function in cirrhotic patients, but the influence of splenectomy on stem cell transplantation is poorly understood. This study investigated the effect of splenectomy on stem cell infusion and elucidated its mechanism. METHODS: Rat adipose tissue-derived mesenchymal stem cells were infused into cirrhosis rats with or without splenectomy, followed by the assessment of the in vivo distribution of stem cells and pathological changes. Stromal cell-derived factor-1 and hepatocyte growth factor expression were also investigated in splenectomized cirrhosis patients and rats. RESULTS: Splenectomy, prior to cell infusion, improved liver function and suppressed fibrosis progression more efficiently than cell infusion alone in the experimental cirrhosis model. Stromal cell-derived factor-1 and hepatocyte growth factor levels after splenectomy were increased in patients and rats. These upregulated cytokines significantly facilitated stem cell motility, migration and proliferation in vitro. C-X-C chemokine receptor type 4 neutralization weakened the promotion of cell migration by these cytokines. The infused cells integrated into liver fibrosis septa and participated in regeneration more efficiently in splenectomized rats. Direct coculture with stem cells led to inhibition of hepatic stellate cell proliferation. In addition, hepatocyte growth factor induced hepatic stellate cell apoptosis via the c-jun N-terminal kinase-p53 pathway. CONCLUSIONS: Splenectomy prior to cell infusion enhanced the therapeutic effect of stem cells on cirrhosis, which involved upregulation of stromal cell-derived factor-1 and hepatocyte growth factor after splenectomy.

Review of experimental models for inducing hepatic cirrhosis by bile duct ligation and carbon tetrachloride injection.

PURPOSE: To present a review about a comparative study of bile duct ligation versus carbon tetrachloride Injection for inducing experimental liver cirrhosis. METHODS: This research was made through Medline/PubMed and SciELO web sites looking for papers on the content "induction of liver cirrhosis in rats". We have found 107 articles but only 30 were selected from 2004 to 2011. RESULTS: The most common methods used for inducing liver cirrhosis in the rat were administration of carbon tetrachloride (CCl4) and bile duct ligation (BDL). CCl4 has induced cirrhosis from 36 hours to 18 weeks after injection and BDL from seven days to four weeks after surgery. CONCLUSION: For a safer inducing cirrhosis method BDL is better than CCl4 because of the absence of toxicity for researches and shorter time for achieving it.

Review of experimental models for inducing hepatic cirrhosis by bile duct ligation and carbon tetrachloride injection / Revisão de modelos experimentais de cirrose hepática induzida por ligadura do ducto biliar e por injeção de tetracloreto de carbono

PURPOSE: To present a review about a comparative study of bile duct ligation versus carbon tetrachloride Injection for inducing experimental liver cirrhosis. METHODS: This research was made through Medline/PubMed and SciELO web sites looking for papers on the content "induction of liver cirrhosis in rats". We have found 107 articles but only 30 were selected from 2004 to 2011. RESULTS: The most common methods used for inducing liver cirrhosis in the rat were administration of carbon tetrachloride (CCl4) and bile duct ligation (BDL). CCl4 has induced cirrhosis from 36 hours to 18 weeks after injection and BDL from seven days to four weeks after surgery. CONCLUSION: For a safer inducing cirrhosis method BDL is better than CCl4 because of the absence of toxicity for researches and shorter time for achieving it.

OBJETIVO: Apresentar revisão sobre estudo comparativo da indução de cirrose hepática (CH) experimental com a injeção de tetra-cloreto de carbono (CCl4) comparado à ligadura do ducto biliar (BDL). MÉTODOS: A pesquisa foi realizada nas bases de dados do Medline/PubMed e SciELO procurando trabalhos com as palavras indução de CH e ratos. Foram encontrados 107 artigos, mas somente 30 foram selecionados no período de 2004 à 2011. RESULTADOS: Os procedimentos mais comum para indução de CH em ratos foram a injeção de CCl4 e a BDL. O CCl4 induzia CH no período de 36 horas após a injeção e a DBL de sete dias à quatro semanas após a cirurgia. CONCLUSÃO: A BDL é o método mais seguro para indução de CH quando comparado a injeção de CCl4 pela ausência de toxicidade para os pesquisadores e o menor tempo para se obter a lesão hepática.

Embryonic hepatocyte transplantation for hepatic cirrhosis: efficacy and mechanism of action.

AIM: To investigate the efficacy and mechanism of action of allogeneic embryonic hepatocyte transplantation for the treatment of hepatic cirrhosis. METHODS: Rat embryonic hepatocytes were characterized by examining cell markers. Wistar rats with CCl(4)-induced cirrhosis were randomly divided into two groups: a model group receiving continuous CCl(4), and a cell transplantation group receiving continuous CCl(4) and transplanted with embryonic fluorescent-labeled hepatocytes. In addition, a normal control group was composed of healthy rats. All rats were sacrificed after 2 wk following the initiation of the cell transplant. Ultrasound, pathological analyses and serum biochemical tests were used to evaluate the efficacy of embryonic hepatocyte transplantation. To analyze the recovery status of cirrhotic hepatocytes and the signaling pathways influenced by embryonic hepatocyte transplantation, real-time polymerase chain reaction was performed to examine the mRNA expression of stellate activation-associated protein (STAP), c-myb, α smooth muscle actin (α-SMA) and endothelin-1 (ET-1). Western blotting and immunohistochemistry were employed to detect α-SMA and ET-1 protein expression in hepatic tissues. RESULTS: Gross morphological, ultrasound and histopathological examinations, serum biochemical tests and radioimmunoassays demonstrated that hepatic cirrhosis was successfully established in the Wistar rats. Stem cell factor receptor (c-kit), hepatocyte growth factor receptor (c-Met), Nestin, α fetal protein, albumin and cytokeratin19 markers were observed in the rat embryonic hepatocytes. Following embryonic hepatocyte transplantation, there was a significant reversal in the gross appearance, ultrasound findings, histopathological properties, and serum biochemical parameters of the rat liver. In addition, after the activation of hepatic stellate cells and STAP signaling, α-SMA, c-myb and ET-1 mRNA levels became significantly lower than in the untreated cirrhotic group (P < 0.05). These levels, however, were not statistically different from those of the normal healthy group. Immunohistochemical staining and Western blot analyses revealed that α-SMA and ET-1 protein expression levels in the transplantation group were significantly lower than in the untreated cirrhotic group, but being not statistically different from the normal group. CONCLUSION: Transplantation of embryonic hepatocytes in rats has therapeutic effects on cirrhosis. The described treatment may significantly reduce the expression of STAP and ET-1.

Autologous bone marrow cell infusions suppress tumor initiation in hepatocarcinogenic mice with liver cirrhosis.

We have previously reported the efficacy and safety of autologous bone marrow cell infusion (ABMi) therapy for liver cirrhosis patients without hepatocellular carcinoma in a multicenter clinical trial. However, since liver cirrhosis is highly oncogenic, evaluation of the effects of ABMi on the mechanisms of hepatocarcinogenesis is of great importance. Therefore, frequent ABMi was performed in hepatocarcinogenic mice, and its effects on hepatocarcinogenesis were analyzed. The N-nitrosodiethylamine (DEN)/green fluorescent protein (GFP)-carbon tetrachloride (CCl(4) ) model was developed by administering DEN once, followed by repeated administration of CCl(4) intraperitoneally as for the control group. In the administration (ABMi) group, GFP-positive bone marrow cells were infused through a tail vein. The kinetics of hepatocarcinogenesis were evaluated histologically 4.5 months after DEN treatment. At 4.5 months, there was significantly lower incidence of foci and tumors in the ABMi group, and they were smaller in number, while their size was almost equal. No GFP-positive tumors were found in ABMi livers. Moreover, ABMi livers showed significantly reduced liver fibrosis, consistent with significantly lower 8-hydroxy-2'-deoxyguanosine levels, higher superoxide dismutase activity, and increased nuclear translocation of nuclear factor-erythroid 2 p45-related factor 2. These results demonstrate that frequent ABMi might contribute to suppressed tumor initiation during stages of hepatocarcinogenesis, consistent with improvements in liver fibrosis and stabilization of redox homeostasis.

The microenvironment in hepatocyte regeneration and function in rats with advanced cirrhosis.

UNLABELLED: In advanced cirrhosis, impaired function is caused by intrinsic damage to the native liver cells and from the abnormal microenvironment in which the cells reside. The extent to which each plays a role in liver failure and regeneration is unknown. To examine this issue, hepatocytes from cirrhotic and age-matched control rats were isolated, characterized, and transplanted into the livers of noncirrhotic hosts whose livers permit extensive repopulation with donor cells. Primary hepatocytes derived from livers with advanced cirrhosis and compensated function maintained metabolic activity and the ability to secrete liver-specific proteins, whereas hepatocytes derived from cirrhotic livers with decompensated function failed to maintain metabolic or secretory activity. Telomere studies and transcriptomic analysis of hepatocytes recovered from progressively worsening cirrhotic livers suggest that hepatocytes from irreversibly failing livers show signs of replicative senescence and express genes that simultaneously drive both proliferation and apoptosis, with a later effect on metabolism, all under the control of a central cluster of regulatory genes, including nuclear factor κB and hepatocyte nuclear factor 4α. Cells from cirrhotic and control livers engrafted equally well, but those from animals with cirrhosis and failing livers showed little initial evidence of proliferative capacity or function. Both, however, recovered more than 2 months after transplantation, indicating that either mature hepatocytes or a subpopulation of adult stem cells are capable of full recovery in severe cirrhosis. CONCLUSION: Transplantation studies indicate that the state of the host microenvironment is critical to the regenerative potential of hepatocytes, and that a change in the extracellular matrix can lead to regeneration and restoration of function by cells derived from livers with end-stage organ failure.

Modulation of AP-endonuclease1 levels associated with hepatic cirrhosis in rat model treated with human umbilical cord blood mononuclear stem cells.

Oxidative stress in liver cells may contribute to the etiology of hepatic diseases, as in liver cirrhosis. AP-Endonuclease1 (APE1/Ref-1) is essential for cell protection toward oxidative stress by acting as a transcriptional regulator of pro-survival genes and as a redox sensitive protein. The aim of this study was to critically analyze the various parameters governing the success of human umbilical cord blood mononuclear stem cell-based (MNCs) therapy without the use of an immunosuppressant and to investigate for the first time the expression of APE1 during thioacetamide (TAA)-induced cirrhosis and MNCs therapy in a rat model. Umbilical cord blood samples from full-term deliveries were collected. Lethal fulminant hepatic cirrhosis in rats was induced by intraperitoneal injection of thio-acetamide. MNCs were then intrahepatically transplanted. We measured APE1 expression at mRNA and protein levels, mRNA expression of TGF-ß, α-SMA, STAP, CTGF, MMP-9 and TIMP-1 in a follow up study. Histopathological and immunohistochemical analyses were performed 10 weeks after intrahepatic injection of the cells. Transdifferentiated cells could be efficiently stained with antihuman hepatocytes. Interestingly, human hepatocyte-specific markers, human albumin, cytokeratin-18 and cytokeratin-19 mRNAs were detected in rat liver after 10 days of MNCs infusion. MNC transplanted by intrahepatic route, could engraft recipient liver, differentiated into functional hepatocytes, and rescued liver failure. Moreover up regulation of APE1 expression confirmed by marked immunohistochemical staining may be involved in MNCs-induced hepatocytes regeneration suggesting that maintaining high level of APE1 has protective effect as pro-survival signal.

Role of angiotensin-1 receptor blockade in cirrhotic liver resection.

BACKGROUND: The regeneration capacity of cirrhotic livers might be affected by angiotensin-1 (AT1) receptors located on hepatic stellate cells (HSC). The effect of AT1 receptor blockade on microcirculation, fibrosis and liver regeneration was investigated. MATERIALS AND METHODS: In 112 Lewis rats, cirrhosis was induced by repetitive intraperitoneal injections of CCl(4) . Six hours, 3, 7 and 14 days after partial hepatectomy or sham operation, rats were sacrificed for analysis. Animals were treated with either vehicle or 5 mg/kg body weight losartan pre-operatively and once daily after surgery by gavage. Microcirculation and portal vein flow were investigated at 6 h. The degree of cirrhosis was assessed by Azan Heidenhein staining, activation of HSC by desmin staining, apoptosis by ssDNA detection and liver regeneration by Ki-67 staining. Changes in expression of various genes important for liver regeneration and fibrosis were analysed at 6 h and 3 days. Haemodynamic parameters and liver enzymes were monitored. RESULTS: Losartan treatment increased sinusoidal diameter, sinusoidal blood flow and portal vein flow after partial hepatectomy (P<0.05), but not after sham operation. AT1 receptor blockade resulted in increased apoptosis early after resection. HSC activation was reduced and after 7 days, a significantly lower degree of cirrhosis in resected animals was observed. Losartan increased the proliferation of hepatocytes at late time-points and of non-parenchymal cells early after partial hepatectomy (P<0.05). Tumour necrosis factor (TNF)-α was significantly upregulated at 6 h and stem cell growth factor (SCF) was downregulated at 3 days (P<0.05). CONCLUSION: Losartan increased hepatic blood flow, reduced HSC activation and liver fibrosis, but interfered with hepatocyte proliferation after partial hepatectomy in cirrhotic livers.

Decreased collagen types I and IV, laminin, CK-19 and α-SMA expression after bone marrow cell transplantation in rats with liver fibrosis.

Bone marrow cells have frequently been tested in animal models of liver fibrosis to assess their role in hepatic regeneration. The mononuclear fraction of bone marrow cells is of particular interest, as many studies show that these cells may be beneficial to treat hepatic fibrosis. In this study, we used the bile duct ligation model to induce hepatic fibrosis in an irreversible manner, and rats were treated with bone marrow mononuclear (BMMN) cells after fibrosis was established. Analysis of collagen types I and IV, laminin and α-SMA showed a decreased expression of these proteins in fibrotic livers after 7 days of BMMN cell injection. Moreover, cytokeratin-19 analysis showed a reduction in bile ducts in the BMMN cell-treated group. These results were accompanied by ameliorated levels of hepatic enzymes GPT (Glutamic-pyruvic transaminase), GOT (glutamic-oxaloacetic transaminase) and alkaline phosphatase (AP). Therefore, we showed that BMMN cells decrease hepatic fibrosis by significantly reducing myofibroblast numbers and through reduction of the collagen and laminin-rich extracellular matrix of fibrotic septa and hepatic sinusoids.

[Cryosurgery in diffuse hepatic diseases].

Comparative studying, using histological and biomicroscopic methods, of the dosed cryohepatodestruction (CHD), periarterial cryodenervation of hepatic artery (CDHA) and their concomitant application influence on the dynamics of hepatic restoration processes in experimental cirrhosis was performed. The investigations were done on 215 male rats owing body mass 200-280 g in a not changed and pathologically changed liver. There was shown, that CDHA promotes changes in hepatic tissue microhemocirculation, as well as the enhancement of the sinusoidal vessels diameter and relative square of vascular bed. CHD stimulates the reparative processes course in a pathologically changed organ. There was established, that while simultaneous application of two cryosurgical methods, the velocity and grade of restoration processes in cirrhotically-changed liver are enhanced in comparison with such indices changes while separate usage of these two methods.

Macrophage-mediated phagocytosis of apoptotic cholangiocytes contributes to reversal of experimental biliary fibrosis.

Studies have suggested the reversibility of liver fibrosis, but the mechanisms of fibrosis reversal are poorly understood. We investigated the possible functional link between apoptosis, macrophages, and matrix turnover in rat liver during reversal of fibrosis secondary to bile duct ligation (BDL). Biliary fibrosis was induced by BDL for 4 wk. After Roux-en-Y (RY)-bilio-jejunal-anastomosis, resolution of fibrosis was monitored for up to 12 wk by hepatic collagen content, matrix metalloproteinase (MMP) expression and activities, and fibrosis-related gene expression. MMP expression and activities were studied in macrophages after engulfment of apoptotic cholangiocytes in vitro. Hepatic collagen decreased to near normal at 12 wk after RY-anastomosis. During reversal, profibrogenic mRNA declined, whereas expression of several profibrolytic MMPs increased. Fibrotic septa showed fragmentation at week 4 and disappeared at week 12. Peak histological remodeling at week 4 was characterized by massive apoptosis of cytokeratin 19+ cholangiocytes, >90% in colocalization with CD68+ macrophages, and a 2- to 7.5-fold increase in matrix-degrading activities. In vitro, phagocytosis of apoptotic cholangiocytes induced matrix-degrading activities and MMP-3, -8, and -9 in rat peritoneal macrophages. We concluded that reconstruction of bile flow after BDL leads to an orchestrated fibrolytic program that results in near complete reversal of advanced fibrosis. The peak of connective tissue remodeling and fibrolytic activity is associated with massive apoptosis of cholangiocytes and their phagocytic clearance by macrophages in vivo. Macrophages upregulate MMPs and become fibrolytic effector cells upon apoptotic cholangiocyte engulfment in vitro, suggesting that phagocytosis-associated MMP induction in macrophages significantly contributes to biliary fibrosis reversal.

Transplanted endothelial progenitor cells ameliorate carbon tetrachloride-induced liver cirrhosis in rats.

Cirrhosis is the most common end stage of liver diseases, and there are no effective treatment methods. Here we evaluated the effect of endothelial progenitor cell (EPC) transplantation from rat bone marrow (BM) on the development of cirrhosis induced by carbon tetrachloride (CCl(4)). Ex vivo generated, characterized, and cultivated rat BM-derived EPCs were identified by their vasculogenic properties in vitro. EPCs from male rats were transplanted into female rats via the intraportal vein 12 weeks after they had been challenged with CCl(4), and the rats were killed 16 weeks later. The control rats received only a saline infusion. The fibrosis index and donor cell engraftment were assessed after EPC transplantation. After transplantation via the portal vein, PKH26 labeling, polymerase chain reaction, and in situ hybridization analysis revealed that the donor EPCs had adhered to the vasolateral surfaces of blood vessels and established in the liver. EPCs reduced the expression of alpha-smooth muscle actin, collagen III, and transforming growth factor beta (P < 0.05) as well as levels of aspartate aminotransferase, alanine aminotransferase, and total bilirubin in the serum (P < 0.05), but at the same time they increased the levels of albumin and Ki67. CCl(4) treatment increased the international prothrombin ratio (P < 0.05) and reduced albumin levels, whereas EPCs restored these parameters to normal levels. These results suggest that EPC transplantation could play a role in regulating hepatocyte regeneration and ameliorating established liver cirrhosis.