- Email: [email protected]
705 Development of reporter gene PET imaging techniques for long-term assessment of transplanted human circulating progenitor cells
S321
Abstracts
porting the use of biatrial ganglionated plexus ablation as an adjunct to myocardial targets in the surgery of AF.
704 LOW DOSE NEBIVOLOL PRESERVED BRAIN PERFUSION IN HEMODILUTED RATS T Hu Toronto, Ontario
Perioperative -blockade has reduced the incidence of myocardial infarctions, but is associated with an increase in ischemic stroke in specific clinical contexts (acute blood loss). Studies have demonstrated that metoprolol, a relatively poor 1-specific antagonist, impairs cerebral perfusion during a model of acute blood loss and fluid resuscitation (hemodilution). This may be due to a combined impairment in cardiac output (1-effect) and cerebral vasodilation (2-effect). Therefore, we hypothesized that cerebral perfusion would be maintained during acute hemodilution after treatment with a highly 1-selective antagonist (nebivolol). METHODS: Anesthetised rats were treated with vehicle (control) or nebivolol (1.25 or 2.5 mg/kg iv). The following outcomes were measured at baseline, after drug or vehicle administration, and after hemodilution to a target hemoglobin concentration near 60 g/L (50% estimated blood volume exchange with 10% pentastarch): nebivolol drug levels, heart rate (HR), mean arterial pressure, cardiac output (CO, echocardiography), and cerebral blood flow (CBF, laser Doppler). Data are presented as the mean ⫾ standard deviation and statistical significance is determined by two-way ANOVA. RESULTS: After treatment with 1.25 and 2.5 mg/kg of nebivolol, drug levels reached peak values of 48 ⫾ 15 ng/mL and 88 ⫾ 9.4 ng/mL respectively and HR decreased comparably (251 ⫾ 19 and 252 ⫾ 27 bpm respectively, n ⫽ 17, P ⬍ 0.05 for both), relative to baseline (301 ⫾ 20 bpm) and controls. In controls, hemodilution caused an increase in HR and stroke volume. The increase in CO observed in controls after hemodilution (205 ⫾ 41 vs. 122 ⫾ 9.5 mL/minute, n ⫽ 5, P ⬍ 0.05) was attenuated by both doses of BACKGROUND:
nebivolol (161 ⫾ 24 mL/minute and 151 ⫾ 13 mL/minute, n ⫽ 5, P ⬍ 0.05). In controls and rats treated with 1.25 mg/kg of nebivolol, the relative CBF increased after hemodilution (1.90 ⫾ 0.49 and 1.80 ⫾ 0.53 respectively, n ⫽ 6, P ⬍ 0.05). In contrast, the CBF response was attenuated after treatment with 2.5 mg/kg of nebivolol (1.09 ⫾ 0.12, n ⫽ 6, P ⬍ 0.05). CONCLUSION: Nebivolol treatment caused a clinically relevant reduction in HR. In controls, hemodilution caused an increase in CO and CBF to maintain cerebral oxygen delivery. Both doses of nebivolol attenuated the CO increase associated with hemodilution, but only the higher dose of nebivolol impaired the CBF response. These data support the hypothesis that cerebral perfusion is maintained during hemodilution with nebivolol in the dose range that is consistent with 1-specific receptor binding (1.25 mg/kg), but becomes jeopardized when the dose enters the non-specific range for nebivolol (1⫹2, 2.5 mg/kg). We conclude that the low dose of nebivolol may provide the positive therapeutic effects (cardiac protection) without the risk of ischemic organ injury. Forest Research Institute, St. Michael’s Hospital and University of Toronto Departments of Anesthesia, CAS 705 DEVELOPMENT OF REPORTER GENE PET IMAGING TECHNIQUES FOR LONG-TERM ASSESSMENT OF TRANSPLANTED HUMAN CIRCULATING PROGENITOR CELLS Y Zhang, D Kuraitis, P Burgon, S Crowe, B Vulesevic, S Thorn, JC Wu, JN DaSilva, RA deKemp, RS Beanlands, EJ Suuronen, M Ruel Ottawa, Ontario
Direct cell radiolabeling methods with positron emission tomography (PET) have been used to track transplanted stem cells noninvasively. However, these techniques can only monitor cells for several hours due to the short half-life of PET radioisotopes. To further investigate cell fate and function in vivo, we transduced human circulating progenitor cells (CPCs) with reporter genes for long-term assessment of transplanted cells. METHODS/RESULTS: A self-inactivating lentiviral particle, carrying a triple-fusion reporter (TFR) construct consisting of PET (herpes simplex virus type 1 truncated thymidine kinase; HSV1-sr39tk), fluorescence (monomeric red fluorescence protein; mRFP), and bioluminescence (firefly luciferase) reporter genes, was produced by transient co-transfection into 293FT cells with pFUUFRTW, psPAX2, and pMD2.G plasmids, using the calcium-phosphate precipitation method. Human CPCs were transduced with the created viral particle at increasing multiplicities of infection (MOI) on RetroNectin-coated plates. The transduction efficiency was determined by flow cytometry analysis of RFP expression. The mean transduction efficiency was 2.2%, 2.6%, 3.5%, 6.9%, 9.9%, 13.3% and 16.0% at MOIs of 0.1, 0.5, 2.5, 12.5, 25, 50 and 100, respectively. The expression of RFP in transduced CPCs was visualized under the fluorescence microscope. Western blot analysis confirmed HSV1-tk protein expression in transduced CPCs. Both transduced and control CPCs exhibited similar rounded or spindle shapes. No significant differBACKGROUND:
S322
Canadian Journal of Cardiology Volume 27 2011
ence was observed in cell viability between the transduced CPCs and the untreated controls at the low level of MOIs (ⱕ50); however, there was a reduction in transduced CPC viability at a MOI of 100 (66.2 ⫾ 9.5% versus 82.8 ⫾ 10.3%, P ⬍ 0.05). The effect of TFR transduction on cellular functions was evaluated by in vitro assays. The migration potential (66.2 ⫾ 21.3 cells/field of view (FOV)) and capillary tube length of cells (5.6 ⫾ 0.6mm) were not adversely affected by the transduction process compared to the control (61.1 ⫾ 19.6 cells/FOV and 5.7 ⫾ 0.5mm; P ⫽ 0.7 and P ⫽ 0.8, respectively). Fluorescence-activated cell sorting analysis isolated the RFP⫹ transduced CPCs. After 4 weeks, 80.3 ⫾ 8.4% of the sorted cells continued to express RFP. CONCLUSION: Quiescent human CPCs transduced with a lentiviral vector show stable expression of the triple reporter genes. The TFR gene approach can be developed for tracking transplanted CPCs, while preserving the cells’ morphology, viability, migration and angiogenesis capabilities. This technique may be used for the development of reporter gene PET imaging, to monitor the fate of transplanted cells noninvasively, longitudinally, and quantitatively, and thereby help elucidate the mechanisms and effectiveness of cell-based therapies. Heart & Stroke, Canadian Institutes of Health Research (CIHR)
Canadian Society of Cardiac Surgeons (CSCS) CSCS022 Highlighted Poster CSCS/SURG HIGHLIGHTED POSTERS Tuesday, October 25, 2011 Featured Research
708 MICRORNA-145 IS A NOVEL REGULATOR OF ATHEROSCLEROSIS AND PLAQUE STABILITY F Lovren, Y Pan, A Quan, B Yanagawa, A Creighton, N Gupta, P Shukla, K Singh, H Teoh, S Verma
mm2; P ⬍ 0.05) and brachiocephalic arteries (59.35ì m2 vs. 169.23ì m2; P ⬍ 0.05). MiRNA-145 lentivirus treatment significantly increased fibrous cap area, profoundly reduced the necrotic core area/ total plaque area ratio, and increased plaque collagen content, suggesting an overall increase in plaque stability. Relative content of Mac-3 macrophages in lesioned brachiocephalic arterial areas was also significantly lower in miRNA-145 lentivirus-treated ApoE-/- mice relative to that in the corresponding samples from control lentivirus-treated mice. These differential effects were associated with significant differences in serum MCP-1 levels (115⫹24 pg/mL vs. 91 ⫾ 10 pg/mL for miRNA-145 vs. control lentivirus group, P ⬍ 0.05). ApoE-/- mice treated with the miRNA-145 lentivirus demonstrated a marked increase in ␣-smooth muscle actin (SMA)-positive area in their atherosclerotic lesions. ␣-SMA-positive plaque areas also stained positive for calponin. MiRNA-145 overexpression in human aortic SMCs (HASMCs) triggered a marked increase in myocardin expression with concomitant decreases in KLF4 expression, consistent with a role for miRNA-145 in promoting the contractile phenotype of VSMCs, an observation corroborated by the enhanced protein expressions of ␣-SMA, calponin and collagen types I and III in HASMCs. CONCLUSION: This is the first evaluation of a miRNA-based SMC targeting strategy to limit atherosclerosis. SMC-specific miRNA-145 overexpression in atherosclerosis prone ApoE-/- mice not only limited atherosclerotic plaque burden but also increased features of plaque stability including increased fibrous cap area, áSMA-positive SMC and collagen content, and reduced necrotic core, while attenuating local inflammation and cytokine elaboration. These effects appeared to occur via miRNA-145-mediated modulation of the VSMC differentiation transcription factors, myocardin and KLF4, in a fashion consistent with promotion of the contractile phenotype of VSMCs. Strategies that augment vascular miRNA-145 levels may serve to limit atherosclerosis and its associated complications. 709 MITRAL STENOSIS AFTER SUCCESSFUL REPAIR FOR DEGENERATIVE MITRAL REGURGITATION: AN UNDERRECOGNIZED SEQUELA OF MITRAL VALVE REPAIR
Toronto, Ontario
S Chen, B Lam, T Mesana, V Chan, K Hay, K Chan
BACKGROUND: Vascular smooth muscle cell (VSMC) activation and remodeling are critical events in the pathophysiology and natural course of atherosclerotic plaque development. In addition to plaque size, the flux of VSMC proliferation also influences plaque stability. MicroRNAs (miRNAs) are essential post-transcriptional modulators of gene expression which coordinate and integrate multiple regulatory pathways involved in normal and abnormal physiology. Since miRNA-145 is highly and selectively expressed in VSMCs and regulates VSMC fate and plasticity, we hypothesized that it may also be a novel regulator of atherosclerosis and plaque stability. -/METHODS & RESULTS: MiRNA-145 lentivirus-treated ApoE mice exhibited significantly attenuated atherosclerotic burden compared to their control lentivirus-treated counterparts as evidenced by the reduced cross sectional plaque area within their aortic sinuses (0.33 mm2 vs. 0.49 mm2; P ⬍ 0.05), ascending aortas (0.17 mm2 vs. 0.36
Ottawa, Ontario BACKGROUND: Mitral valve repair (MVr) has become the stan-
dard treatment for most cases of severe myxomatous mitral regurgitation (MMR); however, some patients have exhibited evidence of significant mitral stenosis (MS) after MVr. The aim of this study was to assess the prevalence and clinical significance of this type of MS following successful MVr. METHODS: 85 patients who had MVr for MMR in 2001-2009 were recruited and assessed with bicycle stress echocardiogram, 6 minute walk test, SF-36 health survey and BNP measurements. We excluded patients with significant residual MMR, aortic valve disease or ventricular dysfunction. The age was 69 ⫾ 20 yrs; 59 (69 %) were male; 83 (98 %) had mitral annuloplasty with 57 (69 %) receiving a band and 26 (31 %) a full ring. In this study, MS was defined as a resting mean mitral gradient (MMG) ⬎ 4 mmHG.
Abstracts
porting the use of biatrial ganglionated plexus ablation as an adjunct to myocardial targets in the surgery of AF.
704 LOW DOSE NEBIVOLOL PRESERVED BRAIN PERFUSION IN HEMODILUTED RATS T Hu Toronto, Ontario
Perioperative -blockade has reduced the incidence of myocardial infarctions, but is associated with an increase in ischemic stroke in specific clinical contexts (acute blood loss). Studies have demonstrated that metoprolol, a relatively poor 1-specific antagonist, impairs cerebral perfusion during a model of acute blood loss and fluid resuscitation (hemodilution). This may be due to a combined impairment in cardiac output (1-effect) and cerebral vasodilation (2-effect). Therefore, we hypothesized that cerebral perfusion would be maintained during acute hemodilution after treatment with a highly 1-selective antagonist (nebivolol). METHODS: Anesthetised rats were treated with vehicle (control) or nebivolol (1.25 or 2.5 mg/kg iv). The following outcomes were measured at baseline, after drug or vehicle administration, and after hemodilution to a target hemoglobin concentration near 60 g/L (50% estimated blood volume exchange with 10% pentastarch): nebivolol drug levels, heart rate (HR), mean arterial pressure, cardiac output (CO, echocardiography), and cerebral blood flow (CBF, laser Doppler). Data are presented as the mean ⫾ standard deviation and statistical significance is determined by two-way ANOVA. RESULTS: After treatment with 1.25 and 2.5 mg/kg of nebivolol, drug levels reached peak values of 48 ⫾ 15 ng/mL and 88 ⫾ 9.4 ng/mL respectively and HR decreased comparably (251 ⫾ 19 and 252 ⫾ 27 bpm respectively, n ⫽ 17, P ⬍ 0.05 for both), relative to baseline (301 ⫾ 20 bpm) and controls. In controls, hemodilution caused an increase in HR and stroke volume. The increase in CO observed in controls after hemodilution (205 ⫾ 41 vs. 122 ⫾ 9.5 mL/minute, n ⫽ 5, P ⬍ 0.05) was attenuated by both doses of BACKGROUND:
nebivolol (161 ⫾ 24 mL/minute and 151 ⫾ 13 mL/minute, n ⫽ 5, P ⬍ 0.05). In controls and rats treated with 1.25 mg/kg of nebivolol, the relative CBF increased after hemodilution (1.90 ⫾ 0.49 and 1.80 ⫾ 0.53 respectively, n ⫽ 6, P ⬍ 0.05). In contrast, the CBF response was attenuated after treatment with 2.5 mg/kg of nebivolol (1.09 ⫾ 0.12, n ⫽ 6, P ⬍ 0.05). CONCLUSION: Nebivolol treatment caused a clinically relevant reduction in HR. In controls, hemodilution caused an increase in CO and CBF to maintain cerebral oxygen delivery. Both doses of nebivolol attenuated the CO increase associated with hemodilution, but only the higher dose of nebivolol impaired the CBF response. These data support the hypothesis that cerebral perfusion is maintained during hemodilution with nebivolol in the dose range that is consistent with 1-specific receptor binding (1.25 mg/kg), but becomes jeopardized when the dose enters the non-specific range for nebivolol (1⫹2, 2.5 mg/kg). We conclude that the low dose of nebivolol may provide the positive therapeutic effects (cardiac protection) without the risk of ischemic organ injury. Forest Research Institute, St. Michael’s Hospital and University of Toronto Departments of Anesthesia, CAS 705 DEVELOPMENT OF REPORTER GENE PET IMAGING TECHNIQUES FOR LONG-TERM ASSESSMENT OF TRANSPLANTED HUMAN CIRCULATING PROGENITOR CELLS Y Zhang, D Kuraitis, P Burgon, S Crowe, B Vulesevic, S Thorn, JC Wu, JN DaSilva, RA deKemp, RS Beanlands, EJ Suuronen, M Ruel Ottawa, Ontario
Direct cell radiolabeling methods with positron emission tomography (PET) have been used to track transplanted stem cells noninvasively. However, these techniques can only monitor cells for several hours due to the short half-life of PET radioisotopes. To further investigate cell fate and function in vivo, we transduced human circulating progenitor cells (CPCs) with reporter genes for long-term assessment of transplanted cells. METHODS/RESULTS: A self-inactivating lentiviral particle, carrying a triple-fusion reporter (TFR) construct consisting of PET (herpes simplex virus type 1 truncated thymidine kinase; HSV1-sr39tk), fluorescence (monomeric red fluorescence protein; mRFP), and bioluminescence (firefly luciferase) reporter genes, was produced by transient co-transfection into 293FT cells with pFUUFRTW, psPAX2, and pMD2.G plasmids, using the calcium-phosphate precipitation method. Human CPCs were transduced with the created viral particle at increasing multiplicities of infection (MOI) on RetroNectin-coated plates. The transduction efficiency was determined by flow cytometry analysis of RFP expression. The mean transduction efficiency was 2.2%, 2.6%, 3.5%, 6.9%, 9.9%, 13.3% and 16.0% at MOIs of 0.1, 0.5, 2.5, 12.5, 25, 50 and 100, respectively. The expression of RFP in transduced CPCs was visualized under the fluorescence microscope. Western blot analysis confirmed HSV1-tk protein expression in transduced CPCs. Both transduced and control CPCs exhibited similar rounded or spindle shapes. No significant differBACKGROUND:
S322
Canadian Journal of Cardiology Volume 27 2011
ence was observed in cell viability between the transduced CPCs and the untreated controls at the low level of MOIs (ⱕ50); however, there was a reduction in transduced CPC viability at a MOI of 100 (66.2 ⫾ 9.5% versus 82.8 ⫾ 10.3%, P ⬍ 0.05). The effect of TFR transduction on cellular functions was evaluated by in vitro assays. The migration potential (66.2 ⫾ 21.3 cells/field of view (FOV)) and capillary tube length of cells (5.6 ⫾ 0.6mm) were not adversely affected by the transduction process compared to the control (61.1 ⫾ 19.6 cells/FOV and 5.7 ⫾ 0.5mm; P ⫽ 0.7 and P ⫽ 0.8, respectively). Fluorescence-activated cell sorting analysis isolated the RFP⫹ transduced CPCs. After 4 weeks, 80.3 ⫾ 8.4% of the sorted cells continued to express RFP. CONCLUSION: Quiescent human CPCs transduced with a lentiviral vector show stable expression of the triple reporter genes. The TFR gene approach can be developed for tracking transplanted CPCs, while preserving the cells’ morphology, viability, migration and angiogenesis capabilities. This technique may be used for the development of reporter gene PET imaging, to monitor the fate of transplanted cells noninvasively, longitudinally, and quantitatively, and thereby help elucidate the mechanisms and effectiveness of cell-based therapies. Heart & Stroke, Canadian Institutes of Health Research (CIHR)
Canadian Society of Cardiac Surgeons (CSCS) CSCS022 Highlighted Poster CSCS/SURG HIGHLIGHTED POSTERS Tuesday, October 25, 2011 Featured Research
708 MICRORNA-145 IS A NOVEL REGULATOR OF ATHEROSCLEROSIS AND PLAQUE STABILITY F Lovren, Y Pan, A Quan, B Yanagawa, A Creighton, N Gupta, P Shukla, K Singh, H Teoh, S Verma
mm2; P ⬍ 0.05) and brachiocephalic arteries (59.35ì m2 vs. 169.23ì m2; P ⬍ 0.05). MiRNA-145 lentivirus treatment significantly increased fibrous cap area, profoundly reduced the necrotic core area/ total plaque area ratio, and increased plaque collagen content, suggesting an overall increase in plaque stability. Relative content of Mac-3 macrophages in lesioned brachiocephalic arterial areas was also significantly lower in miRNA-145 lentivirus-treated ApoE-/- mice relative to that in the corresponding samples from control lentivirus-treated mice. These differential effects were associated with significant differences in serum MCP-1 levels (115⫹24 pg/mL vs. 91 ⫾ 10 pg/mL for miRNA-145 vs. control lentivirus group, P ⬍ 0.05). ApoE-/- mice treated with the miRNA-145 lentivirus demonstrated a marked increase in ␣-smooth muscle actin (SMA)-positive area in their atherosclerotic lesions. ␣-SMA-positive plaque areas also stained positive for calponin. MiRNA-145 overexpression in human aortic SMCs (HASMCs) triggered a marked increase in myocardin expression with concomitant decreases in KLF4 expression, consistent with a role for miRNA-145 in promoting the contractile phenotype of VSMCs, an observation corroborated by the enhanced protein expressions of ␣-SMA, calponin and collagen types I and III in HASMCs. CONCLUSION: This is the first evaluation of a miRNA-based SMC targeting strategy to limit atherosclerosis. SMC-specific miRNA-145 overexpression in atherosclerosis prone ApoE-/- mice not only limited atherosclerotic plaque burden but also increased features of plaque stability including increased fibrous cap area, áSMA-positive SMC and collagen content, and reduced necrotic core, while attenuating local inflammation and cytokine elaboration. These effects appeared to occur via miRNA-145-mediated modulation of the VSMC differentiation transcription factors, myocardin and KLF4, in a fashion consistent with promotion of the contractile phenotype of VSMCs. Strategies that augment vascular miRNA-145 levels may serve to limit atherosclerosis and its associated complications. 709 MITRAL STENOSIS AFTER SUCCESSFUL REPAIR FOR DEGENERATIVE MITRAL REGURGITATION: AN UNDERRECOGNIZED SEQUELA OF MITRAL VALVE REPAIR
Toronto, Ontario
S Chen, B Lam, T Mesana, V Chan, K Hay, K Chan
BACKGROUND: Vascular smooth muscle cell (VSMC) activation and remodeling are critical events in the pathophysiology and natural course of atherosclerotic plaque development. In addition to plaque size, the flux of VSMC proliferation also influences plaque stability. MicroRNAs (miRNAs) are essential post-transcriptional modulators of gene expression which coordinate and integrate multiple regulatory pathways involved in normal and abnormal physiology. Since miRNA-145 is highly and selectively expressed in VSMCs and regulates VSMC fate and plasticity, we hypothesized that it may also be a novel regulator of atherosclerosis and plaque stability. -/METHODS & RESULTS: MiRNA-145 lentivirus-treated ApoE mice exhibited significantly attenuated atherosclerotic burden compared to their control lentivirus-treated counterparts as evidenced by the reduced cross sectional plaque area within their aortic sinuses (0.33 mm2 vs. 0.49 mm2; P ⬍ 0.05), ascending aortas (0.17 mm2 vs. 0.36
Ottawa, Ontario BACKGROUND: Mitral valve repair (MVr) has become the stan-
dard treatment for most cases of severe myxomatous mitral regurgitation (MMR); however, some patients have exhibited evidence of significant mitral stenosis (MS) after MVr. The aim of this study was to assess the prevalence and clinical significance of this type of MS following successful MVr. METHODS: 85 patients who had MVr for MMR in 2001-2009 were recruited and assessed with bicycle stress echocardiogram, 6 minute walk test, SF-36 health survey and BNP measurements. We excluded patients with significant residual MMR, aortic valve disease or ventricular dysfunction. The age was 69 ⫾ 20 yrs; 59 (69 %) were male; 83 (98 %) had mitral annuloplasty with 57 (69 %) receiving a band and 26 (31 %) a full ring. In this study, MS was defined as a resting mean mitral gradient (MMG) ⬎ 4 mmHG.