J.P. Werba

Common carotid intima-media thickness measurement. A method to improve accuracy and precision.

Background and Purpose High-resolution ultrasonographic imaging is a nonnvasive method that allows estimation of the thickness of the intima-media complex in human carotid arteries. The determination of intima-media thickness involves several steps, each of which may introduce an error that influences the reproducibility of the method. In the present study, apart from the general reproducibility of the determination of intima-media thickness, the error introduced by each step was evaluated. Methods B-mode scans were performed on 14 randomly selected patients. The common carotid arteries were examined in anterior, lateral, and posterior planes, with a standard methodology and by a new method, making use of external reference points. Results The error in general reproducibility in determination of the subjects mean intima-media thickness was 5.9%. This parameter was also evaluated in a paired manner after dividing the whole artery into sectors; with this protocol, the percent error in general reproducibility was 15%. The main source of variability in the evaluation of common carotid intima-media thickness was found to lie in the operators subjectivity in the choice of the carotid sector to be processed (percent error, 10.27%). A method was therefore designed that used external reference points, resulting in reduction of this error by 38.2%. Conclusions While the mean intima-media thickness might be considered a reproducible parameter to evaluate differences between populations exposed to diverse risk factors, evolutional or therapy-induced changes in the individual may be better monitored on defined carotid sectors. This may be achieved with a high reproducibility by use of the proposed method based on external reference points.

Transcriptional regulation of the human FPR2/ALX gene: evidence of a heritable genetic variant that impairs promoter activity

Lipoxin (LX) A4 a main endogenous stop‐signal of inflammation, activates the G‐protein‐coupled receptor FPR2/ALX, which triggers potent anti‐inflammatory signaling in vivo. Thus, the regulation of FPR2/ALX expression may have pathophysiological and therapeutic relevance. Here, we mapped a nucleotide sequence with strong FPR2/ALX promoter activity. Chromatin immuno‐precipitation revealed specificity protein 1 (Sp1) binding to the core promoter. Site‐directed mutagenesis of the Sp1 cis‐acting element and Sp1 overexpression established that this transcription factor is key for maximal promoter activity, which is instead suppressed by DNA methylation. LXA4 enhanced FPR2/ALX promoter activity (+74%) and mRNA expression (+87.5%) in MDA‐MB231 cells. A single nucleotide mutation (A/G) was detected in the core promoter of one subject with history of cardiovascular disease and of his two daughters. This mutation reduced by ~35‐90% the promoter activity in vitro. Moreover, neutrophils from individuals carrying the A/G variant displayed ~10‐ and 3‐fold reduction in FPR2/ALX mRNA and protein, respectively, compared with cells from their relatives or healthy volunteers expressing the wild‐type allele. These results uncover FPR2/ALX transcriptional regulation and provide the first evidence of mutations that affect FPR2/ALX transcription, thus opening new opportunities for the understanding of the LXA4‐FPR2/ALX axis in human disease —Simiele, F., Recchiuti, A., Mattoscio, D., De Luca, A., Cianci, E., Franchi, S., Gatta, V., Parolari, A., Werba, J. P., Camera, M., Favaloro, B., Romano, M. Transcriptional regulation of the human FPR2/ALX gene: Evidence of a heritable genetic variant that impairs promoter activity. FASEB J. 26, 1323‐1333 (2012). www.fasebj.org

Effects of Timing and Extent of Smoking, Type of Cigarettes, and Concomitant Risk Factors on the Association Between Smoking and Subclinical Atherosclerosis

Background and Purpose— The purpose of this study was to evaluate the effects of timing and extent of smoking, type of cigarettes, and concomitant vascular risk factors (VRFs) on the association between smoking and carotid intima-media thickness (C-IMT) in a lipid clinic population. Methods— 1804 patients (869 men, age 21 to 85 year) participated in the study. Smoking habits were recorded and C-IMTs were measured by B-mode ultrasound. The associations of C-IMT with smoking status (never, former, and current) and with the cigarettes’ content of tar, nicotine, and carbon monoxide (alone or combined to define “light” or “regular” cigarettes) as well as the interactions between smoking status, gender, and VRFs were evaluated before and after adjustment for confounders. Results— C-IMT was highest in current smokers, lower in former, and lowest in never smokers. C-IMT of former and current smokers differed only after data adjustment for variables describing the extent and timing of smoking exposure. C-IMT was positively related to the number of pack-years (number of cigarettes smoked per day [cigarettes/d] multiplied by number of years smoked/20) in both former and current smokers. There were no differences in C-IMT between smokers of cigarettes with high or low nicotine, tar, or carbon monoxide content. Both diabetes and hypertension interacted positively with smoking in determining C-IMTs. Conclusions— In the present cross-sectional observational investigation, carried out in a cohort of patients attending a lipid clinic, consumption of light cigarettes does not reduce the atherogenic effect of smoking on C-IMT. The number of pack-years, cigarettes/d, and years of smoking are relevant covariates in evaluating the effects of smoking on vascular health. The presence of diabetes or hypertension strengthens the association between smoking and cardiovascular risk.

Statins in coronary bypass surgery: rationale and clinical use.

Statin therapy prevents the first occurrence and recurrence of coronary events and reduces cardiovascular and general mortality in patients with coronary artery disease. These compounds modulate a variety of processes involved in the pathophysiology of arteriosclerosis and vascular graft disease by lipid-dependent and lipid-independent (pleiotropic) mechanisms. As a result, statins produce angiographic and clinical benefits in patients undergoing coronary bypass surgery. We review the present knowledge about the effects of statins on this pathologic condition and the evidence supporting an early treatment initiation.

A meta-analysis of randomized controlled trials on statins for the prevention of contrast-induced acute kidney injury in patients with and without acute coronary syndromes.

OBJECTIVES We assessed whether short-term, pre-procedural, intensive statin treatment may reduce contrast-induced acute kidney injury (CI-AKI) incidence in patients with and without acute coronary syndromes (ACS) undergoing coronary angiography (CA) and percutaneous coronary intervention (PCI). BACKGROUND Statins may exert renal-protective effects through their pleiotropic properties. However, there have been conflicting reports on the CI-AKI preventive effect of pre-procedural statin administration. METHODS Randomized controlled trials published between January 1st, 2003 and February 28th, 2014 comparing the preventive effects against CI-AKI of pre-procedural statins vs. control (lower statin dose, no statin, or placebo) in patients undergoing CA/PCI were included. RESULTS Data were combined from 9 clinical trials enrolling 5212 patients (age 65 ± 5 years, 63% males). Pooled analysis showed that intensive, short-term statin pre-treatment significantly reduced the risk of CI-AKI as compared to control (relative risk [RR] 0.50; 95% confidence interval [CI] 0.39 to 0.64; P<0.001). Pre-specified subgroup analysis showed that intensive statin pre-treatment significantly reduced CI-AKI risk in patients with ACS (RR 0.37; 95% CI 0.25 to 0.55; P<0.0001), with only a non-significant positive trend in patients without ACS (RR 0.65; 95% CI 0.41 to 1.03; P=0.07). No evidence of publication bias was detected. CONCLUSIONS Short-term, pre-procedural, intensive statin treatment significantly reduced CI-AKI incidence in ACS patients, and may contribute to the overall clinical benefit associated with the early use of these drugs in this clinical setting. Its role in non-ACS patients warrants further investigation.

Nitric Oxide Synthetic Pathway in Red Blood Cells Is Impaired in Coronary Artery Disease

Background All the enzymatic factors/cofactors involved in nitric oxide (NO) metabolism have been recently found in red blood cells. Increased oxidative stress impairs NO bioavailability and has been described in plasma of coronary artery disease (CAD) patients. The aim of the study was to highlight a potential dysfunction of the metabolic profile of NO in red blood cells and in plasma from CAD patients compared with healthy controls. Methods We determined L-arginine/NO pathway by liquid-chromatography tandem mass spectrometry and high performance liquid chromatography methods. The ratio of oxidized and reduced forms of glutathione, as index of oxidative stress, was measured by liquid-chromatography tandem mass spectrometry method. NO synthase expression and activity were evaluated by immunofluorescence staining and ex-vivo experiments of L-[15N2]arginine conversion to L-[15N]citrulline respectively. Results Increased amounts of asymmetric and symmetric dimethylarginines were found both in red blood cells and in plasma of CAD patients in respect to controls. Interestingly NO synthase expression and activity were reduced in CAD red blood cells. In contrast, oxidized/reduced glutathione ratio was increased in CAD and was associated to arginase activity. Conclusion Our study analyzed for the first time the whole metabolic pathway of L-arginine/NO, both in red blood cells and in plasma, highlighting an impairment of NO pathway in erythrocytes from CAD patients, associated with decreased NO synthase expression/activity and increased oxidative stress.

Tolerability of fibric acids. Comparative data and biochemical bases

Fibric acids are an established class of drugs for the treatment of hyperlipoproteinaemias. Although they have been in use for 30 years or longer, some doubts remain as to their relative tolerability, both as a class and as single agents. Some side effects, e.g. lithogenicity, may be related to their mode of action, while others, e.g. the acute muscular syndrome, may be linked to the spatial conformation of the molecule. These disadvantages should, however, be weighed against the additional, potentially therapeutic properties shown by these compounds. In particular, effects on maturity onset diabetes and hyperuricaemia, as well as a very interesting fibrinolytic potential, have been described for some of them. A painstaking comparative analysis of the major literature data pertaining to the clinical toxicological profile of these agents allow to conclude that, while belonging to a chemical class, fibric acids show dramatic differences from one another, in terms of side effects and of additional pharmacodynamic activities. Moreover, in the case of lithogenicity for example, considerable differences exist between normo- and hyperlipidaemic subjects. Overall, newer molecules of more sophisticated design have a significantly improved tolerability profile vs the old clofibrate.

Effects of smoking regular or light cigarettes on brachial artery flow-mediated dilation

BACKGROUND AND PURPOSE To compare the effects of regular cigarettes (RCs) and light cigarettes (LCs) on brachial artery flow-mediated dilation (FMD) and sublingual glyceryl trinitrate-induced dilation (GTN), markers of endothelial dependant and independent function, respectively. METHODS 206 subjects (age 51.5 ± 12.8 yr, 122 men) had their smoking habits recorded and FMD and GTN measured by B-mode ultrasound. Cigarettes were categorized as RCs or LCs according to their content of tar, nicotine and CO. The chronic effect was assessed in current smokers of RCs (n = 85) or LCs (n = 53) and in never smokers (NS; n = 68). The acute effect was assessed in current smokers by measuring FMD before and 10-min after smoking a single regular (n = 29) or light (n = 51) cigarette. RESULTS FMD was significantly lower in consumers of RCs (6.26%, 95% C.I. 5.58, 6.94) or LCs (5.59%, 95% C.I. 4.74, 6.45) compared to NS (8.68%, 95% C.I. 7.92, 9.44) (both P < 0.0001), but did not differ (P > 0.05) when compared to each other. GTN was similar in the three groups. Analyses adjusted for clinical confounders and for markers involved in oxidative stress, arginine/nitric oxide pathway, and inflammation provided identical results. Smoking a single cigarette, either regular or light, reduced FMD (-0.88% and -1.17%, respectively, both P < 0.05), without significant difference between cigarette type. RCs and LCs produced analogous chronic and acute effects when FMD was calculated with respect to the last 60 s of the low-flow phase (FMD60s). CONCLUSIONS LCs impair endothelial-dependant vasodilation as much as RCs. Thus, smoking LCs cannot be considered an alternative to the only safe choice of a complete and permanent smoking cessation.

BDNFVal66met polymorphism: a potential bridge between depression and thrombosis.

Aims Epidemiological studies strongly suggest a link between stress, depression, and cardiovascular diseases (CVDs); the mechanistic correlation, however, is poorly understood. A single-nucleotide polymorphism in the BDNF gene (BDNFVal66Met), associated with depression and anxiety, has been proposed as a genetic risk factor for CVD. Using a knock-in mouse carrying the BDNFVal66Met human polymorphism, which phenocopies psychiatric-related symptoms found in humans, we investigated the impact of this SNP on thrombosis. Methods and results BDNFMet/Met mice displayed a depressive-like phenotype concomitantly with hypercoagulable state and platelet hyperreactivity. Proteomic analysis of aorta secretome from BDNFMet/Met and wild-type (WT) mice showed differential expression of proteins involved in the coagulation and inflammatory cascades. The BDNF Met allele predisposed to carotid artery thrombosis FeCl3-induced and to death after collagen/epinephrine injection. Interestingly, transfection with BDNFMet construct induced a prothrombotic/proinflammatory phenotype in WT cells. SIRT1 activation, using resveratrol and/or CAY10591, prevented thrombus formation and restored the physiological levels of coagulation and of platelet markers in BDNFMet/Met mice and/or cells transfected with the Met allele. Conversely, inhibition of SIRT1 by sirtinol and/or by specific siRNA induced the prothrombotic/proinflammatory phenotype in WT mice and cells. Finally, we found that BDNF Met homozygosity is associated with increased risk of acute myocardial infarction (AMI) in humans. Conclusion Activation of platelets, alteration in coagulation pathways, and changes in vessel wall protein expression in BDNFMet/Met mice recapitulate well the features occurring in the anxiety/depression condition. Furthermore, our data suggest that the BDNFVal66Met polymorphism contribute to the individual propensity for arterial thrombosis related to AMI.

Microsomal enzyme inducers raise plasma high-density lipoprotein cholesterol levels in healthy control subjects but not in patients with primary hypoalphalipoproteinemia

In this study we compared the ability of phenytoin, a microsomal enzyme inducer, to raise plasma high‐density lipoprotein (HDL) levels in normolipidemic subjects and patients with primary hypoalphalipoproteinemia. In healthy control subjects, phenytoin caused a dose‐dependent increase of plasma HDL, HDL2, and HDL3 cholesterol levels, up to 40% to 50%. Minor changes were recorded in the plasma concentrations of apolipoprotein (apo) A‐I and apo A‐II; the plasma level of the cholesteryl ester transfer protein (CETP) decreased by 42%. In contrast, none of the patients with hypoalphalipoproteinemia had changes in plasma HDL, HDL2, or HDL3 cholesterol, apo A‐I, apo A‐II, or CETP levels. These findings indicate that microsomal enzyme inducers are unsuitable to increase plasma HDL levels in high‐risk patients with primary hypoalphalipoproteinemia, and they disclose a new mechanism, that is, decreased CETP‐mediated transfer of cholesterol out of HDL, for the HDL‐raising effect of microsomal enzyme inducers in healthy individuals.