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The effect of melatonin on dimethylnitrosamine-induced liver fibrosis in rats
394A
AASLD ABSTRACTS
HEPATOLOGYOctober 2001
887
888
INCREASED SECRETION OF UROKINASE-TYPE PLASMINOGEN ACTIVATOR BY MALONDIALDEHYDE-ACETALDEIgYDE-PROTEIN ADDUCTS IN HEPATIC STELLATE CELLS. Kusum K Kharbanda, Kris A Shu-
INCREASED HEPATIC FIBROSIS IN VERY YOUNG MICE IS ASSOCIATED W I T H INDUCTION OF LIVER NATURAL KILLER CELLS. Cather-
bert, Michael F Sorrell, Dean J Tuma, Department of Veterans Affairs, Nebraska/Western Iowa Health Care System, Omaha, NE Hepatic stellate cells (HSCs) play a central role in the pathogenesis of alcoholinduced fibrosis. Following hepatic injury, HSCs become activated and participate in matrix remodeling through synthesis of matrix metalloproteinases, tissue inhibitors of metafloproteinases, cytokines and components of the plasminogen-activating systems. Studies from our laboratory have shown that malondialdehyde-acetaldehyde-protein adducts (MAA adducts) are formed in the livers of ethanol-fed rats and induce the secretion of two chemokines, MCP-1 and MIP-2, and increase the expression of ICAM-1 in HSCs. Our data also show that MAA adducts activate PKC-a in HSCs. In macrophages, a classical PKC activator, 4]3-phorbo112jS-myristate 13-acetate, has been shown to upregulate a key component of the plasminogen activating system, urokinase-type plasminogen activator (uPA). Because both human as well as rat HSCs have been shown to produce uPA, in this study we investigated the effect of MAA adducts on uPA secretion by these cells. HSCs, isolated from male Wistar rats, in passages 2-6 were exposed to BSA-MAA, and the secretion of uPA, a key component of plasmin-generating system, was quantified by caseinplasminogen zymography. Densitometric analysis revealed a 2.2-fold increase (p < 0.05) in uPA secretion by HSCs treated with BSA-MAA as compared to unmodified BSA. We also observed that calphostin C, a PKC inhibitor, was able to significantly abrogate the BSA-MAA-induceduPA secretion by 90%, but had no effect on the constitutive secretion ofuPA. These results indicated that PKC activation is necessary for this enhanced secretion. We further showed a timedependent effect of MAA adduct-induced uPA secretion by HSCs. Casein zymographs revealed that uPA level becomes detectable in BSA-MAA treated samples at 3 h of exposure, whereas, it is still undetected in control samples. Thereafter, there is a steady increase in uPA with peak levels being reached at 12-16 h of exposure to BSA-MAA. A dose-dependent effect of BSA-MAA (062.5 ~g/ml) on uPA secretion by HSCs was also observed. Taken together, our results indicate that MAA adducts, via a PKC-mediated pathway, may regulate plasmin-mediated matrix degradation by HSCs in the liver, thereby contributing to the progression of hepatic fibrosis.
ine Choy Smith, Gregory D Sempowski, Don C Roekey, Duke University Medical Center, Durham, NC The liver responds to injury by a wound heafing response typified by inflammation, cytokine production, and fibrogenesis. Previous studies indicate that the fibrogenic response varies with age, although the mechanism underlying this phenomenon is unknown. We postulated divergent immune-related responses to be responsible for age-related differences in fibrosis. Methods: Liver injury was induced in six-week (very young) and nine-month-old (adult) male mice by weekly carbon tetrachloride (CC14) (1.5 ml/kg) gavage. Liver fibrosis was quantitated by morphometric analysis. Hepatic lymphocyte populations, isolated by Percoll gradient centrifugation, were phenotyped by four-color flow cytometry. Results: Young livers displayed quantitatively greater fibrosis than adult livers after injury (2.1-+0.2 vs. 1.3-+0.1 percent total liver area, young vs. adult; p < 0.01). Despite differing levels of fibrosis, acute injury, assessed by measurement of serum alanine aminotransferase (ALT) levels after one dose and six doses of CC14,was identical. The fraction of liver natural killer (NK) cells and CD3+ T lymphocytes were equal in adult and young control animals. However, after liver injury, lymphocyte profiles varied substantially with age. In young animals, liver fibrosis was associated with induction of NK cells (22 ±2 vs. 36 -+3 percent, control vs. injured; p < 0.005) and reduction of CD3+ T lymphocytes (48 -+4 vs. 29 ±3 percent, control vs. injured; p < 0.005). In contrast, in adult injured mouse livers, there was no induction of NK cells or reduction of CD3 + T lymphocytes. Conclusions: These data elucidate experimentally induced, age-associated, differences in the fibrogenic response to liver injury in very young and adult mice. This response is associated with a preferential shift in liver lymphocyte populations, which leads us to speculate that the exaggerated hepatic fibrogenic response is the result of recruitment or activation of liver NK ceils, known to produce cytokines known to be important in the fibrogenic response to injury.
889
890
REGULATION OF ADAMS FAMILY MEMBERS IN HEPATIC STELLATE CELLS BY SOLUBLE GRO~/VTH FACTORS: EVIDENCE FOR THEIR ROLE IN HEPATIC FIBROGENESIS. Maria J Lorite, Debbie J Bevitt, Jo Benitez,
THE EFFECT OF MELATONIN ON DIMETI:IYLNITROSAMINE-INDUCED LIVER FIBROSIS IN RATS. Veysel Tahan, Resat Ozaras, Hafize
Norman McKie, Christopher P Day, Univ of Newcastle upon Tyne, Newcastle upon Tyne Uk We have previously reported that a new group of matrix metafloproteinase (MMP)-related enzymes known as ADAMs (A Disintegrin And a Metalloproteinase) are present in hepatic stellate cells (HSC). 1 We have cloned several ADAM family members from primary HSC whose properties suggest their potential role in fibrogenesis including: ADAM10 (a type IV collagenase in vitro), ADAM17 (a TNFa convertase) and ADAMTS-1 and TS-5 (proteoglycanases capable of releasing bound growth factors from the extracellular matrix [ECM]). We have also shown that, in contrast to "normal" MMPs, ADAMs activities are largely insensitive to inhibition by tissue inhibitors of metalloproteinases (TIMPs) which is important in view of the secretion of TIMPs by HSCs during fibrogenesis. To seek further evidence of a role for ADAM proteins in fibrogenesis we have now examined the regulation of ADAM10, ADAM17 and ADAMTS 1 mRNA expression by soluble factors with established profibrogenic (PDGF, TGF]3) or antifibrogenic (IL-10) effects. For comparison the effects of these factors on MMP2 and TIMP1 were also determined. HSCs were isolated from male rats by pronase/collagenase digestion/density gradient centrifugation. Experiments were performed on 2 week old passage 1 cells. Growth factors were present for 1 or 24h after 24h in serum free medium. mRNA was quantified by Northern blotting using labelled PCR products from our previous cloning experiments. PDGF up-regulated the expression of ADAMTS-1, ADAM10, ADAM17 and TIMP-1 in a dose-dependent manner and down-regulated MMP2 expression. IL-10 down-regulated the expression of ADAMTS-1 and ADAM10 and had no effect on ADAM17. TGF~8 had no consistent effect on any ADAM. These data support a role for ADAM proteins in liver fibrogenesis potentially either by (i) releasing TGF]3 from decorin in the ECM (ADAMTS-1); (ii) Degrading type IV collagen and altering HSC-ECM interactions (ADAM10); and/or (iii) activating TNFa (ADAM 17). 1Hepatology 2000;32:188A
Uzun, Seval Aydin, Safiye Dondurmaci, GuLsen Ozbay, Hakan Senturk, Cerrahpasa Medical Faculty, Istanbul Turkey Increased deposition of the extracellular matrix components, particularly colhgen is a central phenomenon in liver fbrosis. Stellate cells, in the space of Disse are responsible for collagen hypersecretion. Stellate ceils are shown to be activated by free radical damage, as well as by malondialdehyde (MDA) to stimulate the synthesis of collagen and this activation has been shown to be blocked by antioxidants. Melatonin represents the most potent physiological scavenger of hydroxyl radicals found to date, Also it has been shown to be involved in the inhibitory regulation of collagen content in tissues. At present, no effective treatment of liver fibrosis is available for clinical use. We aimed to use melatonin in experimental model of dimethylnitrosamine (DMN)-induced liver fibrosis in rats. Male Wistar Albino rats, 3.5-4 months old, were divided into four groups. Melatonin (in 1% ethanol, 100 mg/kg/day, sc) was given in DMN + mdatonin and Melatonin groups for 14 day. A single injection of DMN (in saline, 40 mg/kg, intraperitoneal) was given to DMN and DMN + melatonin groups whereas Normal-control group rats were given vehicle alone. While a single administration of DMN caused hepatic fibrotie changes in all DMN group rats (14/14), these fibrotic changes were suppressed in liver histopathology of 5/14 DMN+melatonin group rats (p<0.05). Hepatic fibrosis was also quantified by the measurement of tissue levels of hydroxyproline. The mean hydroxyproline level of DMN group was significantly higher than those of DMN+melatonin (p<0.00001), normal-control and Melatonin groups (p=0.0003 for both). But mean hydroxyproline level of DMN+melatonin group was still higher than both control groups (p=0.0003 for both). The mean plasma and liver MDA levels of DMN group were significantly higher than those of DMN+melatonin (p<0.00001 for both)', and also normal-control and Melatonin groups (p=0.0002), (p =0.0003). The mean hepatic superoxide dismutase (SOD) levelof DMN group was significantly higher than those of DMN+melatonin (p<0.00001), normal-control and Melatonin groups (p=0.0003 for both), while the mean plasma SOD levels of groups were not statistically significant. The mean plasma and liver reduced glutathione levels in DMN group were significantly higher than those in DMN+melatonin (p<0.00001), normal-control and Melatonin groups (p=0.0002 for both). The mean erythrocyte glutathione peroxidase and plasma alkaline phosphatase levels in DMN group were significantly higher than those in DMN+melatonin (p<0.00001), (p=0.0008); normal-control (p=0.0005), (p=0.001); and Melatonin groups (p=0.0005), (p=0.0022) respectively. Melatonin was used first time for inhibition of fibrosis in the liver in this study. These findings suggest that melatonin functions as a potent fibrosuppressant by inhibiting lipid peroxidation in hepatocytes and stellate cells in vivo.
AASLD ABSTRACTS
HEPATOLOGYOctober 2001
887
888
INCREASED SECRETION OF UROKINASE-TYPE PLASMINOGEN ACTIVATOR BY MALONDIALDEHYDE-ACETALDEIgYDE-PROTEIN ADDUCTS IN HEPATIC STELLATE CELLS. Kusum K Kharbanda, Kris A Shu-
INCREASED HEPATIC FIBROSIS IN VERY YOUNG MICE IS ASSOCIATED W I T H INDUCTION OF LIVER NATURAL KILLER CELLS. Cather-
bert, Michael F Sorrell, Dean J Tuma, Department of Veterans Affairs, Nebraska/Western Iowa Health Care System, Omaha, NE Hepatic stellate cells (HSCs) play a central role in the pathogenesis of alcoholinduced fibrosis. Following hepatic injury, HSCs become activated and participate in matrix remodeling through synthesis of matrix metalloproteinases, tissue inhibitors of metafloproteinases, cytokines and components of the plasminogen-activating systems. Studies from our laboratory have shown that malondialdehyde-acetaldehyde-protein adducts (MAA adducts) are formed in the livers of ethanol-fed rats and induce the secretion of two chemokines, MCP-1 and MIP-2, and increase the expression of ICAM-1 in HSCs. Our data also show that MAA adducts activate PKC-a in HSCs. In macrophages, a classical PKC activator, 4]3-phorbo112jS-myristate 13-acetate, has been shown to upregulate a key component of the plasminogen activating system, urokinase-type plasminogen activator (uPA). Because both human as well as rat HSCs have been shown to produce uPA, in this study we investigated the effect of MAA adducts on uPA secretion by these cells. HSCs, isolated from male Wistar rats, in passages 2-6 were exposed to BSA-MAA, and the secretion of uPA, a key component of plasmin-generating system, was quantified by caseinplasminogen zymography. Densitometric analysis revealed a 2.2-fold increase (p < 0.05) in uPA secretion by HSCs treated with BSA-MAA as compared to unmodified BSA. We also observed that calphostin C, a PKC inhibitor, was able to significantly abrogate the BSA-MAA-induceduPA secretion by 90%, but had no effect on the constitutive secretion ofuPA. These results indicated that PKC activation is necessary for this enhanced secretion. We further showed a timedependent effect of MAA adduct-induced uPA secretion by HSCs. Casein zymographs revealed that uPA level becomes detectable in BSA-MAA treated samples at 3 h of exposure, whereas, it is still undetected in control samples. Thereafter, there is a steady increase in uPA with peak levels being reached at 12-16 h of exposure to BSA-MAA. A dose-dependent effect of BSA-MAA (062.5 ~g/ml) on uPA secretion by HSCs was also observed. Taken together, our results indicate that MAA adducts, via a PKC-mediated pathway, may regulate plasmin-mediated matrix degradation by HSCs in the liver, thereby contributing to the progression of hepatic fibrosis.
ine Choy Smith, Gregory D Sempowski, Don C Roekey, Duke University Medical Center, Durham, NC The liver responds to injury by a wound heafing response typified by inflammation, cytokine production, and fibrogenesis. Previous studies indicate that the fibrogenic response varies with age, although the mechanism underlying this phenomenon is unknown. We postulated divergent immune-related responses to be responsible for age-related differences in fibrosis. Methods: Liver injury was induced in six-week (very young) and nine-month-old (adult) male mice by weekly carbon tetrachloride (CC14) (1.5 ml/kg) gavage. Liver fibrosis was quantitated by morphometric analysis. Hepatic lymphocyte populations, isolated by Percoll gradient centrifugation, were phenotyped by four-color flow cytometry. Results: Young livers displayed quantitatively greater fibrosis than adult livers after injury (2.1-+0.2 vs. 1.3-+0.1 percent total liver area, young vs. adult; p < 0.01). Despite differing levels of fibrosis, acute injury, assessed by measurement of serum alanine aminotransferase (ALT) levels after one dose and six doses of CC14,was identical. The fraction of liver natural killer (NK) cells and CD3+ T lymphocytes were equal in adult and young control animals. However, after liver injury, lymphocyte profiles varied substantially with age. In young animals, liver fibrosis was associated with induction of NK cells (22 ±2 vs. 36 -+3 percent, control vs. injured; p < 0.005) and reduction of CD3+ T lymphocytes (48 -+4 vs. 29 ±3 percent, control vs. injured; p < 0.005). In contrast, in adult injured mouse livers, there was no induction of NK cells or reduction of CD3 + T lymphocytes. Conclusions: These data elucidate experimentally induced, age-associated, differences in the fibrogenic response to liver injury in very young and adult mice. This response is associated with a preferential shift in liver lymphocyte populations, which leads us to speculate that the exaggerated hepatic fibrogenic response is the result of recruitment or activation of liver NK ceils, known to produce cytokines known to be important in the fibrogenic response to injury.
889
890
REGULATION OF ADAMS FAMILY MEMBERS IN HEPATIC STELLATE CELLS BY SOLUBLE GRO~/VTH FACTORS: EVIDENCE FOR THEIR ROLE IN HEPATIC FIBROGENESIS. Maria J Lorite, Debbie J Bevitt, Jo Benitez,
THE EFFECT OF MELATONIN ON DIMETI:IYLNITROSAMINE-INDUCED LIVER FIBROSIS IN RATS. Veysel Tahan, Resat Ozaras, Hafize
Norman McKie, Christopher P Day, Univ of Newcastle upon Tyne, Newcastle upon Tyne Uk We have previously reported that a new group of matrix metafloproteinase (MMP)-related enzymes known as ADAMs (A Disintegrin And a Metalloproteinase) are present in hepatic stellate cells (HSC). 1 We have cloned several ADAM family members from primary HSC whose properties suggest their potential role in fibrogenesis including: ADAM10 (a type IV collagenase in vitro), ADAM17 (a TNFa convertase) and ADAMTS-1 and TS-5 (proteoglycanases capable of releasing bound growth factors from the extracellular matrix [ECM]). We have also shown that, in contrast to "normal" MMPs, ADAMs activities are largely insensitive to inhibition by tissue inhibitors of metalloproteinases (TIMPs) which is important in view of the secretion of TIMPs by HSCs during fibrogenesis. To seek further evidence of a role for ADAM proteins in fibrogenesis we have now examined the regulation of ADAM10, ADAM17 and ADAMTS 1 mRNA expression by soluble factors with established profibrogenic (PDGF, TGF]3) or antifibrogenic (IL-10) effects. For comparison the effects of these factors on MMP2 and TIMP1 were also determined. HSCs were isolated from male rats by pronase/collagenase digestion/density gradient centrifugation. Experiments were performed on 2 week old passage 1 cells. Growth factors were present for 1 or 24h after 24h in serum free medium. mRNA was quantified by Northern blotting using labelled PCR products from our previous cloning experiments. PDGF up-regulated the expression of ADAMTS-1, ADAM10, ADAM17 and TIMP-1 in a dose-dependent manner and down-regulated MMP2 expression. IL-10 down-regulated the expression of ADAMTS-1 and ADAM10 and had no effect on ADAM17. TGF~8 had no consistent effect on any ADAM. These data support a role for ADAM proteins in liver fibrogenesis potentially either by (i) releasing TGF]3 from decorin in the ECM (ADAMTS-1); (ii) Degrading type IV collagen and altering HSC-ECM interactions (ADAM10); and/or (iii) activating TNFa (ADAM 17). 1Hepatology 2000;32:188A
Uzun, Seval Aydin, Safiye Dondurmaci, GuLsen Ozbay, Hakan Senturk, Cerrahpasa Medical Faculty, Istanbul Turkey Increased deposition of the extracellular matrix components, particularly colhgen is a central phenomenon in liver fbrosis. Stellate cells, in the space of Disse are responsible for collagen hypersecretion. Stellate ceils are shown to be activated by free radical damage, as well as by malondialdehyde (MDA) to stimulate the synthesis of collagen and this activation has been shown to be blocked by antioxidants. Melatonin represents the most potent physiological scavenger of hydroxyl radicals found to date, Also it has been shown to be involved in the inhibitory regulation of collagen content in tissues. At present, no effective treatment of liver fibrosis is available for clinical use. We aimed to use melatonin in experimental model of dimethylnitrosamine (DMN)-induced liver fibrosis in rats. Male Wistar Albino rats, 3.5-4 months old, were divided into four groups. Melatonin (in 1% ethanol, 100 mg/kg/day, sc) was given in DMN + mdatonin and Melatonin groups for 14 day. A single injection of DMN (in saline, 40 mg/kg, intraperitoneal) was given to DMN and DMN + melatonin groups whereas Normal-control group rats were given vehicle alone. While a single administration of DMN caused hepatic fibrotie changes in all DMN group rats (14/14), these fibrotic changes were suppressed in liver histopathology of 5/14 DMN+melatonin group rats (p<0.05). Hepatic fibrosis was also quantified by the measurement of tissue levels of hydroxyproline. The mean hydroxyproline level of DMN group was significantly higher than those of DMN+melatonin (p<0.00001), normal-control and Melatonin groups (p=0.0003 for both). But mean hydroxyproline level of DMN+melatonin group was still higher than both control groups (p=0.0003 for both). The mean plasma and liver MDA levels of DMN group were significantly higher than those of DMN+melatonin (p<0.00001 for both)', and also normal-control and Melatonin groups (p=0.0002), (p =0.0003). The mean hepatic superoxide dismutase (SOD) levelof DMN group was significantly higher than those of DMN+melatonin (p<0.00001), normal-control and Melatonin groups (p=0.0003 for both), while the mean plasma SOD levels of groups were not statistically significant. The mean plasma and liver reduced glutathione levels in DMN group were significantly higher than those in DMN+melatonin (p<0.00001), normal-control and Melatonin groups (p=0.0002 for both). The mean erythrocyte glutathione peroxidase and plasma alkaline phosphatase levels in DMN group were significantly higher than those in DMN+melatonin (p<0.00001), (p=0.0008); normal-control (p=0.0005), (p=0.001); and Melatonin groups (p=0.0005), (p=0.0022) respectively. Melatonin was used first time for inhibition of fibrosis in the liver in this study. These findings suggest that melatonin functions as a potent fibrosuppressant by inhibiting lipid peroxidation in hepatocytes and stellate cells in vivo.