Circulating miR-184 is a potential predictive biomarker of cardiac damage in Anderson-Fabry disease.

Enzyme replacement therapy (ERT) is a mainstay of treatment for Anderson-Fabry disease (AFD), a pathology with negative effects on the heart and kidneys. However, no reliable biomarkers are available to monitor its efficacy. Therefore, we tested a panel of four microRNAs linked with cardiac and renal damage in order to identify a novel biomarker associated with AFD and modulated by ERT. To this end, 60 patients with a definite diagnosis of AFD and on chronic ERT, and 29 age- and sex-matched healthy individuals, were enrolled by two Italian university hospitals. Only miR-184 met both conditions: its level discriminated untreated AFD patients from healthy individuals (c-statistic = 0.7522), and it was upregulated upon ERT (P < 0.001). On multivariable analysis, miR-184 was independently and inversely associated with a higher risk of cardiac damage (odds ratio = 0.86; 95% confidence interval [CI] = 0.76-0.98; P = 0.026). Adding miR-184 to a comprehensive clinical model improved the prediction of cardiac damage in terms of global model fit, calibration, discrimination, and classification accuracy (continuous net reclassification improvement = 0.917, P < 0.001; integrated discrimination improvement [IDI] = 0.105, P = 0.017; relative IDI = 0.221, 95% CI = 0.002-0.356). Thus, miR-184 is a circulating biomarker of AFD that changes after ERT. Assessment of its level in plasma could be clinically valuable in improving the prediction of cardiac damage in AFD patients.

In vitro thrombogenicity of drug-eluting and bare metal stents.

AIMS: We sought to investigate the thrombogenicity of different DES and BMS in an in vitro system of stent perfusion. MATERIAL AND METHODS: The experimental model consisted of a peristaltic pump connected to 4 parallel silicone tubes in which different stents were deployed. Blood was drawn from healthy volunteers and the amount of stent surfaced-induced thrombus deposition was determined using 125I-fibrinogen. RESULTS: Compared to Resolute, Biomatrix and Vision, Xience was associated with the lowest amount of stent surface-induced thrombus formation, with a significant difference compared to Vision (125I-fibrinogen median value deposition [IQ range]: 50 ng [25-98] versus 560 ng [320-1520], respectively, p < 0.05), but not to other DES. In the second set of experiments Fluoropolymer-coated BMS not eluting drug was associated with a significant 3-fold reduction in 125I-fibrinogen deposition (245 ng [80-300]) compared to Vision (625 ng [320-760], p < 0.05), but a 7-fold increase compared to Xience (35 ng [20-60], p < 0.05). Finally Xience was associated with a significantly greater absorption of albumin compared to BMS. CONCLUSIONS: In an in vitro system of stent perfusion, Xience was associated with the lowest amount of stent surface-induced thrombus formation compared with Resolute, Biomatrix and Vision, with a noted synergistic effect between the fluoropolymer and the drug.

A Combined Targeted and Whole Exome Sequencing Approach Identified Novel Candidate Genes Involved in Heritable Pulmonary Arterial Hypertension.

The pathogenesis of idiopathic and heritable forms of pulmonary arterial hypertension is still not completely understood, even though several causative genes have been proposed, so that a third of patients remains genetically unresolved. Here we applied a multistep approach to extend identification of the genetic bases of such a disease by searching for novel candidate genes/pathways. Twenty-eight patients belonging to 18 families were screened for BMPR2 mutations and BMPR2-negative samples were tested for 12 additional candidate genes by means of a specific massive parallel sequencing-based assay. Finally, whole exome sequencing was performed on four patients showing no mutations at known disease genes, as well as on their unaffected parents. In addition to EIF2AK4, which has been already suggested to be associated with pulmonary veno-occlusive disease, we identified the novel candidate genes ATP13A3, CD248, EFCAB4B, involved in lung vascular remodeling that represent reliable drivers contributing to the disease according to their biological functions/inheritance patterns. Therefore, our results suggest that combining gene panel and whole exome sequencing provides new insights useful for the genetic diagnosis of familial and idiopathic pulmonary arterial hypertension, as well as for the identification of biological pathways that will be potentially targeted by new therapeutic strategies.

Effects of cardiac resynchronization therapy on right ventricular function during rest and exercise, as assessed by radionuclide angiography, and on NT-proBNP levels.

AIM: We carried out this study to investigate mid-term effects of cardiac resynchronization therapy (CRT) on right ventricular (RV) function and neurohormonal response, expressed by N-terminal pro-brain natriuretic peptide (NT-proBNP), in heart failure patients stratified by baseline RV ejection fraction (RVEF). METHODS AND RESULTS: Thirty-six patients with nonischemic dilated cardiomyopathy underwent technetium-99m radionuclide angiography with bicycle exercise immediately after CRT implantation (during spontaneous rhythm and after CRT activation) and 3 months later. Plasma NT proBNP was assessed before implantation and after 3 months. At baseline, RVEF was impaired (≤35%) in 14 patients, preserved (>35%) in 22. At 3 months, RVEF improved during rest and exercise (P = .02) in patients with impaired RV function, while remaining unchanged in patients with preserved RV function. Rest and exercise RV dyssynchrony decreased in both groups at follow-up (P < .05). A similar mid-term improvement in left ventricular (LV) function and NT-proBNP was observed in patients with impaired and preserved RVEF. In the former, the decrease in NT-proBNP correlated with the improvements both in LV and RV dyssynchrony and functions. CONCLUSION: CRT may improve RV performance, during rest and exercise, and neurohormonal response in heart failure patients with nonischemic dilated cardiomyopathy and baseline RV dysfunction. RV dysfunction should not be considered per se a primary criterion for excluding candidacy to CRT.

Effects of statin therapy on platelet reactivity after percutaneous coronary revascularization in patients with acute coronary syndrome.

Statin use is associated with enhanced pharmacodynamic response to clopidogrel in patients with stable coronary artery disease undergoing percutaneous coronary intervention (PCI). However, the impact of statin therapy on clopidogrel response profiles in patients with acute coronary syndrome (ACS) undergoing PCI has not been established and represents the objective of this investigation. On-treatment P2Y12 platelet reactivity was measured using the vasodilator stimulated phosphoprotein (VASP) phosphorylation assay before PCI, at hospital discharge, and at 1 month after PCI in ACS patients enrolled in the multicenter, prospective GEne polymorphisms, Platelet Reactivity, and Syntax Score (GEPRESS) study (n = 962). High platelet reactivity (HPR) was defined as platelet reactivity index ≥50%. Statins were prescribed at hospital discharge in 87% (n = 835) of patients. All patients were followed for 1 year. The 1-month HPR rate was lower in statin than in non-statin treated patients (39.6 vs 52%, respectively, p = 0.009). This finding was confirmed also among statin-treated patients with high Syntax score (≥15). After adjustment for differences in baseline characteristics, statin use at discharge was independently associated with 1-month HPR rate (odds ratio, 0.58, 95% confidence interval, 0.38-0.89; p = 0.015). In ACS patients undergoing PCI treated with clopidogrel the use of statins at discharge was associated with significantly lower 1-month HPR rates compared with patients not treated with statins.

Relationship between diabetes, platelet reactivity, and the SYNTAX score to one-year clinical outcome in patients with non-ST-segment elevation acute coronary syndrome undergoing percutaneous coronary intervention.

AIMS: In patients with non-ST-elevation acute coronary syndromes (NSTE-ACS) treated with PCI, high (H) platelet reactivity (PR) significantly affects one-year outcome. The aim of this report was to analyse the relationships between HPR, the SYNTAX score (SS) and one-year major adverse cardiac events (MACE: cardiac death, myocardial infarction, stent thrombosis) according to diabetes mellitus (DM) status in patients included in the GEne Polymorphism, Platelet REactivity, and the Syntax Score (GEPRESS) study. METHODS AND RESULTS: PR was measured using the vasodilator-stimulated phosphoprotein (VASP) assay at three time points (before PCI, at hospital discharge and at one month after PCI), with HPR defined as >50% PR index in 1,042 patients treated with aspirin and clopidogrel for one year after PCI. Patients with DM and an SS ≥15 had the highest MACE rate between one month and one year, further increased by the presence of HPR (16.4%). On the other hand, among all patients with an SS <15, MACE rates remained low (<3%), irrespective of DM status and PR. CONCLUSIONS: Among NSTE-ACS patients treated with PCI, the combination of DM, an SS ≥15 and HPR characterised a cohort with the highest MACE rate from one month to one year. In such high-risk patients, careful clinical monitoring and implementation of secondary prevention measures, including the use of potent P2Y12 inhibitors, are strongly advised.

Impact of gene polymorphisms, platelet reactivity, and the SYNTAX score on 1-year clinical outcomes in patients with non-ST-segment elevation acute coronary syndrome undergoing percutaneous coronary intervention: the GEPRESS study.

OBJECTIVES: The aim of this study was to investigate the association between high on-treatment platelet reactivity (HPR) and the SYNTAX (Synergy Between Percutaneous Coronary Intervention With Taxus and Cardiac Surgery) score (SS) for risk prediction of major adverse cardiovascular events (MACE) in patients with non-ST-segment elevation acute coronary syndrome (NSTEACS) undergoing percutaneous coronary intervention (PCI). BACKGROUND: Platelet function testing may be used to optimize antiplatelet therapy in high-risk patients, but identification of this category of patients remains challenging. METHODS: The GEPRESS (Gene Polymorphism, Platelet Reactivity, and the Syntax Score) study was a prospective, multicenter, observational study enrolling 1,053 patients with NSTEACS undergoing PCI and treated with clopidogrel. The platelet reactivity index (PRI) was measured at 3 time points: before PCI, at hospital discharge, and 1 month after PCI. Genetic variants of clopidogrel metabolism were determined in 750 patients. Patients were stratified by the presence of HPR (PRI >50%) and by tertile of the SS (upper SS tertile ≥15). The primary objective of this study was the risk of MACE in the period between 1 month and 1 year. RESULTS: Between 1 month and 1 year, 1-month HPR was an independent predictor of MACE in patients with an SS ≥15, but not in those with an SS <15, displaying a 5-fold increase in event rates (10.4% vs. 2.5%; p < 0.0001). CYP2C19*2 was the only single nucleotide polymorphism associated with HPR, but it was not associated with MACE. Although there was a significant variability in the PRI across the 1-month period, predischarge HPR and SS effectively stratified the risk of subsequent MACE up to 1-year follow-up. CONCLUSIONS: In clopidogrel-treated patients with NSTEACS undergoing PCI, HPR was independently associated with an increased risk of MACE only in the presence of a high SS.

Detection of tissue factor antigen and coagulation activity in coronary artery thrombi isolated from patients with ST-segment elevation acute myocardial infarction.

INTRODUCTION: Although ruptured atherosclerotic plaques have been extensively analyzed, the composition of thrombi causing arterial occlusion in patients with ST-segment elevation acute myocardial infarction has been less thoroughly investigated. We sought to investigate whether coagulant active tissue factor can be retrieved in thrombi of patients with STEMI undergoing primary percutaneous coronary intervention. METHODS: Nineteen patients with ST-segment elevation acute myocardial infarction referred for primary percutaneous coronary intervention were enrolled in this study. Coronary thrombi aspirated from coronary arteries were routinely processed for paraffin embedding and histological evaluation (4 patients) or immediately snap frozen for evaluation of tissue factor activity using a modified aPTT test (15 patients). Immunoprecipitation followed by immunoblotting was also performed in 12 patients. RESULTS: Thrombi aspirated from coronary arteries showed large and irregular areas of tissue factor staining within platelet aggregates, and in close contact with inflammatory cells. Some platelet aggregates stained positive for tissue factor, whereas others did not. Monocytes consistently stained strongly for tissue factor, neutrophils had a more variable and irregular tissue factor staining, and red blood cells did not demonstrate staining for tissue factor. Median clotting time of plasma samples containing homogenized thrombi incubated with a monoclonal antibody that specifically inhibits tissue factor-mediated coagulation activity (mAb 5G9) were significantly longer than their respective controls (88.9 seconds versus 76.5 seconds, respectively; p<0.001). Tissue factor was also identified by immunoprecipitation in 10 patients, with significant variability among band intensities. CONCLUSIONS: Active tissue factor is present in coronary artery thrombi of patients with ST-segment elevation acute myocardial infarction, suggesting that it contributes to activate the coagulation cascade ensuing in coronary thrombosis.

A randomised study comparing the antiplatelet and antiinflammatory effect of clopidogrel 150 mg/day versus 75 mg/day in patients with ST-segment elevation acute myocardial infarction and poor responsiveness to clopidogrel: results from the DOUBLE study.

INTRODUCTION: The antiplatelet effect of standard or increased clopidogrel doses in patients with ST- segment elevation acute myocardial infarction (STEMI) has never been studied. In this study we compared the antiplatelet effect of a 75 mg daily maintenance dose of clopidogrel with 150 mg in patients with STEMI undergoing primary percutaneous coronary intervention (PCI). MATERIALS AND METHODS: Fifty-four patients with STEMI undergoing PCI were randomly allocated to receive either 75 mg/day clopidogrel (group 1) or 150 mg/day (group 2) for 1 month. Platelet function, measured by 5 different assays, was determined at 3 time points: 38+/-8 hours after the procedure, 1 week and 1 month after randomization. RESULTS: In group 1, mean +/- SD platelet reactivity index (PRI) measured with the VASP assay was 57.7+/-15.7% and 46.9+/-15.7% at 1 week and 1 month, respectively, compared to 38.8+/-15.7% and 34.9+/-12.6% in group 2 (p=0.0001). Same results were observed for light transmittance aggregometry, whole blood aggregometry and VerifyNow, but not for thromboelastometry. In contrast to what may be expected, the 75 mg daily maintenance dose took longer than 1-week to provide the full clopidogrel antiplatelet effect. Furthermore, patients in group 2 had a nearly 50% reduction in C-reactive protein levels both at 1 week and 1 month. CONCLUSION: In patients with STEMI and poor responsiveness to clopidogrel a 150 mg daily maintenance dose of clopidogrel is associated with a significant reduction of platelet aggregation and a trend towards reduced inflammation.

Matrix metalloproteinases in premature coronary atherosclerosis: influence of inhibitors, inflammation, and genetic polymorphisms.

Matrix metalloproteinases (MMPs) are thought to participate in the pathogenesis of coronary artery disease (CAD), particularly in the occurrence of acute coronary syndrome (ACS). Little is known about human in vivo MMP regulation in CAD. The expression and regulation of MMPs and their tissue inhibitors (TIMPs) were evaluated in premature CAD. The distribution of MMP-3 5A/6A and MMP-9 C/T promoter polymorphisms and MMP-9 A/G exon-6 polymorphism were investigated in 200 consecutive male premature CAD patients (aged < or = 55 years) and 201 age-matched male blood donors. Plasma concentrations/activities of MMP-2 and MMP-9 were also measured, as were plasma concentrations of MMP-3, TIMP-1, and TIMP-2 in 80 patients (49 with ACSs and 31 with stable CAD) and 40 controls. Inflammation markers were also obtained. MMP genetic polymorphism distributions did not vary between patients and controls and did not seem to influence their respective MMP plasma levels. Patients showed increased MMP-9 and TIMP-1 concentrations and decreased TIMP-2 concentration and MMP-2 total activity (all P < or = 0.002). Overall, TIMP-1 correlated with C-reactive protein (CPR) (r = 0.594, P < 0.001) and haptoglobin (r = 0.276, P = 0.005), whereas MMP-2 activity correlated inversely with haptoglobin (r = -0.195, P = 0.032). Blood glucose correlated positively with TIMP-1 concentration (r = 0.711, P < 0.001) and negatively with MMP-2 activity (r = -0.250, P = 0.006). In conclusion, MMP and TIMP plasma levels in premature CAD are linked to clinical presentation and markers of inflammation and metabolic disorders rather than to genetic polymorphisms.

Monocyte-derived tissue factor contributes to stent thrombosis in an in vitro system.

OBJECTIVES: This study evaluated the role of circulating tissue factor (TF) in mediating thrombus formation on stents in an in vitro model of stent perfusion. BACKGROUND: The traditional view of coagulation has recently been challenged by the demonstration that TF is present in circulating blood. The potential contribution of this intravascular pool of TF to thrombus formation on stents is not known. METHODS: Coronary stents were placed in parallel silicone tubes connected to a roller pump that was set to pump blood at a flow rate of 10 ml/min. Stents were then exposed to heparinized blood from healthy volunteers for 120 min. RESULTS: The presence of the stent in the circuit caused a significant increase in monocyte TF expression, but only monocytes with attached platelets stained positive for TF. Thrombi formed on stents and the thrombi stained positive for TF. Pretreatment of blood with a monoclonal antibody against TF (cH36) caused a 56% reduction in (125)I-fibrin(ogen) deposition on stents compared with controls (p = 0.002). Monocyte depletion of blood reduced (125)I-fibrin(ogen) deposition by 45% (p = 0.01) and TF staining in the thrombus by 83% (p = 0.01). Pretreatment of blood with a monoclonal antibody against P-selectin reduced (125)I-fibrin(ogen) deposition by 24% (p = 0.04). Perfusion of stents with leukocyte-reduced platelet-rich plasma (PRP) produced small thrombi and treatment of PRP with cH36 reduced (125)I-fibrin(ogen) deposition by 43% (p = 0.01). CONCLUSIONS: Circulating TF plays a pivotal role in thrombus formation on stents. Monocytes appear to be the main, but not only, source of TF depositing in the thrombus.

Hydroxymethyl-glutaryl coenzyme a reductase inhibition limits cytomegalovirus infection in human endothelial cells.

BACKGROUND: Statins exert anti-inflammatory effects independently of cholesterol-lowering properties. Cytomegalovirus (CMV) infection appears to be implicated in the pathophysiology of atherosclerosis by inducing inflammatory modifications in endothelial cells, especially in immunosuppressed patients. We investigated whether the activity of statins can inhibit replication of CMV in human endothelial cells. METHODS AND RESULTS: Human umbilical vein endothelial cells (HUVECs) were infected with CMV and coincubated with fluvastatin at 0.1 and 0.2 micromol/L. Fluvastatin inhibited (P<0.001) CMV antigen expression, and this effect was dose related (P<0.001). Quantitative polymerase chain reaction showed that CMV DNA concentration was consistently lower in supernatants from fluvastatin-treated cells than in infected controls, and viral particle concentration was up to 30 times lower in 0.2 micromol/L fluvastatin-treated cells than in infected controls (10.5+/-0.9 versus 0.34+/-0.03 per 10(3) pfu/mL, P<0.001). Addition of mevalonate to treated cultures almost completely abolished fluvastatin inhibition of viral growth. Electrophoretic mobility shift assay showed that fluvastatin reduced nuclear factor-kappaB binding activity in CMV-infected cells. CONCLUSIONS: HMG-CoA inhibition by fluvastatin restrains CMV replication in HUVECs by inhibiting viral antigen expression, DNA synthesis, and viral particle production, conceivably by involving a reduction of nuclear factor-kappaB binding activity.

Residues of oxytetracycline and its 4'-epimer in edible tissues from turkeys.

Residues of oxytetracycline (OTC) in edible tissues (muscle, liver, and kidney) of 18 turkeys were determined after continuous administration of the drug for 3 days in drinking water at the maximum recommended concentration of 400 mg/L. The European Union (EU) maximum residue limits (MRLs) set for OTC are 100 microg/kg in muscle tissues, 300 microg/kg in liver, and 600 microg/kg in kidney, as the sum of the parent compound and its derivative 4'-epi-oxytetracycline (4-epi-OTC). Cleanup of tissue samples was performed by metal chelate affinity chromatography (MCAC), but the original technique was miniaturized by the adoption of a mini solid-phase extraction column, allowing reduction of solvents, time, and hazardous waste. OTC and its 4'-epimer were quantitated by an isocratic liquid chromatography elution with UV detection. After 1 day of withdrawal, OTC plus 4-epi-OTC residues were greater than MRL values in muscle and liver; 3 days after the end of treatment, all tissue residues were far lower than the MRL values. At the first day after the end of treatment, 4-epi-OTC was detected at very low concentrations only in muscle, in liver after 1 and 3 days of withdrawal, and in kidney at all sampling times. The withdrawal time was calculated according to EU recommendations and was set at 5 days.