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Nogo-B Modulates Intimal Thickening during Vein Graft Adaptation
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ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS
49.5. Inhaled CO Decreases Fibrin Deposition and Activates Fibrinolysis: A Potential Mechanism of Vasoprotection. F. Entabi, M. Alef, G. Hong, P. Loughran, D. Stolz, E. Tzeng; University of Pittsburgh, Pittsburgh, PA Introduction: We have previously reported that inhaled carbon monoxide (CO) dramatically inhibits intimal hyperplasia (IH) in both rodent and porcine models of angioplasty injury. The most attractive feature of inhaled CO as a potential clinical therapy is that a brief exposure to inhaled CO mediates a prolonged vasoprotective effect. While the vasoprotection offered by CO is in part through its potent anti-inflammatory properties, CO may also impact one of the earliest responses to vascular injury including thrombus formation and fibrin deposition, both being very strong stimuli for IH. CO has been reported to improve the outcome of lung injury by enhancing fibrinolysis through the down-regulation of plasminogen activator inhibitor-1 (PAI-1) to reverse microvascular thrombosis. Thus, we hypothesize that CO may mediate vasoprotection through the upregulation of fibrinolysis. Methods: Sprague Dawley rats were treated with inhaled CO at 250 ppm for 1 hr or maintained in room air (RA). The rats then underwent unilateral carotid artery injury by balloon angioplasty and were sacrificed at 24 hrs. The carotid arteries were collected for scanning electron microscopy (SEM). In another experiment, rats were also treated with inhaled CO (250 ppm for 1 hr) or maintained in RA. Resident peritoneal macrophages (PM) were collected, washed, and cultured under standard conditions overnight. Cells were then cultured with or without LPS (100 ng/ml) for 24 hrs. Protein was collected for Western blot analysis for PAI-1 and actin. Results: Carotid injury in rats maintained in RA exhibited an extensive amount of platelet adherence and fibrin deposition at 24 hrs post-injury by SEM (Figure 1A). Injury following inhaled CO treatment, however, did not reduce platelet adherence to the arterial wall. However, fibrin deposition on the luminal surface appeared to be significantly decreased (N ¼ 3/treatment). Resident PMs isolated from RA rats expressed baseline levels of PAI-1 (Figure 1B). PM from CO treated rats, however, had significantly reduced PAI-1 levels. LPS treatment increased PAI-1 in PMs from both RA and CO treated rats but to a lesser extent in those from CO rats. Culturing PMs in a CO incubator also inhibited PAI-1 expression but not as dramatically as the PMs treated with CO in vivo. Conclusions: Our findings demonstrate that a brief exposure to inhaled CO did not change platelet adherence to injured carotid arteries 1 day post-injury but did reduce fibrin deposition compared with RA treated rats. This brief inhaled CO treatment was also associated with a prolonged reduction in PAI-1 expression that was more dramatic than achieved with in vitro
CO treatment. Taken together, inhaled CO reduces fibrin deposition following vascular injury potentially by increasing fibrinolytic activity through the prolonged down-regulation of PAI-1. This may be represent another mechanism by which brief CO treatment can have such a dramatic effect on inhibiting IH.
49.6. Nitric Oxide Increases Breakdown of the UbiquitinConjugating Enzyme UbcH10 in Vivo. N. D. Tsihlis, C. S. Oustwani, A. K. Vavra, Q. Jiang, M. R. Kibbe; Northwestern University, Chicago, IL Introduction: Nitric oxide (NO) has been shown to limit the formation of neointimal hyperplasia in animal models of arterial injury. NO is also known to arrest vascular smooth muscle cells (VSMC) in the G0/G1 phase of the cell cycle, and the ubiquitin-proteasome pathway is responsible for degradation of proteins that regulate cell cycle progression. We previously reported that NO reduced the expression of UbcH3, 10, and 12 in VSMC, all of which are essential for proper cell cycle advancement. We also showed that knockdown of UbcH10 expression by siRNA caused a decrease in VSMC proliferation in vitro. Thus, the aim of this study is to evaluate the effect of NO on UbcH10 expression in vivo. We hypothesize that NO prevents VSMC proliferation, and hence neointimal hyperplasia, by decreasing the expression of UbcH10 in vivo. Methods: VSMC harvested from aortas of male Sprague Dawley rats were exposed to the NO donor DETA/NO or transfected with a plasmid bearing UbcH10. VSMC were also treated with the proteasome inhibitor MG132 (1 mM) in the presence or absence of DETA/NO, and expression of UbcH10 was assessed by Western blot analysis. The levels of free and ubiquitin-bound UbcH10 were quantified using ImageJ. The rat carotid artery injury model was performed in male Sprague Dawley rats. Treatment groups included injury or injury þ PROLI/NO (20 mg). UbcH10 expression was assessed by Western blot analysis on homogenized arteries excised at 3 days. Results: Here we show that overexpression of UbcH10 in VSMC resulted in increased cellular proliferation in vitro (100% vs. 117%, P ¼ 0.037). We also report that NO increased the ratio of ubiquitinated UbcH10 to free UbcH10 in NO-treated VSMC. To gain insights into the mechanism by which NO acts to prevent neointimal hyperplasia, we assessed the effects of arterial injury and exogenous NO administration on UbcH10 expression in rat carotid arteries. We found an increase in UbcH10 expression in the injury alone group and a significant decrease in UbcH10 expression in the injury þ PROLI/NO group. Interestingly, we also observed a marked increase in the level of polyubiquitinated UbcH10 in the injury þ PROLI/NO group. Specifically, the bands in the ladder of ubiquitinated UbcH10 observed in the uninjured and injury alone groups were shifted to a weight consistent with the addition of 5 ubiquitin molecules to UbcH10 in the injury þ PROLI/NO group. To determine whether NO was preventing UbcH10 degradation as suggested by the increase in polyubiquitinated UbcH10, we treated VSMC with the proteasome inhibitor MG132. MG132 caused an increase in ubiquitinated proteins, but a decrease in UbcH10 expression, similar to that observed with NO. Conclusions: We report that increased expression of UbcH10 caused an increase in VSMC proliferation, and that administration of the NO donor PROLI/NO after arterial injury leads to an increase in polyubiquitinated UbcH10. However, inhibition of the proteasome in VSMC decreased UbcH10 expression, indicating that NO may inhibit VSMC proliferation by causing increased breakdown of UbcH10 independent of the proteasome. Thus, UbcH10 may be a promising therapeutic target for inhibiting neointimal hyperplasia without causing side effects associated with proteasome inhibition.
49.7. Nogo-B Modulates Intimal Thickening during Vein Graft Adaptation. Y. Kondo, A. Muto, T. Yai, N. Hibino, A. Feigel, K. Ziegler, L. Model, S. Eghbalieh, J. Yu, W. Sessa, A. Dardik; Yale University School of Medicine, New Haven, CT
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS Background: Vein graft adaptation to the arterial environment results in vein wall thickening, cell proliferation and migration, as well as synthesis of extracellular matrix. Nogo-B has been recently implicated as a protective factor in the vascular system, limiting the response to injury, with diminished levels after angioplasty. We hypothesized that Nogo-B levels would diminish in a vein graft after exposure to arterial levels of pressure and shear stress. We examined the expression of Nogo-B, as well as its three receptors, Nogo-receptor (NgR), Nogo-B-receptor (NgBR), and PirB (Paired immunoglobulinlike receptor B, Lilrb3), in a mouse model of vein graft adaptation. Methods: The intrathoracic inferior vena cava of donor C57Bl/6 or Nogo-A/B knockout (Nogo-KO) mice was harvested and implanted as an interposition vein graft into the infrarenal abdominal aorta of recipient C57Bl/6 mice. Mice were sacrificed at 3weeks after implantation. Specimens were evaluated for morphology using computerized morphometry (NIH Image J). mRNA transcript levels of Nogo-B, NgR, NgBR, PirB and GAPDH were measured by quantitative realtime PCR (qPCR). Immunohistological analysis and Western blotting were performed using the antibodies: Nogo (N18: Santa Cruz), NogoB (1761), F4/80 (AbD serotec). Results: After three weeks all vein grafts showed significant intimal thickening, with increased intimal thickening in Nogo-KO mice (0.125 6 .025 mm) compared to control mice (0.043 6 0.003 mm)(n ¼ 4; p < 0.05). There was increased monocyte infiltration in vein grafts derived from Nogo-KO mice compared to vein grafts derived from control mice (2.5-fold; n ¼ 4; p < 0.01). In control vein grafts, Nogo-B protein levels were strongly increased compared to preimplantation vein (immunohistochemistry density approximately 6-fold, Western blot density approximately 10-fold). Similarly, Nogo-B RNA transcripts were increased 2-fold in the vein graft compared to preimplantation vein. Transcript levels of the NgR and NgBR receptors were unchanged in vein grafts compared to preimplantation veins, whereas the PirB receptor transcript level was increased approximately 20-fold (n ¼ 3; P < 0.05). Discussion: Nogo-B transcripts and protein, as well as transcripts for its PirB receptor, were increased during vein graft adaptation, suggesting that vein graft adaptation is not a response to injury, but an adaptive remodeling response. Nogo-B is a factor limiting this adaptive response, and may signal via the PirB receptor. These results suggest that Nogo-B signaling is a mechanism of vein graft adaptation and might be a therapeutic target for human trials targeting vein graft disease.
49.8. A Possible Role for MAP Kinases, MARCKS Interaction in the Development of Intimal Hyperplasia and Graft Failure. L. M. Korepta,1 M. C. Martin,2 P. Guthrie,2 L. Pradhan,1 C. Ferran,1 F. W. LoGerfo1; 1Harvard Medical School/Beth Israel Deaconess Medical Center, Boston, MA; 2Beth Israel Deaconess Medical Center, Boston, MA Introduction: The myrisolated, alanine-rich C-kinase substrate (MARCKS), a major protein kinase C substrate, has been verified to be upregulated in intimal hyperplasia (IH). However, the signaling pathway of MARKCS has yet to be elucidated. Through inhibition of vascular smooth muscle cell (VSMC) migration and proliferation, p27kip1, a cyclin dependent kinase inhibitor, is known to regulate IH. Previous work in our lab demonstrated that MARCKS knockdown using MARCKS siRNA decreases VSMC migration and proliferation and increases p27kip1 gene and protein expression. The MAP kinases, SAP/JNK and p38 that lead to cell cycle arrest and apoptosis and p42/ 44 that leads to progression of cell cycle and cell proliferation, are known to be regulated by p27kip1. The goal of the present study is to monitor the protein expression of p38, p42/44, and SAP-JNK MAP kinases in the setting of MARCKS siRNA knockdown. Methods: Human Coronary Artery Smooth Muscle Cells (HCASMC) were transfected with 50 nm control (scrambled) or MARCKS small interfering RNA (siRNA) or vehicle alone for 24 hours. The cells were serum starved for 24 hours by placing them in 0.1% fetal bovine serum (FBS) growth media, in order to synchronize all of the cells to the G0 phase. HCASMCs were then stimulated with a 10% FBS
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growth media and cells were harvested for total protein extraction at different time points post-stimulation (0 and 30 mins, 18 and 24 hours). Western blot analysis was performed to monitor the protein expression of MARCKS, p27kip1, and the MAP kinases p38, p42/44, and SAP/JNK. Results: All of the following results are presented as percent change in protein expression in MARCKS siRNA treated HCASMCs compared to control siRNA treated HCASMCs. MARCKS siRNA treatment resulted in: 1) 40-75% decrease in MARCKS expression at all different time points 2) 30% increase in p27kip1 at all different time points 3) no significant change in p38 expression at 0 and 30 minutes, but increase by 25% at 18 and 24 hours 3) no significant change in SAP-JNK expression at 0 and 30 minutes, but increase by 30% at 18 hours and 78% at 24 hours and 4) no significant change in p42/44 expression at all different time points. Conclusions: The results of this study shed light on a possible IH signaling pathway mediated by MARCKS via p27kip1, p38, and SAP-JNK MAP kinase regulation and thus supporting a role for MARCKS as a therapeutic target for prevention of IH in venous bypass graft failure.
ORAL SESSION: FRIDAY 2/5 3:30 PM CARDIOTHORACIC AND VASCULAR 5: CARDIAC & VASCULAR DISEASE 50.1. Ischemic Preconditioning Preserves Respiratory Complex Activity and Decreases Reactive Oxygen Species Production in Rat Heart Mitochondria. D. S. Lee,1 G. E. Steinbaugh,1 R. Quarrie,1 B. M. Cramer,1 D. R. Pfeiffer,2 J. L. Zweier,1 J. A. Crestanello1; 1The Ohio State University Medical Center, Columbus, OH; 2The Ohio State University, Columbus, OH Mitochondria (mito) are a major source of reactive oxygen species (ROS) during ischemia reperfusion (IR). IR damages mito respiratory complexes and increases mitochondrial ROS production. Ischemic preconditioning (IPC) protects myocardium from IR injury. It is unclear what the effect of IPC is on mito ROS generation. The purpose of this study was to evaluate the effect of IPC on mito ROS production and respiratory complex activity. Isolated rat hearts (n ¼ 6/group) were subjected to either CONTROL: 30 minutes (min) of equilibration (EQ), 30 min of ischemia (I), and 30 min of reperfusion (RP), or IPC: 10 min of EQ, preconditioning was then induced by two 5 min episodes of ischemia separated by 5 minutes of reperfusion, 30 min I, and 30 min of RP. Mitochondria were isolated at end EQ, at end I, and at end RP. Superoxide production by complexes I and III was assessed by electron paramagnetic resonance (EPR) spectroscopy using the spin trap 5,5-dimethylpyrroline N-oxide (DMPO). Respiratory complex I activity was measured by polarography. Data is expressed as mean6sem.
Mitochondrial superoxide production increases during ischemia and reperfusion. Induction of preconditioning increases mito superoxide production at end EQ. IPC does not prevent the increase in superoxide during ischemia, but decreases mito superoxide production at end RP. IR decreases mito respiratory complex I activity. Induction of preconditioning does not impair complex I activity at end EQ, but preserves it at end RP. We conclude that IPC triggers mitochondrial superoxide production before ischemia and results in respiratory complex protection and lower ROS production during reperfusion.
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS
49.5. Inhaled CO Decreases Fibrin Deposition and Activates Fibrinolysis: A Potential Mechanism of Vasoprotection. F. Entabi, M. Alef, G. Hong, P. Loughran, D. Stolz, E. Tzeng; University of Pittsburgh, Pittsburgh, PA Introduction: We have previously reported that inhaled carbon monoxide (CO) dramatically inhibits intimal hyperplasia (IH) in both rodent and porcine models of angioplasty injury. The most attractive feature of inhaled CO as a potential clinical therapy is that a brief exposure to inhaled CO mediates a prolonged vasoprotective effect. While the vasoprotection offered by CO is in part through its potent anti-inflammatory properties, CO may also impact one of the earliest responses to vascular injury including thrombus formation and fibrin deposition, both being very strong stimuli for IH. CO has been reported to improve the outcome of lung injury by enhancing fibrinolysis through the down-regulation of plasminogen activator inhibitor-1 (PAI-1) to reverse microvascular thrombosis. Thus, we hypothesize that CO may mediate vasoprotection through the upregulation of fibrinolysis. Methods: Sprague Dawley rats were treated with inhaled CO at 250 ppm for 1 hr or maintained in room air (RA). The rats then underwent unilateral carotid artery injury by balloon angioplasty and were sacrificed at 24 hrs. The carotid arteries were collected for scanning electron microscopy (SEM). In another experiment, rats were also treated with inhaled CO (250 ppm for 1 hr) or maintained in RA. Resident peritoneal macrophages (PM) were collected, washed, and cultured under standard conditions overnight. Cells were then cultured with or without LPS (100 ng/ml) for 24 hrs. Protein was collected for Western blot analysis for PAI-1 and actin. Results: Carotid injury in rats maintained in RA exhibited an extensive amount of platelet adherence and fibrin deposition at 24 hrs post-injury by SEM (Figure 1A). Injury following inhaled CO treatment, however, did not reduce platelet adherence to the arterial wall. However, fibrin deposition on the luminal surface appeared to be significantly decreased (N ¼ 3/treatment). Resident PMs isolated from RA rats expressed baseline levels of PAI-1 (Figure 1B). PM from CO treated rats, however, had significantly reduced PAI-1 levels. LPS treatment increased PAI-1 in PMs from both RA and CO treated rats but to a lesser extent in those from CO rats. Culturing PMs in a CO incubator also inhibited PAI-1 expression but not as dramatically as the PMs treated with CO in vivo. Conclusions: Our findings demonstrate that a brief exposure to inhaled CO did not change platelet adherence to injured carotid arteries 1 day post-injury but did reduce fibrin deposition compared with RA treated rats. This brief inhaled CO treatment was also associated with a prolonged reduction in PAI-1 expression that was more dramatic than achieved with in vitro
CO treatment. Taken together, inhaled CO reduces fibrin deposition following vascular injury potentially by increasing fibrinolytic activity through the prolonged down-regulation of PAI-1. This may be represent another mechanism by which brief CO treatment can have such a dramatic effect on inhibiting IH.
49.6. Nitric Oxide Increases Breakdown of the UbiquitinConjugating Enzyme UbcH10 in Vivo. N. D. Tsihlis, C. S. Oustwani, A. K. Vavra, Q. Jiang, M. R. Kibbe; Northwestern University, Chicago, IL Introduction: Nitric oxide (NO) has been shown to limit the formation of neointimal hyperplasia in animal models of arterial injury. NO is also known to arrest vascular smooth muscle cells (VSMC) in the G0/G1 phase of the cell cycle, and the ubiquitin-proteasome pathway is responsible for degradation of proteins that regulate cell cycle progression. We previously reported that NO reduced the expression of UbcH3, 10, and 12 in VSMC, all of which are essential for proper cell cycle advancement. We also showed that knockdown of UbcH10 expression by siRNA caused a decrease in VSMC proliferation in vitro. Thus, the aim of this study is to evaluate the effect of NO on UbcH10 expression in vivo. We hypothesize that NO prevents VSMC proliferation, and hence neointimal hyperplasia, by decreasing the expression of UbcH10 in vivo. Methods: VSMC harvested from aortas of male Sprague Dawley rats were exposed to the NO donor DETA/NO or transfected with a plasmid bearing UbcH10. VSMC were also treated with the proteasome inhibitor MG132 (1 mM) in the presence or absence of DETA/NO, and expression of UbcH10 was assessed by Western blot analysis. The levels of free and ubiquitin-bound UbcH10 were quantified using ImageJ. The rat carotid artery injury model was performed in male Sprague Dawley rats. Treatment groups included injury or injury þ PROLI/NO (20 mg). UbcH10 expression was assessed by Western blot analysis on homogenized arteries excised at 3 days. Results: Here we show that overexpression of UbcH10 in VSMC resulted in increased cellular proliferation in vitro (100% vs. 117%, P ¼ 0.037). We also report that NO increased the ratio of ubiquitinated UbcH10 to free UbcH10 in NO-treated VSMC. To gain insights into the mechanism by which NO acts to prevent neointimal hyperplasia, we assessed the effects of arterial injury and exogenous NO administration on UbcH10 expression in rat carotid arteries. We found an increase in UbcH10 expression in the injury alone group and a significant decrease in UbcH10 expression in the injury þ PROLI/NO group. Interestingly, we also observed a marked increase in the level of polyubiquitinated UbcH10 in the injury þ PROLI/NO group. Specifically, the bands in the ladder of ubiquitinated UbcH10 observed in the uninjured and injury alone groups were shifted to a weight consistent with the addition of 5 ubiquitin molecules to UbcH10 in the injury þ PROLI/NO group. To determine whether NO was preventing UbcH10 degradation as suggested by the increase in polyubiquitinated UbcH10, we treated VSMC with the proteasome inhibitor MG132. MG132 caused an increase in ubiquitinated proteins, but a decrease in UbcH10 expression, similar to that observed with NO. Conclusions: We report that increased expression of UbcH10 caused an increase in VSMC proliferation, and that administration of the NO donor PROLI/NO after arterial injury leads to an increase in polyubiquitinated UbcH10. However, inhibition of the proteasome in VSMC decreased UbcH10 expression, indicating that NO may inhibit VSMC proliferation by causing increased breakdown of UbcH10 independent of the proteasome. Thus, UbcH10 may be a promising therapeutic target for inhibiting neointimal hyperplasia without causing side effects associated with proteasome inhibition.
49.7. Nogo-B Modulates Intimal Thickening during Vein Graft Adaptation. Y. Kondo, A. Muto, T. Yai, N. Hibino, A. Feigel, K. Ziegler, L. Model, S. Eghbalieh, J. Yu, W. Sessa, A. Dardik; Yale University School of Medicine, New Haven, CT
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS Background: Vein graft adaptation to the arterial environment results in vein wall thickening, cell proliferation and migration, as well as synthesis of extracellular matrix. Nogo-B has been recently implicated as a protective factor in the vascular system, limiting the response to injury, with diminished levels after angioplasty. We hypothesized that Nogo-B levels would diminish in a vein graft after exposure to arterial levels of pressure and shear stress. We examined the expression of Nogo-B, as well as its three receptors, Nogo-receptor (NgR), Nogo-B-receptor (NgBR), and PirB (Paired immunoglobulinlike receptor B, Lilrb3), in a mouse model of vein graft adaptation. Methods: The intrathoracic inferior vena cava of donor C57Bl/6 or Nogo-A/B knockout (Nogo-KO) mice was harvested and implanted as an interposition vein graft into the infrarenal abdominal aorta of recipient C57Bl/6 mice. Mice were sacrificed at 3weeks after implantation. Specimens were evaluated for morphology using computerized morphometry (NIH Image J). mRNA transcript levels of Nogo-B, NgR, NgBR, PirB and GAPDH were measured by quantitative realtime PCR (qPCR). Immunohistological analysis and Western blotting were performed using the antibodies: Nogo (N18: Santa Cruz), NogoB (1761), F4/80 (AbD serotec). Results: After three weeks all vein grafts showed significant intimal thickening, with increased intimal thickening in Nogo-KO mice (0.125 6 .025 mm) compared to control mice (0.043 6 0.003 mm)(n ¼ 4; p < 0.05). There was increased monocyte infiltration in vein grafts derived from Nogo-KO mice compared to vein grafts derived from control mice (2.5-fold; n ¼ 4; p < 0.01). In control vein grafts, Nogo-B protein levels were strongly increased compared to preimplantation vein (immunohistochemistry density approximately 6-fold, Western blot density approximately 10-fold). Similarly, Nogo-B RNA transcripts were increased 2-fold in the vein graft compared to preimplantation vein. Transcript levels of the NgR and NgBR receptors were unchanged in vein grafts compared to preimplantation veins, whereas the PirB receptor transcript level was increased approximately 20-fold (n ¼ 3; P < 0.05). Discussion: Nogo-B transcripts and protein, as well as transcripts for its PirB receptor, were increased during vein graft adaptation, suggesting that vein graft adaptation is not a response to injury, but an adaptive remodeling response. Nogo-B is a factor limiting this adaptive response, and may signal via the PirB receptor. These results suggest that Nogo-B signaling is a mechanism of vein graft adaptation and might be a therapeutic target for human trials targeting vein graft disease.
49.8. A Possible Role for MAP Kinases, MARCKS Interaction in the Development of Intimal Hyperplasia and Graft Failure. L. M. Korepta,1 M. C. Martin,2 P. Guthrie,2 L. Pradhan,1 C. Ferran,1 F. W. LoGerfo1; 1Harvard Medical School/Beth Israel Deaconess Medical Center, Boston, MA; 2Beth Israel Deaconess Medical Center, Boston, MA Introduction: The myrisolated, alanine-rich C-kinase substrate (MARCKS), a major protein kinase C substrate, has been verified to be upregulated in intimal hyperplasia (IH). However, the signaling pathway of MARKCS has yet to be elucidated. Through inhibition of vascular smooth muscle cell (VSMC) migration and proliferation, p27kip1, a cyclin dependent kinase inhibitor, is known to regulate IH. Previous work in our lab demonstrated that MARCKS knockdown using MARCKS siRNA decreases VSMC migration and proliferation and increases p27kip1 gene and protein expression. The MAP kinases, SAP/JNK and p38 that lead to cell cycle arrest and apoptosis and p42/ 44 that leads to progression of cell cycle and cell proliferation, are known to be regulated by p27kip1. The goal of the present study is to monitor the protein expression of p38, p42/44, and SAP-JNK MAP kinases in the setting of MARCKS siRNA knockdown. Methods: Human Coronary Artery Smooth Muscle Cells (HCASMC) were transfected with 50 nm control (scrambled) or MARCKS small interfering RNA (siRNA) or vehicle alone for 24 hours. The cells were serum starved for 24 hours by placing them in 0.1% fetal bovine serum (FBS) growth media, in order to synchronize all of the cells to the G0 phase. HCASMCs were then stimulated with a 10% FBS
357
growth media and cells were harvested for total protein extraction at different time points post-stimulation (0 and 30 mins, 18 and 24 hours). Western blot analysis was performed to monitor the protein expression of MARCKS, p27kip1, and the MAP kinases p38, p42/44, and SAP/JNK. Results: All of the following results are presented as percent change in protein expression in MARCKS siRNA treated HCASMCs compared to control siRNA treated HCASMCs. MARCKS siRNA treatment resulted in: 1) 40-75% decrease in MARCKS expression at all different time points 2) 30% increase in p27kip1 at all different time points 3) no significant change in p38 expression at 0 and 30 minutes, but increase by 25% at 18 and 24 hours 3) no significant change in SAP-JNK expression at 0 and 30 minutes, but increase by 30% at 18 hours and 78% at 24 hours and 4) no significant change in p42/44 expression at all different time points. Conclusions: The results of this study shed light on a possible IH signaling pathway mediated by MARCKS via p27kip1, p38, and SAP-JNK MAP kinase regulation and thus supporting a role for MARCKS as a therapeutic target for prevention of IH in venous bypass graft failure.
ORAL SESSION: FRIDAY 2/5 3:30 PM CARDIOTHORACIC AND VASCULAR 5: CARDIAC & VASCULAR DISEASE 50.1. Ischemic Preconditioning Preserves Respiratory Complex Activity and Decreases Reactive Oxygen Species Production in Rat Heart Mitochondria. D. S. Lee,1 G. E. Steinbaugh,1 R. Quarrie,1 B. M. Cramer,1 D. R. Pfeiffer,2 J. L. Zweier,1 J. A. Crestanello1; 1The Ohio State University Medical Center, Columbus, OH; 2The Ohio State University, Columbus, OH Mitochondria (mito) are a major source of reactive oxygen species (ROS) during ischemia reperfusion (IR). IR damages mito respiratory complexes and increases mitochondrial ROS production. Ischemic preconditioning (IPC) protects myocardium from IR injury. It is unclear what the effect of IPC is on mito ROS generation. The purpose of this study was to evaluate the effect of IPC on mito ROS production and respiratory complex activity. Isolated rat hearts (n ¼ 6/group) were subjected to either CONTROL: 30 minutes (min) of equilibration (EQ), 30 min of ischemia (I), and 30 min of reperfusion (RP), or IPC: 10 min of EQ, preconditioning was then induced by two 5 min episodes of ischemia separated by 5 minutes of reperfusion, 30 min I, and 30 min of RP. Mitochondria were isolated at end EQ, at end I, and at end RP. Superoxide production by complexes I and III was assessed by electron paramagnetic resonance (EPR) spectroscopy using the spin trap 5,5-dimethylpyrroline N-oxide (DMPO). Respiratory complex I activity was measured by polarography. Data is expressed as mean6sem.
Mitochondrial superoxide production increases during ischemia and reperfusion. Induction of preconditioning increases mito superoxide production at end EQ. IPC does not prevent the increase in superoxide during ischemia, but decreases mito superoxide production at end RP. IR decreases mito respiratory complex I activity. Induction of preconditioning does not impair complex I activity at end EQ, but preserves it at end RP. We conclude that IPC triggers mitochondrial superoxide production before ischemia and results in respiratory complex protection and lower ROS production during reperfusion.