Cinzia Perrino

Intermittent pressure overload triggers hypertrophy-independent cardiac dysfunction and vascular rarefaction

For over a century, there has been intense debate as to the reason why some cardiac stresses are pathological and others are physiological. One long-standing theory is that physiological overloads such as exercise are intermittent, while pathological overloads such as hypertension are chronic. In this study, we hypothesized that the nature of the stress on the heart, rather than its duration, is the key determinant of the maladaptive phenotype. To test this, we applied intermittent pressure overload on the hearts of mice and tested the roles of duration and nature of the stress on the development of cardiac failure. Despite a mild hypertrophic response, preserved systolic function, and a favorable fetal gene expression profile, hearts exposed to intermittent pressure overload displayed pathological features. Importantly, intermittent pressure overload caused diastolic dysfunction, altered beta-adrenergic receptor (betaAR) function, and vascular rarefaction before the development of cardiac hypertrophy, which were largely normalized by preventing the recruitment of PI3K by betaAR kinase 1 to ligand-activated receptors. Thus stress-induced activation of pathogenic signaling pathways, not the duration of stress or the hypertrophic growth per se, is the molecular trigger of cardiac dysfunction.

Hydroxymethylglutaryl Coenzyme A Reductase Inhibitor Simvastatin Prevents Cardiac Hypertrophy Induced by Pressure Overload and Inhibits p21ras Activation

Background—Patients with cardiac hypertrophy are at increased cardiovascular risk. It has been hypothesized that hydroxymethylglutaryl coenzyme A reductase inhibitors may exert beneficial effects other than their cholesterol-lowering actions. The aims of the study were to assess the in vivo effects of simvastatin (SIM) on cardiac hypertrophy and on Ras signaling in rats with ascending aorta banding. Methods and Results—Wistar rats were randomized to receive either treatment with SIM or placebo, and then short-term (group I) and long-term (group II) left ventricular pressure overload was performed by placing a tantalum clip on ascending aorta. At the end of treatment period, left and right ventricular weight, body weight, and tibial length were measured and echocardiographic evaluations were performed. Ras signaling was investigated by analyzing Ras membrane localization and activation, ERK2 phosphorylation, and p27kip1 and cdk4 levels. In SIM-treated rats, a significant reduction of left ventricular weight/body weight, echocardiographic left ventricular mass, and left ventricular end-diastolic diameter and end-diastolic pressure was found. In rats with pressure overload, SIM treatment significantly reduced Ras membrane targeting, Ras in vivo activation, ERK2 phosphorylation, and the ratio cdk4/p27kip1. Conclusions—HMG CoA inhibitor SIM inhibits in vivo Ras signaling and prevents left ventricular hypertrophy development in aortic-banded animals.

Effects of Balloon Injury on Neointimal Hyperplasia in Streptozotocin-Induced Diabetes and in Hyperinsulinemic Nondiabetic Pancreatic Islet–Transplanted Rats

Background—The mechanisms of increased neointimal hyperplasia after coronary interventions in diabetic patients are still unknown. Methods and Results—Glucose and insulin effects on in vitro vascular smooth muscle cell (VSMC) proliferation and migration were assessed. The effect of balloon injury on neointimal hyperplasia was studied in streptozotocin-induced diabetic rats with or without adjunct insulin therapy. To study the effect of balloon injury in nondiabetic rats with hyperinsulinemia, pancreatic islets were transplanted under the kidney capsule in normal rats. Glucose did not increase VSMC proliferation and migration in vitro. In contrast, insulin induced a significant increase in VSMC proliferation and migration in cell cultures. Furthermore, in VSMC culture, insulin increased MAPK activation. A reduction in neointimal hyperplasia was consistently documented after vascular injury in hyperglycemic streptozotocin-induced diabetic rats. Insulin therapy significantly increased neointimal hyperplasia in these rats. This effect of hyperinsulinemia was totally abolished by transfection on the arterial wall of the N17H-ras–negative mutant gene. Finally, after experimental balloon angioplasty in hyperinsulinemic nondiabetic islet-transplanted rats, a significant increase in neointimal hyperplasia was observed. Conclusions—In rats with streptozotocin-induced diabetes, balloon injury was not associated with an increase in neointimal formation. Exogenous insulin administration in diabetic rats and islet transplantation in nondiabetic rats increased both blood insulin levels and neointimal hyperplasia after balloon injury. Hyperinsulinemia through activation of the ras/MAPK pathway, rather than hyperglycemia per se, seems to be of crucial importance in determining the exaggerated neointimal hyperplasia after balloon angioplasty in diabetic animals.

Novel targets and future strategies for acute cardioprotection: Position Paper of the European Society of Cardiology Working Group on Cellular Biology of the Heart

Ischaemic heart disease and the heart failure that often results, remain the leading causes of death and disability in Europe and worldwide. As such, in order to prevent heart failure and improve clinical outcomes in patients presenting with an acute ST-segment elevation myocardial infarction and patients undergoing coronary artery bypass graft surgery, novel therapies are required to protect the heart against the detrimental effects of acute ischaemia/reperfusion injury (IRI). During the last three decades, a wide variety of ischaemic conditioning strategies and pharmacological treatments have been tested in the clinic-however, their translation from experimental to clinical studies for improving patient outcomes has been both challenging and disappointing. Therefore, in this Position Paper of the European Society of Cardiology Working Group on Cellular Biology of the Heart, we critically analyse the current state of ischaemic conditioning in both the experimental and clinical settings, provide recommendations for improving its translation into the clinical setting, and highlight novel therapeutic targets and new treatment strategies for reducing acute myocardial IRI.Ischaemic heart disease and the heart failure that often results, remain the leading causes of death and disability in Europe and worldwide. As such, in order to prevent heart failure and improve clinical outcomes in patients presenting with an acute ST-segment elevation myocardial infarction and patients undergoing coronary artery bypass graft surgery, novel therapies are required to protect the heart against the detrimental effects of acute ischaemia/reperfusion injury. During the last three decades, a wide variety of ischaemic conditioning strategies and pharmacological treatments have been tested in the clinic - however, their translation from experimental to clinical studies for improving patient outcomes has been both challenging and disappointing. Therefore, in this Position Paper of the European Society of Cardiology Working Group on Cellular Biology of the Heart, we critically analyse the current state of ischaemic conditioning in both the experimental and clinical settings, provide recommendations for improving its translation into the clinical setting, and highlight novel therapeutic targets and new treatment strategies for reducing acute myocardial ischaemia/reperfusion injury.

Position Paper of the European Society of Cardiology Working Group Cellular Biology of the Heart: Cell-based therapies for myocardial repair and regeneration in ischemic heart disease and heart failure

Despite improvements in modern cardiovascular therapy, the morbidity and mortality of ischaemic heart disease (IHD) and heart failure (HF) remain significant in Europe and worldwide. Patients with IHD may benefit from therapies that would accelerate natural processes of postnatal collateral vessel formation and/or muscle regeneration. Here, we discuss the use of cells in the context of heart repair, and the most relevant results and current limitations from clinical trials using cell-based therapies to treat IHD and HF. We identify and discuss promising potential new therapeutic strategies that include ex vivo cell-mediated gene therapy, the use of biomaterials and cell-free therapies aimed at increasing the success rates of therapy for IHD and HF. The overall aim of this Position Paper of the ESC Working Group Cellular Biology of the Heart is to provide recommendations on how to improve the therapeutic application of cell-based therapies for cardiac regeneration and repair.

Restoration of β-Adrenergic Receptor Signaling and Contractile Function in Heart Failure by Disruption of the βARK1/Phosphoinositide 3-Kinase Complex

Background—Desensitization and downregulation of myocardial &bgr;-adrenergic receptors (&bgr;ARs) are initiated by the increase in &bgr;AR kinase 1 (&bgr;ARK1) levels. By interacting with &bgr;ARK1 through the phosphoinositide kinase (PIK) domain, phosphoinositide 3-kinase (PI3K) is targeted to agonist-stimulated &bgr;ARs, where it regulates endocytosis. We tested the hypothesis that inhibition of receptor-targeted PI3K activity would alter receptor trafficking and ameliorate &bgr;AR signaling, ultimately improving contractility of failing cardiomyocytes. Methods and Results—To competitively displace PI3K from &bgr;ARK1, we generated mice with cardiac-specific overexpression of the PIK domain. Seven-day isoproterenol administration in wild-type mice induced desensitization of &bgr;ARs and their redistribution from the plasma membrane to early and late endosomes. In contrast, transgenic PIK overexpression prevented the redistribution of &bgr;ARs away from the plasma membrane and preserved their responsiveness to agonist. We further tested whether PIK overexpression could normalize already established &bgr;AR abnormalities and ameliorate contractile dysfunction in a large animal model of heart failure induced by rapid ventricular pacing in pigs. Failing porcine hearts showed increased &bgr;ARK1-associated PI3K activity and marked desensitization and redistribution of &bgr;ARs to endosomal compartments. Importantly, adenoviral gene transfer of the PIK domain in failing pig myocytes resulted in reduced receptor-localized PI3K activity and restored to nearly normal agonist-stimulated cardiomyocyte contractility. Conclusions—These data indicate that the heart failure state is associated with a maladaptive redistribution of &bgr;ARs away from the plasma membrane that can be counteracted through a strategy that targets the &bgr;ARK1/PI3K complex.

Placental Growth Factor Regulates Cardiac Inflammation Through the Tissue Inhibitor of Metalloproteinases-3/Tumor Necrosis Factor-α–Converting Enzyme Axis Crucial Role for Adaptive Cardiac Remodeling During Cardiac Pressure Overload

Background—Heart failure is one of the leading causes of mortality and is primarily the final stage of several overload cardiomyopathies, preceded by an early adaptive hypertrophic response and characterized by coordinated cardiomyocyte growth, angiogenesis, and inflammation. Therefore, growth factors and cytokines have to be critically regulated during cardiac response to transverse aortic constriction. Interestingly, the dual properties of placental growth factor as an angiogenic factor and cytokine make it a candidate to participate in cardiac remodeling in response to hemodynamic overload. Methods and Results—After transverse aortic constriction, placental growth factor knockout mice displayed a dysregulation of cardiac remodeling, negatively affecting muscle growth. Molecular insights underscored that this effect was ascribable mainly to a failure in the establishment of adequate inflammatory response owing to an impaired activity of tumor necrosis factor-α–converting enzyme. Interestingly, after tra...Background— Heart failure is one of the leading causes of mortality and is primarily the final stage of several overload cardiomyopathies, preceded by an early adaptive hypertrophic response and characterized by coordinated cardiomyocyte growth, angiogenesis, and inflammation. Therefore, growth factors and cytokines have to be critically regulated during cardiac response to transverse aortic constriction. Interestingly, the dual properties of placental growth factor as an angiogenic factor and cytokine make it a candidate to participate in cardiac remodeling in response to hemodynamic overload. Methods and Results— After transverse aortic constriction, placental growth factor knockout mice displayed a dysregulation of cardiac remodeling, negatively affecting muscle growth. Molecular insights underscored that this effect was ascribable mainly to a failure in the establishment of adequate inflammatory response owing to an impaired activity of tumor necrosis factor-&agr;–converting enzyme. Interestingly, after transverse aortic constriction, placental growth factor knockout mice had strongly increased levels of tissue inhibitor of metalloproteinases-3, the main natural TACE inhibitor, thus indicating an unbalance of the tissue inhibitor of metalloproteinases-3/tumor necrosis factor-&agr;–converting enzyme axis. Strikingly, when we used an in vivo RNA interference approach to reduce tissue inhibitor of metalloproteinases-3 levels in placental growth factor knockout mice during transverse aortic constriction, we obtained a complete phenotype rescue of early dilated cardiomyopathy. Conclusions— Our results demonstrate that placental growth factor finely tunes a balanced regulation of the tissue inhibitor of metalloproteinases-3/tumor necrosis factor-&agr;–converting enzyme axis and the consequent TNF-&agr; activation in response to transverse aortic constriction, thus allowing the establishment of an inflammatory response necessary for adaptive cardiac remodeling.

Dynamic Regulation of Phosphoinositide 3-Kinase-γ Activity and β-Adrenergic Receptor Trafficking in End-Stage Human Heart Failure

Background— Downregulation of &bgr;-adrenergic receptors (&bgr;ARs) under conditions of heart failure requires receptor targeting of phosphoinositide 3-kinase (PI3K)–&ggr; and redistribution of &bgr;ARs into endosomal compartments. Because support with a left ventricular assist device (LVAD) results in significant improvement of cardiac function in humans, we investigated the effects of mechanical unloading on regulation of PI3K&ggr; activity and intracellular distribution of &bgr;ARs. Additionally, we tested whether displacement of PI3K&ggr; from activated &bgr;ARs would restore agonist responsiveness in failing human cardiomyocytes. Methods and Results— To test the role of PI3K on &bgr;AR endocytosis in failing human hearts, we assayed for PI3K activity in human left ventricular samples before and after mechanical unloading (LVAD). Before LVAD, failing human hearts displayed a marked increase in &bgr;AR kinase 1 (&bgr;ARK1)–associated PI3K activity that was attributed exclusively to enhanced activity of the PI3K&ggr; isoform. Increased &bgr;ARK1-coupled PI3K activity in the failing hearts was associated with downregulation of &bgr;ARs from the plasma membrane and enhanced sequestration into early and late endosomes compared with unmatched nonfailing controls. Importantly, LVAD support reversed PI3K&ggr; activation, normalized the levels of agonist-responsive &bgr;ARs at the plasma membrane, and depleted the &bgr;ARs from the endosomal compartments without changing the total number of receptors (sum of plasma membrane and early and late endosome receptors). To test whether the competitive displacement of PI3K from the &bgr;AR complex restored receptor responsiveness, we overexpressed the phosphoinositide kinase domain of PI3K (which disrupts &bgr;ARK1/PI3K interaction) in primary cultures of failing human cardiomyocytes. Adenoviral-mediated phosphoinositide kinase overexpression significantly increased basal contractility and rapidly reconstituted responsiveness to &bgr;-agonist. Conclusions— These results suggest a novel paradigm in which human &bgr;ARs undergo a process of intracellular sequestration that is dynamically reversed after LVAD support. Importantly, mechanical unloading leads to complete reversal in PI3K&ggr; and &bgr;ARK1-associated PI3K activation. Furthermore, displacement of active PI3K from &bgr;ARK1 restores &bgr;AR responsiveness in failing myocytes.

A new rat model of small vessel stenting.

AbstractObjectives: Restenosis is the major complication of coronary angioplasty and stenting. In addition, the small vessel diameter represents a major limitation to the wide use of the technology. The aim of this study was to assess the feasibility and the vascular response of stent deployment in rat small vessels. Methods: In 40 Wistar rats (500–550 g) a Nir stent crimped on a 1.5 mm Comet angioplasty balloon catheter was deployed at high pressure in the common carotid artery. Neointimal area, neointima/media ratio and the arterial dimension were assessed immediately and at 7, 14, 21, and 28 days after stenting. Results: After stent deployment, the neointimal area and the neointima/media ratio increased progressively and peaked at 14 days (p < 0.05 vs 0 and 7 days). Alpha-actin-positive cells were found circumferentially organized on the lumen surface. At 21 and 28 days after stenting, the neointima and the neointima/media ratio were not statistically different compared with the results obtained fourteen days after stent deployment. No significant differences in the area of external elastic lamina were observed during the study period. In contrast, the internal lumen area was reduced significantly at 14, 21, and 28 days after the stent deployment. Subacute thrombosis rate after stent implantation was 26.5%. Conclusions: The results of this study demonstrated that the balloon expandable stents can be safely placed into rat arteries and the reduction of the internal arterial lumen observed after stent deployment was only due to the neointima formation whereas remodeling did not occur.

Distinct Effects of Leukocyte and Cardiac Phosphoinositide 3-Kinase γ Activity in Pressure Overload–Induced Cardiac Failure

Background— Signaling from phosphoinositide 3-kinase &ggr; (PI3K&ggr;) is crucial for leukocyte recruitment and inflammation but also contributes to cardiac maladaptive remodeling. To better understand the translational potential of these findings, this study investigates the role of PI3K&ggr; activity in pressure overload–induced heart failure, addressing the distinct contributions of bone marrow–derived and cardiac cells. Methods and Results— After transverse aortic constriction, mice knock-in for a catalytically inactive PI3K&ggr; (PI3K&ggr; KD) showed reduced fibrosis and normalized cardiac function up to 16 weeks. Accordingly, treatment with a selective PI3K&ggr; inhibitor prevented transverse aortic constriction–induced fibrosis. To define the cell types involved in this protection, bone marrow chimeras, lacking kinase activity in the immune system or the heart, were studied after transverse aortic constriction. Bone marrow–derived cells from PI3K&ggr; KD mice were not recruited to wild-type hearts, thus preventing fibrosis and preserving diastolic function. After prolonged pressure overload, chimeras with PI3K&ggr; KD bone marrow–derived cells showed slower development of left ventricular dilation and higher fractional shortening than controls. Conversely, in the presence of a wild-type immune system, KD hearts displayed bone marrow–derived cell infiltration and fibrosis at early stages but reduced left ventricular dilation and preserved contractile function at later time points. Conclusions— Together, these data demonstrate that, in response to transverse aortic constriction, PI3K&ggr; contributes to maladaptive remodeling at multiple levels by modulating both cardiac and immune cell functions.