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BNP Convertase” Is Up-Regulated in Human Heart Failure, but Inversely Correlated with BNP Expression
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Journal of Cardiac Failure Vol. 11 No. 6 Suppl. 2005
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Safety Study for Intracoronary Delivery of Autologous Bone Marrow Cells in Chronic Chagas Disease Dogs Model Joao A. Assad1, Suzana A. Silva1, Rodrigo C. Moreira1, Fabio A. Tuche1, Antonio A. Camacho2, Rodrigo V. Branco1, Radovan Borojevic3, Hans F. Dohmann1; 1Cath Lab, Pro´-Cardı´aco, Rio de Janeiro, Brazil; 2Veterinary Clinic, UNESP, Jaboticabal, Sa˜o Paulo, Brazil; 3Histology, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil
Corin, the “pro-ANP/BNP Convertase” Is Up-Regulated in Human Heart Failure, but Inversely Correlated with BNP Expression Daniel L. Dries1, Thomas P. Cappola1, Arun Changolkar1, Kenneth B. Margulies1; 1 Heart Failure/Transplant Group, Division of Cardiology, Hospital of the University of Pennsylvania, Philadelphia, PA
Background: Chagas disease is an endemic infectious (T. Cruzi) in South America. There are very few new acute cases but millions of chronic symptomatic cardiomyopathy patients characterized by ventricular dysfunction and arrhythmias. Purpose: Evaluate the safety and feasibility of intracoronary delivery of autologous bone marrow mononuclear cells (ABMNC) in a chronic Chagas cardiomyopathy dog model. Methods: Six dogs, which were inoculated via intraperitoneal with T.Cruzi 11 years ago. All of them had positive laboratory tests confirming Chagas disease development. Animals were divided in two groups: Four animals in the treated group and two in the control group. The treated group underwent intracoronary delivery of 50 millions ABMMNC. All animals were followed by echocardiograms. Results: At baseline, animals have showed a subtle systolic dysfunction and mild diastolic dysfunction. There were neither adverse effects related to intracoronary route, nor clinical deterioration after the procedure. Ejection fraction showed an improvement in treated group (61.0 ⫾ 13% to 69.3 ⫾ 8.4%) not observed in control group, as well as in cardiac volumes (fig 1). Diastolic parameters, Isovolumetric Relaxing Time (IVRT) and Propagation Velocity M mode E/A wave, varied from 77⫹-23 to 58⫹-6,56 and from 0,81⫹-0,10 to 0,75⫹-0,11 on the treated group, respectively. Control group did not improve. We did not perform statistical analysis due to the small number of animals. Conclusion: In this very rare chronic model (11 years) of Chagas cardiomyopathy, we have showed for the first time in an animal model that intracoronary delivery of ABMMNC had no adverse effects, suggesting safety. On the other hand, treated animals have improved cardiac performance, which was not observed in controls.
Background: Corin is a type II transmembrane serine protease expressed in cardiomyocytes. Both in vitro and in vivo data demonstrate that corin converts pro-ANP and pro-BNP into smaller biologically active molecules. There are only two studies examining corin gene expression in heart failure, both are in rat models of heart failure, and they report conflicting results. One study reports increased myocardial corin gene expression that parallels increased BNP expression, whereas the other study demonstrates decreased myocardial corin gene expression. We are not aware of any published data examining corin gene expression and its correlation with BNP gene expression in human heart failure. Methods: Myocardial RNA from end-stage failing human hearts (n ⫽ 173) and non-failing controls (n ⫽ 13) were hybridized with individual Affymetrix HU133A arrays. Data were normalized and filtered using RNA (www.bioconductor.org) resulting in 13,639 transcripts present above background. Corin and BNP transcript expression levels were measured in failing hearts, stratified by etiology (ischemic vs. non-ischemic) and compared to control heart expression levels. Affymetrix HU 133A array data regarding corin and BNP gene expression was confirmed by RT-PCR. Corin protein levels in the myocardium were examined by Western blot analysis using a monoclonal antibody to human corin. Results: Corin gene expression was increased approximately two-fold in both ischemic (P ⫽ 0.027) and non-ischemic (P ⫽ 0.030) heart failure compared to controls. The increased expression of corin was confirmed by RT-PCR. Western blot analysis demonstrated that myocardial corin protein correlated with corin gene expression levels. BNP gene expression was also increased in both ischemic and non-ischemic cardiomyopathy compared to controls. However, the plot of BNP expression (x-axis) versus corin expression (y-axis) demonstrated an inverse relationship. Conclusions: These data indicate that corin gene expression is upregulated in human heart failure and this correlates with increased myocardial corin protein levels. However, there is an inverse relationship between BNP and corin gene expression in ischemic and nonischemic cardiomyopathy. These results suggest that in advanced heart failure the ability of corin to efficiently process pro-BNP may become rate-limiting.
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Robust RNA Amplification for Gene Expression Analysis from Biopsy-Sized Human Heart Tissue Brian R. Barrows1, Agnes A. Azimzadeh5, Hui Zhou5, Gloria Vives-Rodriguez1, Stacey L. McCulle3, Wayne N. Stark, Jr.1, Nicholas Ambulos4, Jing Yin4, Richard N. Pierson III5, Frances L. Johnson3, Stephen S. Gottlieb3, Meredith Bond1; 1Physiology, University of Maryland, Baltimore, Baltimore, MD; 2School of Medicine, Cardiology, University of Maryland, Baltimore, Baltimore, MD; 3Genomics Core Facility, University of Maryland, Baltimore, Baltimore, MD; 4UMMC, Department of Surgery, University of Maryland, Baltimore, Baltimore, MD
Manganese superoxide dismutase (SOD2) A16V Polymorphism Is Not Associated with Increased Mortality in Heart Failure: A Retrospective Study from the Bucindolol Evaluation and Survival (BEST) Trial’s DNA-Substudy Daniel L. Dries1, Thomas P. Cappola1, Ken B. Margulies1, Surai Thaneemit-Chen2, Philip Lavori2; 1Cardiology Division, Hospital of the University of Pennsylvania, Philadelphia, PA; 2Palo Alto VA, DNA Bank Coordinating Center, Stanford, Menlo Park, CA
Introduction: Gene expression experiments are emerging as a method for elucidating biological pathways that contribute to disease states, such as cancer and heart failure. In the case of heart failure, gene array experiments are limited by the amount of tissue that can be obtained. Hypothesis: Both one- and two-cycle amplification of RNA from biopsy-sized pieces of human heart will produce sufficient high-quality cDNA or cRNA for gene expression analysis. Methods: RNA amplification was performed on 10ng, 20ng, and 50ng samples of total RNA obtained from an explanted heart and amplified two ways: 1) one-cycle amplification producing cDNA (NuGEN Ovation), and 2) two-cycle amplification producing cRNA (Affymetrix쑓 Two-Cycle Target Labeling). For gene expression analysis, at least 2.2ug of cDNA or 20ug of cRNA is needed for each Affymetrix GeneChip쑓 array (Human Genome U133 Plus 2.0 Array). GeneChip쑓 data were used to assess the variability within each amplification method and between both one- and two-cycle methods. Patient consent was received for this study. Results: Both one- and two-cycle methods produced high-quality nucleotide samples (260:280nm ⬎ 1.8). The one-cycle method yielded enough cDNA for hybridization to one GeneChip쑓 (all values are mean ⫾ SD; 2.5 ⫾ 0.2ug). The two-cycle method yielded enough cRNA for hybridization to up to four GeneChips쑓 (61 ⫾ 27ug). Gene expression data were used to determine variance among the 10, 20, and 50ng RNA samples amplified by either one- or two-cycle methods. Expression values for 15 selected genes (12000 ⬎ values ⬎ 50) showed an average variance of 18 ⫾ 14% and 20 ⫾ 12% for the one- and two-cycle methods, respectively (P ⬎ 0.05). Conclusions: These results indicate that as little as 10ng of total RNA can be amplified by either one- or two-cycle kits to provide sufficient cDNA or cRNA for Affymetrix GeneChip쑓 experiments. This now opens opportunities to analyze gene expression patterns from hearts of living patients using but a few mg of tissue. Such samples could be obtained during regular cardiac surgery or possibly by less invasive biopsy methods.
Background: SOD2 catalyzes the dismutation of superoxide radicals into hydrogen peroxide in the mitochondria. SOD2 is targeted to the inner mitochondrial by an amino acid leader sequence that contains the A16V mutation. Substitution of valine for alanine at position 16 alters the secondary structure of the leader sequence resulting in impaired targeting of SOD2 to the mitochondria. The resulting increased mitochondrial free radical damage may increase progression of heart failure by an increase in mitochondrial DNA damage, impaired oxidative phosphorylation, and promotion of mitochondrial-mediated apoptosis. Although one report in Japanese pateints demonstrated an association of the SOD2-16 VV genotype with NIDCM, there are no studies examining the prognostic impact of the SOD2-A16V mutation in patients with systolic heart failure. Methods: A total of 755 non-black and 206 black participants in the BEST DNA-Substudy were genotyped for the SOD2-A16V polymorphism. Survival free from the endpoint of all-cause mortality (ACM) and the composite endpoint, death or CHF hospitalization, was compared between the three genotype groups using Kaplan-Meier (KM) survival analysis. Results: The mean follow-up in the BEST DNA-Substudy participants was 768.4 days. The SOD2-A16V genotype prevalences were: SOD2-AA (26%), SOD2-AV (52%), SOD2-VV (22%). There were no significant baseline differences between the three genotype groups. ACM did not differ between the three genotype groups: SOD2-16AA (43/251 or 17.1%), SOD2-16AV (94/502 or 18.7%) and SOD2-16VV (43/208 or 20.7%; P ⫽ 0.5). KM survival analysis demonstrated no significant differences between the three genotype groups defined by the SOD2-A16V polymorphism in survival free from the endpoint of ACM (P ⫽ 0.49) nor the composite endpoint, death or first CHF hospitalization (P ⫽ 0.11). There was no evidence of significant interaction between the SOD2-A16V polymorphism and treatment (placebo vs. bucindolol), etiology of heart failure (ischemic vs. nonischemic) or self-reported race (black vs. non-black). Conclusion: In the BEST DNA-Substudy cohort, largely consisting of white males with NYHA Class III or IV heart failure, the SOD2-A16V polymorphism is not associated with ACM nor the composite endpoint, ACM or first CHF hospitalization.
Journal of Cardiac Failure Vol. 11 No. 6 Suppl. 2005
138
140
Safety Study for Intracoronary Delivery of Autologous Bone Marrow Cells in Chronic Chagas Disease Dogs Model Joao A. Assad1, Suzana A. Silva1, Rodrigo C. Moreira1, Fabio A. Tuche1, Antonio A. Camacho2, Rodrigo V. Branco1, Radovan Borojevic3, Hans F. Dohmann1; 1Cath Lab, Pro´-Cardı´aco, Rio de Janeiro, Brazil; 2Veterinary Clinic, UNESP, Jaboticabal, Sa˜o Paulo, Brazil; 3Histology, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil
Corin, the “pro-ANP/BNP Convertase” Is Up-Regulated in Human Heart Failure, but Inversely Correlated with BNP Expression Daniel L. Dries1, Thomas P. Cappola1, Arun Changolkar1, Kenneth B. Margulies1; 1 Heart Failure/Transplant Group, Division of Cardiology, Hospital of the University of Pennsylvania, Philadelphia, PA
Background: Chagas disease is an endemic infectious (T. Cruzi) in South America. There are very few new acute cases but millions of chronic symptomatic cardiomyopathy patients characterized by ventricular dysfunction and arrhythmias. Purpose: Evaluate the safety and feasibility of intracoronary delivery of autologous bone marrow mononuclear cells (ABMNC) in a chronic Chagas cardiomyopathy dog model. Methods: Six dogs, which were inoculated via intraperitoneal with T.Cruzi 11 years ago. All of them had positive laboratory tests confirming Chagas disease development. Animals were divided in two groups: Four animals in the treated group and two in the control group. The treated group underwent intracoronary delivery of 50 millions ABMMNC. All animals were followed by echocardiograms. Results: At baseline, animals have showed a subtle systolic dysfunction and mild diastolic dysfunction. There were neither adverse effects related to intracoronary route, nor clinical deterioration after the procedure. Ejection fraction showed an improvement in treated group (61.0 ⫾ 13% to 69.3 ⫾ 8.4%) not observed in control group, as well as in cardiac volumes (fig 1). Diastolic parameters, Isovolumetric Relaxing Time (IVRT) and Propagation Velocity M mode E/A wave, varied from 77⫹-23 to 58⫹-6,56 and from 0,81⫹-0,10 to 0,75⫹-0,11 on the treated group, respectively. Control group did not improve. We did not perform statistical analysis due to the small number of animals. Conclusion: In this very rare chronic model (11 years) of Chagas cardiomyopathy, we have showed for the first time in an animal model that intracoronary delivery of ABMMNC had no adverse effects, suggesting safety. On the other hand, treated animals have improved cardiac performance, which was not observed in controls.
Background: Corin is a type II transmembrane serine protease expressed in cardiomyocytes. Both in vitro and in vivo data demonstrate that corin converts pro-ANP and pro-BNP into smaller biologically active molecules. There are only two studies examining corin gene expression in heart failure, both are in rat models of heart failure, and they report conflicting results. One study reports increased myocardial corin gene expression that parallels increased BNP expression, whereas the other study demonstrates decreased myocardial corin gene expression. We are not aware of any published data examining corin gene expression and its correlation with BNP gene expression in human heart failure. Methods: Myocardial RNA from end-stage failing human hearts (n ⫽ 173) and non-failing controls (n ⫽ 13) were hybridized with individual Affymetrix HU133A arrays. Data were normalized and filtered using RNA (www.bioconductor.org) resulting in 13,639 transcripts present above background. Corin and BNP transcript expression levels were measured in failing hearts, stratified by etiology (ischemic vs. non-ischemic) and compared to control heart expression levels. Affymetrix HU 133A array data regarding corin and BNP gene expression was confirmed by RT-PCR. Corin protein levels in the myocardium were examined by Western blot analysis using a monoclonal antibody to human corin. Results: Corin gene expression was increased approximately two-fold in both ischemic (P ⫽ 0.027) and non-ischemic (P ⫽ 0.030) heart failure compared to controls. The increased expression of corin was confirmed by RT-PCR. Western blot analysis demonstrated that myocardial corin protein correlated with corin gene expression levels. BNP gene expression was also increased in both ischemic and non-ischemic cardiomyopathy compared to controls. However, the plot of BNP expression (x-axis) versus corin expression (y-axis) demonstrated an inverse relationship. Conclusions: These data indicate that corin gene expression is upregulated in human heart failure and this correlates with increased myocardial corin protein levels. However, there is an inverse relationship between BNP and corin gene expression in ischemic and nonischemic cardiomyopathy. These results suggest that in advanced heart failure the ability of corin to efficiently process pro-BNP may become rate-limiting.
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141
Robust RNA Amplification for Gene Expression Analysis from Biopsy-Sized Human Heart Tissue Brian R. Barrows1, Agnes A. Azimzadeh5, Hui Zhou5, Gloria Vives-Rodriguez1, Stacey L. McCulle3, Wayne N. Stark, Jr.1, Nicholas Ambulos4, Jing Yin4, Richard N. Pierson III5, Frances L. Johnson3, Stephen S. Gottlieb3, Meredith Bond1; 1Physiology, University of Maryland, Baltimore, Baltimore, MD; 2School of Medicine, Cardiology, University of Maryland, Baltimore, Baltimore, MD; 3Genomics Core Facility, University of Maryland, Baltimore, Baltimore, MD; 4UMMC, Department of Surgery, University of Maryland, Baltimore, Baltimore, MD
Manganese superoxide dismutase (SOD2) A16V Polymorphism Is Not Associated with Increased Mortality in Heart Failure: A Retrospective Study from the Bucindolol Evaluation and Survival (BEST) Trial’s DNA-Substudy Daniel L. Dries1, Thomas P. Cappola1, Ken B. Margulies1, Surai Thaneemit-Chen2, Philip Lavori2; 1Cardiology Division, Hospital of the University of Pennsylvania, Philadelphia, PA; 2Palo Alto VA, DNA Bank Coordinating Center, Stanford, Menlo Park, CA
Introduction: Gene expression experiments are emerging as a method for elucidating biological pathways that contribute to disease states, such as cancer and heart failure. In the case of heart failure, gene array experiments are limited by the amount of tissue that can be obtained. Hypothesis: Both one- and two-cycle amplification of RNA from biopsy-sized pieces of human heart will produce sufficient high-quality cDNA or cRNA for gene expression analysis. Methods: RNA amplification was performed on 10ng, 20ng, and 50ng samples of total RNA obtained from an explanted heart and amplified two ways: 1) one-cycle amplification producing cDNA (NuGEN Ovation), and 2) two-cycle amplification producing cRNA (Affymetrix쑓 Two-Cycle Target Labeling). For gene expression analysis, at least 2.2ug of cDNA or 20ug of cRNA is needed for each Affymetrix GeneChip쑓 array (Human Genome U133 Plus 2.0 Array). GeneChip쑓 data were used to assess the variability within each amplification method and between both one- and two-cycle methods. Patient consent was received for this study. Results: Both one- and two-cycle methods produced high-quality nucleotide samples (260:280nm ⬎ 1.8). The one-cycle method yielded enough cDNA for hybridization to one GeneChip쑓 (all values are mean ⫾ SD; 2.5 ⫾ 0.2ug). The two-cycle method yielded enough cRNA for hybridization to up to four GeneChips쑓 (61 ⫾ 27ug). Gene expression data were used to determine variance among the 10, 20, and 50ng RNA samples amplified by either one- or two-cycle methods. Expression values for 15 selected genes (12000 ⬎ values ⬎ 50) showed an average variance of 18 ⫾ 14% and 20 ⫾ 12% for the one- and two-cycle methods, respectively (P ⬎ 0.05). Conclusions: These results indicate that as little as 10ng of total RNA can be amplified by either one- or two-cycle kits to provide sufficient cDNA or cRNA for Affymetrix GeneChip쑓 experiments. This now opens opportunities to analyze gene expression patterns from hearts of living patients using but a few mg of tissue. Such samples could be obtained during regular cardiac surgery or possibly by less invasive biopsy methods.
Background: SOD2 catalyzes the dismutation of superoxide radicals into hydrogen peroxide in the mitochondria. SOD2 is targeted to the inner mitochondrial by an amino acid leader sequence that contains the A16V mutation. Substitution of valine for alanine at position 16 alters the secondary structure of the leader sequence resulting in impaired targeting of SOD2 to the mitochondria. The resulting increased mitochondrial free radical damage may increase progression of heart failure by an increase in mitochondrial DNA damage, impaired oxidative phosphorylation, and promotion of mitochondrial-mediated apoptosis. Although one report in Japanese pateints demonstrated an association of the SOD2-16 VV genotype with NIDCM, there are no studies examining the prognostic impact of the SOD2-A16V mutation in patients with systolic heart failure. Methods: A total of 755 non-black and 206 black participants in the BEST DNA-Substudy were genotyped for the SOD2-A16V polymorphism. Survival free from the endpoint of all-cause mortality (ACM) and the composite endpoint, death or CHF hospitalization, was compared between the three genotype groups using Kaplan-Meier (KM) survival analysis. Results: The mean follow-up in the BEST DNA-Substudy participants was 768.4 days. The SOD2-A16V genotype prevalences were: SOD2-AA (26%), SOD2-AV (52%), SOD2-VV (22%). There were no significant baseline differences between the three genotype groups. ACM did not differ between the three genotype groups: SOD2-16AA (43/251 or 17.1%), SOD2-16AV (94/502 or 18.7%) and SOD2-16VV (43/208 or 20.7%; P ⫽ 0.5). KM survival analysis demonstrated no significant differences between the three genotype groups defined by the SOD2-A16V polymorphism in survival free from the endpoint of ACM (P ⫽ 0.49) nor the composite endpoint, death or first CHF hospitalization (P ⫽ 0.11). There was no evidence of significant interaction between the SOD2-A16V polymorphism and treatment (placebo vs. bucindolol), etiology of heart failure (ischemic vs. nonischemic) or self-reported race (black vs. non-black). Conclusion: In the BEST DNA-Substudy cohort, largely consisting of white males with NYHA Class III or IV heart failure, the SOD2-A16V polymorphism is not associated with ACM nor the composite endpoint, ACM or first CHF hospitalization.