- Email: [email protected]
Nitric Oxide Inhibits Neointimal Hyperplasia More Effectively In A Type 1 Diabetic Model With Tight Glucose Control
254
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS
(P<0.05). PCNA staining was reduced substantially in the arteries of mice treated with GA-GNPs. Conclusions: GA-GNPs reduce vascular SMC proliferation and migration in vitro. These effects may be mediated through reduced activation of ERK1/2. Meanwhile, local treatment with GA-GNPs in areas of arterial injury prone to neointimal hyperplasia and subsequent stenosis arrests the progress of this disease in mice. This study suggests a novel therapeutic strategy for the vascular lession formation.
29.9. Nitric Oxide Inhibits Neointimal Hyperplasia More Effectively In A Type 1 Diabetic Model With Tight Glucose Control. Z. M. Emond, Z. Wang, G. E. Havelka, J. Martinez, M. R. Kibbe; Northwestern University, Chicago, IL Introduction: Diabetic patients have been shown to develop greater restenosis from neointimal hyperplasia after vascular interventions. Our lab recently reported that nitric oxide (NO) loses its ability to inhibit neointimal hyperplasia in type 1 diabetic rats. However, the efficacy of NO was restored in type 1 diabetic rats when given exogenous insulin. Unfortunately, the method of insulin delivery did not reduce blood glucose levels to normoglycemic levels, thus differentiating the effect of insulin from glucose on the efficacy of NO was difficult. The aim of the current study was to evaluate the efficacy of NO in type 1 diabetic rats with no, moderate, and tight glucose control. We hypothesized that NO will be more effective in the diabetic environments with the addition of exogenous insulin. Methods: Type 1 diabetes was induced in Lean Zucker (LZ) rats with a single injection of streptozotosin (60mg/kg IP). After one week, rats with glucose>300mg/dL were included in the study. Groups include uncontrolled, moderately controlled, and tightly controlled glucose using subcutaneously implanted insulin pellets (2U/pellet/24hr). Blood glucose levels were evaluated throughout the 6 week treatment and insulin dosage were adjusted accordingly to maintain group assignment. After 4 weeks, the carotid artery injury model was performed. Treatment groups included: injury and injury+PROLI/NO (20 mg) [n¼6/group]. Contralateral arteries served as controls. Arteries were harvested 2 weeks following carotid balloon injury and processed histologically for morphometric assessment. Results: Glucose levels were significantly different between all groups (p<0.05). Glucose levels for uncontrolled rats averaged 487653, moderately controlled rats 280683, and tightly controlled rats 173616. Insulin administration for the moderately controlled rats averaged 2.8 units/24 hours and for the tightly controlled rats averaged 3.2 units/24 hours. In the type 1 diabetic rats, insulin induced a dose-dependent increase in neointimal hyperplasia, with the greatest effect seen in the tightly controlled group (1.049 vs 0.337, p<0.001). Interestingly, NO significantly inhibited intimal area in the moderate and tightly controlled groups (72% vs. 89%, respectively, p¼0.01), but not in the uncontrolled group, and the efficacy of NO was greatest in the tightly controlled vs. moderately controlled group (I/M 81% vs. 63%, respectively, p<0.05). NO was noted to have a modest effect on the media in all three treatment groups (24 - 32% inhibition), but there were not statistically significant differences between the groups. Conclusions: In this study we show that the efficacy of NO is clearly dependent on the metabolic environment, with the greatest efficacy of NO observed in rats with tight glucose control. These data provide important insights into the biology of NO and will lead to the development of better NO-based therapies that will be equally effective in all patients.
29.10. The Smooth Muscle Myosin Heavy Chain Cre-loxp System Definitively Demonstrates Medial Smooth Muscle Involvement in Neointimal Hyperplasia With Ligation and Wire Injury Models. M. S. Hong,1,2 T. Gupta,1,2 S. A. Berceli,1,2 Z. Jiang1,2; 1University of Florida College of Medicine, Gainesville, FL; 2Malcom Randall Veterans Affairs Medical Center, Gainesville, FL
Introduction: Intimal hyperplasia is a maladaptive response involving improper organization of cellular and matrix components by smooth muscle cells (SMCs). While research efforts have focused on the regulation of the phenotype and function of neointimal SMCs, a critical and unresolved issue is the origin of these cells. Although medial SMCs (mSMCs) have traditionally been considered as the main source, direct evidence remains lacking. Challenging this paradigm, recent studies suggest the involvement of bone marrow cells in neointimal hyperplasia. Uncertainty remains on whether these bone marrow cells can fully mature to functional SMCs, due to difficulty in characterizing the full repertoire of the SMC proteome, particularly in acquiring the most definitive SMC marker: smooth muscle myosin heavy chain (SMMHC). In order to address these issues, we established a Cre-loxP system to trace the destination of mSMCs in two models of intimal hyperplasia. Methods: We employed a Cre-loxP system consisting of Rosa26R and SMMHC-CreER components that allows exclusive tagging of mSMCs with b-galactosidase following tamoxifen induction. Male R26R;SMMHC-CreER mice (8-12 week old) received intra-peritoneal tamoxifen injections (1.0mg/10g body weight 3 10 d). On the day following the last tamoxifen administration, unmanipulated arteries (aorta, carotid, femoral) were collected for evaluation of baseline mSMC labeling. In a separate group of mice, we induced intimal hyperplasia in the common carotid and femoral artery via complete ligation and wire injury, respectively. The injured and contralateral uninjured vessels were both perfusion-fixed at one month after surgery, and samples were stained with X-gal to identify the neointimal cells of mSMC origin. Results: Uniform X-gal staining was observed exclusively in medial SMCs of all baseline and contralateral uninjured arteries, demonstrating homogeneous and durable labeling of mSMCs via the Cre-loxP technique. Both the injured carotid and femoral arteries developed robust neointimal formation. Staining with X-gal revealed relatively more b-galactosidase positive cells in the neointima formed in the ligation model compared to the wire injury model. However, the fraction of b-galactosidase positive neointimal cells varied widely among vessels with either type of injury, indicating significant heterogeneity in the relative contribution of mSMCs in the neointima among different models and between individual animals. Conclusions: Using a genetic cell tracing method, we provide direct evidence for the contribution of mSMCs to neointimal hyperplasia following arterial injury. Notable variation exists in the presence of mSMCs in the neointima among different models and individual animals. Further studies will be aimed at
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS
(P<0.05). PCNA staining was reduced substantially in the arteries of mice treated with GA-GNPs. Conclusions: GA-GNPs reduce vascular SMC proliferation and migration in vitro. These effects may be mediated through reduced activation of ERK1/2. Meanwhile, local treatment with GA-GNPs in areas of arterial injury prone to neointimal hyperplasia and subsequent stenosis arrests the progress of this disease in mice. This study suggests a novel therapeutic strategy for the vascular lession formation.
29.9. Nitric Oxide Inhibits Neointimal Hyperplasia More Effectively In A Type 1 Diabetic Model With Tight Glucose Control. Z. M. Emond, Z. Wang, G. E. Havelka, J. Martinez, M. R. Kibbe; Northwestern University, Chicago, IL Introduction: Diabetic patients have been shown to develop greater restenosis from neointimal hyperplasia after vascular interventions. Our lab recently reported that nitric oxide (NO) loses its ability to inhibit neointimal hyperplasia in type 1 diabetic rats. However, the efficacy of NO was restored in type 1 diabetic rats when given exogenous insulin. Unfortunately, the method of insulin delivery did not reduce blood glucose levels to normoglycemic levels, thus differentiating the effect of insulin from glucose on the efficacy of NO was difficult. The aim of the current study was to evaluate the efficacy of NO in type 1 diabetic rats with no, moderate, and tight glucose control. We hypothesized that NO will be more effective in the diabetic environments with the addition of exogenous insulin. Methods: Type 1 diabetes was induced in Lean Zucker (LZ) rats with a single injection of streptozotosin (60mg/kg IP). After one week, rats with glucose>300mg/dL were included in the study. Groups include uncontrolled, moderately controlled, and tightly controlled glucose using subcutaneously implanted insulin pellets (2U/pellet/24hr). Blood glucose levels were evaluated throughout the 6 week treatment and insulin dosage were adjusted accordingly to maintain group assignment. After 4 weeks, the carotid artery injury model was performed. Treatment groups included: injury and injury+PROLI/NO (20 mg) [n¼6/group]. Contralateral arteries served as controls. Arteries were harvested 2 weeks following carotid balloon injury and processed histologically for morphometric assessment. Results: Glucose levels were significantly different between all groups (p<0.05). Glucose levels for uncontrolled rats averaged 487653, moderately controlled rats 280683, and tightly controlled rats 173616. Insulin administration for the moderately controlled rats averaged 2.8 units/24 hours and for the tightly controlled rats averaged 3.2 units/24 hours. In the type 1 diabetic rats, insulin induced a dose-dependent increase in neointimal hyperplasia, with the greatest effect seen in the tightly controlled group (1.049 vs 0.337, p<0.001). Interestingly, NO significantly inhibited intimal area in the moderate and tightly controlled groups (72% vs. 89%, respectively, p¼0.01), but not in the uncontrolled group, and the efficacy of NO was greatest in the tightly controlled vs. moderately controlled group (I/M 81% vs. 63%, respectively, p<0.05). NO was noted to have a modest effect on the media in all three treatment groups (24 - 32% inhibition), but there were not statistically significant differences between the groups. Conclusions: In this study we show that the efficacy of NO is clearly dependent on the metabolic environment, with the greatest efficacy of NO observed in rats with tight glucose control. These data provide important insights into the biology of NO and will lead to the development of better NO-based therapies that will be equally effective in all patients.
29.10. The Smooth Muscle Myosin Heavy Chain Cre-loxp System Definitively Demonstrates Medial Smooth Muscle Involvement in Neointimal Hyperplasia With Ligation and Wire Injury Models. M. S. Hong,1,2 T. Gupta,1,2 S. A. Berceli,1,2 Z. Jiang1,2; 1University of Florida College of Medicine, Gainesville, FL; 2Malcom Randall Veterans Affairs Medical Center, Gainesville, FL
Introduction: Intimal hyperplasia is a maladaptive response involving improper organization of cellular and matrix components by smooth muscle cells (SMCs). While research efforts have focused on the regulation of the phenotype and function of neointimal SMCs, a critical and unresolved issue is the origin of these cells. Although medial SMCs (mSMCs) have traditionally been considered as the main source, direct evidence remains lacking. Challenging this paradigm, recent studies suggest the involvement of bone marrow cells in neointimal hyperplasia. Uncertainty remains on whether these bone marrow cells can fully mature to functional SMCs, due to difficulty in characterizing the full repertoire of the SMC proteome, particularly in acquiring the most definitive SMC marker: smooth muscle myosin heavy chain (SMMHC). In order to address these issues, we established a Cre-loxP system to trace the destination of mSMCs in two models of intimal hyperplasia. Methods: We employed a Cre-loxP system consisting of Rosa26R and SMMHC-CreER components that allows exclusive tagging of mSMCs with b-galactosidase following tamoxifen induction. Male R26R;SMMHC-CreER mice (8-12 week old) received intra-peritoneal tamoxifen injections (1.0mg/10g body weight 3 10 d). On the day following the last tamoxifen administration, unmanipulated arteries (aorta, carotid, femoral) were collected for evaluation of baseline mSMC labeling. In a separate group of mice, we induced intimal hyperplasia in the common carotid and femoral artery via complete ligation and wire injury, respectively. The injured and contralateral uninjured vessels were both perfusion-fixed at one month after surgery, and samples were stained with X-gal to identify the neointimal cells of mSMC origin. Results: Uniform X-gal staining was observed exclusively in medial SMCs of all baseline and contralateral uninjured arteries, demonstrating homogeneous and durable labeling of mSMCs via the Cre-loxP technique. Both the injured carotid and femoral arteries developed robust neointimal formation. Staining with X-gal revealed relatively more b-galactosidase positive cells in the neointima formed in the ligation model compared to the wire injury model. However, the fraction of b-galactosidase positive neointimal cells varied widely among vessels with either type of injury, indicating significant heterogeneity in the relative contribution of mSMCs in the neointima among different models and between individual animals. Conclusions: Using a genetic cell tracing method, we provide direct evidence for the contribution of mSMCs to neointimal hyperplasia following arterial injury. Notable variation exists in the presence of mSMCs in the neointima among different models and individual animals. Further studies will be aimed at