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Combined use of adenosine and phosphodiesterase inhibitor inhibits reperfusion injury of rat liver
I-IEPATOLOGYVol. 34, No. 4, Pt. 2, 2001
AASLD ABSTRACTS
305A
531
532
ESTABLISHMENT OF HTERT-IMMORTALIZED HUMAN HEPATOCYTES AS A SOURCE FOR A HYBRID BIOARTIFICIAL LIVER. Toshinori Totsugawa, Okayama University Graduate School of Medicine and Dentistry, Okayama Japan; Naoya Kobayashi, Hirofumi Noguchi, Takamasa Watanabe, Toshihisa Matsumura, Masanobu Maruyama, Tomoko Matsumoto, Noriaki Tanaka, Okayama University Grad, OkayamaJapan; Karen A Westerman, Harvard-Massachusetts Institute of Technology, Boston, MA; Philippe Leboulch, Harvard-Massachusetts I, Boston, MA
ADENOVIRUS-MEDIATED P 2 I GENE TRANSFER ENHANCES DIFFERENTIATED HEPATIC PHENOTYPES OF IMMORTALIZED HUMAN HEPATOCYTES. Naoya Kobayashi, Hirofumi Noguchi, Toshinori Totsugawa, Takamasa Watanabe, Toshihisa Matsumura, Masanobu Maruyama, Tomoko Matsumoto, Toshiyoshi Fujiwara, Noriaki Tanaka, Masakiyo Sakaguchi, Okayama University Graduate School of Medicine and Dentistry, Okayama ]apan
Purpose: The shortage of donor livers for hepatocyte isolation is one of the major limiting factors for developing bioartificial liver (BAL) therapy. The present study describes a novel strategy for constructing a human hepatocyte cell line as a source in BAL. Methods: Adult human hepatocytes were trarlsduced with a retroviral vector SSR#197 encoding the genes of telomerase reverse transcriptase (hTERT) and positive selectable marker, green fluorescent protein (GFP), which were flanked by a pair of recombination targets of loxP. After the transduction of hepatocytes with SSR#197, ceils were prepared with FACS to obtain GFP-positive populations. Expression of differentiated liver functions and efficacy of CreAoxP recombination mediated by an adenoviral vector producing Cre (AxCANCre) were evaluated in SSR#197-transduced hepatocytes. Results: TTNT-1, one of SSR#197-immortalized human hepatocytes, grew steadily in tissue culture and demonstrated morphological characteristics of liver parenchymal cells. The cells showed hepatocyte-specific gene expression. When transplanted into immunodeficient mice, TTNT-1 cells were not mrmorgenic. Efficient Cre/loxP recombination in TTNT-1 cells was performed following AxCANCre infection. Subsequent GFP-negative cell sorting allowed reversible immortalization system in human hepatocytes. Differentiated hepatic functions were enhanced in reverted TTNT-1 cells. Conclusion: We have developed an innovative method of reversible immortalization of human hepatocytes. This work represents a potentially novel strategy for addressing the problem of donor liver shortage that currently limits the use of human hepatocytes for BAL therapy.
Purpose: The p21 molecule, a potent cyclin-dependent kinase inhibitor, regulates the transition from the G1 phase to the S phase of the cell cycle. Overexpression of p21 has been shown to induce differentiation in various cell lines. Previously we have established an immortalized human hepatocyte cell line NKNT-3 to develop a bioartificial liver (BAL) (N.Kobayashi et al., Science 1252; 1246-1262, 2000). The findings prompted us to investigate the effect of p21 on hepatic phenotypes in NKNT-3 cells. Methods: NKNT-3 ceils, simian virus 40 T antigen-immortalized human hepatocytes, were used in the present study. A replication-deficient adenovirus vector encoding a p21 gene under the control of the CMV promoter (Ad5CMVp21) was utilized to achieve efficient gene transfer into NKNT-3 cells. Expression of p-450 associated enzymes, cell-cycle progression, and morphology were analyzed in NKNT-3 cells infected with Ad5CMVp21. Results: Efficient adenoviral gene transfer of p21 into NKNT-3 cells were confirmed by immunohistochemistry and Western blotting analysis. Overexpression of p21 predominantly arrested the cell cycle at the G1 checkpoint in NKNT-3 cells, resulting in differentiated hepatic phenotvpes including a decreased nuclear-to cytoplasmic ratio, a decreased cell density, increased glycogen synthesis, and enhanced expression of CYP3A4 and 2C9. Conclusion: We demonstrate that p21 gene transfer augments the differentiated hepatic phenotypes in NKNT-3 ceils. The strategy would be useful to increase hepatic functions of immortalized human hepatocytes in BAL therapy.
533
534
COMBINED USE OF ADENOSINE AND PHOSPHODIESTERASE INHIBITOR INHIBITS REPERFUSION INJURY OF RAT LIVER. Shigekazu Takemura, Kunihiro Katsuragi, Yukiko Minamiyama, Hiromu Tanaka, Kazuhiro Hirohashi, Osaka City University Medical School, Osaka Japan; Shigeru Okada, Okayama University, Okayama Japan; Masayasu lnoue, Hiroaki Kinoshita, Osaka City University Medical School, Osaka Japan
INTRAHEPATIC AUGMENTED IFN-GAMMA EXPRESSION AND T CELL INFLUX PRECEEDE RECOVERY FROM ACUTE VIRAL HEPATITIS AND ACCOMPANY OCCULT LIFELONG VIRUS PERSISTENCE AFTER THE ADULT ONSET OF WOODCHUCK HEPATITIS VIRUS INFECTION. Paul D Hodgson, Thomas I Michalak, Memorial University of Newfoundland, St. John's, NF Canada
lschemia-reperfnsion injury is one of the major problems in liver transplantation. We recently reported that intraportal infusion of adenosine inhibited the activation of neutrophils, thereby suppressing reperfusion injury of canine liver (J Hepatobiliary Pancreat Snrg. 7: 78, 2000). Post-ischemic perfusion of a tissue decreases extracellular levels of adenosine. Since the lifetime of adenosine in the circulation is extremely short, a fairly large dose is required to obtain its pharmacological effect. However, high doses of adenosine elicit systemic hypotension. The present work demonstrates that combined use of low doses of adenosine and amrinone, a phosphodiesterase inhibitor, snongly inhibited reperfusion injury of the liverwithout elicitinghypotension. The portal vein branches supplying the lateral, medial left and medial right lobes and the hepatic artery were occluded to induce 73% isehemia of the rat liver. After 45 rain ischemia, the reperfused liver was infused with adenosine (1 or 10 tzmol/kg/min) and amrinone (0.05 ftmol/kg/min) for 60 rain. Sham operated animals were infused with the same volume (1 m//hr) of saline. After reperfusion for 60 min, blood samples were obtained from the aorta. After centrifugation at 3000 g for 10 rain, plasma was obtained. Plasma and liver specimens were immediately frozen in liquid nitrogen and stored at -80°C until used. Some pieces of the liver were fixed with 10% formalin, cut into thin sections, and stained by hematoxylin-eosin. After 45 rain ischemia fol|owed by 60 rain reperfusion of rat liver, low doses of adenosine and amrinone were administrated intraportally, resulting in significantly increased hepatic levels of cGMP, cAMP, nitrite plus nitrate in plasma, and decreased alanine aminotransferase and lactate dehydrogenase in plasma without changing hemodynamics. After 60 rain of reperfusion, necrosis as weI1as ballooning of hepatocytes was apparent particularly around the midzonal area of hepatic Iobules. Congestion of hepatic sinusoids and many infdtrated cells were also apparent in the reperfnsed liver. Low dose of either adenosine or amrinone was found only congestion but not necrosis. However, the combined use of adenosine and amrinone markedly protected hepatocytes from reperfusion injury. Intraportal administration of low doses of adenosine and amrinone increased the cyclic nucleotides, thereby improving microcirculation and attenuating reperfusion injury of the liver. Thus, the combined administration of low doses of adenosine and amrinone may have therapeutic potential for the prevention of reperfusion injury of the liver without causing hemodynamic changes.
Woodchucks infected with woodchuck hepatitis virus (WHV) demonstrate profiles of liver disease and age dependent rates of progression to chronic hepatitis comparable to those seen in human hepatitis B. The mechanism of recovery from acute hepadnaviral infection or its evolution to chronicity remains unknown, although the liver local immune cell and cytokine responses are expected to play an important role. To determine intrahepatic dynamics of cytokine expression and T cell involvement, and to assess their value in prediction of the outcome of acute hepatitis, we evaluated levels of liver transcription of interferon gamma (1FN'y), tumor necrosis factor alpha (TNFc¢) and interleukins (IL) 2, 4 and 6, and the T cell influx in serial liver biopsies collected during the lifespan of woodchucks with experimental acute WHV infection who either ceased hepatitis or developed chronic liver disease. Our results show that recovery from acute viral hepatitis in adulthood is preceded by a significantly greater hepatic expression of IFN'y and CD3, an increased TNF~ transcription, lower hepatic WHV load and by a greater degree of liver inflammation than those seen in adult acute infection with chronic hepatitis outcome. Furthermore, we have learned that the elevated liver IFN% TNFc~ and CD3 expression endures for years not only in the animals with evident chronic hepatitis but also, although to a lesser extent, in those which apparently completely resolved acute infection. This is consistent with our previous findings that residual, serologically silent WHV replication and remnant liver inflammation continue for life after recovery from acute infection in adult woodchucks (Hepatology, 1999;29:928-938). The current study provides evidence that antiviral cytokines, in particular IFNy, may play a central role in the long-term control of occult hepadnavirus persistence in the liver.
Changes in hepatic cyclic nucleotides,serum Nox, and ALT after reperfusion Sham
Control
Adenosine Amdnone (0.05 Adenosine ~mol/kg.!mio) + amrinone
_(lt~mottkglmin) cAMP (pmoltg) 618 -+ 11 198 -+ 32 cGMP (pmoltg) 8.03 -+ 0.12 4,79-+0.43 NOF + NO3- (IJM) 8.0±0.5 6,2-+1,8 ALT(IUIL) 54 ± 10 1832 -+ 144
329 -+ 55 6.02±0,84 4.3-+1.2 162'~ -+ 135
302 -+ 29 5,47-+0,30 7.9_+1,4 "1784_+163
468 _+27 7.36-+0.86 15.5 ~ 2,8 1010 -+ 62
AASLD ABSTRACTS
305A
531
532
ESTABLISHMENT OF HTERT-IMMORTALIZED HUMAN HEPATOCYTES AS A SOURCE FOR A HYBRID BIOARTIFICIAL LIVER. Toshinori Totsugawa, Okayama University Graduate School of Medicine and Dentistry, Okayama Japan; Naoya Kobayashi, Hirofumi Noguchi, Takamasa Watanabe, Toshihisa Matsumura, Masanobu Maruyama, Tomoko Matsumoto, Noriaki Tanaka, Okayama University Grad, OkayamaJapan; Karen A Westerman, Harvard-Massachusetts Institute of Technology, Boston, MA; Philippe Leboulch, Harvard-Massachusetts I, Boston, MA
ADENOVIRUS-MEDIATED P 2 I GENE TRANSFER ENHANCES DIFFERENTIATED HEPATIC PHENOTYPES OF IMMORTALIZED HUMAN HEPATOCYTES. Naoya Kobayashi, Hirofumi Noguchi, Toshinori Totsugawa, Takamasa Watanabe, Toshihisa Matsumura, Masanobu Maruyama, Tomoko Matsumoto, Toshiyoshi Fujiwara, Noriaki Tanaka, Masakiyo Sakaguchi, Okayama University Graduate School of Medicine and Dentistry, Okayama ]apan
Purpose: The shortage of donor livers for hepatocyte isolation is one of the major limiting factors for developing bioartificial liver (BAL) therapy. The present study describes a novel strategy for constructing a human hepatocyte cell line as a source in BAL. Methods: Adult human hepatocytes were trarlsduced with a retroviral vector SSR#197 encoding the genes of telomerase reverse transcriptase (hTERT) and positive selectable marker, green fluorescent protein (GFP), which were flanked by a pair of recombination targets of loxP. After the transduction of hepatocytes with SSR#197, ceils were prepared with FACS to obtain GFP-positive populations. Expression of differentiated liver functions and efficacy of CreAoxP recombination mediated by an adenoviral vector producing Cre (AxCANCre) were evaluated in SSR#197-transduced hepatocytes. Results: TTNT-1, one of SSR#197-immortalized human hepatocytes, grew steadily in tissue culture and demonstrated morphological characteristics of liver parenchymal cells. The cells showed hepatocyte-specific gene expression. When transplanted into immunodeficient mice, TTNT-1 cells were not mrmorgenic. Efficient Cre/loxP recombination in TTNT-1 cells was performed following AxCANCre infection. Subsequent GFP-negative cell sorting allowed reversible immortalization system in human hepatocytes. Differentiated hepatic functions were enhanced in reverted TTNT-1 cells. Conclusion: We have developed an innovative method of reversible immortalization of human hepatocytes. This work represents a potentially novel strategy for addressing the problem of donor liver shortage that currently limits the use of human hepatocytes for BAL therapy.
Purpose: The p21 molecule, a potent cyclin-dependent kinase inhibitor, regulates the transition from the G1 phase to the S phase of the cell cycle. Overexpression of p21 has been shown to induce differentiation in various cell lines. Previously we have established an immortalized human hepatocyte cell line NKNT-3 to develop a bioartificial liver (BAL) (N.Kobayashi et al., Science 1252; 1246-1262, 2000). The findings prompted us to investigate the effect of p21 on hepatic phenotypes in NKNT-3 cells. Methods: NKNT-3 ceils, simian virus 40 T antigen-immortalized human hepatocytes, were used in the present study. A replication-deficient adenovirus vector encoding a p21 gene under the control of the CMV promoter (Ad5CMVp21) was utilized to achieve efficient gene transfer into NKNT-3 cells. Expression of p-450 associated enzymes, cell-cycle progression, and morphology were analyzed in NKNT-3 cells infected with Ad5CMVp21. Results: Efficient adenoviral gene transfer of p21 into NKNT-3 cells were confirmed by immunohistochemistry and Western blotting analysis. Overexpression of p21 predominantly arrested the cell cycle at the G1 checkpoint in NKNT-3 cells, resulting in differentiated hepatic phenotvpes including a decreased nuclear-to cytoplasmic ratio, a decreased cell density, increased glycogen synthesis, and enhanced expression of CYP3A4 and 2C9. Conclusion: We demonstrate that p21 gene transfer augments the differentiated hepatic phenotypes in NKNT-3 ceils. The strategy would be useful to increase hepatic functions of immortalized human hepatocytes in BAL therapy.
533
534
COMBINED USE OF ADENOSINE AND PHOSPHODIESTERASE INHIBITOR INHIBITS REPERFUSION INJURY OF RAT LIVER. Shigekazu Takemura, Kunihiro Katsuragi, Yukiko Minamiyama, Hiromu Tanaka, Kazuhiro Hirohashi, Osaka City University Medical School, Osaka Japan; Shigeru Okada, Okayama University, Okayama Japan; Masayasu lnoue, Hiroaki Kinoshita, Osaka City University Medical School, Osaka Japan
INTRAHEPATIC AUGMENTED IFN-GAMMA EXPRESSION AND T CELL INFLUX PRECEEDE RECOVERY FROM ACUTE VIRAL HEPATITIS AND ACCOMPANY OCCULT LIFELONG VIRUS PERSISTENCE AFTER THE ADULT ONSET OF WOODCHUCK HEPATITIS VIRUS INFECTION. Paul D Hodgson, Thomas I Michalak, Memorial University of Newfoundland, St. John's, NF Canada
lschemia-reperfnsion injury is one of the major problems in liver transplantation. We recently reported that intraportal infusion of adenosine inhibited the activation of neutrophils, thereby suppressing reperfusion injury of canine liver (J Hepatobiliary Pancreat Snrg. 7: 78, 2000). Post-ischemic perfusion of a tissue decreases extracellular levels of adenosine. Since the lifetime of adenosine in the circulation is extremely short, a fairly large dose is required to obtain its pharmacological effect. However, high doses of adenosine elicit systemic hypotension. The present work demonstrates that combined use of low doses of adenosine and amrinone, a phosphodiesterase inhibitor, snongly inhibited reperfusion injury of the liverwithout elicitinghypotension. The portal vein branches supplying the lateral, medial left and medial right lobes and the hepatic artery were occluded to induce 73% isehemia of the rat liver. After 45 rain ischemia, the reperfused liver was infused with adenosine (1 or 10 tzmol/kg/min) and amrinone (0.05 ftmol/kg/min) for 60 rain. Sham operated animals were infused with the same volume (1 m//hr) of saline. After reperfusion for 60 min, blood samples were obtained from the aorta. After centrifugation at 3000 g for 10 rain, plasma was obtained. Plasma and liver specimens were immediately frozen in liquid nitrogen and stored at -80°C until used. Some pieces of the liver were fixed with 10% formalin, cut into thin sections, and stained by hematoxylin-eosin. After 45 rain ischemia fol|owed by 60 rain reperfusion of rat liver, low doses of adenosine and amrinone were administrated intraportally, resulting in significantly increased hepatic levels of cGMP, cAMP, nitrite plus nitrate in plasma, and decreased alanine aminotransferase and lactate dehydrogenase in plasma without changing hemodynamics. After 60 rain of reperfusion, necrosis as weI1as ballooning of hepatocytes was apparent particularly around the midzonal area of hepatic Iobules. Congestion of hepatic sinusoids and many infdtrated cells were also apparent in the reperfnsed liver. Low dose of either adenosine or amrinone was found only congestion but not necrosis. However, the combined use of adenosine and amrinone markedly protected hepatocytes from reperfusion injury. Intraportal administration of low doses of adenosine and amrinone increased the cyclic nucleotides, thereby improving microcirculation and attenuating reperfusion injury of the liver. Thus, the combined administration of low doses of adenosine and amrinone may have therapeutic potential for the prevention of reperfusion injury of the liver without causing hemodynamic changes.
Woodchucks infected with woodchuck hepatitis virus (WHV) demonstrate profiles of liver disease and age dependent rates of progression to chronic hepatitis comparable to those seen in human hepatitis B. The mechanism of recovery from acute hepadnaviral infection or its evolution to chronicity remains unknown, although the liver local immune cell and cytokine responses are expected to play an important role. To determine intrahepatic dynamics of cytokine expression and T cell involvement, and to assess their value in prediction of the outcome of acute hepatitis, we evaluated levels of liver transcription of interferon gamma (1FN'y), tumor necrosis factor alpha (TNFc¢) and interleukins (IL) 2, 4 and 6, and the T cell influx in serial liver biopsies collected during the lifespan of woodchucks with experimental acute WHV infection who either ceased hepatitis or developed chronic liver disease. Our results show that recovery from acute viral hepatitis in adulthood is preceded by a significantly greater hepatic expression of IFN'y and CD3, an increased TNF~ transcription, lower hepatic WHV load and by a greater degree of liver inflammation than those seen in adult acute infection with chronic hepatitis outcome. Furthermore, we have learned that the elevated liver IFN% TNFc~ and CD3 expression endures for years not only in the animals with evident chronic hepatitis but also, although to a lesser extent, in those which apparently completely resolved acute infection. This is consistent with our previous findings that residual, serologically silent WHV replication and remnant liver inflammation continue for life after recovery from acute infection in adult woodchucks (Hepatology, 1999;29:928-938). The current study provides evidence that antiviral cytokines, in particular IFNy, may play a central role in the long-term control of occult hepadnavirus persistence in the liver.
Changes in hepatic cyclic nucleotides,serum Nox, and ALT after reperfusion Sham
Control
Adenosine Amdnone (0.05 Adenosine ~mol/kg.!mio) + amrinone
_(lt~mottkglmin) cAMP (pmoltg) 618 -+ 11 198 -+ 32 cGMP (pmoltg) 8.03 -+ 0.12 4,79-+0.43 NOF + NO3- (IJM) 8.0±0.5 6,2-+1,8 ALT(IUIL) 54 ± 10 1832 -+ 144
329 -+ 55 6.02±0,84 4.3-+1.2 162'~ -+ 135
302 -+ 29 5,47-+0,30 7.9_+1,4 "1784_+163
468 _+27 7.36-+0.86 15.5 ~ 2,8 1010 -+ 62