Emily E. Mackey

Reduced Endoglin Activity Limits Cardiac Fibrosis and Improves Survival in Heart Failure

Background— Heart failure is a major cause of morbidity and mortality worldwide. The ubiquitously expressed cytokine transforming growth factor-&bgr;1 (TGF&bgr;1) promotes cardiac fibrosis, an important component of progressive heart failure. Membrane-associated endoglin is a coreceptor for TGF&bgr;1 signaling and has been studied in vascular remodeling and preeclampsia. We hypothesized that reduced endoglin expression may limit cardiac fibrosis in heart failure. Methods and Results— We first report that endoglin expression is increased in the left ventricle of human subjects with heart failure and determined that endoglin is required for TGF&bgr;1 signaling in human cardiac fibroblasts using neutralizing antibodies and an siRNA approach. We further identified that reduced endoglin expression attenuates cardiac fibrosis, preserves left ventricular function, and improves survival in a mouse model of pressure-overload–induced heart failure. Prior studies have shown that the extracellular domain of endoglin can be cleaved and released into the circulation as soluble endoglin, which disrupts TGF&bgr;1 signaling in endothelium. We now demonstrate that soluble endoglin limits TGF&bgr;1 signaling and type I collagen synthesis in cardiac fibroblasts and further show that soluble endoglin treatment attenuates cardiac fibrosis in an in vivo model of heart failure. Conclusion— Our results identify endoglin as a critical component of TGF&bgr;1 signaling in the cardiac fibroblast and show that targeting endoglin attenuates cardiac fibrosis, thereby providing a potentially novel therapeutic approach for individuals with heart failure.

Mechanically Unloading the Left Ventricle Before Coronary Reperfusion Reduces Left Ventricular Wall Stress and Myocardial Infarct Size

Background— Ischemia/reperfusion injury worsens infarct size, a major determinant of morbidity and mortality after acute myocardial infarction (MI). We tested the hypothesis that reducing left ventricular wall stress with a percutaneous left atrial-to-femoral artery centrifugal bypass system while delaying coronary reperfusion limits myocardial injury in a model of acute MI. Methods and Results— MI was induced by balloon occlusion of the left anterior descending artery in adult male swine. In the MI group (n=4), 120 minutes of left anterior descending artery occlusion was followed by 120 minutes of reperfusion without mechanical support. In the mechanically supported group (MI+unload; n=4), percutaneous left atrial–to–femoral artery bypass was initiated after 120 minutes of ischemia, and left anterior descending artery occlusion was prolonged for an additional 30 minutes, followed by 120 minutes of reperfusion with device support. All animals were euthanized after reperfusion, and infarct size was quantified by triphenyltetrazolium chloride staining. Compared with baseline, mean left ventricular wall stress and stroke work were not changed at any point in the MI group but were decreased after reperfusion in the MI+unload group (mean left ventricular wall stress, 44 658 versus 22 963 dynes/cm2; stroke work, 2823 versus 655 mm Hg·mL, MI versus MI+unload). Phosphorylation of reperfusion injury salvage kinase pathway proteins from noninfarcted left ventricular tissue was unchanged in the MI group but was increased in the MI+unload group. Compared with the MI group, total infarct size was reduced in the MI+unload group (49% versus 28%, MI versus MI+unload). Conclusions— These data support that first unloading the left ventricle despite delaying coronary reperfusion during an acute MI reduces myocardial injury.

Biventricular Remodeling in Murine Models of Right Ventricular Pressure Overload

Right ventricular (RV) failure is a major cause of mortality in acute or chronic lung disease and left heart failure. The objective of this study was to demonstrate a percutaneous approach to study biventricular hemodynamics in murine models of primary and secondary RV pressure overload (RVPO) and further explore biventricular expression of two key proteins that regulate cardiac remodeling: calcineurin and transforming growth factor beta 1 (TGFβ1). Methods Adult, male mice underwent constriction of the pulmonary artery or thoracic aorta as models of primary and secondary RVPO, respectively. Conductance catheterization was performed followed by tissue analysis for changes in myocyte hypertrophy and fibrosis. Results Both primary and secondary RVPO decreased biventricular stroke work however RV instantaneous peak pressure (dP/dtmax) and end-systolic elastance (Ees) were preserved in both groups compared to controls. In contrast, left ventricular (LV) dP/dtmax and LV-Ees were unchanged by primary, but reduced in the secondary RVPO group. The ratio of RV:LV ventriculo-arterial coupling was increased in primary and reduced in secondary RVPO. Primary and secondary RVPO increased RV mass, while LV mass decreased in primary and increased in the secondary RVPO groups. RV fibrosis and hypertrophy were increased in both groups, while LV fibrosis and hypertrophy were increased in secondary RVPO only. RV calcineurin expression was increased in both groups, while LV expression increased in secondary RVPO only. Biventricular TGFβ1 expression was increased in both groups. Conclusion These data identify distinct effects of primary and secondary RVPO on biventricular structure, function, and expression of key remodeling pathways.

Reducing Endoglin Activity Limits Calcineurin and TRPC-6 Expression and Improves Survival in a Mouse Model of Right Ventricular Pressure Overload

Background Right ventricular (RV) failure is a major cause of mortality worldwide and is often a consequence of RV pressure overload (RVPO). Endoglin is a coreceptor for the profibrogenic cytokine, transforming growth factor beta 1 (TGF‐β1). TGF‐β1 signaling by the canonical transient receptor protein channel 6 (TRPC‐6) was recently reported to stimulate calcineurin‐mediated myofibroblast transformation, a critical component of cardiac fibrosis. We hypothesized that reduced activity of the TGF‐β1 coreceptor, endoglin, limits RV calcineurin expression and improves survival in RVPO. Methods and Results We first demonstrate that endoglin is required for TGF‐β1‐mediated calcineurin/TRPC‐6 expression and up‐regulation of alpha‐smooth muscle antigen (α‐SMA), a marker of myofibroblast transformation, in human RV fibroblasts. Using endoglin haploinsufficient mice (Eng+/−) we show that reduced endoglin activity preserves RV function, limits RV fibrosis, and attenuates activation of the calcineurin/TRPC‐6/α‐SMA pathway in a model of angio‐obliterative pulmonary hypertension. Next, using Eng+/− mice or a neutralizing antibody (Ab) against endoglin (N‐Eng) in wild‐type mice, we show that reduced endoglin activity improves survival and attenuates RV fibrosis in models of RVPO induced by pulmonary artery constriction. To explore the utility of targeting endoglin, we observed a reversal of RV fibrosis and calcineurin levels in wild‐type mice treated with a N‐Eng Ab, compared to an immunoglobulin G control. Conclusion These data establish endoglin as a regulator of TGF‐β1 signaling by calcineurin and TRPC‐6 in the RV and identify it as a potential therapeutic target to limit RV fibrosis and improve survival in RVPO, a common cause of death in cardiac and pulmonary disease.

Distinct effects of unfractionated heparin versus bivalirudin on circulating angiogenic peptides.

Background Human studies of therapeutic angiogenesis, stem-cell, and progenitor-cell therapy have failed to demonstrate consistent clinical benefit. Recent studies have shown that heparin increases circulating levels of anti-angiogenic peptides. Given the widely prevalent use of heparin in percutaneous and surgical procedures including those performed as part of studies examining the benefit of therapeutic angiogenesis and cell-based therapy, we compared the effects of unfractionated heparin (UFH) on angiogenic peptides with those of bivalirudin, a relatively newer anticoagulant whose effects on angiogenic peptides have not been studied. Methodology/Principal Findings We measured soluble fms-like tyrosine kinase-1 (sFLT1), placental growth factor (PlGF), vascular endothelial growth factor (VEGF), and soluble Endoglin (sEng) serum levels by enzyme linked immunosorbent assays (ELISA) in 16 patients undergoing elective percutaneous coronary intervention. Compared to baseline values, sFLT1 and PlGF levels increased by 2629±313% and 253±54%, respectively, within 30 minutes of UFH therapy (p<0.01 for both; n = 8). VEGF levels decreased by 93.2±5% in patients treated with UFH (p<0.01 versus baseline). No change in sEng levels were observed after UFH therapy. No changes in sFLT1, PlGF, VEGF, or sEng levels were observed in any patients receiving bivalirudin (n = 8). To further explore the direct effect of anticoagulation on circulating angiogenic peptides, adult, male wild-type mice received venous injections of clinically dosed UFH or bivalirudin. Compared to saline controls, sFLT1 and PlGF levels increased by >500% (p<0.01, for both) and VEGF levels increased by 221±101% (p<0.05) 30 minutes after UFH treatment. Bivalirudin had no effect on peptide levels. To study the cellular origin of peptides after anticoagulant therapy, human coronary endothelial cells were treated with UFH and demonstrated increased sFLT1 and PlGF levels (ANOVA p<0.01 for both) with reduced VEGF levels (ANOVA p<0.05). Bivalirudin had no effect on peptide levels in vitro. Conclusions/Significance Circulating levels of sFLT1, PlGF, and VEGF are significantly altered by UFH, while bivalirudin therapy has no effect. These findings may have significant implications for clinical studies of therapeutic angiogenesis, stem-cell and progenitor-cell therapy.

Reduced activin receptor-like kinase 1 activity promotes cardiac fibrosis in heart failure

INTRODUCTION Activin receptor-like kinase 1 (ALK1) mediates signaling via the transforming growth factor beta-1 (TGFβ1), a pro-fibrogenic cytokine. No studies have defined a role for ALK1 in heart failure. HYPOTHESIS We tested the hypothesis that reduced ALK1 expression promotes maladaptive cardiac remodeling in heart failure. METHODS AND RESULTS In patients with advanced heart failure referred for left ventricular (LV) assist device implantation, LV Alk1 mRNA and protein levels were lower than control LV obtained from patients without heart failure. To investigate the role of ALK1 in heart failure, Alk1 haploinsufficient (Alk1+/-) and wild-type (WT) mice were studied 2 weeks after severe transverse aortic constriction (TAC). LV and lung weights were higher in Alk1+/- mice after TAC. Cardiomyocyte area and LV mRNA levels of brain natriuretic peptide and β-myosin heavy chain were increased similarly in Alk1+/- and WT mice after TAC. Alk-1 mice exhibited reduced Smad 1 phosphorylation and signaling compared to WT mice after TAC. Compared to WT, LV fibrosis and Type 1 collagen mRNA and protein levels were higher in Alk1+/- mice. LV fractional shortening was lower in Alk1+/- mice after TAC. CONCLUSIONS Reduced expression of ALK1 promotes cardiac fibrosis and impaired LV function in a murine model of heart failure. Further studies examining the role of ALK1 and ALK1 inhibitors on cardiac remodeling are required.

TCT-196 From Door to Balloon to Door to Unload: A Transcriptomic Analysis Identifies that Primary Unloading Globally Shifts Gene Expression and Preserves Mitochondrial Integrity within the Infarct Zone during the Acute Phase of AMI

We recently reported that compared to Primary Reperfusion (PR), first reducing myocardial oxygen demand by activating an acute mechanical circulatory support (AMCS) pump while delaying coronary reperfusion (Primary Unloading; PU) reduces myocardial damage in models of acute myocardial infarction (