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Ischemic preconditioning protects liver grafts via tyrosine kinases
Vol. 191, No. 4S, October 2000
Surgical Forum Abstracts
strate that intestinal I/R causes an alteration of the intestinal absorptive function. PY treatment completely prevented such alterations maintaining D-xylose absorption within normal limits.
Ischemic preconditioning protects liver grafts via tyrosine kinases Rocco Ricciardi MD, Ravi Chari MD, Bradley Schaffer MD, Robin Kim MD, Shimul Shah MD, Michael Potter MD, Steven Quarfordt MD, Mark Callery MD, William Meyers MD. Surgery, UMass Medical School, 55 Lake Avenue North, Worcester, MA 01655. USA (508) 856-5622 Introduction: Ischemic preconditioning (IPC) protects the heart via tyrosine kinases. Presently, the effect of IPC on liver preservation is unclear. We evaluated the effect of IPC on liver preservation and investigated the role of tyrosine kinases in response to IPC. Methods: Control pig livers underwent routine harvest (n ⫽ 6). IPC livers underwent 15 min ischemia and 15 min in situ perfusion before harvest, with (n ⫽ 5) or without (n ⫽ 5) pretreatment with a tyrosine kinase inhibitor (Genistein). After cold storage and reperfusion, graft function was determined by bile flow, response to bile acid challenge, and graft O2 consumption. Graft circulatory impairment was estimated with tissue blood flow (thermistors) and vascular resistance. Cellular damage was estimated with ALT and LDH. Endothelial preservation was determined by Factor VIII immunostain (graded 0 to 2). Tissue extracts were analyzed for tyrosine phosphorylation by Western blot. Statistical significance was confirmed with t tests. Results: The table values are expressed as mean ⫾ standard error of the mean. Western blot demonstrated increased tyrosine phosphorylation in IPC grafts following preconditioning. This increase was not noted in Genistein pretreated grafts. Control
IPC alone
IPC ⴙ Genistein
Bile Flow (mL/15min) 1.1 ⫾ 0.1 2.5 ⫾ 0.5* 0.7 ⫾ 0.3 Bile Acid Response (mL/15min) 3.5 ⫾ 0.6 7.6 ⫾ 1.1* 3.3 ⫾ 1.4 0.47 ⫾ 0.07 0.72 ⫾ 0.06* 0.57 ⫾ 0.11 O2 Consumption (mL/100g/min) Tissue Blood Flow (mL/100g/min) 25 ⫾ 4.6 56 ⫾ 3.7* 28 ⫾ 4.2 Vascular Resistance (mmHg) 0.021 ⫾ 0.0008 0.015 ⫾ 0.0007* 0.020 ⫾ 0.0008 ALT (Fold rise from preharvest) 8 ⫾ 1.4 5.6 ⫾ 2.1 6.4 ⫾ 0.6 LDH (Fold rise from preharvest) 3.9 ⫾ 1.1 3.7 ⫾ 0.3 3.2 ⫾ 0.2 Endothelial Cell Grade 0.53 ⫾ 0.08 1.3 ⫾ 0.06* 0.67 ⫾ 0.25 * p ⬍ 0.05
Conclusions: IPC grafts demonstrated improved function, tissue circulation, and endothelial preservation. These improvements were reversed by Genistein pretreatment. The data suggest that IPC has a protective effect on the liver, which is tyrosine kinase dependent.
Differential activity of the Ca2ⴙ-ATPases in porcine hepatocytes and sinusoidal endothelial cells
S51
tion of the membranes with 45Ca2⫹ and ATP. O-vanadate, inhibitor of all CA, provided background levels of uptake (blanks). PMCA and SERCA activity was selectively measured by 45Ca2⫹-uptake inhibition by eosin (EO) and thapsigargin (THAP), respectively. PMCA and SERCA activity was then calculated as a percent of the total inhibition by o-vanadate. Results: Total SEC CA activity of 1.4 ⫾ 0.6 nmoleCa2/mg protein/min was significantly higher (p ⬍ 0.05 by ANOVA and Least Significant Difference) than the HEP activity of 0.49 ⫾ 0.18 nmoleCa2⫹/mg protein/min. PMCA and SERCA activity in the HEP was 53% and 50% of the total CA activity, respectively. PMCA activity in the SEC was 89% of the total CA activity while SERCA activity represented only 21%. The combined measured activities of PMCA and SERCA in HEP and SEC are ⬎ 100% of the total CA activity due to some crossover inhibition by EO and THAP. Conclusions: There is higher activity of the CA in the porcine SEC compared to HEP, suggesting an increased number or function of these proteins. HEP have an equivalent activity of PMCA and SERCA while SEC have a four-fold higher activity of PMCA versus SERCA. If the PMCA proteins are more sensitive to cold ischemia than SERCA, the fact that the SEC has a greater activity of PMCA relative to the activity SERCA may explain the greater SEC vulnerability to cold ischemia in comparison to the HEP.
Hypoxia during cold preservation activates NF-B rat primary hepatocytes Bradley K Schaffer MD, Robin D Kim MD, Rocco Ricciardi MD, Chad E Darling MD, Ravi S Chari, MD. Surgery, UMass Medical School, 55 Lake Ave. North, Worcester, MA 01655. USA (508) 856-8473. Introduction: The reperfusion phase of cold ischemia-reperfusion (I/R) is known to activate the transcription factor NF-B. Previous in vivo studies have correlated NF-B activation with improved postreperfusion graft function. The relative contributions of cold or hypoxia to the activation of NF-B during the cold ischemic (C/I) phase is unclear. Using an in vitro model, we studied hypothermia and hypoxia in NF-B translocation during cold ischemia. Methods: Quiescent primary rat hepatocytes were exposed for one hour to the following conditions: Group 1 was exposed to cold media and refrigeration at 4°C. Group 2 received hypoxic media (95% N2, 5% CO2) and were kept in a sealed hypoxic chamber at 37°C. TNF-␣ (400 U/mL) and normothermic media at 5% CO2 served as positive and negative controls, respectively. Nuclear proteins were extracted at 0,30,60 minutes. Relative densitometry of Western blots measured the p65 subunit of NF-B. Experiments were performed in triplicate. Results: There was no change in levels of expression of NF-B in response to cold, hypoxia or control at 30 min. At 60 min hypoxia increased nuclear NF-B to levels similar to TNF-␣.
Paul E Wise, MD, Piotr K Janicki, MD, PhD, Andrey E Belous, MD, PhD, C Wright Pinson, MD, MBA. Vanderbilt University Medical Center, Department of Liver Transplantation 801 Oxford House, 131221st Ave, South, Nashville, TN, 37232-4753 USA; phone: (615) 343-6990 Introduction: The Ca2⫹-ATPases (CA), both plasma membrane (PMCA) and endoplasmic reticular (SERCA), are the primary ‘‘pumps’’ that maintain intracellular calcium homeostasis. Excessive accumulation of intracellular calcium in hepatocytes (HEP) and sinusoidal endothelial cells (SEC) after ischemia/reperfusion has been implicated as a factor leading to cellular necrosis/apoptosis and eventual hepatic graft dysfunction in liver transplantation. SEC are known to be more sensitive to cold ischemia/reperfusion than are the HEP. Because of the differences in the sensitivities of these liver cells to cold ischemia, we hypothesized that the porcine SEC would have a greater activity of PMCA and SERCA when compared to the HEP. Methods: Pig livers were perfused with a collagenase solution for separation. HEP and SEC were separated by differential centrifugation and Percoll gradient, respectively. HEP were confirmed by morphology and SEC by uptake of acetylated LDL. The cells were lysed and spun to pellet membrane proteins. CA activity was measured after incuba-
Conclusions: In this in vitro model of the C/I phase of I/R injury, hypoxia translocated NF-B, while hypothermia did not alter NF-B. Hypoxia alone alters NF-B in the C/I phase of I/R injury. This modulation during C/I may have significant impact on graft response during reperfusion.
Cobalt protoporphyrin-induced overexpression of heme oxygenase-1 protects rat hearts from cold ischemia/reperfusion injury via anti-apoptotic pathway Masamichi Katori, MD, Roland Buelow, MD, PhD, Bibo Ke, PhD, Ana J Coito, MD, PhD, Ronald W Busuttil, MD, PhD, Jerzy W Kupiec-
Surgical Forum Abstracts
strate that intestinal I/R causes an alteration of the intestinal absorptive function. PY treatment completely prevented such alterations maintaining D-xylose absorption within normal limits.
Ischemic preconditioning protects liver grafts via tyrosine kinases Rocco Ricciardi MD, Ravi Chari MD, Bradley Schaffer MD, Robin Kim MD, Shimul Shah MD, Michael Potter MD, Steven Quarfordt MD, Mark Callery MD, William Meyers MD. Surgery, UMass Medical School, 55 Lake Avenue North, Worcester, MA 01655. USA (508) 856-5622 Introduction: Ischemic preconditioning (IPC) protects the heart via tyrosine kinases. Presently, the effect of IPC on liver preservation is unclear. We evaluated the effect of IPC on liver preservation and investigated the role of tyrosine kinases in response to IPC. Methods: Control pig livers underwent routine harvest (n ⫽ 6). IPC livers underwent 15 min ischemia and 15 min in situ perfusion before harvest, with (n ⫽ 5) or without (n ⫽ 5) pretreatment with a tyrosine kinase inhibitor (Genistein). After cold storage and reperfusion, graft function was determined by bile flow, response to bile acid challenge, and graft O2 consumption. Graft circulatory impairment was estimated with tissue blood flow (thermistors) and vascular resistance. Cellular damage was estimated with ALT and LDH. Endothelial preservation was determined by Factor VIII immunostain (graded 0 to 2). Tissue extracts were analyzed for tyrosine phosphorylation by Western blot. Statistical significance was confirmed with t tests. Results: The table values are expressed as mean ⫾ standard error of the mean. Western blot demonstrated increased tyrosine phosphorylation in IPC grafts following preconditioning. This increase was not noted in Genistein pretreated grafts. Control
IPC alone
IPC ⴙ Genistein
Bile Flow (mL/15min) 1.1 ⫾ 0.1 2.5 ⫾ 0.5* 0.7 ⫾ 0.3 Bile Acid Response (mL/15min) 3.5 ⫾ 0.6 7.6 ⫾ 1.1* 3.3 ⫾ 1.4 0.47 ⫾ 0.07 0.72 ⫾ 0.06* 0.57 ⫾ 0.11 O2 Consumption (mL/100g/min) Tissue Blood Flow (mL/100g/min) 25 ⫾ 4.6 56 ⫾ 3.7* 28 ⫾ 4.2 Vascular Resistance (mmHg) 0.021 ⫾ 0.0008 0.015 ⫾ 0.0007* 0.020 ⫾ 0.0008 ALT (Fold rise from preharvest) 8 ⫾ 1.4 5.6 ⫾ 2.1 6.4 ⫾ 0.6 LDH (Fold rise from preharvest) 3.9 ⫾ 1.1 3.7 ⫾ 0.3 3.2 ⫾ 0.2 Endothelial Cell Grade 0.53 ⫾ 0.08 1.3 ⫾ 0.06* 0.67 ⫾ 0.25 * p ⬍ 0.05
Conclusions: IPC grafts demonstrated improved function, tissue circulation, and endothelial preservation. These improvements were reversed by Genistein pretreatment. The data suggest that IPC has a protective effect on the liver, which is tyrosine kinase dependent.
Differential activity of the Ca2ⴙ-ATPases in porcine hepatocytes and sinusoidal endothelial cells
S51
tion of the membranes with 45Ca2⫹ and ATP. O-vanadate, inhibitor of all CA, provided background levels of uptake (blanks). PMCA and SERCA activity was selectively measured by 45Ca2⫹-uptake inhibition by eosin (EO) and thapsigargin (THAP), respectively. PMCA and SERCA activity was then calculated as a percent of the total inhibition by o-vanadate. Results: Total SEC CA activity of 1.4 ⫾ 0.6 nmoleCa2/mg protein/min was significantly higher (p ⬍ 0.05 by ANOVA and Least Significant Difference) than the HEP activity of 0.49 ⫾ 0.18 nmoleCa2⫹/mg protein/min. PMCA and SERCA activity in the HEP was 53% and 50% of the total CA activity, respectively. PMCA activity in the SEC was 89% of the total CA activity while SERCA activity represented only 21%. The combined measured activities of PMCA and SERCA in HEP and SEC are ⬎ 100% of the total CA activity due to some crossover inhibition by EO and THAP. Conclusions: There is higher activity of the CA in the porcine SEC compared to HEP, suggesting an increased number or function of these proteins. HEP have an equivalent activity of PMCA and SERCA while SEC have a four-fold higher activity of PMCA versus SERCA. If the PMCA proteins are more sensitive to cold ischemia than SERCA, the fact that the SEC has a greater activity of PMCA relative to the activity SERCA may explain the greater SEC vulnerability to cold ischemia in comparison to the HEP.
Hypoxia during cold preservation activates NF-B rat primary hepatocytes Bradley K Schaffer MD, Robin D Kim MD, Rocco Ricciardi MD, Chad E Darling MD, Ravi S Chari, MD. Surgery, UMass Medical School, 55 Lake Ave. North, Worcester, MA 01655. USA (508) 856-8473. Introduction: The reperfusion phase of cold ischemia-reperfusion (I/R) is known to activate the transcription factor NF-B. Previous in vivo studies have correlated NF-B activation with improved postreperfusion graft function. The relative contributions of cold or hypoxia to the activation of NF-B during the cold ischemic (C/I) phase is unclear. Using an in vitro model, we studied hypothermia and hypoxia in NF-B translocation during cold ischemia. Methods: Quiescent primary rat hepatocytes were exposed for one hour to the following conditions: Group 1 was exposed to cold media and refrigeration at 4°C. Group 2 received hypoxic media (95% N2, 5% CO2) and were kept in a sealed hypoxic chamber at 37°C. TNF-␣ (400 U/mL) and normothermic media at 5% CO2 served as positive and negative controls, respectively. Nuclear proteins were extracted at 0,30,60 minutes. Relative densitometry of Western blots measured the p65 subunit of NF-B. Experiments were performed in triplicate. Results: There was no change in levels of expression of NF-B in response to cold, hypoxia or control at 30 min. At 60 min hypoxia increased nuclear NF-B to levels similar to TNF-␣.
Paul E Wise, MD, Piotr K Janicki, MD, PhD, Andrey E Belous, MD, PhD, C Wright Pinson, MD, MBA. Vanderbilt University Medical Center, Department of Liver Transplantation 801 Oxford House, 131221st Ave, South, Nashville, TN, 37232-4753 USA; phone: (615) 343-6990 Introduction: The Ca2⫹-ATPases (CA), both plasma membrane (PMCA) and endoplasmic reticular (SERCA), are the primary ‘‘pumps’’ that maintain intracellular calcium homeostasis. Excessive accumulation of intracellular calcium in hepatocytes (HEP) and sinusoidal endothelial cells (SEC) after ischemia/reperfusion has been implicated as a factor leading to cellular necrosis/apoptosis and eventual hepatic graft dysfunction in liver transplantation. SEC are known to be more sensitive to cold ischemia/reperfusion than are the HEP. Because of the differences in the sensitivities of these liver cells to cold ischemia, we hypothesized that the porcine SEC would have a greater activity of PMCA and SERCA when compared to the HEP. Methods: Pig livers were perfused with a collagenase solution for separation. HEP and SEC were separated by differential centrifugation and Percoll gradient, respectively. HEP were confirmed by morphology and SEC by uptake of acetylated LDL. The cells were lysed and spun to pellet membrane proteins. CA activity was measured after incuba-
Conclusions: In this in vitro model of the C/I phase of I/R injury, hypoxia translocated NF-B, while hypothermia did not alter NF-B. Hypoxia alone alters NF-B in the C/I phase of I/R injury. This modulation during C/I may have significant impact on graft response during reperfusion.
Cobalt protoporphyrin-induced overexpression of heme oxygenase-1 protects rat hearts from cold ischemia/reperfusion injury via anti-apoptotic pathway Masamichi Katori, MD, Roland Buelow, MD, PhD, Bibo Ke, PhD, Ana J Coito, MD, PhD, Ronald W Busuttil, MD, PhD, Jerzy W Kupiec-