- Email: [email protected]
GP×1-double knockout mouse
S107
Abstracts
co-SMAD-4 that enhances TGF- signaling were also upregulated in Ex- compared to Sed-db/db mice hearts, implying further activation of fibrosis in these aged diabetic hearts. Finally, augmented collagen 1a and fibronectin levels, products of TGF- activity, were confirmed with immunofluoresecence and western blotting respectively in Ex-db/db mice hearts. CONCLUSION: These studies demonstrate that TGF- mediated cardiac profibrotic signaling may be upregulated with forced exercise in aged T2D hearts. Although the reasons are currently unknown, it is plausible that excess oxidative stress evident with such forced exercise paradigms may worsen cardiac prognosis in older T2D patients. Canadian Diabetes Association
124 USE OF GPⴛ-1 MIMETICS TO REDUCE DIABETICASSOCIATED ATHEROSCLEROSIS IN THE ApoE/GPⴛ1DOUBLE KNOCKOUT MOUSE
of the pro-inflammatory mediators P-IKK, P-JNK and TNF-␣ with GP⫻1 mimetic pre-treatment. FUTURE DIRECTIONS: Currently, we are performing oxidative stress analysis, immunohistochemistry and RT-PCR analysis of pro-inflammatory markers to gain an understanding of the mechanisms by which the GP⫻1 mimetic reduces DAA. CONCLUSION: Novel GP⫻1-mimetics show promise as an antiatherogenic therapeutic target via signalling pathways that limit inflammation. LEARNING INITIATIVE: This study is aimed at characterizing a therapeutic anti-oxidant drug that limits oxidative stress in cardiovascular complications associated with diabetes. Canadian Cardiovascular Society (CCS) CCS088 Highlighted Poster HEART FAILURE POTPOURRI Sunday, October 23, 2011
A Sharma, N Stefanovic, D Yuen, P Chew, M Tan, P Bernatchez, J de Haan Vancouver, British Columbia INTRODUCTION: Diabetes is a major risk factor for cardiovascular dis-
eases, in particular atherosclerosis. Oxidative stress contributes significantly to the pathogenesis of diabetic-associated atherosclerosis (DAA), predominantly by inducing pro-inflammatory pathways. Oxidative stress occurs through the upregulation of reactive oxygen species (ROS), such as superoxide, hydrogen peroxide (H2O2) and peroxynitrite (ONOO-) and their inadequate removal by anti-oxidant enzymes. Glutathione peroxidase 1 (GP⫻1) is a ubiquitously expressed anti-oxidant enzyme, which is involved in the reduction of H2O2 to water and also functions as an ONOO- reductase. Previously, we have shown that genetic deletion of the GP⫻1 gene in diabetic ApoE knockout (KO) mice augmented the atherosclerotic burden. In addition, administration of ebselen, a synthetic compound that mimics the action of GP⫻1, significantly reduces DAA in diabetic ApoE KO and ApoE/GP⫻1 double KO mice. In the current proposal, our aim is to extend these findings to newer GP⫻1 mimetics, which have been generated for their greater efficacy, potency and their potential to behave in a superior fashion to ebselen in vivo. OBJECTIVE: To investigate whether GP⫻1 mimetics can substitute for GP⫻1 in ApoE/GP⫻1 double KO mice and reduce atherosclerosis. Secondly, we aim to examine if GP⫻1 mimetics reduce proinflammatory pathways in vivo and in vitro. METHODS AND RESULTS: 8-week old male ApoE/GP⫻1 double KO mice were made diabetic with streptozotocin. Diabetic and non-diabetic control mice received the GP⫻1 mimetic by twice daily gavage (10mg/kg body weight) or vehicle only. After 20 weeks of diabetes, lesions were assessed by Sudan IV staining. The lesions were significantly increased in diabetic ApoE KO aortas compared with non-diabetic controls (total plaque: 15% ⫹/⫺3% vs 1%⫹/⫺ 0.5%; P ⬍ 0.001). GP⫻1 mimetic significantly reduced diabetic lesions by 50% (P ⬍ 0.001). For in vitro assays, endothelial cells were pre-treated with 0.03M GP⫻-1 mimetic and stimulated with 100M H2O2 thereafter. Western blots analysis showed reduced levels
127 INNATE IMMUNE EFFECTORS ARE NECESSARY AND SUFFICIENT TO CAUSE EARLY POST TRANSPLANT FAILURE OF MEDIAL SMOOTH MUSCLE CELL RECOVERY IN MURINE AORTIC ALLOGRAFTS CL Hancock Friesen, M So, TD Lee Halifax, Nova Scotia BACKGROUND: Ischemia reperfusion injury is inherent and important in the initiation of allograft vasculopathy (AV). Despite evidence that prolonged cold ischemia (CI) exacerbates AV, organ demand has driven clinician acceptance of significantly prolonged CI duration. We have shown that CI is associated with neutrophil-mediated medial smooth muscle cell (SMC) loss. Incomplete recovery of medial SMC is associated with AV lesion formation and is inversely proportional to the duration of CI. We hypothesize that innate, not adaptive immune effectors, are responsible for early changes that predate AV. METHODS: Aortic transplants were performed between fully disparate mouse strains under CyA immunosuppression, C3H/HeJ to C57BL/6 (wildtype (WT) recipient; intact innate and adaptive immunity) or B6.RAG1⫺/⫺ (immunodeficient (RAG) recipient; intact innate immunity, no adaptive immunity). Grafts were subjected to 20min (control) or 60min (experimental) CI pre-transplant, harvested at 7d and 14d posttransplant, formalin-fixed, and sectioned for H&E (medial SMC quantification) and immunohistochemistry (macrophage (MØ), neutrophil (NØ)). RESULTS: There is similar failure of medial SMC repopulation in experimental vs control grafts at 2wk post-transplant (WT P ⫽ 0.0006; RAG: P ⫽ 0.0003). Coincident with SMC loss is a profound NØ influx in experimental grafts at 1wk vs 2wk, similar in magnitude regardless of the presence (WT P ⬍ 0.01) or absence (RAG P ⬍ 0.01) of adaptive immune effectors. Similar trends in MØ influx were observed in experimental WT and RAG recipients at 1wk vs 2wk post-transplant.
Abstracts
co-SMAD-4 that enhances TGF- signaling were also upregulated in Ex- compared to Sed-db/db mice hearts, implying further activation of fibrosis in these aged diabetic hearts. Finally, augmented collagen 1a and fibronectin levels, products of TGF- activity, were confirmed with immunofluoresecence and western blotting respectively in Ex-db/db mice hearts. CONCLUSION: These studies demonstrate that TGF- mediated cardiac profibrotic signaling may be upregulated with forced exercise in aged T2D hearts. Although the reasons are currently unknown, it is plausible that excess oxidative stress evident with such forced exercise paradigms may worsen cardiac prognosis in older T2D patients. Canadian Diabetes Association
124 USE OF GPⴛ-1 MIMETICS TO REDUCE DIABETICASSOCIATED ATHEROSCLEROSIS IN THE ApoE/GPⴛ1DOUBLE KNOCKOUT MOUSE
of the pro-inflammatory mediators P-IKK, P-JNK and TNF-␣ with GP⫻1 mimetic pre-treatment. FUTURE DIRECTIONS: Currently, we are performing oxidative stress analysis, immunohistochemistry and RT-PCR analysis of pro-inflammatory markers to gain an understanding of the mechanisms by which the GP⫻1 mimetic reduces DAA. CONCLUSION: Novel GP⫻1-mimetics show promise as an antiatherogenic therapeutic target via signalling pathways that limit inflammation. LEARNING INITIATIVE: This study is aimed at characterizing a therapeutic anti-oxidant drug that limits oxidative stress in cardiovascular complications associated with diabetes. Canadian Cardiovascular Society (CCS) CCS088 Highlighted Poster HEART FAILURE POTPOURRI Sunday, October 23, 2011
A Sharma, N Stefanovic, D Yuen, P Chew, M Tan, P Bernatchez, J de Haan Vancouver, British Columbia INTRODUCTION: Diabetes is a major risk factor for cardiovascular dis-
eases, in particular atherosclerosis. Oxidative stress contributes significantly to the pathogenesis of diabetic-associated atherosclerosis (DAA), predominantly by inducing pro-inflammatory pathways. Oxidative stress occurs through the upregulation of reactive oxygen species (ROS), such as superoxide, hydrogen peroxide (H2O2) and peroxynitrite (ONOO-) and their inadequate removal by anti-oxidant enzymes. Glutathione peroxidase 1 (GP⫻1) is a ubiquitously expressed anti-oxidant enzyme, which is involved in the reduction of H2O2 to water and also functions as an ONOO- reductase. Previously, we have shown that genetic deletion of the GP⫻1 gene in diabetic ApoE knockout (KO) mice augmented the atherosclerotic burden. In addition, administration of ebselen, a synthetic compound that mimics the action of GP⫻1, significantly reduces DAA in diabetic ApoE KO and ApoE/GP⫻1 double KO mice. In the current proposal, our aim is to extend these findings to newer GP⫻1 mimetics, which have been generated for their greater efficacy, potency and their potential to behave in a superior fashion to ebselen in vivo. OBJECTIVE: To investigate whether GP⫻1 mimetics can substitute for GP⫻1 in ApoE/GP⫻1 double KO mice and reduce atherosclerosis. Secondly, we aim to examine if GP⫻1 mimetics reduce proinflammatory pathways in vivo and in vitro. METHODS AND RESULTS: 8-week old male ApoE/GP⫻1 double KO mice were made diabetic with streptozotocin. Diabetic and non-diabetic control mice received the GP⫻1 mimetic by twice daily gavage (10mg/kg body weight) or vehicle only. After 20 weeks of diabetes, lesions were assessed by Sudan IV staining. The lesions were significantly increased in diabetic ApoE KO aortas compared with non-diabetic controls (total plaque: 15% ⫹/⫺3% vs 1%⫹/⫺ 0.5%; P ⬍ 0.001). GP⫻1 mimetic significantly reduced diabetic lesions by 50% (P ⬍ 0.001). For in vitro assays, endothelial cells were pre-treated with 0.03M GP⫻-1 mimetic and stimulated with 100M H2O2 thereafter. Western blots analysis showed reduced levels
127 INNATE IMMUNE EFFECTORS ARE NECESSARY AND SUFFICIENT TO CAUSE EARLY POST TRANSPLANT FAILURE OF MEDIAL SMOOTH MUSCLE CELL RECOVERY IN MURINE AORTIC ALLOGRAFTS CL Hancock Friesen, M So, TD Lee Halifax, Nova Scotia BACKGROUND: Ischemia reperfusion injury is inherent and important in the initiation of allograft vasculopathy (AV). Despite evidence that prolonged cold ischemia (CI) exacerbates AV, organ demand has driven clinician acceptance of significantly prolonged CI duration. We have shown that CI is associated with neutrophil-mediated medial smooth muscle cell (SMC) loss. Incomplete recovery of medial SMC is associated with AV lesion formation and is inversely proportional to the duration of CI. We hypothesize that innate, not adaptive immune effectors, are responsible for early changes that predate AV. METHODS: Aortic transplants were performed between fully disparate mouse strains under CyA immunosuppression, C3H/HeJ to C57BL/6 (wildtype (WT) recipient; intact innate and adaptive immunity) or B6.RAG1⫺/⫺ (immunodeficient (RAG) recipient; intact innate immunity, no adaptive immunity). Grafts were subjected to 20min (control) or 60min (experimental) CI pre-transplant, harvested at 7d and 14d posttransplant, formalin-fixed, and sectioned for H&E (medial SMC quantification) and immunohistochemistry (macrophage (MØ), neutrophil (NØ)). RESULTS: There is similar failure of medial SMC repopulation in experimental vs control grafts at 2wk post-transplant (WT P ⫽ 0.0006; RAG: P ⫽ 0.0003). Coincident with SMC loss is a profound NØ influx in experimental grafts at 1wk vs 2wk, similar in magnitude regardless of the presence (WT P ⬍ 0.01) or absence (RAG P ⬍ 0.01) of adaptive immune effectors. Similar trends in MØ influx were observed in experimental WT and RAG recipients at 1wk vs 2wk post-transplant.