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Neutrophil-derived reactive oxygen species (ROS) induce lipid peroxidation and collagen synthesis in human ITO cells
$74
Posters 2
[ P2 C6/193 ]
[ P2C6/195 [
NEUTROPHIL-DERIVED REACTIVE OXYGEN SPECIES (ROS) INDUCE LIPID PEROXIDATION AND
THE MAGNITUDE OF THE REGENERATIVE RESPONSE AFTER PARTIAL HEPATECTOMY 18 REGULATED IN MID GI
COLLAGEN SYNTHESIS IN HUMAN ITO CELLS A.._~.Casini, E__~.Ceni, R__~.Salzano, M___:Parola, M___~Romano, . C.__~. Surrenti. Dept. of Clinical Pathophysiology, Gastroenterology
L. ~.ambotte, A, ~aliez, J.P. Gona. B. Li. Laboratop/ of Experimental Surgery, University of Louvain, 1200 Brussels, Belgium. The magnitude of the regenerative response after a partial hepatectomy (PH) is exponentially related to the size of the resection by a mechanism which is quite unexplored. In an attempt to determine if this regulation is obtained in the eady phase by "priming" an adequate number of cells or later in the cycle, male Wistar rats were submitted to a 1/3rd PH (removal of the left lateral lobe) which was converted, at s later time, into a 2/3rd PH by removing the median lobe (as control, a sham operation was performed at an equivalent time). When this conversion is performed 10 hours after the initial 1/3rd PH, the H3-thymidine uptake into the DNA has the same magnitude at the 24th and 28th hours as after a classical 2/3rd PH at the same times (315 +_ 27 and 176 _+ 14 versus 333 +_ 18 and 245 +_ 88 DPM/pg DNA), markedly greater than after ll3rd PH, unmodified or submitted to a sham at 10 hours (41 + 3 and 67 + 20 at the 24th hour). When the conversion is made at the 14th hour, the response is smaller and ~ppears only at the 28th hour. 1o explain the unexpected short time needed to reach the peak thymidine uptake after the conversion at 10 hours (14 instead of 24 hours) it is proposed that the effect of the second reduction of liver mass acts on cells which are not only "primed" but have already accomplished various preparative steps through G1. The initial stimulus of a l/3rd PH seems thus to induce a regenerative response in an excessive number of cells which will be submitted to some form of negative control or selection in mid G1 in order to ,roduce a response appropriate to the reduction of the liver mass ~resent at that time.
Unit; University of Florence, Florence; and Dept. of Experimental Medicine, University of Turin, Turin, Italy.
Alcoholic hepatitis is characterized b~, liver neutrophil infiltration followed by excessive matrtx deposition. Ito cells (also known as hepatic fat-storing cells, FSC) are nonparenchymal liver cells of the Disse space that play a major role in the development of alcohohc liver fibrosis. Aim of this study was to evaluate if neutrophil-derived ROS may induce fipid peroxidation and collagen synthesis by human FSC in a co-culture system. Neutrophils were isolated from peripheral blood of normal donors and stimulated in vitro by N-Forpayl-Met-Leu-Phenylalanine to produce ROS (26 nmol/10 ~ cells, by cyt. C oxidation) in a co-culture system with human FSC. Exposure of FSC to ROS resulted in a early induction of lipid peroxidation, as indicated by malonildialdehyde (MDA) production (4.6 nmol/mg protein) and formation of fluorescent protein adduct with MDA or 4-hydroxynonenal (HNE). MDA and HNE formation was associated with a significant increase of procollagen al(I) mRNA expression in FSC cultures (4fold after 3 hs, by Northern blotting) paralleled by an increase (3-fold) of collagen type I accumulation in the culture medium after 24 hs, by an ELISA method. In conclusion, these results indicate that neutrophil-derived ROS and the consequent lipid peroxidation phenomena may contribute to the excessive deposition of extracellular matrix that occurs in the liver after alcoholic hepatitis.
[ P2 C6/194 I CONTINUOUS VS INTERMITTENT ANOXlA/REOXYGENATION IN HEPATOCYTES:EFFECTON OXYGENFREE RADICALSFORMATIONAND CYTOTOXlClTY. A. Gasbarrini,W. A. Colantoni^. P. Caraceni^. F. Trevisani^. M. Masetti'. A. Facchini#. M. Bernardi^. PatologiaMedica I^, Laboratoriodi Immunologiae Genetics#(IOR) ClinicaChrurgicaI1",Universita'di Bologna; The temporary clamping of the hepatic pedicle according to Pringle (Pringle manouvre) is the most used technique to control intraoperatory bleeding during hepatoresection. However, liver damage due to ischemia/reperfusionis a main problem with prolongedclamping. Intermittent clamping has been proposedas a method to reduceorgan injury. Aim of this study was to assess oxygenfree radicalsformation(OFR)and cell injury during conhnuous or intermitent anoxia/reoxygenationin rat hepatocytes. After isolation, the cells were cast in agarosegel threadsand continuouslyperfused with oxigenated (95%02-5%C02) Krebs-Henseleit bicarbonate buffer. Hepatocyteswereexposedto 2 h continuousor intermittent(4 periodsof 30 rain of anoxia followed by 10 min of reoxygenation)anoxia obtained by 95%N25%C02 pedusionand to a 1 h reoxygenationperiod.Cell injury was evaluated by LDH release;OFR by enhanced-chemiluminescenca:lucigeninand luminol were utilized to enhance anion superoxide (02-) and hydrogen peroxide (H202) formalJon,respectively.After 2 h of anoxia,OFR formationwas reduced to barely measurablelevel in beth groups. The increasein LDH releasewas significantely greater in hepatocytes exposed to continuous compared to intermittentanoxia:580_+60vs 310-+40% (p<0.01);H202:83_+12vs 38_+10 nA (p<0.01). During the early phase of reoxy~enation,LDH release parallely increased significantly in both groups but Jt was higher in cells exposedtocontinuousanoxia:900-~_40vs 550-~-65%(p<0.01).In both groupsthe lucigeninchemiluminescencepeak, expressionof 02 formation,occurred 10-15 m in beforemaximumLDH release. These data suggestthat: 1) OFR productionseemsto be the causeand not the consequenceof reoxygenationinjury since LDH release is temporally delayed with respect to 02- formation, ; 2) intermittent oxygen depnvation reduces liver cell injury and oxygen free radicals formation determinedby anoxia/reoxygenation;3) intermittentrather than continuous Pringlemanouvre may be preferablyused tin liver resection.
P2 C6/196 I E F F E C T O F I S C H E M I A ON I C A M - I E X P R E S S I O N BY R A T H E F A T O C Y T E S . AN IN VIVO I M M U N O H I S T O C H E M I C A L STUDY. C RodL J-Y Scoazec. J Bei=ltifi. G Feldmarm. INSERM U327, Facolt~ de Mbi~cine Xavier Bichat, Paris. and Hfpiud Beaujon. Clichy. France. ICAM-I is an inducible adhesion molecule mediating leukocyte interactions with endothelial and epithelial cells during inflammation and immune reactions. In heart and kidney, warm ischemia up-regulates ICAM-1 expression on parenchymal cells, through a cytokine-dopendent mechanism. In these organs, ICAM-I induction is delayed for 24 to 48 hours afrer the end of warm ischemia. In the liver, the situation might be different, because of the presence of Kupffer cells, whose activation during ischemia might result in the local releese of cytokines. We therefore investigated by immunohistochemistry the effect of warm and cold ischemia on the expression of ICAM-I by rat hepatocytes. Warm ischemia (WI) was obtained by complete exclusion of the left part of the liver for periods of 10, 20, 30 and 45 minutes. Animals were sacrificed: (a) immediately at the end of WI without reflow, (b) immediately after reflow or after liver reperf~sion for periods of 4, 24, 48, or 72 hours. Livers from sham-operated animals mind samples from the right part of the liver served as controls. For comparison purposes, in some animals, WI of the right kidney was performed simultaneously to liver ischemia. Cold ischemia was obtained by immersing rat liver pieces in UW-solution for 0 to 24 hours at +4°C. At least 3 animals were included in each experimental group. In control animals, ICAM-1 was detected only on sinusoidal endothelial cells. D u r i n g W l without renow, the expression of ICAM-I depended on the duration of ischemia. No induction was detexted after 10 minutes. ICAM-1 was detected on a few hepatocytes after 20 minutes, on large clusters of hepatocytes after 30 minutes and was diffusely induced after 45 minutes. In kidneys simultaneously submitted to WI, no ICAM1 induction was detected in any case. After WI followed by reperfusion, the pattern of 1CAM-I induction depended on the duration of WI. After 10 or 20 minutes of WI followed by reperfusion. ICAM-1 expression was detectehle on a significant number of hepatucytes only after 48 hours of reperfusion. After 30 or 45 minutes of WI followed by reperfusion, ICAM-1 expression was retained by hepatocytes after raf]ow and at 4 hours, was low or undeteclable at 24 hours, a~d was secondarily induced after 48 hours. During cold lschemla, ICAM-I was detectable on clusters of hepatucytes after 6 to 8 hours of preservation at +40C. In conclusion, our study shows that: (a) ischemia of the liver, both warm or cold, might induce ICAM-I expression by rat hepatocytes, (b) in conlrast to other organs, induction of ICAM-I in the liver might occur very early after prolonged W1, likely as a result of Kupffer cell activation. This might be of physiopathological relevance in surgical and grafting procedures.
Posters 2
[ P2 C6/193 ]
[ P2C6/195 [
NEUTROPHIL-DERIVED REACTIVE OXYGEN SPECIES (ROS) INDUCE LIPID PEROXIDATION AND
THE MAGNITUDE OF THE REGENERATIVE RESPONSE AFTER PARTIAL HEPATECTOMY 18 REGULATED IN MID GI
COLLAGEN SYNTHESIS IN HUMAN ITO CELLS A.._~.Casini, E__~.Ceni, R__~.Salzano, M___:Parola, M___~Romano, . C.__~. Surrenti. Dept. of Clinical Pathophysiology, Gastroenterology
L. ~.ambotte, A, ~aliez, J.P. Gona. B. Li. Laboratop/ of Experimental Surgery, University of Louvain, 1200 Brussels, Belgium. The magnitude of the regenerative response after a partial hepatectomy (PH) is exponentially related to the size of the resection by a mechanism which is quite unexplored. In an attempt to determine if this regulation is obtained in the eady phase by "priming" an adequate number of cells or later in the cycle, male Wistar rats were submitted to a 1/3rd PH (removal of the left lateral lobe) which was converted, at s later time, into a 2/3rd PH by removing the median lobe (as control, a sham operation was performed at an equivalent time). When this conversion is performed 10 hours after the initial 1/3rd PH, the H3-thymidine uptake into the DNA has the same magnitude at the 24th and 28th hours as after a classical 2/3rd PH at the same times (315 +_ 27 and 176 _+ 14 versus 333 +_ 18 and 245 +_ 88 DPM/pg DNA), markedly greater than after ll3rd PH, unmodified or submitted to a sham at 10 hours (41 + 3 and 67 + 20 at the 24th hour). When the conversion is made at the 14th hour, the response is smaller and ~ppears only at the 28th hour. 1o explain the unexpected short time needed to reach the peak thymidine uptake after the conversion at 10 hours (14 instead of 24 hours) it is proposed that the effect of the second reduction of liver mass acts on cells which are not only "primed" but have already accomplished various preparative steps through G1. The initial stimulus of a l/3rd PH seems thus to induce a regenerative response in an excessive number of cells which will be submitted to some form of negative control or selection in mid G1 in order to ,roduce a response appropriate to the reduction of the liver mass ~resent at that time.
Unit; University of Florence, Florence; and Dept. of Experimental Medicine, University of Turin, Turin, Italy.
Alcoholic hepatitis is characterized b~, liver neutrophil infiltration followed by excessive matrtx deposition. Ito cells (also known as hepatic fat-storing cells, FSC) are nonparenchymal liver cells of the Disse space that play a major role in the development of alcohohc liver fibrosis. Aim of this study was to evaluate if neutrophil-derived ROS may induce fipid peroxidation and collagen synthesis by human FSC in a co-culture system. Neutrophils were isolated from peripheral blood of normal donors and stimulated in vitro by N-Forpayl-Met-Leu-Phenylalanine to produce ROS (26 nmol/10 ~ cells, by cyt. C oxidation) in a co-culture system with human FSC. Exposure of FSC to ROS resulted in a early induction of lipid peroxidation, as indicated by malonildialdehyde (MDA) production (4.6 nmol/mg protein) and formation of fluorescent protein adduct with MDA or 4-hydroxynonenal (HNE). MDA and HNE formation was associated with a significant increase of procollagen al(I) mRNA expression in FSC cultures (4fold after 3 hs, by Northern blotting) paralleled by an increase (3-fold) of collagen type I accumulation in the culture medium after 24 hs, by an ELISA method. In conclusion, these results indicate that neutrophil-derived ROS and the consequent lipid peroxidation phenomena may contribute to the excessive deposition of extracellular matrix that occurs in the liver after alcoholic hepatitis.
[ P2 C6/194 I CONTINUOUS VS INTERMITTENT ANOXlA/REOXYGENATION IN HEPATOCYTES:EFFECTON OXYGENFREE RADICALSFORMATIONAND CYTOTOXlClTY. A. Gasbarrini,W. A. Colantoni^. P. Caraceni^. F. Trevisani^. M. Masetti'. A. Facchini#. M. Bernardi^. PatologiaMedica I^, Laboratoriodi Immunologiae Genetics#(IOR) ClinicaChrurgicaI1",Universita'di Bologna; The temporary clamping of the hepatic pedicle according to Pringle (Pringle manouvre) is the most used technique to control intraoperatory bleeding during hepatoresection. However, liver damage due to ischemia/reperfusionis a main problem with prolongedclamping. Intermittent clamping has been proposedas a method to reduceorgan injury. Aim of this study was to assess oxygenfree radicalsformation(OFR)and cell injury during conhnuous or intermitent anoxia/reoxygenationin rat hepatocytes. After isolation, the cells were cast in agarosegel threadsand continuouslyperfused with oxigenated (95%02-5%C02) Krebs-Henseleit bicarbonate buffer. Hepatocyteswereexposedto 2 h continuousor intermittent(4 periodsof 30 rain of anoxia followed by 10 min of reoxygenation)anoxia obtained by 95%N25%C02 pedusionand to a 1 h reoxygenationperiod.Cell injury was evaluated by LDH release;OFR by enhanced-chemiluminescenca:lucigeninand luminol were utilized to enhance anion superoxide (02-) and hydrogen peroxide (H202) formalJon,respectively.After 2 h of anoxia,OFR formationwas reduced to barely measurablelevel in beth groups. The increasein LDH releasewas significantely greater in hepatocytes exposed to continuous compared to intermittentanoxia:580_+60vs 310-+40% (p
P2 C6/196 I E F F E C T O F I S C H E M I A ON I C A M - I E X P R E S S I O N BY R A T H E F A T O C Y T E S . AN IN VIVO I M M U N O H I S T O C H E M I C A L STUDY. C RodL J-Y Scoazec. J Bei=ltifi. G Feldmarm. INSERM U327, Facolt~ de Mbi~cine Xavier Bichat, Paris. and Hfpiud Beaujon. Clichy. France. ICAM-I is an inducible adhesion molecule mediating leukocyte interactions with endothelial and epithelial cells during inflammation and immune reactions. In heart and kidney, warm ischemia up-regulates ICAM-1 expression on parenchymal cells, through a cytokine-dopendent mechanism. In these organs, ICAM-I induction is delayed for 24 to 48 hours afrer the end of warm ischemia. In the liver, the situation might be different, because of the presence of Kupffer cells, whose activation during ischemia might result in the local releese of cytokines. We therefore investigated by immunohistochemistry the effect of warm and cold ischemia on the expression of ICAM-I by rat hepatocytes. Warm ischemia (WI) was obtained by complete exclusion of the left part of the liver for periods of 10, 20, 30 and 45 minutes. Animals were sacrificed: (a) immediately at the end of WI without reflow, (b) immediately after reflow or after liver reperf~sion for periods of 4, 24, 48, or 72 hours. Livers from sham-operated animals mind samples from the right part of the liver served as controls. For comparison purposes, in some animals, WI of the right kidney was performed simultaneously to liver ischemia. Cold ischemia was obtained by immersing rat liver pieces in UW-solution for 0 to 24 hours at +4°C. At least 3 animals were included in each experimental group. In control animals, ICAM-1 was detected only on sinusoidal endothelial cells. D u r i n g W l without renow, the expression of ICAM-I depended on the duration of ischemia. No induction was detexted after 10 minutes. ICAM-1 was detected on a few hepatocytes after 20 minutes, on large clusters of hepatocytes after 30 minutes and was diffusely induced after 45 minutes. In kidneys simultaneously submitted to WI, no ICAM1 induction was detected in any case. After WI followed by reperfusion, the pattern of 1CAM-I induction depended on the duration of WI. After 10 or 20 minutes of WI followed by reperfusion. ICAM-1 expression was detectehle on a significant number of hepatucytes only after 48 hours of reperfusion. After 30 or 45 minutes of WI followed by reperfusion, ICAM-1 expression was retained by hepatocytes after raf]ow and at 4 hours, was low or undeteclable at 24 hours, a~d was secondarily induced after 48 hours. During cold lschemla, ICAM-I was detectable on clusters of hepatucytes after 6 to 8 hours of preservation at +40C. In conclusion, our study shows that: (a) ischemia of the liver, both warm or cold, might induce ICAM-I expression by rat hepatocytes, (b) in conlrast to other organs, induction of ICAM-I in the liver might occur very early after prolonged W1, likely as a result of Kupffer cell activation. This might be of physiopathological relevance in surgical and grafting procedures.