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P294
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ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS
P294. ACTIVATION OF THE ALPHA ESTROGEN RECEPTOR STIMULATES STEM CELL PRODUCTION OF VEGF AND HGF, BUT NOT IGF-1 OR SCF, IN FEMALES. T. Markel, M. Wang, C. Herring, P. Crisostomo, K. D. Lillemoe, D. R. Meldrum; Indiana University, Indianapolis, IN Background: Stem cells effects may enhance neovascularization through the production of local factors. Understanding the mechanisms by which they produce these factors is important to maximizing their benefit. Bone marrow mesenchymal stem cells (BMSC) derived from females release more vascular endothelial growth factor (VEGF) in response to LPS and hypoxia when compared to males. However, it is unknown whether estrogen has an effect on BMSC production of VEGF, hepatocyte growth factor (HGF), stem cell factor (SCF) or insulin like growth factor (IGF-1), and if so, whether this effect is mediated by the alpha estrogen receptor (ER1). Methods: Mouse BMSCs from female wild type (WT) and female ER1 knockout mice (ER1KO) were incubated with or without 1 microM of estradiol (E2). After 24-hours of incubation, the supernatants were collected and measured for production of VEGF, HGF, IGF-1 and SCF (ELISA). The experiment was repeated on three individual times (n⫽6-9/group). Data (means⫹/-SEM) were analyzed with t-test, p⬍0.05⫽statistically significant. Results: E2 significantly stimulated BMSC release of VEGF (pg/1,000,000 cells) from 2979⫹/-53.3 to 4666⫹/-324.7, and HGF (pg/1,000,000 cells) from 81.3⫹/-23.7 to 174.3⫹/-29 in WT, whereas BMSC production of VEGF (3487⫹/-184 vs. E2 3738⫹/261) and HGF (65.4⫹/-21.2 vs. E2 70.4⫹/-14.4) was not increased by E2 in ER1KO. However, E2 did not affect BMSC secretion of IGF-1 (WT: 107⫹/-23 vs. E2 87⫹/-21.6, ER1KO: 180.5⫹/-30 vs. E2 217⫹/-57) and SCF (WT: 136⫹/-32 vs. E2 129⫹/-31, ER1KO: 92⫹/6.8 vs. E2 112⫹/-18) in both WT and ER1KO. Conclusions: 1) BMSCs are a potent source of VEGF and HGF; 2) E2 positively affected BMSC production of VEGF and HGF, but not SCF or IGF-1, through ER1 in female BMSCs. This understanding may allow for the ex vivo priming of MSCs for maximal growth factor production prior to and during therapeutic use.
P295. TNF ALPHA INDUCES MCP 1 EXPRESSION IN VASCULAR SMOOTH MUSCLE CELLS THROUGH A MECHANISM INVOLVING PKC DELTA. B. Liu, C. Wang, J. Hu, K. Kamiya, K. Kent; Weill Cornell Medical College, New York, NY Introduction: Monocycle chemo attractant protein 1 (MCP-1), produced by many types of vascular cells including smooth muscle cells (SMCs), plays an early and critical role in the development of vascular inflammation through the recruitment of monocytes and macrophages to the arterial wall. Targeted gene deletion of MCP-1 inhibits the development of intimal hyperplasia or abdominal aortic aneurysms in animal models. In this study, we investigated the molecular mechanism underlying the regulation of MCP-1 expression in SMCs, in particular the role of signaling molecule protein kinase C delta (PKC␦), which is known to be activated by pro-inflammation cytokines. Methods: The pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-␣) was used as a prototype to stimulate rat aortic SMC line A10 as well as primary SMCs isolated from PKC␦ knock-out mice and their wild-type littermates. MCP-1 protein in cell culture media was determined by an ELISA assay and MCP-1 mRNA by the semi-quantitative RT-PCR. Results: TNF-␣, dose-dependently, elicited a marked increase in the amount of MCP-1 protein produced by A10 SMCs. A time-couse analysis showed that the induction was evident at 2 h and persistent up to 48 h. Similarly, TNF-␣ rapidly upregulated the level of MCP-1 mRNA in the cells, suggesting the induction may take place at the gene expression level. To understand the role of PKC␦ in regulation of MCP-1 expression, we pretreated
A10 cells with an increasing amount of rottlerin (0, 0.1, 0.5, 1 and 2 M), a selective inhibitor to PKC␦. Rottlerin dose-dependently blocked the induction of MCP-1 protein and mRNA. To confirm these results obtained with the chemical inhibitor, we employed PKC␦ knock-out mice. Aortic SMCs isolated from PKC␦ null mice or their wild-type littermates were treated with TNF-␣ as above. PKC␦ gene deficiency led to a complete loss of ability of SMCs to express MCP-1. Finally, we tested whether molecular activation of PKC␦ is sufficient to induce MCP-1 expression. To this end, we ectopically expressed PKC␦ in A10 cells via an adenoviral vector that expresses the wild-type PKC␦. Compared to the viral vector, Ad-PKC␦ elicited an about two-fold increase in MCP-1 expression. Conclusions: Taken together, our data show that the proinflammatory cytokine TNF-␣ is a potent inducer of MCP-1 expression in vascular SMCs. TNF-␣ stimulates MCP-1 expression through a signaling pathway that involves PKC␦. Further studies are mandated to test whether inhibition of PKC␦ could prevent MCP-1 expression thus monocytes recruitment in animal models following arterial injury.
P296. A NEW RABBIT MODEL OF DIABETIC ISCHEMIC WOUND HEALING USING HYPERSPECTRAL IMAGING AND MOLECULAR ANALYSIS. L. Pradhan1, X. Cai 2, N. Andersen2, M. Jain1, J. Malek1, M. Contreras1, A. Veves1, F. W. Logerfo1; 1Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, 2Harvard Medical School, Boston, MA Background: Diabetic foot ulceration (DFU) significantly impairs the quality of life of patients, leads to prolonged hospitalization, and commonly leads to major amputation. Abnormal wound healing is a key cause of the chronic non-healing wound. Inflammation, angiogenesis, and re-epithelization are essential phases of normal wound healing. The cytokines secreted in the wound microenvironment play a role in the inflammatory and angiogenesis phases of wound healing. Abnormalities in the expression and activity of inflammatory cytokines, followed by reduced infiltration and angiogenesis, can disrupt normal wound healing, contributing both to the development of DFU and its failure to heal. Of late, dysregulation of neuropeptides secreted from peripheral nerves has also been implicated in abnormal wound healing. The exact mechanisms by which cytokines and neuropeptides affect wound healing are not well understood. To systematically understand the pathobiology of diabetic wound healing, it is vital to develop in vivo models that can mimic the complexity of DFU. Model: Rabbits are made diabetic using alloxan monohydrate (100 mg/kg i.v.). After the induction of diabetes, surgery is performed on the rabbit ears to create wounds. Dermal circulation to the left ear is interrupted by ligating the rostral and central arteries, thus modeling ischemia. In the right
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS
P294. ACTIVATION OF THE ALPHA ESTROGEN RECEPTOR STIMULATES STEM CELL PRODUCTION OF VEGF AND HGF, BUT NOT IGF-1 OR SCF, IN FEMALES. T. Markel, M. Wang, C. Herring, P. Crisostomo, K. D. Lillemoe, D. R. Meldrum; Indiana University, Indianapolis, IN Background: Stem cells effects may enhance neovascularization through the production of local factors. Understanding the mechanisms by which they produce these factors is important to maximizing their benefit. Bone marrow mesenchymal stem cells (BMSC) derived from females release more vascular endothelial growth factor (VEGF) in response to LPS and hypoxia when compared to males. However, it is unknown whether estrogen has an effect on BMSC production of VEGF, hepatocyte growth factor (HGF), stem cell factor (SCF) or insulin like growth factor (IGF-1), and if so, whether this effect is mediated by the alpha estrogen receptor (ER1). Methods: Mouse BMSCs from female wild type (WT) and female ER1 knockout mice (ER1KO) were incubated with or without 1 microM of estradiol (E2). After 24-hours of incubation, the supernatants were collected and measured for production of VEGF, HGF, IGF-1 and SCF (ELISA). The experiment was repeated on three individual times (n⫽6-9/group). Data (means⫹/-SEM) were analyzed with t-test, p⬍0.05⫽statistically significant. Results: E2 significantly stimulated BMSC release of VEGF (pg/1,000,000 cells) from 2979⫹/-53.3 to 4666⫹/-324.7, and HGF (pg/1,000,000 cells) from 81.3⫹/-23.7 to 174.3⫹/-29 in WT, whereas BMSC production of VEGF (3487⫹/-184 vs. E2 3738⫹/261) and HGF (65.4⫹/-21.2 vs. E2 70.4⫹/-14.4) was not increased by E2 in ER1KO. However, E2 did not affect BMSC secretion of IGF-1 (WT: 107⫹/-23 vs. E2 87⫹/-21.6, ER1KO: 180.5⫹/-30 vs. E2 217⫹/-57) and SCF (WT: 136⫹/-32 vs. E2 129⫹/-31, ER1KO: 92⫹/6.8 vs. E2 112⫹/-18) in both WT and ER1KO. Conclusions: 1) BMSCs are a potent source of VEGF and HGF; 2) E2 positively affected BMSC production of VEGF and HGF, but not SCF or IGF-1, through ER1 in female BMSCs. This understanding may allow for the ex vivo priming of MSCs for maximal growth factor production prior to and during therapeutic use.
P295. TNF ALPHA INDUCES MCP 1 EXPRESSION IN VASCULAR SMOOTH MUSCLE CELLS THROUGH A MECHANISM INVOLVING PKC DELTA. B. Liu, C. Wang, J. Hu, K. Kamiya, K. Kent; Weill Cornell Medical College, New York, NY Introduction: Monocycle chemo attractant protein 1 (MCP-1), produced by many types of vascular cells including smooth muscle cells (SMCs), plays an early and critical role in the development of vascular inflammation through the recruitment of monocytes and macrophages to the arterial wall. Targeted gene deletion of MCP-1 inhibits the development of intimal hyperplasia or abdominal aortic aneurysms in animal models. In this study, we investigated the molecular mechanism underlying the regulation of MCP-1 expression in SMCs, in particular the role of signaling molecule protein kinase C delta (PKC␦), which is known to be activated by pro-inflammation cytokines. Methods: The pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-␣) was used as a prototype to stimulate rat aortic SMC line A10 as well as primary SMCs isolated from PKC␦ knock-out mice and their wild-type littermates. MCP-1 protein in cell culture media was determined by an ELISA assay and MCP-1 mRNA by the semi-quantitative RT-PCR. Results: TNF-␣, dose-dependently, elicited a marked increase in the amount of MCP-1 protein produced by A10 SMCs. A time-couse analysis showed that the induction was evident at 2 h and persistent up to 48 h. Similarly, TNF-␣ rapidly upregulated the level of MCP-1 mRNA in the cells, suggesting the induction may take place at the gene expression level. To understand the role of PKC␦ in regulation of MCP-1 expression, we pretreated
A10 cells with an increasing amount of rottlerin (0, 0.1, 0.5, 1 and 2 M), a selective inhibitor to PKC␦. Rottlerin dose-dependently blocked the induction of MCP-1 protein and mRNA. To confirm these results obtained with the chemical inhibitor, we employed PKC␦ knock-out mice. Aortic SMCs isolated from PKC␦ null mice or their wild-type littermates were treated with TNF-␣ as above. PKC␦ gene deficiency led to a complete loss of ability of SMCs to express MCP-1. Finally, we tested whether molecular activation of PKC␦ is sufficient to induce MCP-1 expression. To this end, we ectopically expressed PKC␦ in A10 cells via an adenoviral vector that expresses the wild-type PKC␦. Compared to the viral vector, Ad-PKC␦ elicited an about two-fold increase in MCP-1 expression. Conclusions: Taken together, our data show that the proinflammatory cytokine TNF-␣ is a potent inducer of MCP-1 expression in vascular SMCs. TNF-␣ stimulates MCP-1 expression through a signaling pathway that involves PKC␦. Further studies are mandated to test whether inhibition of PKC␦ could prevent MCP-1 expression thus monocytes recruitment in animal models following arterial injury.
P296. A NEW RABBIT MODEL OF DIABETIC ISCHEMIC WOUND HEALING USING HYPERSPECTRAL IMAGING AND MOLECULAR ANALYSIS. L. Pradhan1, X. Cai 2, N. Andersen2, M. Jain1, J. Malek1, M. Contreras1, A. Veves1, F. W. Logerfo1; 1Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, 2Harvard Medical School, Boston, MA Background: Diabetic foot ulceration (DFU) significantly impairs the quality of life of patients, leads to prolonged hospitalization, and commonly leads to major amputation. Abnormal wound healing is a key cause of the chronic non-healing wound. Inflammation, angiogenesis, and re-epithelization are essential phases of normal wound healing. The cytokines secreted in the wound microenvironment play a role in the inflammatory and angiogenesis phases of wound healing. Abnormalities in the expression and activity of inflammatory cytokines, followed by reduced infiltration and angiogenesis, can disrupt normal wound healing, contributing both to the development of DFU and its failure to heal. Of late, dysregulation of neuropeptides secreted from peripheral nerves has also been implicated in abnormal wound healing. The exact mechanisms by which cytokines and neuropeptides affect wound healing are not well understood. To systematically understand the pathobiology of diabetic wound healing, it is vital to develop in vivo models that can mimic the complexity of DFU. Model: Rabbits are made diabetic using alloxan monohydrate (100 mg/kg i.v.). After the induction of diabetes, surgery is performed on the rabbit ears to create wounds. Dermal circulation to the left ear is interrupted by ligating the rostral and central arteries, thus modeling ischemia. In the right