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STAT3 and p38 MAPK dependent VEGF production by bone marrow stem cells following stimulation with TNF or LPS
TRANSPLANTATION reperfusion prior to the prolonged ischemic insult. Each group was then subdivided (7 rats each) into control (IV normal saline) and IV methylprednisolone (MP) groups. Forty-five min before ischemia induction, rats were pretreated with intravenous methylprednisolone (2 mg/kg) or normal saline (NS). Warm I/R injury was evaluated using serum levels of aspartate aminotransferase (AST), serum IL-6, expression of TNF-alpha gene (PCR), hematoxylin and eosin staining, and a marker of apoptosis (TUNEL staining).
STAT3 and p38 MAPK dependent VEGF production by bone marrow stem cells following stimulation with TNF or LPS Meijing Wang MD, MS, Paul Crisostomo MD, Keith Lillemoe MD, FACS, Daniel Meldrum MD, FACS Indiana University Medical Center, Indianpolis, IN INTRODUCTION: Progenitor cells may in part mediate their beneficial effects, limit apoptosis, and modulate inflammation via production of local factors. Understanding the mechanisms by which they produce these factors is important to maximizing their benefit. We hypothesized that tumor necrosis factor alpha (TNF) and lipopolysaccharide (LPS): 1) activates bone marrow mesenchymal stem cells (BMSCs) to release vascular endothelial growth factor (VEGF) by STAT3 and p38 MAPK dependent mechanisms.
RESULTS: Histology, serum IL-6 and AST release revealed that MP treatment provided significant protection compared with ischemic controls (both CC and IP groups) only in the normal, not steatotic, liver. TUNEL-positive cells, as well as TNF expression, were clearly reduced in hepatic tissue of MP-treated animals with normal liver and not in the Zucker rats. In general, IP groups showed decreased l/R injury compared to the CC group. MP attenuated the postischemic apoptotic and inflammatory response in the normal and not steatotic, liver.
METHODS: Mouse BMSCs from wild type (WT) and STAT3 knockout mice (STAT3KO) were plated at 100,000 cells/ml/well and each were divided into groups: control, p38 MAPK inhibitor (10 microM of SB203580⫽p38MKI), TNF (50ng/ml), TNF⫹ p38MKI, LPS (100ng/ml), LPS⫹p38MKI. After 24-hour incubation, the supernatants were collected for production of VEGF (ELISA). Data (means⫹/⫺SEM) were analyzed with t-test, p⬍0.05⫽statistically significant.
CONCLUSIONS: MP pretreatment might be effective in reducing warm I/R injury to livers without steatosis.
RESULTS: VEGF production was significantly lower in all STAT3KO groups compared to WT. TNF and LPS significantly increased VEGF production in mBMSCs (WT: TNF 897⫾41.9, LPS 772⫾15.6 vs. control 533⫾11.9; STAT3KO: TNF 314⫾5.7, LPS 349⫾13.2 vs. control 242⫾5.9 pg/ml). The p38 MAPK inhibitor decreased TNF and LPS induced VEGF production (TNF⫹p38MKI: WT 570⫾6.4, STAT3KO 244⫾11.1; LPS⫹p38MKI: WT 548⫾38.7, STAT3KO 319⫾4.6 pg/ml). p38MKI alone had no effect.
Arginase blockade as a strategy to mitigate liver ischemia-reperfusion (I/R) injury Geetha Jeyabalan MD, Allan Tsung MD, John Klune BS, Timothy Billiar MD, David Geller MD University of Pittsburgh, Pittsburgh, PA INTRODUCTION: Liver I/R injury is associated with profound arginine depletion due to arginase release from injured hepatocytes. Nitric oxide (NO), shown to have protective effects in I/R, is produced by nitric oxide synthases (NOS) from the substrate arginine. The purpose of our study was to determine if norNOHA (NN), a novel arginase inhibitor, would be able to increase circulating arginine levels and decrease hepatic damage after I/R.
CONCLUSIONS: BMSC are a potent source of VEGF, the production of which is mediated by STAT3 and p38 MAPK. This understanding may allow for the ex vivo priming of BMSC for maximal growth factor production prior to and during therapeutic use.
METHODS: C57BL6 mice underwent 60 minutes of partial liver warm I/R. NN (100 mg/kg) was administered 15 minutes before ischemia and immediately after reperfusion. Serum amino acid analysis was performed using HPLC assay.
Effect of methylprednisolone on liver warm ischemia-reperfusion injury
RESULTS: Arginase activity after hepatic I/R peaked at 3-6 h after reperfusion and resulted in profound arginine depletion with circulating levels dropping 10-fold (2105nmol/ml vs. 201nmol/ml). Treatment with NN inhibited arginase activity and reversed the arginine depletion after I/R (175nmol/ml⫾26 vs. 334nmol/ml⫾55). Inhibition of arginase also resulted in protection from hepatic I/Rinduced damage in association with markedly lower hepatic TNF, IL-6, and iNOS mRNA levels compared to controls. Since arginine is the major substrate for nitiric oxide sythases (NOS), serum citrulline, a product of NOS activity, was measured in experimental animals treated with NN or control saline. Circulating citrulline levels were increased in NN-treated animals compared to controls (611nmol/ ml⫾102 vs. 293 ⫾ 23).
Reza F Saidi MD, Yeon-Jeen Chang MD, Steven Verb MD, Steven Brooks MD, Vijay Mittal MD, Michael Jacobs MD Providence Hospital, Southfield, MI INTRODUCTION: Liver ischemia-reperfusion (I/R) injury is a wellrecognized cause of morbidity and mortality in liver surgery and transplantation and is closely related to postoperative mortality and morbidity. METHODS: Male Sprague-Dawley and Zucker (with liver steatosis) rats were subjected to 75 min of 70% hepatic ischemia and three-hours reperfusion. Each ischemic period was evaluated using two different protocols: continuous clamping (CC) and ischemic preconditioning (IP) by clamping for 10 min and 15 min of
© 2006 by the American College of Surgeons Published by Elsevier Inc.
ISSN 1072-7515/06/$32.00
S89
STAT3 and p38 MAPK dependent VEGF production by bone marrow stem cells following stimulation with TNF or LPS Meijing Wang MD, MS, Paul Crisostomo MD, Keith Lillemoe MD, FACS, Daniel Meldrum MD, FACS Indiana University Medical Center, Indianpolis, IN INTRODUCTION: Progenitor cells may in part mediate their beneficial effects, limit apoptosis, and modulate inflammation via production of local factors. Understanding the mechanisms by which they produce these factors is important to maximizing their benefit. We hypothesized that tumor necrosis factor alpha (TNF) and lipopolysaccharide (LPS): 1) activates bone marrow mesenchymal stem cells (BMSCs) to release vascular endothelial growth factor (VEGF) by STAT3 and p38 MAPK dependent mechanisms.
RESULTS: Histology, serum IL-6 and AST release revealed that MP treatment provided significant protection compared with ischemic controls (both CC and IP groups) only in the normal, not steatotic, liver. TUNEL-positive cells, as well as TNF expression, were clearly reduced in hepatic tissue of MP-treated animals with normal liver and not in the Zucker rats. In general, IP groups showed decreased l/R injury compared to the CC group. MP attenuated the postischemic apoptotic and inflammatory response in the normal and not steatotic, liver.
METHODS: Mouse BMSCs from wild type (WT) and STAT3 knockout mice (STAT3KO) were plated at 100,000 cells/ml/well and each were divided into groups: control, p38 MAPK inhibitor (10 microM of SB203580⫽p38MKI), TNF (50ng/ml), TNF⫹ p38MKI, LPS (100ng/ml), LPS⫹p38MKI. After 24-hour incubation, the supernatants were collected for production of VEGF (ELISA). Data (means⫹/⫺SEM) were analyzed with t-test, p⬍0.05⫽statistically significant.
CONCLUSIONS: MP pretreatment might be effective in reducing warm I/R injury to livers without steatosis.
RESULTS: VEGF production was significantly lower in all STAT3KO groups compared to WT. TNF and LPS significantly increased VEGF production in mBMSCs (WT: TNF 897⫾41.9, LPS 772⫾15.6 vs. control 533⫾11.9; STAT3KO: TNF 314⫾5.7, LPS 349⫾13.2 vs. control 242⫾5.9 pg/ml). The p38 MAPK inhibitor decreased TNF and LPS induced VEGF production (TNF⫹p38MKI: WT 570⫾6.4, STAT3KO 244⫾11.1; LPS⫹p38MKI: WT 548⫾38.7, STAT3KO 319⫾4.6 pg/ml). p38MKI alone had no effect.
Arginase blockade as a strategy to mitigate liver ischemia-reperfusion (I/R) injury Geetha Jeyabalan MD, Allan Tsung MD, John Klune BS, Timothy Billiar MD, David Geller MD University of Pittsburgh, Pittsburgh, PA INTRODUCTION: Liver I/R injury is associated with profound arginine depletion due to arginase release from injured hepatocytes. Nitric oxide (NO), shown to have protective effects in I/R, is produced by nitric oxide synthases (NOS) from the substrate arginine. The purpose of our study was to determine if norNOHA (NN), a novel arginase inhibitor, would be able to increase circulating arginine levels and decrease hepatic damage after I/R.
CONCLUSIONS: BMSC are a potent source of VEGF, the production of which is mediated by STAT3 and p38 MAPK. This understanding may allow for the ex vivo priming of BMSC for maximal growth factor production prior to and during therapeutic use.
METHODS: C57BL6 mice underwent 60 minutes of partial liver warm I/R. NN (100 mg/kg) was administered 15 minutes before ischemia and immediately after reperfusion. Serum amino acid analysis was performed using HPLC assay.
Effect of methylprednisolone on liver warm ischemia-reperfusion injury
RESULTS: Arginase activity after hepatic I/R peaked at 3-6 h after reperfusion and resulted in profound arginine depletion with circulating levels dropping 10-fold (2105nmol/ml vs. 201nmol/ml). Treatment with NN inhibited arginase activity and reversed the arginine depletion after I/R (175nmol/ml⫾26 vs. 334nmol/ml⫾55). Inhibition of arginase also resulted in protection from hepatic I/Rinduced damage in association with markedly lower hepatic TNF, IL-6, and iNOS mRNA levels compared to controls. Since arginine is the major substrate for nitiric oxide sythases (NOS), serum citrulline, a product of NOS activity, was measured in experimental animals treated with NN or control saline. Circulating citrulline levels were increased in NN-treated animals compared to controls (611nmol/ ml⫾102 vs. 293 ⫾ 23).
Reza F Saidi MD, Yeon-Jeen Chang MD, Steven Verb MD, Steven Brooks MD, Vijay Mittal MD, Michael Jacobs MD Providence Hospital, Southfield, MI INTRODUCTION: Liver ischemia-reperfusion (I/R) injury is a wellrecognized cause of morbidity and mortality in liver surgery and transplantation and is closely related to postoperative mortality and morbidity. METHODS: Male Sprague-Dawley and Zucker (with liver steatosis) rats were subjected to 75 min of 70% hepatic ischemia and three-hours reperfusion. Each ischemic period was evaluated using two different protocols: continuous clamping (CC) and ischemic preconditioning (IP) by clamping for 10 min and 15 min of
© 2006 by the American College of Surgeons Published by Elsevier Inc.
ISSN 1072-7515/06/$32.00
S89