P.G. Brambilla

Circulating microRNAs are new and sensitive biomarkers of myocardial infarction

Aims Circulating microRNAs (miRNAs) may represent a novel class of biomarkers; therefore, we examined whether acute myocardial infarction (MI) modulates miRNAs plasma levels in humans and mice. Methods and results Healthy donors (n = 17) and patients (n = 33) with acute ST-segment elevation MI (STEMI) were evaluated. In one cohort (n = 25), the first plasma sample was obtained 517 ± 309 min after the onset of MI symptoms and after coronary reperfusion with percutaneous coronary intervention (PCI); miR-1, -133a, -133b, and -499-5p were ∼15- to 140-fold control, whereas miR-122 and -375 were ∼87–90% lower than control; 5 days later, miR-1, -133a, -133b, -499-5p, and -375 were back to baseline, whereas miR-122 remained lower than control through Day 30. In additional patients (n = 8; four treated with thrombolysis and four with PCI), miRNAs and troponin I (TnI) were quantified simultaneously starting 156 ± 72 min after the onset of symptoms and at different times thereafter. Peak miR-1, -133a, and -133b expression and TnI level occurred at a similar time, whereas miR-499-5p exhibited a slower time course. In mice, miRNAs plasma levels and TnI were measured 15 min after coronary ligation and at different times thereafter. The behaviour of miR-1, -133a, -133b, and -499-5p was similar to STEMI patients; further, reciprocal changes in the expression levels of these miRNAs were found in cardiac tissue 3–6 h after coronary ligation. In contrast, miR-122 and -375 exhibited minor changes and no significant modulation. In mice with acute hind-limb ischaemia, there was no increase in the plasma level of the above miRNAs. Conclusion Acute MI up-regulated miR-1, -133a, -133b, and -499-5p plasma levels, both in humans and mice, whereas miR-122 and -375 were lower than control only in STEMI patients. These miRNAs represent novel biomarkers of cardiac damage.

Crossvalidation of Anthropometry Against Magnetic Resonance Imaging for the Assessment of Visceral and Subcutaneous Adipose Tissue in Children

Background:The study of the relationship between anthropometry and visceral adipose tissue (VAT) is of great interest because VAT is associated with many risk factors for noncommunicable diseases and anthropometry is easy to perform in clinical practice. The studies hitherto available for children have, however, been performed on small sample sizes.Design:Pooling of the data of studies published from 1992 to 2004 as indexed on Medline.Aims:To assess the relationship between anthropometry and VAT and subcutaneous adipose tissue (SAT) as measured by magnetic resonance imaging (MRI) in children and to analyze the effect of age, gender, pubertal status and ethnicity.Subjects and methods:Eligible subjects were 7–16 year-old, with availability of VAT and SAT, gender, ethnicity, body mass index (BMI) and waist circumference (WC). A total of 497 subjects were collected from seven different investigators and 407 of them (178 Caucasians and 229 Hispanics) were analyzed.Results:Despite ethnic differences in MRI data, BMI, WC and age, no difference in VAT was found between Caucasians and Hispanics after correction for SAT and BMI. Univariate regression analysis identified WC as the best single predictor of VAT (64.8% of variance) and BMI of SAT (88.9% of variance). The contribution of ethnicity and gender to the unexplained variance of the VAT–WC relationship was low (⩽3%) but significant (P⩽0.002). The different laboratories explained a low (⩽4.8%) but significant (P<0.0001) portion of the unexplained variance of the VAT–WC and SAT–BMI relationships. Prediction equations for VAT (VAT (cm2)=1.1 × WC (cm)−52.9) and SAT (SAT (cm2)=23.2 × BMI (kg/m2)−329) were developed on a randomly chosen half of the population and crossvalidated in the remaining half. The pure error of the estimate was 13 cm2 for VAT and 57 cm2 for SAT.Conclusions:WC can be considered a good predictor of VAT as well as BMI of SAT. The importance of ethnicity and gender on VAT estimation is not negligible.

DHA Effects in Brain Development and Function.

Docosahexaenoic acid (DHA) is a structural constituent of membranes specifically in the central nervous system. Its accumulation in the fetal brain takes place mainly during the last trimester of pregnancy and continues at very high rates up to the end of the second year of life. Since the endogenous formation of DHA seems to be relatively low, DHA intake may contribute to optimal conditions for brain development. We performed a narrative review on research on the associations between DHA levels and brain development and function throughout the lifespan. Data from cell and animal studies justify the indication of DHA in relation to brain function for neuronal cell growth and differentiation as well as in relation to neuronal signaling. Most data from human studies concern the contribution of DHA to optimal visual acuity development. Accumulating data indicate that DHA may have effects on the brain in infancy, and recent studies indicate that the effect of DHA may depend on gender and genotype of genes involved in the endogenous synthesis of DHA. While DHA levels may affect early development, potential effects are also increasingly recognized during childhood and adult life, suggesting a role of DHA in cognitive decline and in relation to major psychiatric disorders.

The effects of sex and method of blood pressure measurement on genetic associations with blood pressure in the PAMELA study.

Background Phenotypic accuracy and specificity are essential for a successful genetic association study. Blood pressure (BP) measurements show heterogeneity depending on the method and time of measurement, sexual dimorphism and measurement errors, making genetic dissection difficult. Methods and results We studied 1550 adults aged 25–74 years, not on any antihypertensive treatment, resident in Monza, Italy (PAMELA study) all of whom had home, clinic and ambulatory BPs measured. We analysed 3705 single nucleotide polymorphisms (SNPs) (1324 typed and 2381 imputed) across 168 genes for association with these traits. No SNP achieved an experiment wide significance level of P less than 3 × 10−4 for any of the phenotypes studied. We selected 28 top candidate SNPs for further analysis of phenotypic heterogeneity and sexual dimorphism using a gene-centric strategy calculating empirical P values by permutations within each gene by including genic SNPs with an r2 less than 0.5. The association signals were not consistent across all the BP phenotypes, whether compared by genes or by physiological pathways. The top SNPs in WNK1, ADRA1A, ADRA1B, DRD1, NOS1 and PON3 showed significant sex interaction for BP and when analysed separately by sex showed evidence of dimorphism with opposite direction of effect for the same allele in the two sexes. Conclusion In the largest study of its kind, we show that sex and BP measurement methods have a significant impact on association signals. These findings might explain previous inconsistencies in studies on cardiovascular candidate genes and should have major implications for the design and interpretation of association studies.

Pulmonic stenosis in dogs: survival and risk factors in a retrospective cohort of patients

OBJECTIVES To assess survival and risk factors in dogs with pulmonic stenosis. METHODS A retrospective review of medical case records of all cases of pulmonic stenosis >50 mmHg, undergoing pulmonary balloon valvuloplasty or not. Survival curves and multivariate analysis were calculated in the overall population and in subgroups. RESULTS One hundred and seventy-two cases were included. Factors negatively affecting survival were clinical signs [hazard ratio (HR) 3 · 44, P < 0 · 001], younger age at diagnosis (HR 3 · 96, P = 0 · 001) and valve morphology type B (HR 3 · 33, P = 0 · 001) in the overall population. In those that had pulmonary balloon valvuloplasty group only clinical signs was significant (HR 3 · 44, P < 0 · 001). In cases that did not undergo pulmonary balloon valvuloplasty group Doppler gradient (HR 1 · 02, P < 0 · 001), clinical signs (HR 5 · 41, P = 0 · 002), valve morphology type B (HR 10 · 20, P = 0 · 001) and younger age at diagnosis (HR 12 · 82, P < 0 · 001) negatively affected survival. Dogs with severe pulmonic stenosis undergoing pulmonary balloon valvuloplasty (HR 0 · 47, P = 0 · 047) and asymptomatic dogs with moderate pulmonic stenosis (HR 0 · 10, P = 0 · 042) had a better outcome. Younger age at diagnosis was correlated with poorer outcome in right-sided congestive heart failure dogs (HR 14 · 02, P = 0 · 01). CLINICAL SIGNIFICANCE Clinical signs, valve morphology type B and age at diagnosis are risk factors in pulmonic stenosis patients. Pulmonary balloon valvuloplasty is a reasonable treatment choice in dogs with severe pulmonic stenosis.

Assessment of Mitral Regurgitation Severity by Doppler Color Flow Mapping of the Vena Contracta in Dogs

Background Quantitative and semiquantitative methods have been proposed for the assessment of MR severity, and though all are associated with limitations. Measurement of vena contracta width (VCW) has been used in clinical practice. Objective To measure the VCW in dogs with different levels of MR severity. Animals Two hundred and seventy‐nine dogs were classified according to 5 levels of MR severity. Methods This was a retrospective study. EROA and regurgitant volume calculated by the PISA method, were measured and indexed to BSA. Descriptive statistics were calculated for VCW and VCW index for all categories of MR severity. Spearmans rank correlation coefficients (ρs) were calculated to compare the results of the different methods (VCW and VCW index vs RV PISA, RV PISA index, EROA, EROA index), and between VCW and VCW index versus MR severity. Results All Spearmans rank correlation coefficients were significant (P < .001). The median values of VCW resulted of 2.9 mm (IQR 3.4–2.5) and of 4.6 mm (IQR 5.4–4.1) in the groups previously classified as mild‐to‐moderate and moderate‐to‐severe, respectively. The median values of VCW index resulted of 4.4 mm/m2 (IQR = 5.5–4.2) in mild‐to‐moderate MR and of 10.8 mm/m2 (IQR = 12.8–9.4) in moderate‐to‐severe MR. Conclusion and Clinical Importance This is not a validation study against any previously validated invasive gold standard, the VCW method has proved easy to employ and it might be an additional tool in quantifying disease severity that supports, rather than replace, data coming from other techniques in daily clinical practice and research.

Echocardiographic values in clinically healthy adult dogue de Bordeaux dogs.

OBJECTIVES To assess the influence of body surface area, age and gender on echocardiographic parameters and to establish echocardiographic reference values for dogue de Bordeaux dogs. METHODS Thirty-nine healthy dogue de Bordeaux dogs of both sexes, older than one year, were recruited and 31 of these were included in the study. The classic linear regression model proved to be the best way to analyse the data. The reference limits of the echocardiographic measurements were calculated using the regression equations. The difference between the mean values of body surface area in both gender groups was evaluated by using one-way ANOVA. RESULTS A significant correlation was seen between several echocardiographic parameters and body surface area or body surface area and age, and high coefficients of determination (R2) were found. No effect of gender was detected on echocardiographic variables, except for the thickness of the left ventricular posterior wall at end diastole. CLINICAL SIGNIFICANCE The echocardiographic parameters related to body surface area, in the absence of correlation with other independent variables (gender and age) should be interpreted with caution because their variation could be significant for the presence of heart disease. The proposed statistical model allows estimation of echocardiographic parameters in dogue de Bordeaux dogs with different body surface areas and ages.

Eight full‐length abelson related gene (Arg) isoforms are constitutively expressed in caki‐1 cell line and cell distribution of two isoforms has been analyzed after transfection

The human Arg (Abl2) nonreceptor tyrosine kinase has a role in cytoskeletal rearrangements by its C‐terminal F‐actin‐ and microtubule‐binding sequences. We have previously identified Arg transcripts with different 5′‐ and 3′‐ends, named respectively long and short 1A and 1B (1AL, 1AS, 1BL, 1BS) and long and short C‐termini (CTL and CTS), that have different expression patterns in various cell types. The combination of the different ends permits to predict eight putative full‐length Arg transcripts and corresponding proteins. By Reverse Transcription‐Long PCR we show here that all eight full‐length transcripts are endogenously expressed in Caki‐1 cells and the two bands, ≃10 kDa different, shown by 1‐D Western blots of Hek293T and Caki‐1 lysates correspond to the full‐length Arg protein isoforms with different C‐termini. 2‐D Western blot analysis evidenced different high molecular weight and slight acidic specific spots in Hek293T and Caki‐1 lysates. The cellular localization of two Arg isoforms (1BLCTL and 1BLCTS) transfected in Caki‐1 and Hek293T cells was cytoplasmic, and some differences in cytoskeleton interactions have been evidenced. Moreover, in Hek293T cells only the transfected 1BLCTS isoform gives rise to a large intracytoplasmic cylindrical structure containing phalloidin‐positive amorphous actin aggregates. The presence of eight full‐length Arg isoforms with different cellular expression may imply a diverse functional role in normal and neoplastic cells. J. Cell. Biochem. 105: 1219–1227, 2008.

Complex Congenital Heart Disease: Prevalence and Clinical Findings

Congenital heart disease represents one of the most frequent causes of mortality in stillborn dogs and puppies younger than 1 year old (Buchanan, 1999). The majority of dogs are affected with only one defect (e.g. aortic stenosis, patent ductus arteriosus, mitral valve dysplasia, etc.). However in some cases two or more alterations may coexist. In these cases the malformations can be definite complex congenital heart disease.

Assessment of right ventricular function by feature-tracking echocardiography in conscious healthy dogs

Advanced two-dimensional echocardiographic techniques allow strain (S) analysis of regional function and thus can provide information on regional myocardial deformation. Feature-tracking echocardiography (FTE) is based on a mono-dimensional technology and may offer more detailed information about septal deformation because it can analyse the activity of left- and right-sided septal fibres separately. The present study aimed to quantify global and regional (free wall and septal) right ventricular (RV) longitudinal S and strain rate (SR). We also investigated the relationships of S and SR with age, sex, weight, breed (sighthound breed vs other breeds), and heart rate. Cine loops were acquired from the left apical four-chamber view, optimized for the RV, in 60 dogs. The within-day and between-day intra-observer coefficient of variation for global RV S and SR in normal dogs using FTE was acceptable (<8.5%). Global longitudinal S (GLS) and SR showed a significant correlation with breed. GLS showed a significant weak positive correlation with weight. Global longitudinal SR showed a significant moderate negative correlation with heart rate. No correlation was found between GLS/SR and age. There was no significant difference between male and female dogs. This study shows, for the first time, that a novel FTE algorithm represents a promising and feasible non-invasive technique to assess RV myocardial function (free wall and septal deformation) in dogs. Based on our results, sighthound breeds appear to need specific reference values.