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Anemia and inflammation in chronic heart failure
The 7th Annual Scientific Meeting
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HFSA
S33
116
117
Anemia and Inflammation in Chronic Heart Failure Aidan Bolger,1 Stephan Haehling,1 Wolfram Doehner,2 Philip A. Poole-Wilson,1 Andrew J.S. Coats,1 Stefan D. Anker1,2—1Clinical Cardiology, NHLI London, Imperial College, London, United Kingdom; 2Division of Applied Cachexia Research, Dept of Cardiology, Charite, Campus Virchow-Klinikum, Berlin, Germany
Preformed Angiotensin II Is Present in Human Mast Cells Masatake Hara,1 Koh Ono,1 Hiromi Wada,1 Shigetake Sasayama,1 Akira Matsumori1—1Cardiovascular Medicine, Kyoto University Graduate School of Medicine, Kyoto, Japan
Background: Plasma levels of tumor necrosis factor (TNF), soluble TNF receptors (sTNFRs) and other mediators of the innate immune response are elevated in chronic heart failure (CHF) and anemia is a poor prognostic feature in this condition. TNF is a negative regulator of hematopoiesis, promoting apoptosis among hematopoietic progenitor cells via TNFR1 and 2. TNF receptors are shed from the cell surface following interaction with TNF. The relationship of TNF and the TNFRs with anemia and changes in leukocyte populations in CHF has not been described. Methods: TNF, sTNFR1, sTNFR2 and soluble CD14 (a marker of the degree of endotoxin-cellular interaction) were quantified by ELISA in 110 patients with stable CHF (NYHA class 2.5 ⫾ 0.7, LVEF 30 ⫾ 9, peak VO2 18.5 ⫾ 5.6, mean ⫾ SD) and in 56 healthy controls. Hemoglobin concentration (Hb), hematocrit (Hct), platelet count and a differential white cell count were established using standard flow cytometric techniques. Results: 28 CHF patients (25.5%) and 3 controls (5.4%) were anemic (Hb ⱕ12.0 g/dL. Across NYHA classes, Hb fell by 16% from 14.3 to 12.0 g/dL and was associated with a 14% drop in hematocrit from 43% to 37% (p ⬎ 0.0001 for trends). There was a stepwise increase in neutrophil and monocyte count across functional class (in 109
cells/L, controls vs class IV: 4.1 ⫾ 1.2 vs 6.5 ⫾ 1.7, p ⬍ 0.0001 and 0.38 ⫾ 0.13 vs 0.54 ⫾ 0.14, p ⫽ 0.001 respectively) but lymphocyte count was lower in patients with the most severe disease (2.1 ⫾ 0.8 vs 1.3 ⫾ 0.5, controls vs class IV, p ⬍ 0.0001). A step-wise increase in plasma levels of TNF, sTNFR1, sTNFR2 and sCD14 was observed with increasing NYHA class (controls vs class IV: 2.6 ⫾ 1.4 vs 5.1 ⫾ 1.8 pg/mL, 808 ⫾ 239 vs 2026 ⫾ 1377 pg/mL, 1773 ⫾ 547 vs 3635 ⫾ 1239 pg/mL and 3606 ⫾ 1162 vs 5390 ⫾ 1386 ng/mL respectively, p ⬍ 0.0001 for trends). All 4 immune parameters had an inverse relationship to Hb (TNF r ⫽ ⫺0.45, sTNFR1 r ⫽ ⫺0.56, sTNFR2 r ⫽ ⫺0.62, sCD14 r ⫽ ⫺0.56, all p ⬍ 0.0001) and similar relationships were seen for Hct (all p ⬍ 0.001). Furthermore, levels of TNF, sTNFR1 and sTNFR2 related inversely to lymphocyte count (r ⫽ ⫺0.23, p ⬍ 0.05; r ⫽ ⫺0.36, p ⬍ 0.001 and r ⫽ ⫺0.31, p ⬍ 0.05 respectively). Conclusions: Patients with advancing CHF frequently have anemia and demonstrate relative neutrophilia, monocytosis and lymphopenia. High circulating levels of TNF, sTNFR1, sTNFR2 and sCD14 relate strongly to decreased hemoglobin levels and falling lymphocyte count. Anemia and inflammatory immune activation relate to mortality in CHF and both might be considered important (and related) therapeutic targets.
Background: Mast cells are multifunctional cells, which contain various bioactive mediators including cytokines, leukotrienes, histamine, proteases such as chymase and tryptase. They are found in the human heart, and their density increases in the myocardium of patients suffering from heart failure. Chymase is a serine protease which participates in inflammation and tissue remodeling . Like angiotensin converting enzyme, human mast cell chymase possesses angiotensin II (A II) forming activity in the heart and blood vessels. In this study, we investigated whether preformed A II was present in human mast cells. Methods and results: To examine whether human mast cells are capable of synthesizing A II, we first examined a human mast cell line (HMC-1) for the expression of mRNA of renin-angiotensin system using RT-PCR analysis. The gene transcripts of renin and angiotensinogen mRNA were detected both in HMC-1 and human myocardium. ACE mRNA was found in human heart tissue, but not in HMC-1. In ELISA, immunoreactive A II protein was detected in HMC-1. Supernatant of sonicated HMC-1 contained 526 ⫾ 11 ng/ml (n ⫽ 3) of A II. On immunohistochemistry, A II was distributed predominantly in the cytosol of HMC-1. Since myocardial mast cells are interfaced with nerve fibers and functionally associated with the calcitonin gene-related peptide (CGRP), the effect of CGRP on A II release from HMC-1 was examined. CGRP induced the release of A II from HMC-1 cells in a concentration dependent manner. In quantitative RT-PCR analysis, CGRP also increased angiotensinogen mRNA. Onset of upregulation of angiotensinogen mRNA was observed after 30 min of incubation with CGRP, and increased 2to 3-fold after 6h of incubation. Conclusions: The presence of preformed A II and gene expression of the renin-angiotensin system were detected in human mast cells. The release and synthesis of A II in mast cells was regulated by CGRP. Since A II plays an important role in the evolution of heart failure, these observations suggest that the release of A II by mast cells, and its neural regulation, are implicated in the pathophysiology of heart failure.
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119
Preoperative Plasma BNP Level as a Predictor of Postoperative Complications and Outcomes in Patients Undergoing Heart Surgery Radmila Kazanegra,1 Ryan Hutfless,1 Meenakshi A. Bhalla,1 Alisi Tulua-Tata,1 Paul Clopton,1 Cherimarie James,1 Alan S. Maisel1—1VA San Diego Healthcare System, San Diego, CA
Aldosterone Directly Stimulates Growth of Neonatal Rat Myocytes Marina P. Okoshi,1 Xinhua Yan,1 Katashi Okoshi,1 Adam J.T. Schuldt,1 Masaharu Nakayama,1 Paul C. Simpson,2 Beverly H. Lorell1—1Cardiovascular Division, Beth Israel Deaconess Medical Center, Harvard University, Boston, MA; 2VA Medical Center, University of California, San Francisco, CA
A variety of multifactor indices have been proposed for preoperative risk assessment of patients undergoing cardiac surgery, but have shown limited ability and utility in accurately predicting postoperative complications, hospital stay, and mortality. The purpose of this study was to assess whether the preoperative plasma BNP measurements could be used as predictors of clinical endpoints following open heart surgery. We hypothesized that BNP could be capable of predicting postoperative outcomes since it has been shown to have a strong positive correlation with ejection fraction and ventricular dysfunction. B-type natriuretic peptide (BNP) is secreted primarily from the left ventricle in response to volume expansion and pressure overload and has been useful in diagnosing congestive heart failure. Subjects were 98 male patients (63 ⫾ 8 years) that underwent open heart surgery at the San Diego Veterans Administration Health System during a 19 month period. Patient data was determined by review of finalized surgical notes and anesthesia records. Samples were analyzed for BNP levels using a fluorescence immunoassay kit (Biosite Diagnostics, San Diego). Preoperative BNP levels showed positive correlation with length of postoperative hospital stay (r ⫽ 0.25, p ⬍ 0.05), and preoperative ejection fraction (r ⫽ 0.20, p ⬍ 0.05). Student’s t-test demonstrated significantly higher mean preoperative BNP measurements in patients requiring the use of an Intra-Aortic-Balloon Pump (387 ⫾ 112 pg/ml vs. 181 ⫾ 25 pg/ml, p ⬍ 0.05). There were significantly higher preoperative plasma BNP levels in patients with postoperative hospital stays in excess of nine days (307 ⫾ 68 pg/ml vs. 179 ⫾ 27 pg/ml, p ⬍ 0.05), and in the group with mortality within 60 days (522 ⫾ 208 pg/ml vs. 190 ⫾ 25 pg/ml, p ⫽ 0.01) and one year (357 ⫾ 93 pg/ml vs. 184 ⫾ 26 pg/ml, p ⬍ 0.05). ROC curves were significant for preoperative BNP levels as predictors of postoperative IABP use, hospital stay ⬎9 days, and mortality ⬍1 year with area under curves of 0.70, 0.64, and 0.70 respectively. A BNP cut-off value above 385 pg/ml demonstratedhigh specificity (90% in each) and accuracy (86%, 79%, 85% respectively) for predicting each of these endpoints. Ejection Fraction, as determined by echocardiogram, was compared to all clinical endpoints by ROC curves displayed acceptable significance. In conclusion, preoperative BNP measurements ⬎385 pg/ml may quickly, accurately, and cost effectively predict the postoperative need for an Intra-Aortic Balloon Pump, prolonged postoperative hospital stay, and mortality within one year following heart surgery. ROC: Preopoperative BNP levels and mortality ⬍1 year (AUC ⫽ 0.7)
Background: Clinical and experimental studies on hypertension and heart failure suggest that aldosterone (ALDO) modulates myocardial hypertrophy. However, in the in vivo studies, it is not possible to distinguish between direct effects on myocyte growth and hemodynamic actions. We tested the hypothesis that ALDO induces myocyte hypertrophy in low-density, serum-free cultures of neonatal rat ventricular myocytes in comparison to vehicle. ALDO (1 nmol/L - 5 µmol/L) was added on day 4 of culture. Phenylephrine (PE, 20 µmol/L) was used as a positive control. Hypertrophy was assessed by [14C]-phenylalanine incorporation and confocal microscopy of myocyte surface area. PKC levels (total PKC, PKC-α, PKC-β1, PKC-δ, and PKC-ε) were evaluated by Western blotting of cell particulate fraction after stimulation for 1 min. All experiments were done at least in triplicate. Results: ALDO caused a dose-dependent increase in cell protein incorporation (p ⬍ 0.001 vs vehicle-treated group, one-way ANOVA), with EC50 ⫽ 40 nmol/L. Table shows time course and magnitude of protein incorporation and change in myocyte surface area with ALDO (5 µmol/L ) and PE (20 µmol/L). Time Course of [14C]-phenylalanine incorporation Aldosterone Phenylephrine
24 h 112 ⫾ 3* 140 ⫾ 3*
48 h 118 ⫾ 2* 162 ⫾ 3*
72 h 127 ⫾ 4* 196 ⫾ 6*
Time Course of Change in Myocyte Area Aldosterone Phenylephrine
24 h – –
48 h 129 ⫾ 4† 133 ⫾ 5*
72 h 125 ⫾ 4† 156 ⫾ 10*
Mean ⫾ SEM as % of vehicle-treated group; *p ⬍ 0.001 † p ⬍ 0.05 vs vehicle. The mineralocorticoid receptor antagonist spironolactone (10 µmol/L) and PD 98059 (1 µmol/L), an inhibitor of extracellular signal-regulated kinase (ERK) activator MEK (mitogen-activated protein kinase kinase), each inhibited ALDO responses, with no effect on PE-induced growth. Total PKC and fractions PKC-α, PKC-β1, and PKCε were acutely activated by PE, but not by Aldo. Summary: Aldosterone directly stimulates myocyte hypertrophy in neonatal rat ventricular myocytes. This growth response is dependent on the activation of the mineralocorticoid receptor and the ERK pathway and is independent of very early PKC activation.
•
HFSA
S33
116
117
Anemia and Inflammation in Chronic Heart Failure Aidan Bolger,1 Stephan Haehling,1 Wolfram Doehner,2 Philip A. Poole-Wilson,1 Andrew J.S. Coats,1 Stefan D. Anker1,2—1Clinical Cardiology, NHLI London, Imperial College, London, United Kingdom; 2Division of Applied Cachexia Research, Dept of Cardiology, Charite, Campus Virchow-Klinikum, Berlin, Germany
Preformed Angiotensin II Is Present in Human Mast Cells Masatake Hara,1 Koh Ono,1 Hiromi Wada,1 Shigetake Sasayama,1 Akira Matsumori1—1Cardiovascular Medicine, Kyoto University Graduate School of Medicine, Kyoto, Japan
Background: Plasma levels of tumor necrosis factor (TNF), soluble TNF receptors (sTNFRs) and other mediators of the innate immune response are elevated in chronic heart failure (CHF) and anemia is a poor prognostic feature in this condition. TNF is a negative regulator of hematopoiesis, promoting apoptosis among hematopoietic progenitor cells via TNFR1 and 2. TNF receptors are shed from the cell surface following interaction with TNF. The relationship of TNF and the TNFRs with anemia and changes in leukocyte populations in CHF has not been described. Methods: TNF, sTNFR1, sTNFR2 and soluble CD14 (a marker of the degree of endotoxin-cellular interaction) were quantified by ELISA in 110 patients with stable CHF (NYHA class 2.5 ⫾ 0.7, LVEF 30 ⫾ 9, peak VO2 18.5 ⫾ 5.6, mean ⫾ SD) and in 56 healthy controls. Hemoglobin concentration (Hb), hematocrit (Hct), platelet count and a differential white cell count were established using standard flow cytometric techniques. Results: 28 CHF patients (25.5%) and 3 controls (5.4%) were anemic (Hb ⱕ12.0 g/dL. Across NYHA classes, Hb fell by 16% from 14.3 to 12.0 g/dL and was associated with a 14% drop in hematocrit from 43% to 37% (p ⬎ 0.0001 for trends). There was a stepwise increase in neutrophil and monocyte count across functional class (in 109
cells/L, controls vs class IV: 4.1 ⫾ 1.2 vs 6.5 ⫾ 1.7, p ⬍ 0.0001 and 0.38 ⫾ 0.13 vs 0.54 ⫾ 0.14, p ⫽ 0.001 respectively) but lymphocyte count was lower in patients with the most severe disease (2.1 ⫾ 0.8 vs 1.3 ⫾ 0.5, controls vs class IV, p ⬍ 0.0001). A step-wise increase in plasma levels of TNF, sTNFR1, sTNFR2 and sCD14 was observed with increasing NYHA class (controls vs class IV: 2.6 ⫾ 1.4 vs 5.1 ⫾ 1.8 pg/mL, 808 ⫾ 239 vs 2026 ⫾ 1377 pg/mL, 1773 ⫾ 547 vs 3635 ⫾ 1239 pg/mL and 3606 ⫾ 1162 vs 5390 ⫾ 1386 ng/mL respectively, p ⬍ 0.0001 for trends). All 4 immune parameters had an inverse relationship to Hb (TNF r ⫽ ⫺0.45, sTNFR1 r ⫽ ⫺0.56, sTNFR2 r ⫽ ⫺0.62, sCD14 r ⫽ ⫺0.56, all p ⬍ 0.0001) and similar relationships were seen for Hct (all p ⬍ 0.001). Furthermore, levels of TNF, sTNFR1 and sTNFR2 related inversely to lymphocyte count (r ⫽ ⫺0.23, p ⬍ 0.05; r ⫽ ⫺0.36, p ⬍ 0.001 and r ⫽ ⫺0.31, p ⬍ 0.05 respectively). Conclusions: Patients with advancing CHF frequently have anemia and demonstrate relative neutrophilia, monocytosis and lymphopenia. High circulating levels of TNF, sTNFR1, sTNFR2 and sCD14 relate strongly to decreased hemoglobin levels and falling lymphocyte count. Anemia and inflammatory immune activation relate to mortality in CHF and both might be considered important (and related) therapeutic targets.
Background: Mast cells are multifunctional cells, which contain various bioactive mediators including cytokines, leukotrienes, histamine, proteases such as chymase and tryptase. They are found in the human heart, and their density increases in the myocardium of patients suffering from heart failure. Chymase is a serine protease which participates in inflammation and tissue remodeling . Like angiotensin converting enzyme, human mast cell chymase possesses angiotensin II (A II) forming activity in the heart and blood vessels. In this study, we investigated whether preformed A II was present in human mast cells. Methods and results: To examine whether human mast cells are capable of synthesizing A II, we first examined a human mast cell line (HMC-1) for the expression of mRNA of renin-angiotensin system using RT-PCR analysis. The gene transcripts of renin and angiotensinogen mRNA were detected both in HMC-1 and human myocardium. ACE mRNA was found in human heart tissue, but not in HMC-1. In ELISA, immunoreactive A II protein was detected in HMC-1. Supernatant of sonicated HMC-1 contained 526 ⫾ 11 ng/ml (n ⫽ 3) of A II. On immunohistochemistry, A II was distributed predominantly in the cytosol of HMC-1. Since myocardial mast cells are interfaced with nerve fibers and functionally associated with the calcitonin gene-related peptide (CGRP), the effect of CGRP on A II release from HMC-1 was examined. CGRP induced the release of A II from HMC-1 cells in a concentration dependent manner. In quantitative RT-PCR analysis, CGRP also increased angiotensinogen mRNA. Onset of upregulation of angiotensinogen mRNA was observed after 30 min of incubation with CGRP, and increased 2to 3-fold after 6h of incubation. Conclusions: The presence of preformed A II and gene expression of the renin-angiotensin system were detected in human mast cells. The release and synthesis of A II in mast cells was regulated by CGRP. Since A II plays an important role in the evolution of heart failure, these observations suggest that the release of A II by mast cells, and its neural regulation, are implicated in the pathophysiology of heart failure.
118
119
Preoperative Plasma BNP Level as a Predictor of Postoperative Complications and Outcomes in Patients Undergoing Heart Surgery Radmila Kazanegra,1 Ryan Hutfless,1 Meenakshi A. Bhalla,1 Alisi Tulua-Tata,1 Paul Clopton,1 Cherimarie James,1 Alan S. Maisel1—1VA San Diego Healthcare System, San Diego, CA
Aldosterone Directly Stimulates Growth of Neonatal Rat Myocytes Marina P. Okoshi,1 Xinhua Yan,1 Katashi Okoshi,1 Adam J.T. Schuldt,1 Masaharu Nakayama,1 Paul C. Simpson,2 Beverly H. Lorell1—1Cardiovascular Division, Beth Israel Deaconess Medical Center, Harvard University, Boston, MA; 2VA Medical Center, University of California, San Francisco, CA
A variety of multifactor indices have been proposed for preoperative risk assessment of patients undergoing cardiac surgery, but have shown limited ability and utility in accurately predicting postoperative complications, hospital stay, and mortality. The purpose of this study was to assess whether the preoperative plasma BNP measurements could be used as predictors of clinical endpoints following open heart surgery. We hypothesized that BNP could be capable of predicting postoperative outcomes since it has been shown to have a strong positive correlation with ejection fraction and ventricular dysfunction. B-type natriuretic peptide (BNP) is secreted primarily from the left ventricle in response to volume expansion and pressure overload and has been useful in diagnosing congestive heart failure. Subjects were 98 male patients (63 ⫾ 8 years) that underwent open heart surgery at the San Diego Veterans Administration Health System during a 19 month period. Patient data was determined by review of finalized surgical notes and anesthesia records. Samples were analyzed for BNP levels using a fluorescence immunoassay kit (Biosite Diagnostics, San Diego). Preoperative BNP levels showed positive correlation with length of postoperative hospital stay (r ⫽ 0.25, p ⬍ 0.05), and preoperative ejection fraction (r ⫽ 0.20, p ⬍ 0.05). Student’s t-test demonstrated significantly higher mean preoperative BNP measurements in patients requiring the use of an Intra-Aortic-Balloon Pump (387 ⫾ 112 pg/ml vs. 181 ⫾ 25 pg/ml, p ⬍ 0.05). There were significantly higher preoperative plasma BNP levels in patients with postoperative hospital stays in excess of nine days (307 ⫾ 68 pg/ml vs. 179 ⫾ 27 pg/ml, p ⬍ 0.05), and in the group with mortality within 60 days (522 ⫾ 208 pg/ml vs. 190 ⫾ 25 pg/ml, p ⫽ 0.01) and one year (357 ⫾ 93 pg/ml vs. 184 ⫾ 26 pg/ml, p ⬍ 0.05). ROC curves were significant for preoperative BNP levels as predictors of postoperative IABP use, hospital stay ⬎9 days, and mortality ⬍1 year with area under curves of 0.70, 0.64, and 0.70 respectively. A BNP cut-off value above 385 pg/ml demonstratedhigh specificity (90% in each) and accuracy (86%, 79%, 85% respectively) for predicting each of these endpoints. Ejection Fraction, as determined by echocardiogram, was compared to all clinical endpoints by ROC curves displayed acceptable significance. In conclusion, preoperative BNP measurements ⬎385 pg/ml may quickly, accurately, and cost effectively predict the postoperative need for an Intra-Aortic Balloon Pump, prolonged postoperative hospital stay, and mortality within one year following heart surgery. ROC: Preopoperative BNP levels and mortality ⬍1 year (AUC ⫽ 0.7)
Background: Clinical and experimental studies on hypertension and heart failure suggest that aldosterone (ALDO) modulates myocardial hypertrophy. However, in the in vivo studies, it is not possible to distinguish between direct effects on myocyte growth and hemodynamic actions. We tested the hypothesis that ALDO induces myocyte hypertrophy in low-density, serum-free cultures of neonatal rat ventricular myocytes in comparison to vehicle. ALDO (1 nmol/L - 5 µmol/L) was added on day 4 of culture. Phenylephrine (PE, 20 µmol/L) was used as a positive control. Hypertrophy was assessed by [14C]-phenylalanine incorporation and confocal microscopy of myocyte surface area. PKC levels (total PKC, PKC-α, PKC-β1, PKC-δ, and PKC-ε) were evaluated by Western blotting of cell particulate fraction after stimulation for 1 min. All experiments were done at least in triplicate. Results: ALDO caused a dose-dependent increase in cell protein incorporation (p ⬍ 0.001 vs vehicle-treated group, one-way ANOVA), with EC50 ⫽ 40 nmol/L. Table shows time course and magnitude of protein incorporation and change in myocyte surface area with ALDO (5 µmol/L ) and PE (20 µmol/L). Time Course of [14C]-phenylalanine incorporation Aldosterone Phenylephrine
24 h 112 ⫾ 3* 140 ⫾ 3*
48 h 118 ⫾ 2* 162 ⫾ 3*
72 h 127 ⫾ 4* 196 ⫾ 6*
Time Course of Change in Myocyte Area Aldosterone Phenylephrine
24 h – –
48 h 129 ⫾ 4† 133 ⫾ 5*
72 h 125 ⫾ 4† 156 ⫾ 10*
Mean ⫾ SEM as % of vehicle-treated group; *p ⬍ 0.001 † p ⬍ 0.05 vs vehicle. The mineralocorticoid receptor antagonist spironolactone (10 µmol/L) and PD 98059 (1 µmol/L), an inhibitor of extracellular signal-regulated kinase (ERK) activator MEK (mitogen-activated protein kinase kinase), each inhibited ALDO responses, with no effect on PE-induced growth. Total PKC and fractions PKC-α, PKC-β1, and PKCε were acutely activated by PE, but not by Aldo. Summary: Aldosterone directly stimulates myocyte hypertrophy in neonatal rat ventricular myocytes. This growth response is dependent on the activation of the mineralocorticoid receptor and the ERK pathway and is independent of very early PKC activation.