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Loss of endothelial cell ecto-ATP diphosphohydrolase activity: Mechanism of abnormal thromboregulation in hepatic preservation injury
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437
AASLD ABSTRACTS LOSS OF ENDOTHELIAL CELL ECTO-ATP DIPHOSPHOHYDROLASE ACTIVITY: MECHANISM OF ABNORMAL THROMBOREGULATIONIN HEPATIC PRESERVATION INJURY. ~;CRobson,D Candinasand FH Bach Divisionof OrganTransplantation,SandozCenter for Immunobiology,Departmentof Surgery,New England DeaconessHospital,Boston.MA.
Platelet aggregation and microvascular damage are important components of hepatic preservation injury. Initiation and progression of platelet aggregation, the local prothrombotic enviroment and associated leukocyte infiltrate are all promoted by the inflammatory mediator ADP. A critical regulatory element in this process may be the expression on the surface of quiescent vascular endothelial cells (EC) of an ecto-ATP diphosphohydrolase that degrades both ATP and ADP. We examined whether the activation of human aortic endothelial cells (HAEC) following exposure to inflammatory mediators, such as TNF alpha or reactive oxygen intermediates (ROD, could modulate the activity of this ectoenzyme as such an effect could contribute to microvascular injury in graft reperfusion injury. HAEC associated ecto-ATP diphosphohydrolase activity was determined by catabolism of 14C-ADP as calculated by thin layer chromatography (TLC), by determination of phosphate release from extracellular supplemental ATP and ADP and by the inhibition of platelet aggregation responses to ADP. Activation of the HAEC by incubation with TNF (5-20ng/mL) which induces oxidative stress, or ROI directly [HOOH (5-10raM) or xanthine (200mM)/xanthine oxidase (100mU/mL) combinations] resulted in significant and rapid reduction of ecto-ATP diphosphohydrolase activity with loss of the anti-aggregatory phenotype of the HAEC. Inhibition and loss of ecto-ATP diphosphohydrolase activity could be prevented by exogenous antioxidants viz. superoxide dismutase Cu-Zn form (330U/mL) and catalase (1,000U/mL) combinations. In conclusion, ecto-ATP diphosphohydrolase activity, a major mechanism for regulating platelet activation, inflammation and thrombosis is inhibited upon exposure to oxidative stress. Hence, stable EC expression of ROI resistant forms of ecto-ATP diphosphohydrolases may be important in abrogating pathological platelet activation associated with preservation injury and potentially other vascular disorders of the liver. This research was funded in part by Sandoz Pharma, Basle. Switzerland.
439
INACTIVATION OF KUPFFER CELLS MINIMIZES GRAFT INJURY AND LIPID HYDROPEROXIDE FORMATION AFTER FATTY LIVER TRANSPLANTATION FROM ETHANOL-TREATED RATS. Z. Zhong~ H. Cannot. W. Ou. R.F. Stachlewit~ G.E. Arteel. J.J. Lemasters. W. Gun and R.G. Thurman Laboratory of Hepatobiology and Toxicology, Department of Pharmacology, University of North Carolina at Chapel Hill Fatty livers fail after transplantation, and we investigated the role of Kupffer cells in graft injury to fatty livers transplanted from ethanoltreated rats. Donor rats were given ethanol (5 g/kg, p.o.) 20 h before explantation, and liver grafts were preserved in UW cold storage solution for 24-42 h prior to implantation. Blood samples were taken from the inferior veaa eava for 3 h after implantation, and liver surface 0 2 tension was measured with a Clark-type 0 2 electrode. Serum AST levels increased gradually from <100 U/L to about 600 U/L in control rats, while values reached about 2200 U/L in ethanol-treated rats, reflecting reperfusion injury. Gadolinium chloride (GdCi3, 20 mg/kg, i.e.), a selective inartivator of Kupffer cells, minimized AST release to values around 1000 U/L in rats receiving fatty livers from ethanol-treated rats. Serum lipid hydroperoxides, which arise from the effects of free radicals on membranes, were increased about 4-fold in ethanol-treated rats, an effect blocked largely by GdCI3. Further, alcohol treatment decreased surface 0 2 tension by about 50% and elevated portal pressure by 23%, indicating disturbances to the hepatic microcireulation. These effects were also reversed by GdCI 3. Adherent white blood cells in grafts were collected by infusion of calcium-free buffer 30 min after implantation. Ethanol treatment increased white blood cell adhesion by about 2-fold, and this process was also blocked by GdCI 3. Survival was 88% in controls, yet values decreased to 33% in fatty grafts from the ethanoltreated group. GdCI 3 significantly improved survival to 75%. Taken together, these data indicate that reperfusion injury after implantation is greater in grafts from fatty livers due to alcohol exposure. Inactivation of Kupffer cells diminished graft damage, most likely by minimizing reperfusion injury and decreasing lipid peroxidntinn (AA-09156).
HEPATOLOGY O c t o b e r 1995
438 ICAM-1 IS ELEVATED DRAMATICALLY IN FATTY LIVER GRAFTS FROM ALCOHOL-TREATED RATS. M.v. Frankmbem. H.K Boies. Y. limuro. R. Sehoonhoven*. J.J. Lemasters# and R.G. Thurman. Dcpts. of Pharmacology, Environmental Sciences and Engineering* and Cell Biology and Anatomy#, Univ. of North Carolina at Chapel Hill, Chapel Hill, NC 27599, U.S.A. Introduction: Up to 28% of potential organ donors have fatty livers mainly due to alcohol, leading to more primary nonfunetion and initial poor fun~ion. While reasons for early graft failure are not yet totally clear, we recently observed massive white cell and platelet sticking shortly after transplantation. To investigate mechanisms of this phenomenon and a possible link with increased failure of fatty grafts, we studied expression of Intraeellular Adhesion Moleenie-1 (ICAM-1) immanohistochemieally. Methods: Male, 200-250g Lewis mrs were used for all studies. Rats were either given a single dose of 5g/kg ethanol i,g. (acute model), fed a liquid high-fat diet containing ethanol (Lieber-DeCarli)or given a liquid ethanol diet continuously via gastric cannula for 12 weeks (Tsukamoto-French). Controls were given saline or highfat diet without ethanol. Transplanted livers were stored for various times in UW at 2-4 °C, harvested 24 hours after transplantation and stained immanohistochemieally with a monoclonal ICAM-I antibody. Results: ICAM-I was detected in rats exposed to alcohol for 12 weeks (Table 1). After transplantation, strong ICAM-I expression in sinusoidal lining cells was seen in fatty liver grafts from alcohol-treated rats stored for 24 hours (Table 2). In controls, ICAM-! expression was much weaker and could not be detected in livers with cold storage times of less than 6 hours. Table 1: Untrans flanted livers Table 2: Transnlanted Lieber-DeCarli livers Acute Lieber- TsukamotoStorage time Ethanol Controls model ! DeCarli French 1 hr + 6 hr 24 hr -H+ Conclusions: ICAM-1 is induced by continuous, long-term alcohol exposure. Alcoholic fatty liver grafts stored for 24 hours express much more ICAM-1 after transplantation than controls (AA-09156).
440 PREDOMINANCE OF IL-4 mRNA EXPRESSION EARLY AFTER
EXPERIMENTAL LIVER TRANSPLANTATION (OLT) NR Krieger, A Yamaguchi. P Huie, RK Sibley. DC Dafoe, El Alfrey. Depts of Surgery and Pathology, Stanford University, Stanford CA.
In the early postoperative period following experimental OLT, lymphocytic cellular infiltrates are present in grafts that will become tolerant. The role of these cells is unclear. The cytokine profile within these allografts in the initial phase following OLT has been previously investigated using PCR, which does not localize message to specific ceils. We have recently reported upregulation of both Thl and Th2 cytokine profiles in spontaneously tolerant rats >100 days after OLT (Lewis, RT11, into Wistar Furth, RT1u) .in hepatocytes and not graft infiltrating cells. To define the kinetics and localization of the Th type cytokine profile early after OLT in these tolerant animals, we examined the cytokine mRNA expression at weekly intervals in allografted tissue compared to naive controls, using non-isotopic in situ hybridization which localizes mRNA to specific cells within tissue. Since Th2 clones produce IL-4 and Thl clones produce IFN-T, the ratio IL-4/IFN-y (Th2/Thl) was assessed to determine whether a specific Th type profile predominated at any time point after OLT. Positive cells/mm 2 tissue were counted. The ratio IL-4/IFN-Tis recorded + SEM at 1, 2, 3 and 6 weeks (wks) after OLT. control (n=3) 1 wk (n=t) 2 wks (n=2) 3 wks (n=2) 6 wks (n=2) 0.6+0.2 15.3 17.5+5.7" 4.5±2.5 1.1+0.5 *p<0.05, ANOVA, 2 wks vs control These data demonstrate that after OLT, the ratio IL-4/IFN-'/peaks at two weeks, gradually decreasing by 6 weeks. Cytokine mRNA expression was localized to portal inflammatory cells, in contrast to our previous experiments (>100 days after OLT) where expression appeared in the hepatocytes and not graft infiltrating cells. These data suggest that the transition of cytokine expression from graft infiltrating cells to hepatocytes occurs after six weeks following OLT. In conclusion, a predominance of IL-4 (Th2) cytoklne mRNA expression occurs in portal inflammatory ceils early after experimental OLT.
437
AASLD ABSTRACTS LOSS OF ENDOTHELIAL CELL ECTO-ATP DIPHOSPHOHYDROLASE ACTIVITY: MECHANISM OF ABNORMAL THROMBOREGULATIONIN HEPATIC PRESERVATION INJURY. ~;CRobson,D Candinasand FH Bach Divisionof OrganTransplantation,SandozCenter for Immunobiology,Departmentof Surgery,New England DeaconessHospital,Boston.MA.
Platelet aggregation and microvascular damage are important components of hepatic preservation injury. Initiation and progression of platelet aggregation, the local prothrombotic enviroment and associated leukocyte infiltrate are all promoted by the inflammatory mediator ADP. A critical regulatory element in this process may be the expression on the surface of quiescent vascular endothelial cells (EC) of an ecto-ATP diphosphohydrolase that degrades both ATP and ADP. We examined whether the activation of human aortic endothelial cells (HAEC) following exposure to inflammatory mediators, such as TNF alpha or reactive oxygen intermediates (ROD, could modulate the activity of this ectoenzyme as such an effect could contribute to microvascular injury in graft reperfusion injury. HAEC associated ecto-ATP diphosphohydrolase activity was determined by catabolism of 14C-ADP as calculated by thin layer chromatography (TLC), by determination of phosphate release from extracellular supplemental ATP and ADP and by the inhibition of platelet aggregation responses to ADP. Activation of the HAEC by incubation with TNF (5-20ng/mL) which induces oxidative stress, or ROI directly [HOOH (5-10raM) or xanthine (200mM)/xanthine oxidase (100mU/mL) combinations] resulted in significant and rapid reduction of ecto-ATP diphosphohydrolase activity with loss of the anti-aggregatory phenotype of the HAEC. Inhibition and loss of ecto-ATP diphosphohydrolase activity could be prevented by exogenous antioxidants viz. superoxide dismutase Cu-Zn form (330U/mL) and catalase (1,000U/mL) combinations. In conclusion, ecto-ATP diphosphohydrolase activity, a major mechanism for regulating platelet activation, inflammation and thrombosis is inhibited upon exposure to oxidative stress. Hence, stable EC expression of ROI resistant forms of ecto-ATP diphosphohydrolases may be important in abrogating pathological platelet activation associated with preservation injury and potentially other vascular disorders of the liver. This research was funded in part by Sandoz Pharma, Basle. Switzerland.
439
INACTIVATION OF KUPFFER CELLS MINIMIZES GRAFT INJURY AND LIPID HYDROPEROXIDE FORMATION AFTER FATTY LIVER TRANSPLANTATION FROM ETHANOL-TREATED RATS. Z. Zhong~ H. Cannot. W. Ou. R.F. Stachlewit~ G.E. Arteel. J.J. Lemasters. W. Gun and R.G. Thurman Laboratory of Hepatobiology and Toxicology, Department of Pharmacology, University of North Carolina at Chapel Hill Fatty livers fail after transplantation, and we investigated the role of Kupffer cells in graft injury to fatty livers transplanted from ethanoltreated rats. Donor rats were given ethanol (5 g/kg, p.o.) 20 h before explantation, and liver grafts were preserved in UW cold storage solution for 24-42 h prior to implantation. Blood samples were taken from the inferior veaa eava for 3 h after implantation, and liver surface 0 2 tension was measured with a Clark-type 0 2 electrode. Serum AST levels increased gradually from <100 U/L to about 600 U/L in control rats, while values reached about 2200 U/L in ethanol-treated rats, reflecting reperfusion injury. Gadolinium chloride (GdCi3, 20 mg/kg, i.e.), a selective inartivator of Kupffer cells, minimized AST release to values around 1000 U/L in rats receiving fatty livers from ethanol-treated rats. Serum lipid hydroperoxides, which arise from the effects of free radicals on membranes, were increased about 4-fold in ethanol-treated rats, an effect blocked largely by GdCI3. Further, alcohol treatment decreased surface 0 2 tension by about 50% and elevated portal pressure by 23%, indicating disturbances to the hepatic microcireulation. These effects were also reversed by GdCI 3. Adherent white blood cells in grafts were collected by infusion of calcium-free buffer 30 min after implantation. Ethanol treatment increased white blood cell adhesion by about 2-fold, and this process was also blocked by GdCI 3. Survival was 88% in controls, yet values decreased to 33% in fatty grafts from the ethanoltreated group. GdCI 3 significantly improved survival to 75%. Taken together, these data indicate that reperfusion injury after implantation is greater in grafts from fatty livers due to alcohol exposure. Inactivation of Kupffer cells diminished graft damage, most likely by minimizing reperfusion injury and decreasing lipid peroxidntinn (AA-09156).
HEPATOLOGY O c t o b e r 1995
438 ICAM-1 IS ELEVATED DRAMATICALLY IN FATTY LIVER GRAFTS FROM ALCOHOL-TREATED RATS. M.v. Frankmbem. H.K Boies. Y. limuro. R. Sehoonhoven*. J.J. Lemasters# and R.G. Thurman. Dcpts. of Pharmacology, Environmental Sciences and Engineering* and Cell Biology and Anatomy#, Univ. of North Carolina at Chapel Hill, Chapel Hill, NC 27599, U.S.A. Introduction: Up to 28% of potential organ donors have fatty livers mainly due to alcohol, leading to more primary nonfunetion and initial poor fun~ion. While reasons for early graft failure are not yet totally clear, we recently observed massive white cell and platelet sticking shortly after transplantation. To investigate mechanisms of this phenomenon and a possible link with increased failure of fatty grafts, we studied expression of Intraeellular Adhesion Moleenie-1 (ICAM-1) immanohistochemieally. Methods: Male, 200-250g Lewis mrs were used for all studies. Rats were either given a single dose of 5g/kg ethanol i,g. (acute model), fed a liquid high-fat diet containing ethanol (Lieber-DeCarli)or given a liquid ethanol diet continuously via gastric cannula for 12 weeks (Tsukamoto-French). Controls were given saline or highfat diet without ethanol. Transplanted livers were stored for various times in UW at 2-4 °C, harvested 24 hours after transplantation and stained immanohistochemieally with a monoclonal ICAM-I antibody. Results: ICAM-I was detected in rats exposed to alcohol for 12 weeks (Table 1). After transplantation, strong ICAM-I expression in sinusoidal lining cells was seen in fatty liver grafts from alcohol-treated rats stored for 24 hours (Table 2). In controls, ICAM-! expression was much weaker and could not be detected in livers with cold storage times of less than 6 hours. Table 1: Untrans flanted livers Table 2: Transnlanted Lieber-DeCarli livers Acute Lieber- TsukamotoStorage time Ethanol Controls model ! DeCarli French 1 hr + 6 hr 24 hr -H+ Conclusions: ICAM-1 is induced by continuous, long-term alcohol exposure. Alcoholic fatty liver grafts stored for 24 hours express much more ICAM-1 after transplantation than controls (AA-09156).
440 PREDOMINANCE OF IL-4 mRNA EXPRESSION EARLY AFTER
EXPERIMENTAL LIVER TRANSPLANTATION (OLT) NR Krieger, A Yamaguchi. P Huie, RK Sibley. DC Dafoe, El Alfrey. Depts of Surgery and Pathology, Stanford University, Stanford CA.
In the early postoperative period following experimental OLT, lymphocytic cellular infiltrates are present in grafts that will become tolerant. The role of these cells is unclear. The cytokine profile within these allografts in the initial phase following OLT has been previously investigated using PCR, which does not localize message to specific ceils. We have recently reported upregulation of both Thl and Th2 cytokine profiles in spontaneously tolerant rats >100 days after OLT (Lewis, RT11, into Wistar Furth, RT1u) .in hepatocytes and not graft infiltrating cells. To define the kinetics and localization of the Th type cytokine profile early after OLT in these tolerant animals, we examined the cytokine mRNA expression at weekly intervals in allografted tissue compared to naive controls, using non-isotopic in situ hybridization which localizes mRNA to specific cells within tissue. Since Th2 clones produce IL-4 and Thl clones produce IFN-T, the ratio IL-4/IFN-y (Th2/Thl) was assessed to determine whether a specific Th type profile predominated at any time point after OLT. Positive cells/mm 2 tissue were counted. The ratio IL-4/IFN-Tis recorded + SEM at 1, 2, 3 and 6 weeks (wks) after OLT. control (n=3) 1 wk (n=t) 2 wks (n=2) 3 wks (n=2) 6 wks (n=2) 0.6+0.2 15.3 17.5+5.7" 4.5±2.5 1.1+0.5 *p<0.05, ANOVA, 2 wks vs control These data demonstrate that after OLT, the ratio IL-4/IFN-'/peaks at two weeks, gradually decreasing by 6 weeks. Cytokine mRNA expression was localized to portal inflammatory cells, in contrast to our previous experiments (>100 days after OLT) where expression appeared in the hepatocytes and not graft infiltrating cells. These data suggest that the transition of cytokine expression from graft infiltrating cells to hepatocytes occurs after six weeks following OLT. In conclusion, a predominance of IL-4 (Th2) cytoklne mRNA expression occurs in portal inflammatory ceils early after experimental OLT.