Carlo Gaudio

Separating the Mechanism-Based and Off-Target Actions of Cholesteryl Ester Transfer Protein Inhibitors With CETP Gene Polymorphisms

Background— Cholesteryl ester transfer protein (CETP) inhibitors raise high-density lipoprotein (HDL) cholesterol, but torcetrapib, the first-in-class inhibitor tested in a large outcome trial, caused an unexpected blood pressure elevation and increased cardiovascular events. Whether the hypertensive effect resulted from CETP inhibition or an off-target action of torcetrapib has been debated. We hypothesized that common single-nucleotide polymorphisms in the CETP gene could help distinguish mechanism-based from off-target actions of CETP inhibitors to inform on the validity of CETP as a therapeutic target. Methods and Results— We compared the effect of CETP single-nucleotide polymorphisms and torcetrapib treatment on lipid fractions, blood pressure, and electrolytes in up to 67 687 individuals from genetic studies and 17 911 from randomized trials. CETP single-nucleotide polymorphisms and torcetrapib treatment reduced CETP activity and had a directionally concordant effect on 8 lipid and lipoprotein traits (total, low-density lipoprotein, and HDL cholesterol; HDL2; HDL3; apolipoproteins A-I and B; and triglycerides), with the genetic effect on HDL cholesterol (0.13 mmol/L, 95% confidence interval [CI] 0.11 to 0.14 mmol/L) being consistent with that expected of a 10-mg dose of torcetrapib (0.13 mmol/L, 95% CI 0.10 to 0.15). In trials, 60 mg of torcetrapib elevated systolic and diastolic blood pressure by 4.47 mm Hg (95% CI 4.10 to 4.84 mm Hg) and 2.08 mm Hg (95% CI 1.84 to 2.31 mm Hg), respectively. However, the effect of CETP single-nucleotide polymorphisms on systolic blood pressure (0.16 mm Hg, 95% CI −0.28 to 0.60 mm Hg) and diastolic blood pressure (−0.04 mm Hg, 95% CI −0.36 to 0.28 mm Hg) was null and significantly different from that expected of 10 mg of torcetrapib. Conclusions— Discordance in the effects of CETP single-nucleotide polymorphisms and torcetrapib treatment on blood pressure despite the concordant effects on lipids indicates the hypertensive action of torcetrapib is unlikely to be due to CETP inhibition or shared by chemically dissimilar CETP inhibitors. Genetic studies could find a place in drug-development programs as a new source of randomized evidence for drug-target validation in humans.

Comorbidities Frequency in Takotsubo Syndrome: An International Collaborative Systematic Review Including 1109 Patients

BACKGROUND To identify predisposing factors that can result in the onset of takotsubo syndrome, we performed an international, collaborative systematic review focusing on clinical characteristics and comorbidities of patients with takotsubo syndrome. METHODS We searched and reviewed cited references up to August 2013 to identify relevant studies. Corresponding authors of selected studies were contacted and asked to provide additional quantitative details. Data from each study were extracted by 2 independent reviewers. The cumulative prevalence of presenting features and comorbidities was assessed. Nineteen studies whose authors sent the requested information were included in the systematic review, with a total of 1109 patients (951 women; mean age, 59-76 years). Evaluation of risk factors showed that obesity was present in 17% of patients (range, 2%-48%), hypertension in 54% (range, 27%-83%), dyslipidemia in 32% (range, 7%-59%), diabetes in 17% (range, 4%-34%), and smoking in 22% (range, 6%-49%). Emotional stressors preceded takotsubo syndrome in 39% of patients and physical stressors in 35%. The most common comorbidities were psychological disorders (24%; range, 0-49%), pulmonary diseases (15%; range, 0-22%), and malignancies (10%; range, 4%-29%). Other common associated disorders were neurologic diseases (7%; range, 0-22%), chronic kidney disease (7%; range, 2%-27%), and thyroid diseases (6%; range, 0-37%). CONCLUSIONS Patients with takotsubo syndrome have a relevant prevalence of cardiovascular risk factors and associated comorbidities. Such of associations needs to be evaluated in further studies.

Aspirin Extrusion From Human Platelets Through Multidrug Resistance Protein-4-Mediated Transport Evidence of a Reduced Drug Action in Patients After Coronary Artery Bypass Grafting

OBJECTIVES In this study we investigate: 1) the role of multidrug resistance protein-4 (MRP4), an organic anion unidirectional transporter, in modulating aspirin action on human platelet cyclooxygenase (COX)-1; and 2) whether the impairment of aspirin-COX-1 interaction, found in coronary artery bypass grafting (CABG) patients, could be dependent on MRP4-mediated transport. BACKGROUND Platelets of CABG patients present a reduced sensitivity to aspirin despite in vivo and in vitro drug treatment. Aspirin is an organic anion and could be a substrate for MRP4. METHODS Intracellular aspirin concentration and drug COX-1 activity, measured by thrombin-induced thromboxane B2 (TxB2) production, were evaluated in platelets obtained from healthy volunteers (HV) and hematopoietic-progenitor cell cultures reducing or not reducing MRP4-mediated transport. Platelet MRP4 expression was evaluated, in platelets from HV and CABG patients, by dot-blot or by immunogold-electromicrographs or immunofluorescence-microscopy analysis. RESULTS Inhibition of MRP4-mediated transport by dipyridamole or Mk-571 increases aspirin entrapment and its in vitro effect on COX-1 activity (142.7 ± 34.6 pg/10(8) cells vs. 343.7 ± 169.3 pg/10⁸ cells TxB2-production). Platelets derived from megakaryocytes transfected with MRP4 small interfering ribonucleic acid have a higher aspirin entrapment and drug COX-1 activity. Platelets from CABG patients showed a high expression of MRP4 whose in vitro inhibition enhanced aspirin effect on COX-1 (349 ± 141 pg/10⁸ cells vs. 1,670 ± 646 pg/10⁸ cells TxB2-production). CONCLUSIONS Aspirin is a substrate for MRP4 and can be extruded from platelet through its transportation. Aspirin effect on COX-1 is little-related to MRP4-mediated aspirin transport in HV, but in CABG patients with MRP4 over-expression, its pharmacological inhibition enhances aspirin action in an efficient way.

Comparison of left ventricular ejection fraction by magnetic resonance imaging and radionuclide ventriculography in idiopathic dilated cardiomyopathy.

To assess the validity of gated magnetic resonance imaging (MRI) in determining left ventricular (LV) ejection fraction (EF), MRI (Spin Echo, multislice-multiphase technique on the short-axis plane) was compared with equilibrium radionuclide ventriculography in 32 patients with idiopathic dilated cardiomyopathy. All patients underwent MRI and radionuclide ventriculography, performed consecutively on the same day (mean time interval between the 2 examinations: 40 minutes). Comparison with LVEF showed a high correlation (y = 0.79 X +3.51, r = 0.91; p less than 0.001). Mean difference between radionuclide ventriculography and MRI data was 1.7, with the 95% confidence interval 0.71 to 2.68: MRI slightly underestimated LVEF. MRI interobserver and intrapatient variability (assessed in 15 of 32 patients) showed a high correlation (r = 0.91, r = 0.98). In conclusion, data suggest that MRI, using the short-axis approach and the multislice-multiphase technique, is an accurate, noninvasive, highly reproducible method of evaluating LVEF in patients with idiopathic dilated cardiomyopathy.

Clinical ResearchAntiplatelet TherapyAspirin Extrusion From Human Platelets Through Multidrug Resistance Protein-4–Mediated Transport: Evidence of a Reduced Drug Action in Patients After Coronary Artery Bypass Grafting

OBJECTIVES In this study we investigate: 1) the role of multidrug resistance protein-4 (MRP4), an organic anion unidirectional transporter, in modulating aspirin action on human platelet cyclooxygenase (COX)-1; and 2) whether the impairment of aspirin-COX-1 interaction, found in coronary artery bypass grafting (CABG) patients, could be dependent on MRP4-mediated transport. BACKGROUND Platelets of CABG patients present a reduced sensitivity to aspirin despite in vivo and in vitro drug treatment. Aspirin is an organic anion and could be a substrate for MRP4. METHODS Intracellular aspirin concentration and drug COX-1 activity, measured by thrombin-induced thromboxane B2 (TxB2) production, were evaluated in platelets obtained from healthy volunteers (HV) and hematopoietic-progenitor cell cultures reducing or not reducing MRP4-mediated transport. Platelet MRP4 expression was evaluated, in platelets from HV and CABG patients, by dot-blot or by immunogold-electromicrographs or immunofluorescence-microscopy analysis. RESULTS Inhibition of MRP4-mediated transport by dipyridamole or Mk-571 increases aspirin entrapment and its in vitro effect on COX-1 activity (142.7 ± 34.6 pg/10(8) cells vs. 343.7 ± 169.3 pg/10⁸ cells TxB2-production). Platelets derived from megakaryocytes transfected with MRP4 small interfering ribonucleic acid have a higher aspirin entrapment and drug COX-1 activity. Platelets from CABG patients showed a high expression of MRP4 whose in vitro inhibition enhanced aspirin effect on COX-1 (349 ± 141 pg/10⁸ cells vs. 1,670 ± 646 pg/10⁸ cells TxB2-production). CONCLUSIONS Aspirin is a substrate for MRP4 and can be extruded from platelet through its transportation. Aspirin effect on COX-1 is little-related to MRP4-mediated aspirin transport in HV, but in CABG patients with MRP4 over-expression, its pharmacological inhibition enhances aspirin action in an efficient way.

Cardiac dysfunction in cirrhosis is not associated with the severity of liver disease

BACKGROUND Cirrhotic cardiomiopathy is described as the presence of cardiac dysfunction in cirrhotic patients. The aim of the study was to investigate factors associated with cardiac dysfunction in cirrhotic patients. PATIENTS AND METHODS Seventy-four cirrhotic patients and twenty-six controls performed a conventional echocardiography and Tissue Doppler Imaging (TDI) for systolic and diastolic function. Results were analyzed by using the Guidelines of American Society of Echocardiography. RESULTS In patients with cirrhosis, left ventricular end-diastolic diameter was increased (p<0.001) , peak systolic velocities were decreased (11.3±2.7 vs 13.9±1.4cm/s; p<0.001) and left atrial volumes were increased (32.7±8.3 vs 24±8.5ml, p<0.001) as well as cardiac mass (90.6±23 vs 70.5±22g/m(2), p<0.001). Forty-seven cirrhotic patients (64%) showed diastolic dysfunction at rest: grade I in 37 and grade II in 10 patients. Systolic and/or diastolic dysfunction were not influenced by a more severe liver impairment. Diastolic dysfunction was more prevalent in patients with ascites vs those without (77% vs 56%; p=0.04). CONCLUSION A mild diastolic dysfunction at rest is frequent in cirrhotic patients but cardiac load conditions are confounding factors in this diagnosis. We did not identify an association between severity of liver disease and cardiac dysfunction.

Angina in Fabry Disease Reflects Coronary Small Vessel Disease

Background—Chest pain is frequently reported in Fabry disease (FD). However, its mechanism and clinical relevance are unclear. Methods and Results—Basal troponin I level, exercise stress test, single-photon emission computed tomography imaging with 99mTc sestamibi, coronary angiography with thrombolysis in myocardial infarction (TIMI) frame count and left ventricular angiography and endomyocardial biopsy were obtained in 13 patients with FD with angina. Ratio of external to lumen diameter of intramural arteries (E/L ratio), myocyte diameter, and extent of fibrosis were morphometrically evaluated by using tissue sections. Controls for coronary angiography and histology were 25 patients with FD without angina and 20 mitral stenosis patients with normal left ventricular function. Troponin I level was elevated in 6 of the 13 patients. Exercise stress test showed evidence of myocardial ischemia, and single-photon emission computed tomography was positive for stress-induced perfusion defects in all patients with FD with angina. Epicardial coronaries were structurally normal but showed slow flow in all and were associated with aneurisms of posterior left ventricular wall in 3 cases. Histology showed remarkable lumen narrowing of most intramural arteries (mean E/L ratio=3.5±1.2; P<0.001 versus both control groups), because of hypertrophy and proliferation of smooth muscle and endothelial cells, both engulfed by glycosphingolipids. Replacement fibrosis exceeded that of both controls (P<0.001). Small vessel disease correlated with coronary slow flow and extent of fibrosis, but did not with patients’ age, sex, and degree of left ventricular hypertrophy. Conclusions—patients with FD with angina have perfusion defects, slow coronary flow, and luminal narrowing of intramural arteries. Small vessel disease may contribute to symptomatic limitation and progressive myocardial dysfunction.

Prognostic relevance of masked hypertension in subjects with prehypertension.

BACKGROUND The prognostic impact of masked hypertension is not yet completely clear. The aim of this study was to evaluate the prognostic relevance of masked hypertension in subjects with prehypertension. METHODS The occurrence of fatal and nonfatal cardiovascular events was evaluated in 591 subjects with prehypertension defined as clinic blood pressure (BP) in the range of 120-139 mm Hg for systolic BP and 80-89 mm Hg for diastolic BP. Among them, 471 were classified as having true prehypertension (clinic BP <140/90 mm Hg and daytime BP <135/85 mm Hg) and 120 as having masked hypertension (clinic BP <140/90 mm Hg and daytime BP > or =135 or 85 mm Hg). RESULTS During the follow-up (6.6 +/- 4.3 years, range 0.5-15.5 years), 29 cardiovascular events occurred. In subjects with true prehypertension and masked hypertension the event-rates per 100 patient-years were 0.57 and 1.51, respectively. Event-free survival was significantly different between the groups (P < 0.005). After adjustment for other covariates, including clinic BP (forced into the model), Cox regression analysis showed that cardiovascular risk was significantly higher in masked hypertension than in true prehypertension (masked vs. true prehypertension, relative risk 2.65, 95% confidence interval 1.18-5.98, P = 0.018). CONCLUSIONS Among subjects with prehypertension, those with masked hypertension are at higher cardiovascular risk than those with true prehypertension. Out-of-office BP should be known in individuals with prehypertension, preferably by ambulatory BP monitoring or alternatively by home BP measurement, to obtain a better prognostic stratification.

Three‐Dimensional Echocardiography and 2D‐3D Speckle‐Tracking Imaging in Chronic Pulmonary Hypertension: Diagnostic Accuracy in Detecting Hemodynamic Signs of Right Ventricular (RV) Failure

Background Our aim was to compare three‐dimensional (3D) and 2D and 3D speckle‐tracking (2D‐STE, 3D‐STE) echocardiographic parameters with conventional right ventricular (RV) indexes in patients with chronic pulmonary hypertension (PH), and investigate whether these techniques could result in better correlation with hemodynamic variables indicative of heart failure. Methods and Results Seventy‐three adult patients (mean age, 53±13 years; 44% male) with chronic PH of different etiologies were studied by echocardiography and cardiac catheterization (25 precapillary PH from pulmonary arterial hypertension, 23 obstructive pulmonary heart disease, and 23 postcapillary PH from mitral regurgitation). Thirty healthy subjects (mean age, 54±15 years; 43% male) served as controls. Standard 2D measurements (RV–fractional area change–tricuspid annular plane systolic excursion) and mitral and tricuspid tissue Doppler annular velocities were obtained. RV 3D volumes and global and regional ejection fraction (3D‐RVEF) were determined. RV strains were calculated by 2D‐STE and 3D‐STE. RV 3D global‐free‐wall longitudinal strain (3DGFW‐RVLS), 2D global‐free‐wall longitudinal strain (GFW‐RVLS), apical‐free‐wall longitudinal strain, basal‐free‐wall longitudinal strain, and 3D‐RVEF were lower in patients with precapillary PH (P<0.0001) and postcapillary PH (P<0.01) compared to controls. 3DGFW‐RVLS (hazard ratio 4.6, 95% CI 2.79 to 8.38, P=0.004) and 3D‐RVEF (hazard ratio 5.3, 95% CI 2.85 to 9.89, P=0.002) were independent predictors of mortality. Receiver operating characteristic curves showed that the thresholds offering an adequate compromise between sensitivity and specificity for detecting hemodynamic signs of RV failure were 39% for 3D‐RVEF (AUC 0.89), −17% for 3DGFW‐RVLS (AUC 0.88), −18% for GFW‐RVLS (AUC 0.88), −16% for apical‐free‐wall longitudinal strain (AUC 0.85), 16 mm for tricuspid annular plane systolic excursion (AUC 0.67), and 38% for RV‐FAC (AUC 0.62). Conclusions In chronic PH, 3D, 2D‐STE and 3D‐STE parameters indicate global and regional RV dysfunction that is associated with RV failure hemodynamics better than conventional echo indices.

Angiotensin II receptor antagonism with telmisartan increases number of endothelial progenitor cells in normotensive patients with coronary artery disease: A randomized, double-blind, placebo-controlled study

INTRODUCTION Circulating endothelial progenitor cells (EPCs) provide an endogenous repair mechanism of the dysfunctional endothelium and therefore can play a crucial role in the pathophysiology of coronary artery disease (CAD). Angiotensin II receptor antagonism has been shown to be able to increase EPCs in hypertension but its effect in patients with CAD is unknown. Aim of this study was to evaluate whether telmisartan, an angiotensin II receptor antagonist, can modify the number of subpopulations of EPCs and may in turn affect the endothelial function of normotensive patients with CAD. METHODS In a prospective double-blind parallel group study, 40 normotensive patients with CAD were randomly treated with telmisartan (80 mg) or placebo for 4 weeks at time of coronary angiography. Measurements of EPCs and assessment of flow-mediated dilatation (FMD) of the brachial artery was performed before and after therapy. RESULTS Absolute number of EPCs was similar at baseline in the telmisartan and placebo groups. After 4 weeks treatment, CD34+/KDR+/CD45- cells increased significantly in the telmisartan group (from 0.010+/-0.003 to 0.014+/-0.004%, P=0.0001) but not in the placebo group (from 0.009+/-0.004 to 0.009+/-0.005%, NS). Similarly, CD133+/KDR+/CD45- cells raised significantly with telmisartan (from 0.003+/-0.002 to 0.006+/-0.002%, P=0.0001) but not with placebo (from 0.004+/-0.003 to 0.003+/-0.002%, NS). Also, CD14+/CD45+ cells increased significantly with telmisartan (from 0.005+/-0.002 to 0.008+/-0.002%, P=0.0001) and were unchanged with placebo (0.006+/-0.002 vs. 0.005+/-0.003%, NS). FMD improved significantly in patients who received telmisartan (10.4+/-3.9%, P=0.0015 vs. baseline) but did not change in the placebo group (5.9+/-2.8%; P=0.32 vs. baseline; telmisartan vs. placebo, P=0.002). A significant positive correlation was found in the telmisartan group between the improvement in FMD and the increase in CD34+/KDR+/CD45- cells and CD133+/KDR+/CD45- cells (r=0.55, P<0.01, and r=0.49, P<0.05, respectively). CONCLUSION Angiotensin II receptor antagonism with telmisartan increases the number of regenerative EPCs and improves endothelial function in normotensive patients with CAD. These novel effects are interrelated and can explain, at least in part, why telmisartan has beneficial cardiovascular effects independent of its blood pressure lowering action.