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153 Treatment of cirrhotic rats with the immunomodulator AM3 ameliorates both their systemic proinflammatory state and their hyperdynamic circulation
Category 2a." Cirrhosis and Complications." Pathophysiology Conclusions: In aseitic cirrhotic rats simvastatin improves endotheliumdependent vasorelaxation o f the liver vasculature, reduces the response to vasoconstrictors and attenuates flow-induced increases in portal pressure, thus improving sinusoidal endothelial dysfunction in the cirrhotic liver.
11531 TREATMENT OF CIRRHOTIC RATS WITH THE IMMUNOMODULATOR AM3 AMELIORATES BOTH THEIR SYSTEMIC PROINFLAMMATORY STATE AND THEIR HYPERDYNAMIC CIRCULATION
M. Nieto 1.2, A. Albillos 1.2, L. Mufioz 2 , E. Reyes2, L. Lled6 3 , M. Ubeda 2, E. Sanz 2, A. de-la-Hera2, M. Alvarez-Mon2'4. 1Serv. Gastroenterologfa,
Hospital Ramdn y Cajal, Madrid, Spain; 2Dpto. Medicina, Unidad I+D Asociada CSIC (CNB), Universidad Alcald, Madrid, Spain," ~Dpto. Microbiologla, Universidad Alcald, Madrid, Spain," 4Hospital Pdncipe de Asturias, Alcald de Henares, Madrid, Spain Traslocation of enteric bacteria and their products (LPS) play a key role in the pathogenesis of the immune dysfunction and increased production of profifftammatory cytoldnes, as tumor necrosis factor alpha (TNF), that are features of advanced drrhosis. TNF is involved in the hemodynamic derangement of cirrhosis. We have recently reported that the immunomodulator AM3 inhibits enteric bacteria-driven TNF production by monocytes, and that monocytes are tile major source of TNF overproduction in drrhotic patients. Aim: To investigate in bile duct-ligated (BDL) cirrhotic tats: 1) the activation status and cytokine production in circulating irnmmle cells (protocol I), and2) file effect of AM3 on the finmune system and hyperdynamic drculation (protocols II and III). In protocol I, sham and BDL tats were studied 6 wk after surgery. In protocols II and III, BDL rats were treated with a 3-wk AM3 course (3 mg/kg.d po) or placebo from 4th wk after surgery on. Results (meand-SEM, cells/~tl): Compared to controls, cirrhotic tats showed monocyte expansion (56174_1254 vs. 452_12, p < 0.01), including a massive increase of TNF-producing monocytes (33702_960 vs. 214_6, p < 0.01). Activated Th-lymphocytes were also expanded (CD4+CD134+, 922-26 vs. 32-0.9, p<0.01) and Thl polarized (interferon-¥ producing, 352-9 vs. 4.22-0.02, p<0.01). AM3 reduced by more that 10-fold the abnormal monocyte expansion (to 552d-139, p<0.01) and their TNF production (to 249d-86, p < 0.01). Activated (to 43d-14, p < 0.01) and interferon-¥ producing (to 102_3, p < 0.01) Th-lymphocytes numbers were also improved. Notably, AM3 reduced serum TNF (654_ 14 vs. 382_9 pg/ml, p<0.05) and cardiac index (46d-9 vs. 52±11ml/min. 100g, p<0.01) and increased SVR by 352-8% (p < 0.01), whereas portal pressure was unchanged. Conclusion: In rats with established cirrhosis, AM3 markedly diminishes the expansion of monocytes and T-lymphocytes activated to TNF and interferon-¥ production, and serum TNE The AM3 immunomodulatory effects assodate with amelioration of tile hyperdynarnic circulation.
I•
OXIDATIVE STRESS CONTRIBUTES TO THE HYPERDYNAMIC CIRCULATION IN PORTAL HYPERTENSIVE RATS
B. Angermavr, M. Mejias, J.C. Garcia-Pagan, J. Rodes, J. Bosch, M. Fernandez. Hepatic Hemodynamic Laboratory, Liver Unit, Hospital
Clinic, IDIBAPS, University of Barcelona, Barcelona, Spain Background: Portal hypertension causes oxidative stress in terms of reactive oxygen species (ROS) in the mesentery of portal hypertensive rats. Since ROS are vasoactive molecules leading directly and indirectly via several pathways to vasodilatation, ROS may also contribute to the hyperdynamic circulation in portal hypertension. Aim of the study was to determine tile effect of inhibition of ROS-production on splanchnic hemodynamics in a prehepatic portal hypertensive rat model.
63
Methods: Twenty male Spragxm-Dawley rats underwent partial portal vein
ligation (PPVL) and 10 rats underwent sham-operation (SO). Ten PPVLanimals and 5 SO rats were treated with apoeynin (30 mg/kg body weight, dissolved in 0.3 ml DMSO, daily, i.p.), an inhibitor of the NAD(P)H oxidase, which is the major source for non-phagocytic ROS-production. All other animals received vehicle treatment. Hemodynamic studies were performed 5 days after PPVL/SO. Student's t-test was the main statistical analysis. Results: Inhibition o f ROS production in PPVL rats resulted in a significant (p=0.018) decrease in blood flow (ml/mirdl00g) in the superior mesenteric artery (SMABF: mean=4.24) and a significant (p=0.001) increase in splanchnic arteriolar resistance (SAR: mean = 19.21), as compared with PPVL rats receiving vehicle (SMABF: mean=5.29, SAR: mean = 14.09). Portal pressure (rnmHg) was lower in PPVL rats undergoing antioxidative treatment (mean= 17.62) than in those treated with vehicle (mean 19.80), although the difference (lid not reach statistical significance (p= 0.081). In SO control animals, inhibition of ROS production had not significant hernodynamic effects, as compared with rats receiving vehicle. Conclusion: Our study demonstrates that the elevated levels of oxidative stress in the splanchnic territory contribute to the hyperdynamic splanchnic circulation of portal hypertensive rats. Inhibition of ROS production could markedly and significantly attenuate the splanchnic hemodynamic abnormalities in portal hypertensive vats, which therefore may become a promising strategy in the therapy of portal hypertension. Acknowledgement: The EASEs Sheila Sherlock Fellowship enabled B.A. to conduct this study. TM
I•1
POLYMORPHIC IMPRINTING OF P E G I / M E S T GENE: A SUSCEPTIBILITY FACTOR FOR LIVER DISEASE?
A. Calvo-Ferrer, E. Petri, J. Castillo, M.A. Avila, J. Prieto, C. Berasain.
Divisio of Hepatology and Gene Therapy, CIMA, Universidad de Naoarra, Pamplona, Spain Background: Genomic imprinting describes the preferential or exchtsive expression of a gene from only one of the two parental alleles based on allele-specific epigenetic modifications. Recent findings demonstrate that imprinting is a polymorphic trait in the population that is associated with a different susceptibility to cancer, as it has been shown for IGF2 gene and colon cancer. Many reports that refer to loss of imprinting (LOI) in tumors really describes tile existence of a subset of individuals with biallelic expression of the gene in both tumoral and non tumoral tissues. Frequent LOI of PEG1/MEST (a putative a/b-hy&olase) has been described in breast, colon and lung cancer, although recent data suggest that this LOI is the result of tile induction of the expression of a nonimprinted or reciprocally imprinted PEG1/MEST isoform. In this study, we have fiwestigated the allelic usage of the two identified isoforms of PEG 1/MEST in cirrhotic and normal livers. Material and Methods: We have genotyped 89 normal and 92 cirrhotic human livers for the AflIII RFLP in the 3/ untranslated region of the PEG1/MEST gene. Using isoform-specific primers and a nested PCR flanking this polymorphism we have studied the allelic usage of isoforros 1 and 2 of PEG1/MEST in the informative 0aeterozygous) samples. Results: Eighteen percent of the samples were informative. Both PEG 1/MEST isoforms were constitutively expressed in tile liver: isoforro 1 was monoallelically expressed and isoforrn 2 was polymorphically imprinted. The allelic usage ofisoforrn 2 is significantly different (p <0.05) in normal and cirrhotic liver. None of tile drrhotic livers versus 12.5% of control livers expressed the isoform 2 monoallelically from tile same allele than the isoform 1, and 44% of cirrhotic livers versus 12.5% of control livers expressed the isoform 2 monoallelically from the opposite allele than the isoform 1. Fifty six percent of cirrhotic livers and 75% of control livers expressed the isoform 2 biallelically.
11531 TREATMENT OF CIRRHOTIC RATS WITH THE IMMUNOMODULATOR AM3 AMELIORATES BOTH THEIR SYSTEMIC PROINFLAMMATORY STATE AND THEIR HYPERDYNAMIC CIRCULATION
M. Nieto 1.2, A. Albillos 1.2, L. Mufioz 2 , E. Reyes2, L. Lled6 3 , M. Ubeda 2, E. Sanz 2, A. de-la-Hera2, M. Alvarez-Mon2'4. 1Serv. Gastroenterologfa,
Hospital Ramdn y Cajal, Madrid, Spain; 2Dpto. Medicina, Unidad I+D Asociada CSIC (CNB), Universidad Alcald, Madrid, Spain," ~Dpto. Microbiologla, Universidad Alcald, Madrid, Spain," 4Hospital Pdncipe de Asturias, Alcald de Henares, Madrid, Spain Traslocation of enteric bacteria and their products (LPS) play a key role in the pathogenesis of the immune dysfunction and increased production of profifftammatory cytoldnes, as tumor necrosis factor alpha (TNF), that are features of advanced drrhosis. TNF is involved in the hemodynamic derangement of cirrhosis. We have recently reported that the immunomodulator AM3 inhibits enteric bacteria-driven TNF production by monocytes, and that monocytes are tile major source of TNF overproduction in drrhotic patients. Aim: To investigate in bile duct-ligated (BDL) cirrhotic tats: 1) the activation status and cytokine production in circulating irnmmle cells (protocol I), and2) file effect of AM3 on the finmune system and hyperdynamic drculation (protocols II and III). In protocol I, sham and BDL tats were studied 6 wk after surgery. In protocols II and III, BDL rats were treated with a 3-wk AM3 course (3 mg/kg.d po) or placebo from 4th wk after surgery on. Results (meand-SEM, cells/~tl): Compared to controls, cirrhotic tats showed monocyte expansion (56174_1254 vs. 452_12, p < 0.01), including a massive increase of TNF-producing monocytes (33702_960 vs. 214_6, p < 0.01). Activated Th-lymphocytes were also expanded (CD4+CD134+, 922-26 vs. 32-0.9, p<0.01) and Thl polarized (interferon-¥ producing, 352-9 vs. 4.22-0.02, p<0.01). AM3 reduced by more that 10-fold the abnormal monocyte expansion (to 552d-139, p<0.01) and their TNF production (to 249d-86, p < 0.01). Activated (to 43d-14, p < 0.01) and interferon-¥ producing (to 102_3, p < 0.01) Th-lymphocytes numbers were also improved. Notably, AM3 reduced serum TNF (654_ 14 vs. 382_9 pg/ml, p<0.05) and cardiac index (46d-9 vs. 52±11ml/min. 100g, p<0.01) and increased SVR by 352-8% (p < 0.01), whereas portal pressure was unchanged. Conclusion: In rats with established cirrhosis, AM3 markedly diminishes the expansion of monocytes and T-lymphocytes activated to TNF and interferon-¥ production, and serum TNE The AM3 immunomodulatory effects assodate with amelioration of tile hyperdynarnic circulation.
I•
OXIDATIVE STRESS CONTRIBUTES TO THE HYPERDYNAMIC CIRCULATION IN PORTAL HYPERTENSIVE RATS
B. Angermavr, M. Mejias, J.C. Garcia-Pagan, J. Rodes, J. Bosch, M. Fernandez. Hepatic Hemodynamic Laboratory, Liver Unit, Hospital
Clinic, IDIBAPS, University of Barcelona, Barcelona, Spain Background: Portal hypertension causes oxidative stress in terms of reactive oxygen species (ROS) in the mesentery of portal hypertensive rats. Since ROS are vasoactive molecules leading directly and indirectly via several pathways to vasodilatation, ROS may also contribute to the hyperdynamic circulation in portal hypertension. Aim of the study was to determine tile effect of inhibition of ROS-production on splanchnic hemodynamics in a prehepatic portal hypertensive rat model.
63
Methods: Twenty male Spragxm-Dawley rats underwent partial portal vein
ligation (PPVL) and 10 rats underwent sham-operation (SO). Ten PPVLanimals and 5 SO rats were treated with apoeynin (30 mg/kg body weight, dissolved in 0.3 ml DMSO, daily, i.p.), an inhibitor of the NAD(P)H oxidase, which is the major source for non-phagocytic ROS-production. All other animals received vehicle treatment. Hemodynamic studies were performed 5 days after PPVL/SO. Student's t-test was the main statistical analysis. Results: Inhibition o f ROS production in PPVL rats resulted in a significant (p=0.018) decrease in blood flow (ml/mirdl00g) in the superior mesenteric artery (SMABF: mean=4.24) and a significant (p=0.001) increase in splanchnic arteriolar resistance (SAR: mean = 19.21), as compared with PPVL rats receiving vehicle (SMABF: mean=5.29, SAR: mean = 14.09). Portal pressure (rnmHg) was lower in PPVL rats undergoing antioxidative treatment (mean= 17.62) than in those treated with vehicle (mean 19.80), although the difference (lid not reach statistical significance (p= 0.081). In SO control animals, inhibition of ROS production had not significant hernodynamic effects, as compared with rats receiving vehicle. Conclusion: Our study demonstrates that the elevated levels of oxidative stress in the splanchnic territory contribute to the hyperdynamic splanchnic circulation of portal hypertensive rats. Inhibition of ROS production could markedly and significantly attenuate the splanchnic hemodynamic abnormalities in portal hypertensive vats, which therefore may become a promising strategy in the therapy of portal hypertension. Acknowledgement: The EASEs Sheila Sherlock Fellowship enabled B.A. to conduct this study. TM
I•1
POLYMORPHIC IMPRINTING OF P E G I / M E S T GENE: A SUSCEPTIBILITY FACTOR FOR LIVER DISEASE?
A. Calvo-Ferrer, E. Petri, J. Castillo, M.A. Avila, J. Prieto, C. Berasain.
Divisio of Hepatology and Gene Therapy, CIMA, Universidad de Naoarra, Pamplona, Spain Background: Genomic imprinting describes the preferential or exchtsive expression of a gene from only one of the two parental alleles based on allele-specific epigenetic modifications. Recent findings demonstrate that imprinting is a polymorphic trait in the population that is associated with a different susceptibility to cancer, as it has been shown for IGF2 gene and colon cancer. Many reports that refer to loss of imprinting (LOI) in tumors really describes tile existence of a subset of individuals with biallelic expression of the gene in both tumoral and non tumoral tissues. Frequent LOI of PEG1/MEST (a putative a/b-hy&olase) has been described in breast, colon and lung cancer, although recent data suggest that this LOI is the result of tile induction of the expression of a nonimprinted or reciprocally imprinted PEG1/MEST isoform. In this study, we have fiwestigated the allelic usage of the two identified isoforms of PEG 1/MEST in cirrhotic and normal livers. Material and Methods: We have genotyped 89 normal and 92 cirrhotic human livers for the AflIII RFLP in the 3/ untranslated region of the PEG1/MEST gene. Using isoform-specific primers and a nested PCR flanking this polymorphism we have studied the allelic usage of isoforros 1 and 2 of PEG1/MEST in the informative 0aeterozygous) samples. Results: Eighteen percent of the samples were informative. Both PEG 1/MEST isoforms were constitutively expressed in tile liver: isoforro 1 was monoallelically expressed and isoforrn 2 was polymorphically imprinted. The allelic usage ofisoforrn 2 is significantly different (p <0.05) in normal and cirrhotic liver. None of tile drrhotic livers versus 12.5% of control livers expressed the isoform 2 monoallelically from tile same allele than the isoform 1, and 44% of cirrhotic livers versus 12.5% of control livers expressed the isoform 2 monoallelically from the opposite allele than the isoform 1. Fifty six percent of cirrhotic livers and 75% of control livers expressed the isoform 2 biallelically.