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Recruitment of hemopoietic stem cells to the liver improves liver function in cirrhotic rats
280A
AASLD ABSTRACTS
HEPATOLOGYOctober 2001
431
432
DIFFERENTIAL MODULATION OF ANGIOPOIETIN-1, ANGIOPOIETIN-2, VASCULAR ENDOTHELIAL GROWTH FACTOR, AND HYPOXIA-INDUCIBLE FACTOR-1 IN RAT HEPATIC STELLATE CELLS DURING HEPATIC W O U N D HEALING. Zhihao Dai, Victor Ankoma-Sey, University
HEPATIC "yA T CELL POPULATIONS CO-EXPRESS NK RECEPTORS AND PREDOMINANTLY SECRETE TH1 CYTOKINES, Tony Kenna Mr, St
of Texas Medical School-Houston, Houston, TX Wound healing is regulated by a variety of angiogenic factors. We have previously shown the induction of vascular endothelial growth factor (VEGF) expression in rat hepatic stellate cells after in vivo injury induced by administration of carbon tetrachloride (CC14). We hypothesize that key mediators of angiogenesis, such as, angiopoietin-1, angiopoietin-2, VEGF and hypoxia-inducible factor I(HIF-1) are modulated in rat hepatic stellate ceils during liver injury and repair. The specific aims: to characterize the pattern and temporal relationship of gene expression of angiopoietin-1, angiopoietin-2, VEGF and HIF-1 after CC14-induced injury. Methods: Retired breeder Sprague-Dawley rats were treated with a single dose of CC14 by gavage and hepatic stellate cells isolated from these rats 3-36 hours later. Total RNA was isolated from the hepatic stefiate cells from CC14 treated rats and controls and the mRNA levels of angiopoietin-1, angiopoietin-2, VEGF and HIF-1 were determined using ribonuclease protection assay. Results: VEGF mRNA expression was induced in hepatic stellate cells after a single dose of CC14, with a maximal four-fold increase after 12 hours, which persists over the 36-hour period compared to controls. In contrast to the pattern of VEGF expression, the high levels of angiopoietin-1 mRNA detected in the control cells was down regulated after a single dose of CC14, with a nadir, 12 hours after CC14 administration. Angiopoietin-2 mRNA was detected in hepatic stellate cel|s from both control and CC14-treated rats, but there was no appreciable changes detected over the 36-hour period after CC14-mediated in vivo injury. Low levels of HIF-1 mRNA was detected in control stellate cells. HIF-1 mRNA expression in stellate cells was induced after a single dose of CC14 treatment, with a maximal four-fold increase after 24 hours, compared to controls. At 36 hours, HIF-1 mRNA returned to a basal level. Conclusions: the in vivo expression of angiopoietin-1, angiopoietin-2, VEGF and HIF-1 in rat hepatic stellate cells is differentially modulated after CC14-induced liver injury. Our data suggest that these angiogenic factors may play a critical role during liver injury and repair.
Vincent's University Hospital, Dublin Ireland; Lucy Golden-Mason Ms, ERC, St Vincent's University Hospital, Dublin Ireland; Suzanne N Norris Dr., King's College Hospital, London United Kingdom; Gerry McEntee Dr, Oscar Traynor Dr, John Hegarty Dr, Liver Unit, St Vincent's University Hospital, Dublin Ireland; Cliona O'Farrelly Dr, ERC, St Vincent's University Hospital, Dublin Ireland; Derek G Doherty Dr, Dept Biology, NUI, Maynooth, Kildare Ireland "y3T cells are a subpopulation of T ceils accounting for approximately 1-5% of circulating lymphocytes. 3'3 T cells are found in highest numbers at epithelial surfaces. 3/6 T cells are believed to play a key role in control of microbiaI infection and tumour immunity. Through the production of a range of cytokines 3'3 T ceils regulate the activity of other immune subsets and thus affect the outcome of an immune response. The present study aimed to characterise hepatic 3'3 T cells in terms of their phenotypes and cytokine secretion profiles in order to assess the role played by these cells in the hepatic immune system. HMNC s were isolated from normal liver specimens (n = 12), obtained prior to transplantation, by mechanical and enzymatic disruption. Isolated HMNC s and matched PBMC s were characterised with respect to natural killer cell marker expression and V6 gene segment usage using flow cytometry. Cytokine production by hepatic 3'6 T cells was determined by intracytoplasmic flow cytometry. A significantly higher proportion of hepatic ~/6 T cells were found (8.96%) compared with matched blood (2.78%), (P = 0.0007). Considerable numbers of hepatic 3'3 T cells express the Natural killer cell markers CD56 (38.86%) and CD 161 (36.15%). Analysis of the V6 chain usage by hepatic 3,3 T cells showed preferential usage of the V33 gene segment almost never seen in circulating y6 T cells (20.51.% Vs. 0.041%, n = 9), suggesting a possible antigen specific expansion of this population within the liver. The frequency of hepatic 3'fi T cells capable of producing the proinflammatory cytokine TNF-c¢ (11.81%), the Thl cytokines IFN-7 (32.7%) and IL-2 (9.02%) and the Th2 cytokine IL-4 (5.21%) upon stimulation was determined (n = 4). The results indicate that 3,6 T cells may act as first line of the adaptive defences against pathogens and tumours and as such as a bridge between the innate and adaptive immune responses.
433
434
INVOLVEMENT OF P-SELECTIN AND oz4-INTEGRIN IN LIVER LEUKOCYTE RECRUITMENT INDUCED BY ADENOVIRUS VECTOR. Yang Li,
RECRUITMENT OF HEMOPOIETIC STEM CELLS TO THE LIVER IMPROVES LIVER FUNCTION IN CIRRHOTIC RATS. Helena Katchman, Isa-
Daniel A Muruve, Paul Kubes, Samuel S Lee, The University of Calgary, Calgary, AB Canada
bel Zvibel, Zamir Halpern, Ran Oren, Shlomo Brill, Tel Aviv Medical Center, Tel Aviv Israel
Backgrounds: The use of adenovirus vector for gene therapy was found to cause serious liver inflammation. The mechanisms underlying this inflammation are poorly understood. Selectins and cr4-integrin are known to mediate both the rolling and adhesion steps of leukocyte recruitment. The present study aimed to investigate the role of selectins and o~4-integrin on the liver leukocyte recruitment induced by an adenovirus vector. Methods: The rolling and adhesion of leukocytes in the liver of wild-type C57B1/6 or E-selectin deficient (E-selectin-/-) mice were observed using intravital microscopy. Adenovirus vector (AdGFP) was administered via the jugular vein. P-selectin mAb (RB40.34, 20 ~g/mouse) and/or ec4-integrin mAb (R1-2, 75/~g/mouse) were given before AdGFP. Results: AdGFP caused a time-dependent increase in leukocyte rolling and adhesion in postsinusoidal venules within 120 min. P-selectin mAb significantly reduced numbers of rolling leukocytes, but not adhesion in wild-type mice. This effect was not enhanced in E-selectin-/- mice. P-selectin mAb combined with c~4-integrin mAb significantly blocked both leukocyte rolling and adhesion in E-selectin-/- mice. Conclusions: Rolfng and adhesion of leukocytes induced by adenovirus vector in the postsinusoidal venules of mouse liver were mediated by P-selectin and a4-integrin, respectively.
Background : Recently, hemopoietic stem cells were shown to be capable to differentiate not only blood cells, but also into muscle cells, neuronal cells, as well as hepatocytes. Several studies have shown that, in addition to G-CSF, the heparin-like sugar polymer fucoidin, increased mobilization of hemopoietic stem cells into the circulation and prevented homing of the cells to the bone marrow. Aim : We tested whether recruitment of hemopoietic stem cells to the liver improves liver functions in cirrhotic rats, Methods : Liver cirrhosis was induced by i.p. injection of dimethylnitrosamine in female Fischer rats and confirmed by histopathology and liver functions as assessed by blood tests. Recruitment of hemopoietic stem cells was induced by treatment with G-CSF, followed by fucoidin administration. Liver functions in control and treated rats was assessed by testing for levels of blood albumin, bilirubin, liver enzymes, prothrombin time and ammonia. Results : We have found that G-CSF alone doubled the WBC count in injected rats, while G-CSF followed by fucoidin injection resulted in a 10-fold increase in circulating WBC. All liver functions in rats treated with G-CSF and fucoidin were significantly better than the same functions tested in control cirrhotic rats. Liver enzymes and ammonia were dramatically reduced in fucoidin-treated rats, while synthetic functions improved: albumin increased substantially while PT was decreased. Conclusion : The plasticity of hemopoietic stem cells, i.e. their ability to differentiate into hepatocytes, may be used to develop alternative approaches that may replace liver transplantation in the future.
AASLD ABSTRACTS
HEPATOLOGYOctober 2001
431
432
DIFFERENTIAL MODULATION OF ANGIOPOIETIN-1, ANGIOPOIETIN-2, VASCULAR ENDOTHELIAL GROWTH FACTOR, AND HYPOXIA-INDUCIBLE FACTOR-1 IN RAT HEPATIC STELLATE CELLS DURING HEPATIC W O U N D HEALING. Zhihao Dai, Victor Ankoma-Sey, University
HEPATIC "yA T CELL POPULATIONS CO-EXPRESS NK RECEPTORS AND PREDOMINANTLY SECRETE TH1 CYTOKINES, Tony Kenna Mr, St
of Texas Medical School-Houston, Houston, TX Wound healing is regulated by a variety of angiogenic factors. We have previously shown the induction of vascular endothelial growth factor (VEGF) expression in rat hepatic stellate cells after in vivo injury induced by administration of carbon tetrachloride (CC14). We hypothesize that key mediators of angiogenesis, such as, angiopoietin-1, angiopoietin-2, VEGF and hypoxia-inducible factor I(HIF-1) are modulated in rat hepatic stellate ceils during liver injury and repair. The specific aims: to characterize the pattern and temporal relationship of gene expression of angiopoietin-1, angiopoietin-2, VEGF and HIF-1 after CC14-induced injury. Methods: Retired breeder Sprague-Dawley rats were treated with a single dose of CC14 by gavage and hepatic stellate cells isolated from these rats 3-36 hours later. Total RNA was isolated from the hepatic stefiate cells from CC14 treated rats and controls and the mRNA levels of angiopoietin-1, angiopoietin-2, VEGF and HIF-1 were determined using ribonuclease protection assay. Results: VEGF mRNA expression was induced in hepatic stellate cells after a single dose of CC14, with a maximal four-fold increase after 12 hours, which persists over the 36-hour period compared to controls. In contrast to the pattern of VEGF expression, the high levels of angiopoietin-1 mRNA detected in the control cells was down regulated after a single dose of CC14, with a nadir, 12 hours after CC14 administration. Angiopoietin-2 mRNA was detected in hepatic stellate cel|s from both control and CC14-treated rats, but there was no appreciable changes detected over the 36-hour period after CC14-mediated in vivo injury. Low levels of HIF-1 mRNA was detected in control stellate cells. HIF-1 mRNA expression in stellate cells was induced after a single dose of CC14 treatment, with a maximal four-fold increase after 24 hours, compared to controls. At 36 hours, HIF-1 mRNA returned to a basal level. Conclusions: the in vivo expression of angiopoietin-1, angiopoietin-2, VEGF and HIF-1 in rat hepatic stellate cells is differentially modulated after CC14-induced liver injury. Our data suggest that these angiogenic factors may play a critical role during liver injury and repair.
Vincent's University Hospital, Dublin Ireland; Lucy Golden-Mason Ms, ERC, St Vincent's University Hospital, Dublin Ireland; Suzanne N Norris Dr., King's College Hospital, London United Kingdom; Gerry McEntee Dr, Oscar Traynor Dr, John Hegarty Dr, Liver Unit, St Vincent's University Hospital, Dublin Ireland; Cliona O'Farrelly Dr, ERC, St Vincent's University Hospital, Dublin Ireland; Derek G Doherty Dr, Dept Biology, NUI, Maynooth, Kildare Ireland "y3T cells are a subpopulation of T ceils accounting for approximately 1-5% of circulating lymphocytes. 3'3 T cells are found in highest numbers at epithelial surfaces. 3/6 T cells are believed to play a key role in control of microbiaI infection and tumour immunity. Through the production of a range of cytokines 3'3 T ceils regulate the activity of other immune subsets and thus affect the outcome of an immune response. The present study aimed to characterise hepatic 3'3 T cells in terms of their phenotypes and cytokine secretion profiles in order to assess the role played by these cells in the hepatic immune system. HMNC s were isolated from normal liver specimens (n = 12), obtained prior to transplantation, by mechanical and enzymatic disruption. Isolated HMNC s and matched PBMC s were characterised with respect to natural killer cell marker expression and V6 gene segment usage using flow cytometry. Cytokine production by hepatic 3'6 T cells was determined by intracytoplasmic flow cytometry. A significantly higher proportion of hepatic ~/6 T cells were found (8.96%) compared with matched blood (2.78%), (P = 0.0007). Considerable numbers of hepatic 3'3 T cells express the Natural killer cell markers CD56 (38.86%) and CD 161 (36.15%). Analysis of the V6 chain usage by hepatic 3,3 T cells showed preferential usage of the V33 gene segment almost never seen in circulating y6 T cells (20.51.% Vs. 0.041%, n = 9), suggesting a possible antigen specific expansion of this population within the liver. The frequency of hepatic 3'fi T cells capable of producing the proinflammatory cytokine TNF-c¢ (11.81%), the Thl cytokines IFN-7 (32.7%) and IL-2 (9.02%) and the Th2 cytokine IL-4 (5.21%) upon stimulation was determined (n = 4). The results indicate that 3,6 T cells may act as first line of the adaptive defences against pathogens and tumours and as such as a bridge between the innate and adaptive immune responses.
433
434
INVOLVEMENT OF P-SELECTIN AND oz4-INTEGRIN IN LIVER LEUKOCYTE RECRUITMENT INDUCED BY ADENOVIRUS VECTOR. Yang Li,
RECRUITMENT OF HEMOPOIETIC STEM CELLS TO THE LIVER IMPROVES LIVER FUNCTION IN CIRRHOTIC RATS. Helena Katchman, Isa-
Daniel A Muruve, Paul Kubes, Samuel S Lee, The University of Calgary, Calgary, AB Canada
bel Zvibel, Zamir Halpern, Ran Oren, Shlomo Brill, Tel Aviv Medical Center, Tel Aviv Israel
Backgrounds: The use of adenovirus vector for gene therapy was found to cause serious liver inflammation. The mechanisms underlying this inflammation are poorly understood. Selectins and cr4-integrin are known to mediate both the rolling and adhesion steps of leukocyte recruitment. The present study aimed to investigate the role of selectins and o~4-integrin on the liver leukocyte recruitment induced by an adenovirus vector. Methods: The rolling and adhesion of leukocytes in the liver of wild-type C57B1/6 or E-selectin deficient (E-selectin-/-) mice were observed using intravital microscopy. Adenovirus vector (AdGFP) was administered via the jugular vein. P-selectin mAb (RB40.34, 20 ~g/mouse) and/or ec4-integrin mAb (R1-2, 75/~g/mouse) were given before AdGFP. Results: AdGFP caused a time-dependent increase in leukocyte rolling and adhesion in postsinusoidal venules within 120 min. P-selectin mAb significantly reduced numbers of rolling leukocytes, but not adhesion in wild-type mice. This effect was not enhanced in E-selectin-/- mice. P-selectin mAb combined with c~4-integrin mAb significantly blocked both leukocyte rolling and adhesion in E-selectin-/- mice. Conclusions: Rolfng and adhesion of leukocytes induced by adenovirus vector in the postsinusoidal venules of mouse liver were mediated by P-selectin and a4-integrin, respectively.
Background : Recently, hemopoietic stem cells were shown to be capable to differentiate not only blood cells, but also into muscle cells, neuronal cells, as well as hepatocytes. Several studies have shown that, in addition to G-CSF, the heparin-like sugar polymer fucoidin, increased mobilization of hemopoietic stem cells into the circulation and prevented homing of the cells to the bone marrow. Aim : We tested whether recruitment of hemopoietic stem cells to the liver improves liver functions in cirrhotic rats, Methods : Liver cirrhosis was induced by i.p. injection of dimethylnitrosamine in female Fischer rats and confirmed by histopathology and liver functions as assessed by blood tests. Recruitment of hemopoietic stem cells was induced by treatment with G-CSF, followed by fucoidin administration. Liver functions in control and treated rats was assessed by testing for levels of blood albumin, bilirubin, liver enzymes, prothrombin time and ammonia. Results : We have found that G-CSF alone doubled the WBC count in injected rats, while G-CSF followed by fucoidin injection resulted in a 10-fold increase in circulating WBC. All liver functions in rats treated with G-CSF and fucoidin were significantly better than the same functions tested in control cirrhotic rats. Liver enzymes and ammonia were dramatically reduced in fucoidin-treated rats, while synthetic functions improved: albumin increased substantially while PT was decreased. Conclusion : The plasticity of hemopoietic stem cells, i.e. their ability to differentiate into hepatocytes, may be used to develop alternative approaches that may replace liver transplantation in the future.