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April 1995 AASLD A1209 SYMPTOMATIC PORTAL HYPERTENSION IN DYSKERATOSIS CONGENITA: MAYO CLINIC EXPERIENCE AND REVIEW OF THE LITERATURE. T,R. Zembower. P. Chareonthaitawee, P.S. Karnes, D.M. Nagorney, H.A. Carpenter, D.D. Douglas, R.L. Phyliky, J.B. Gross, Jr. Mayo Clinic & Foundation, Rochester MN. Dyskeratosis congenita (DC) is a rare hereditary disorder characterized by skin hyperpigmentation, mucous membrane leukoplakia, nail dystrophy, and bone marrow failure. Less common manifestations involve the bones, eyes, teeth, urinary tract, and GI tract. A recent case of DC in which portal hyper- tension was the predominant feature prompted us to review our experience and the literature on hepatic involvement in DC. METHODS: Computer search of all available on-line and secondary medical references. Review of all Mayo Clinic records of patients with DC. CASE REPORT: 27 y.o.WM with recurrent painless melena requiring multiple transfusions over 2 too. History of dysplastic deciduous teeth, pathologic fractures, anemia, hypoal- buminemia, diarrhea. Examination: underweight; sparse, gray hair; gray teeth; hyperpigmentation of neck and upper chest; hyperkeratosis of palms and soles; absence of fingerprints; dysplastic nails; firm liver; marked splenomegaly. Laboratory: Pancytopenia; hypoalbuminemia; normal liver function. Evaluation for chronic liver diseases negative. Endoscopy: multi- ple vascular lesions of stomach and colon; bxs=ectasias, edema, congestion, c/w portal hypertension. Liver Bx: portal fibrosis, portal vascular ectasia, no hepatitis or cirrhosis. Side-to-side portacaval shunt was performed. No bleeding x 3 too. One episode reversible hepatic encephalopathy. Loss of ascites and diarrhea; gain in weight. Follow-up: Died of urosepsis 3 mo af- ter surgery. REVIEW: Nine previous cases of DC seen. One had GI bleeding and bx-proven portal fibrosis; died at age 30 of streptococcal sepsis. A second had hepatosplenomegaly, not further investigated. 200 cases DC published, 8 with documented portal hypertension; both cases with liver biopsies showed portal fibrosis only. 20% of DC had some form of GI bleeding, several cases with multiple vascular lesions. Only previous case of portacaval shunt died 3 yr later from sepsis. CONCLUSIONS: 1) Idiopathic hepatic portal fibrosis is part of the syndrome of DC and may lead to clinically significant portal hypertension. 2) Portal hypertension should be suspected in any patient with DC who has multiple vascular lesions at endoscopy. 3) Portal decompression in DC is effective in relieving GI bleeding but may lead to an increased risk of sepsis or encephalopathy. e MODULATION OF MURINE HEPATIC LIPASE BY CYTOKINES. Ruijun Zh~n and Dennis Black. Dept. of Pediatrics, Univ. of Arkansas for Medical Sciences, Arkansas Children's Hospital, Little Rock, AR and Clinical Nutrition Research Unit, Univ. of Chicago, Chicago, IL Hepatic lipase (HL) is an enzyme produced by hepatocytes which resides on the liver sinusoidal endothelial cell. HL functions in the metabolism of high-density and triglyceride-rich lipoproteins, and hepatic uptake of chylomicron remnants. Both endogenous and exogenous reticuloendothelial system activation, including hepatic Kupffer cell activation, in the mouse in vivo results in a decrease in HL activity without a change in mRNA abundance (Biochem J 292:249, 1993). Purpose: To test the hypothesis that cytokines, specifically tumor necrosis faetor-a (TNF-¢), interleukin-1 (IL-1) and interleukin-6 (IL-6), produced by activated Kupffer cells, modulate HL activity, and to determine the mechanism of this regulation. Methods: Cultured routine hepatocytes were incubated with 1, 10, or 100 ng/ml of each cytokine for 24, 48, and 72 hrs. Culture medium HL activity was determined as ng free fatty acid hydrolyzed from 3H-triolein/well/hr. Culture medium HL mass was measured by ELISA, end secretion was expressed as % of control. HL immunoprecipitation was performed after zSS-methionine radiolabeling using an immunopurified goat anti-rat HL antibody, followed by SDS PAGE, autoradiography, and digital densitometry. Results: TNF-c( reduced HL activity in a dose-response manner with maximal reduction to 166 + 10 (mean + SEM), compared to a control value of 251 + 4, (p<0.Ol). IL-1 reduced activity to 178 +__ 12, compared to a control value of 254 +__ 2, (p<0.Ol). Maximal effect of both cytokines occurred at a concentration of 100 ng/ml at 48 hrs. TNF-c~ and IL-1 reduced HL secretion to 91.3 % (p<0.05) and 84.5% (p<0.005) of control values, respectively, under these conditions. The TNF-a and IL-1 effects were not additive. IL-6 had no effect on HL activity or secretion. A 19% reduction in radiolabeling of both HL bands (Mr 57 kDa matdre and Mr 55 kDa precursor) in cell homogenates occurred with TNF-(z treatment. Conclusions: TNF-c¢ and IL-1, but not IL-6, reduce cultured murine hepatocyte HL activity and protein secretion. TNF-c¢ reduced HL synthesis. Local secretion of these cytokines by activated Kupffer cells in vivo may contribute to the reduction in HL activity and disordered lipoprotein metabolism in acute and chronic inflammatory states. REGULATION OF MURINE HEPATOCYTE APOLIPOPROTEIN SECRETION BY CYTOKINES. Ruiiun Zhan and Dennis Black. Dept. of Pediatrics, Univ. of Arkansas for Medical Sciences, Arkansas Children's Hospital, Little Rock, AR and Clinical Nutrition Research Unit, Univ. of Chicago, Chicago, IL Apolipoproteins (apoLps) are surface peptides of lipoprotein particles. The liver is a major source of circulating lipoproteins and associated apoLps. Conditions associated with reticuloendothelial system activation, such as infection and autoimmune disease, result in disordered lipoprotein metabolism, associated with reduced circulating apo A-I and E levels. Apo A-I is a major component of high-density lipoproteins and is a cofactor for lecithin:cholesterol acyltransferase. Apo E is essential for the clearance of lipopretein remnants. Purpose: To test the hypothesis that cytokines, specifically tumor necrosis factor-c¢ (TNF-c 0 , interleukin-1 (IL-t) and interleukin-6 (IL-6), produced by activated hepatic Kupffer cells, modulate secretion of hepatocyte apo A-I and E. Methods: Cultured murine hepatocytes were incubated with 100 ng/ml of each cytokine for 48 hrs. Culture medium apoLp secretion was determined by ELISA using rabbit anti-rat apo E and anti-mouse apo A-I antibodies. Secretion was measured as Pg apoLp secreted/well/24 hrs, expressed as % of control. There were no differences in viable cell density per well among the experimental groups at the end of the incubation as determined by vital dye staining and cell counting. Results: TNF-ct, IL-1, and IL-6 treatment resulted in apo A-I secretion rates of 107 + 6% (mean + SEM) (pNS), 67 + 5% (p=0.003), and 67 + 9% (p=0.04), respectively, of control cultures. For apo E, secretion rates were 55 +_ 3% (p=0.006), 92 +_ 5% (pNS), and 49 + 4% (p=0.002), respectively, of control cultures. Conclusions: IL-1 and IL-6, but not TNF-c(, both reduce apo A-I secretion by cultured routine hepatocytes. TNF-c( and IL-6, but not IL- l, reduce apo E secretion. This regulation of hepatocyte apoLp secretion by cytokines produced locally by activated Kupffer cells may contribute to the abnormal lipoprotein metabolism observed in conditions characterized by reticuloendothelial activation. • ENDOTHELIN-I INDUCES LIVER VASO- CONSTRICTION THROUGH ET A RECEPTOR, B. Zhang, A.T. Dinh Xuan, P. Sogni, D. Houssin, Y. Calmus. H6pital Cochin, Paris, France. Endothelin- 1 (ET- 1) has potent vasoconstrictive effects on systemic and splanchnic circulations. Vascular smooth muscle cells express both ET A andET e receptors, both of them mediating the vasoactive effects of ET- 1. It has been recently shown that ET- 1 may increase the resfstance of the portal circulation. Using an isolated perfusion model of rat liver, we investigated the effect of ET-1 on the portal circulation to determine which endothelin receptor is mediating this effect. ET-1 induced a dose-dependent decrease of portal flow compared to the control group (2.21 _+0.27 ml.min- l.g foie- 1): ET- 1 at lxl0 -l~ and lxl0 -j° M had no significant effect (-0 and -9%, respectively, when compared to control group), while the portal f19w was significantly decreased (-55 % of control) following the perfusion of ET-1 at lxl0 9 M and was completely interrupted at lxlO 8 M. BQ 123 (2x10 6 M), an antagonist of ET A receptor, partially reversed (-25 % of control) the effect of lxl0 -9 M ET-I, while IRL 1038 (lxl0 -7 M), an antagonist ofET s receptor, did not influcence the effect of I x 10 -9 MET- 1 on the portal circulation (- 55 % of control). Sarafatoxin C (3x10 -6 M), an agonist of ET A receptor, significantly decreased the portal flow (-55 % of control). These results confirm that ET- 1 has potent vasoconstrictive effect on portal circulation and that this effect is mainly mediated through ET A receptor.

Regulation of murine hepatocyte apolipoprotein secretion by cytokines

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Page 1: Regulation of murine hepatocyte apolipoprotein secretion by cytokines

April 1 9 9 5 AASLD A 1 2 0 9

SYMPTOMATIC PORTAL HYPERTENSION IN DYSKERATOSIS CONGENITA: MAYO CLINIC EXPERIENCE AND REVIEW OF THE LITERATURE. T,R. Zembower. P. Chareonthaitawee, P.S. Karnes, D.M. Nagorney, H.A. Carpenter, D.D. Douglas, R.L. Phyliky, J.B. Gross, Jr. Mayo Clinic & Foundation, Rochester MN.

Dyskeratosis congenita (DC) is a rare hereditary disorder characterized by skin hyperpigmentation, mucous membrane leukoplakia, nail dystrophy, and bone marrow failure. Less common manifestations involve the bones, eyes, teeth, urinary tract, and GI tract. A recent case of DC in which portal hyper- tension was the predominant feature prompted us to review our experience and the literature on hepatic involvement in DC. METHODS: Computer search of all available on-line and secondary medical references. Review of all Mayo Clinic records of patients with DC. CASE REPORT: 27 y.o.WM with recurrent painless melena requiring multiple transfusions over 2 too. History of dysplastic deciduous teeth, pathologic fractures, anemia, hypoal- buminemia, diarrhea. Examination: underweight; sparse, gray hair; gray teeth; hyperpigmentation of neck and upper chest; hyperkeratosis of palms and soles; absence of fingerprints; dysplastic nails; firm liver; marked splenomegaly. Laboratory: Pancytopenia; hypoalbuminemia; normal liver function. Evaluation for chronic liver diseases negative. Endoscopy: multi- ple vascular lesions of stomach and colon; bxs=ectasias, edema, congestion, c/w portal hypertension. Liver Bx: portal fibrosis, portal vascular ectasia, no hepatitis or cirrhosis. Side-to-side portacaval shunt was performed. No bleeding x 3 too. One episode reversible hepatic encephalopathy. Loss of ascites and diarrhea; gain in weight. Follow-up: Died of urosepsis 3 mo af- ter surgery. REVIEW: Nine previous cases of DC seen. One had GI bleeding and bx-proven portal fibrosis; died at age 30 of streptococcal sepsis. A second had hepatosplenomegaly, not further investigated. 200 cases DC published, 8 with documented portal hypertension; both cases with liver biopsies showed portal fibrosis only. 20% of DC had some form of GI bleeding, several cases with multiple vascular lesions. Only previous case of portacaval shunt died 3 yr later from sepsis. C O N C L U S I O N S : 1) Idiopathic hepatic portal fibrosis is part of the syndrome of DC and may lead to clinically significant portal hypertension. 2) Portal hypertension should be suspected in any patient with DC who has multiple vascular lesions at endoscopy. 3) Portal decompression in DC is effective in relieving GI bleeding but may lead to an increased risk of sepsis or encephalopathy.

e MODULATION OF MURINE HEPATIC LIPASE BY CYTOKINES. Ruijun Zh~n and Dennis Black. Dept. of Pediatrics, Univ. of Arkansas for Medical Sciences, Arkansas Children's Hospital, Little Rock, AR and Clinical Nutrition Research Unit, Univ. of Chicago, Chicago, IL

Hepatic lipase (HL) is an enzyme produced by hepatocytes which resides on the liver sinusoidal endothelial cell. HL functions in the metabolism of high-density and triglyceride-rich lipoproteins, and hepatic uptake of chylomicron remnants. Both endogenous and exogenous reticuloendothelial system activation, including hepatic Kupffer cell activation, in the mouse in vivo results in a decrease in HL activity without a change in mRNA abundance (Biochem J 292:249, 1993). Purpose: To test the hypothesis that cytokines, specifically tumor necrosis faetor-a (TNF-¢), interleukin-1 (IL-1) and interleukin-6 (IL-6), produced by activated Kupffer cells, modulate HL activity, and to determine the mechanism of this regulation. Methods: Cultured routine hepatocytes were incubated with 1, 10, or 100 ng/ml of each cytokine for 24, 48, and 72 hrs. Culture medium HL activity was determined as ng free fatty acid hydrolyzed from 3H-triolein/well/hr. Culture medium HL mass was measured by ELISA, end secretion was expressed as % of control. HL immunoprecipitation was performed after zSS-methionine radiolabeling using an immunopurified goat anti-rat HL antibody, followed by SDS PAGE, autoradiography, and digital densitometry. Results: TNF-c( reduced HL activity in a dose-response manner with maximal reduction to 166 + 10 (mean + SEM), compared to a control value of 251 + 4, (p<0.Ol). IL-1 reduced activity to 178 +__ 12, compared to a control value of 254 +__ 2, (p<0.Ol). Maximal effect of both cytokines occurred at a concentration of 100 ng/ml at 48 hrs. TNF-c~ and IL-1 reduced HL secretion to 91.3 % (p<0.05) and 84.5% (p<0.005) o f control values, respectively, under these conditions. The TNF-a and IL-1 effects were not additive. IL-6 had no effect on HL activity or secretion. A 19% reduction in radiolabeling of both HL bands (Mr 57 kDa matdre and M r 55 kDa precursor) in cell homogenates occurred with TNF-(z treatment. Conclusions: TNF-c¢ and IL-1, but not IL-6, reduce cultured murine hepatocyte HL activity and protein secretion. TNF-c¢ reduced HL synthesis. Local secretion of these cytokines by activated Kupffer cells in vivo may contribute to the reduction in HL activity and disordered lipoprotein metabolism in acute and chronic inflammatory states.

• REGULATION OF MURINE HEPATOCYTE APOLIPOPROTEIN SECRETION BY CYTOKINES. Ruiiun Zhan and Dennis Black. Dept. of Pediatrics, Univ. of Arkansas for Medical Sciences, Arkansas Children's Hospital, Little Rock, AR and Clinical Nutrition Research Unit, Univ. of Chicago, Chicago, IL

Apolipoproteins (apoLps) are surface peptides of lipoprotein particles. The liver is a major source of circulating lipoproteins and associated apoLps. Conditions associated with reticuloendothelial system activation, such as infection and autoimmune disease, result in disordered lipoprotein metabolism, associated with reduced circulating apo A-I and E levels. Apo A-I is a major component of high-density lipoproteins and is a cofactor for lecithin:cholesterol acyltransferase. Apo E is essential for the clearance of lipopretein remnants. Purpose: To test the hypothesis that cytokines, specifically tumor necrosis factor-c¢ (TNF-c 0, interleukin-1 (IL-t) and interleukin-6 (IL-6), produced by activated hepatic Kupffer cells, modulate secretion of hepatocyte apo A-I and E. Methods: Cultured murine hepatocytes were incubated with 100 ng/ml of each cytokine for 48 hrs. Culture medium apoLp secretion was determined by ELISA using rabbit anti-rat apo E and anti-mouse apo A-I antibodies. Secretion was measured as Pg apoLp secreted/well/24 hrs, expressed as % of control. There were no differences in viable cell density per well among the experimental groups at the end of the incubation as determined by vital dye staining and cell counting. Results: TNF-ct, IL-1, and IL-6 treatment resulted in apo A-I secretion rates of 107 + 6% (mean + SEM) (pNS), 67 + 5% (p=0.003), and 67 + 9% (p=0.04), respectively, of control cultures. For apo E, secretion rates were 55 +_ 3% (p=0.006), 92 +_ 5% (pNS), and 49 + 4% (p=0.002), respectively, of control cultures. Conclusions: IL-1 and IL-6, but not TNF-c(, both reduce apo A-I secretion by cultured routine hepatocytes. TNF-c( and IL-6, but not IL- l, reduce apo E secretion. This regulation of hepatocyte apoLp secretion by cytokines produced locally by activated Kupffer cells may contribute to the abnormal lipoprotein metabolism observed in conditions characterized by reticuloendothelial activation.

• E N D O T H E L I N - I I N D U C E S L I V E R VASO- C O N S T R I C T I O N T H R O U G H ET A R E C E P T O R , B. Zhang, A.T. Dinh Xuan, P. Sogni, D. Houssin, Y. Calmus. H6pital Cochin, Paris, France.

Endothelin- 1 (ET- 1) has potent vasoconstrictive effects on systemic and splanchnic circulations. Vascular smooth muscle cells express both ET A andET e receptors, both of them mediating the vasoactive effects o f ET- 1. It has been recently shown that ET- 1 may increase the resfstance of the portal circulation. Using an isolated perfusion model of rat liver, we investigated the effect o f ET-1 on the portal circulation to determine which endothelin receptor is mediating this effect.

ET-1 induced a dose-dependent decrease o f portal flow compared to the control group (2.21 _+ 0.27 ml.min- l .g foie- 1): ET- 1 at l x l 0 -l~ and l x l 0 -j° M had no significant effect (-0 and -9%, respectively, when compared to control group), while the portal f19 w was significantly decreased (-55 % of control) following the perfusion of ET-1 at l x l 0 9 M and was completely interrupted at l x l O 8 M. BQ 123 (2x10 6 M), an antagonist o f ET A receptor, partially reversed (-25 % o f control) the effect o f l x l 0 -9 M ET-I, while IRL 1038 ( l x l 0 -7 M), an antagonist o f E T s receptor, did not influcence the effect o f I x 10 -9 MET- 1 on the portal circulation (- 55 % o f control). Sarafatoxin C (3x10 -6 M), an agonist o f ET A receptor, significantly decreased the portal flow (-55 % of control).

These results confirm that ET- 1 has potent vasoconstrictive effect on portal circulation and that this effect is mainly mediated through ET A receptor.