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1.  ACTN3 R577X polymorphism and long-term survival in patients with chronic heart failure 
Previous studies have shown the occurrence of actinin-3 deficiency in the presence of the R577X polymorphism in the ACTN3 gene. Our hypothesis is that this deficiency, by interfering with the function of skeletal muscle fiber, can result in a worse prognosis in patients with chronic heart failure.
A prospective cohort study was conducted from 2002 to 2004. The eligibility criteria included diagnosis of chronic heart failure stage C from different etiologies. We excluded all patients with concomitant disease that could be related to poor prognosis. ACTN3 rs1815739 (R577X) polymorphism was detected by high resolution melting analysis. Survival curves were calculated with the Kaplan-Meier method and evaluated with the log-rank statistic. The relationship between the baseline variables and the composite end-point of all-cause death was assessed using a Cox proportional hazards survival model.
A total of 463 patients were included in this study. The frequency of the ACTN3 577X variant allele was 39.0%. The LVEF mean was 45.6 ± 18.7% and the most common etiology of this study was hypertensive. After a follow-up of five years, 239 (51.6%) patients met the pre-defined endpoint. Survival curves showed higher mortality in patients carrying RX or XX genotypes compared with patients carrying RR genotype (p = 0.01).
R577X polymorphism in the ACTN3 gene was independently associated with worse survival in patients with chronic heart failure. Further studies are necessary to ensure its use as a marker of prognosis for this syndrome.
PMCID: PMC4113663  PMID: 25059829
ACTN3; R577X; Polymorphism; Heart failure
2.  Short-term mechanical stretch fails to differentiate human adipose-derived stem cells into cardiovascular cell phenotypes 
We and others have previously demonstrated that adipose-derived stem cells (ASCs) transplantation improve cardiac dysfunction post-myocardium infarction (MI) under hemodynamic stress in rats. The beneficial effects appear to be associated with pleiotropic factors due to a complex interplay between the transplanted ASCs and the microenvironment in the absence of cell transdifferentiation. In the present work, we tested the hypothesis that mechanical stretch per se could change human ASCs (hASCs) into cardiovascular cell phenotypes that might influence post-MI outcomes.
Human ASCs were obtained from patients undergoing liposuction procedures. These cells were stretched 12%, 1Hz up to 96 hours by using Flexercell 4000 system. Protein and gene expression were evaluated to identify cardiovascular cell markers. Culture medium was analyzed to determine cell releasing factors, and contraction potential was also evaluated.
Mechanical stretch, which is associated with extracellular signal-regulated kinase (ERK) phosphorylation, failed to induce the expression of cardiovascular cell markers in human ASCs, and mesenchymal cell surface markers (CD29; CD90) remained unchanged. hASCs and smooth muscle cells (SMCs) displayed comparable contraction ability. In addition, these cells demonstrated a profound ability to secrete an array of cytokines. These two properties of human ASCs were not influenced by mechanical stretch.
Altogether, our findings demonstrate that hASCs secrete an array of cytokines and display contraction ability even in the absence of induction of cardiovascular cell markers or the loss of mesenchymal surface markers when exposed to mechanical stretch. These properties may contribute to beneficial post-MI cardiovascular outcomes and deserve to be further explored under the controlled influence of other microenvironment components associated with myocardial infarction, such as tissue hypoxia.
PMCID: PMC4012171  PMID: 24885410
Adipose-derived stem cell; Cell therapy; Stretch; Cell differentiation
3.  Investigation of Genetic Disturbances in Oxygen Sensing and Erythropoietin Signaling Pathways in Cases of Idiopathic Erythrocytosis 
Background. Idiopathic erythrocytosis is the term reserved for cases with unexplained origins of abnormally increased hemoglobin after initial investigation. Extensive molecular investigation of genes associated with oxygen sensing and erythropoietin signaling pathways, in those cases, usually involves sequencing all of their exons and it may be time consuming. Aim. To perform a strategy for molecular investigation of patients with idiopathic erythrocytosis regarding oxygen sensing and erythropoietin signaling pathways. Methods. Samples of patients with idiopathic erythrocytosis were evaluated for the EPOR, VHL, PHD2, and HIF-2α genes using bidirectional sequencing of their hotspots. Results. One case was associated with HIF-2α mutation. Sequencing did not identify any pathogenic mutation in 4 of 5 cases studied in any of the studied genes. Three known nonpathogenic polymorphisms were found (VHL p.P25L, rs35460768; HIF-2α p.N636N, rs35606117; HIF-2α p.P579P, rs184760160). Conclusion. Extensive molecular investigation of cases considered as idiopathic erythrocytosis does not frequently change the treatment of the patient. However, we propose a complementary molecular investigation of those cases comprising genes associated with erythrocytosis phenotype to meet both academic and genetic counseling purposes.
PMCID: PMC3864166  PMID: 24363938
4.  Development of a New Approach to Aid in Visual Identification of Murine iPS Colonies Using a Fuzzy Logic Decision Support System 
PLoS ONE  2013;8(8):e70605.
The a priori identification of induced pluripotent stem cells remains a challenge. Being able to quickly identify the most embryonic stem cell-similar induced pluripotent stem cells when validating results could help to reduce costs and save time. In this context, tools based on non-classic logic can be useful in creating aid-systems based on visual criteria. True colonies when viewed at 100x magnification have been found to have the following 3 characteristics: a high degree of border delineation, a more uniform texture, and the absence of a cracked texture. These visual criteria were used for fuzzy logic modeling. We investigated the possibility of predicting the presence of alkaline phosphatase activity, typical of true induced pluripotent stem cell colonies, after 25 individuals, with varying degrees of experience in working with murine iPS cells, categorized the images of 136 colonies based on visual criteria. Intriguingly, the performance evaluation by area under the ROC curve (16 individuals with satisfactory performance), Spearman correlation (all statistically significant), and Cohen's Kappa agreement analysis (all statistically significant) demonstrates that the discriminatory capacity of different evaluators are similar, even those who have never cultivated cells. Thus, we report on a new system to facilitate visual identification of murine- induced pluripotent stem cell colonies that can be useful for staff training and opens the possibility of exploring visual characteristics of induced pluripotent stem cell colonies with their functional peculiarities. The fuzzy model has been integrated as a web-based tool named “2see-iPS” which is freely accessed at
PMCID: PMC3738584  PMID: 23950970
5.  Higher incidence of death in multi-vessel coronary artery disease patients associated with polymorphisms in chromosome 9p21 
We investigated whether 9p21 polymorphisms are associated with cardiovascular events in a group of 611 patients enrolled in the Medical, Angioplasty or Surgery Study II (MASS II), a randomized trial comparing treatments for patients with coronary artery disease (CAD) and preserved left ventricular function.
The participants of the MASS II were genotyped for 9p21 polymorphisms (rs10757274, rs2383206, rs10757278 and rs1333049). Survival curves were calculated with the Kaplan–Meier method and compared with the log-rank statistic. We assessed the relationship between baseline variables and the composite end-point of death, death from cardiac causes and myocardial infarction using a Cox proportional hazards survival model.
We observed significant differences between patients within each polymorphism genotype group for baseline characteristics. The frequency of diabetes was lower in patients carrying GG genotype for rs10757274, rs2383206 and rs10757278 (29.4%, 32.8%, 32.0%) compared to patients carrying AA or AG genotypes (49.1% and 39.2%, p = 0.01; 52.4% and 40.1%, p = 0.01; 47.8% and 37.9%, p = 0.04; respectively).
Significant differences in genotype frequencies between double and triple vessel disease patients were observed for the rs10757274, rs10757278 and rs1333049. Finally, there was a higher incidence of overall mortality in patients with the GG genotype for rs2383206 compared to patients with AA and AG genotypes (19.5%, 11.9%, 11.0%, respectively; p = 0.04). Moreover, the rs2383206 was still significantly associated with a 1.75-fold increased risk of overall mortality (p = 0.02) even after adjustment of a Cox multivariate model for age, previous myocardial infarction, diabetes, smoking and type of coronary anatomy.
Our data are in accordance to previous evidence that chromosome 9p21 genetic variation may constitute a genetic modulator in the cardiovascular system in different scenarios. In patients with established CAD, we observed an association between the rs2383206 and higher incidence of overall mortality and death from cardiac causes in patients with multi-vessel CAD.
PMCID: PMC3469382  PMID: 22856518
Coronary artery disease; Polymorphism; Genetics; Chromosome 9p21
6.  ACE as a Mechanosensor to Shear Stress Influences the Control of Its Own Regulation via Phosphorylation of Cytoplasmic Ser1270 
PLoS ONE  2011;6(8):e22803.
We tested whether angiotensin converting enzyme (ACE) and phosphorylation of Ser1270 are involved in shear-stress (SS)-induced downregulation of the enzyme.
Methods and Results
Western blotting analysis showed that SS (18 h, 15 dyn/cm2) decreases ACE expression and phosphorylation as well as p-JNK inhibition in human primary endothelial cells (EC). CHO cells expressing wild-type ACE (wt-ACE) also displayed SS-induced decrease in ACE and p-JNK. Moreover, SS decreased ACE promoter activity in wt-ACE, but had no effect in wild type CHO or CHO expressing ACE without either the extra- or the intracellular domains, and decreased less in CHO expressing a mutated ACE at Ser1270 compared to wt-ACE (13 vs. 40%, respectively). The JNK inhibitor (SP600125, 18 h), in absence of SS, also decreased ACE promoter activity in wt-ACE. Finally, SS-induced inhibition of ACE expression and phosphorylation in EC was counteracted by simultaneous exposure to an ACE inhibitor.
ACE displays a key role on its own downregulation in response to SS. This response requires both the extra- and the intracellular domains and ACE Ser1270, consistent with the idea that the extracellular domain behaves as a mechanosensor while the cytoplasmic domain elicits the downstream intracellular signaling by phosphorylation on Ser1270.
PMCID: PMC3161988  PMID: 21901117
7.  Human Saphenous Vein Organ Culture Under Controlled Hemodynamic Conditions 
Clinics (Sao Paulo, Brazil)  2008;63(5):683-688.
Saphenous vein grafting is still widely used to revascularize ischemic myocardium. The effectiveness of this procedure is limited by neointima formation and accelerated atherosclerosis, which frequently leads to graft occlusion. A better understanding of this process is important to clarify the mechanisms of vein graft disease and to aid in the formulation of strategies for prevention and/or therapeutics.
To develop an ex vivo flow system that allows for controlled hemodynamics in order to mimic arterial and venous conditions.
Human saphenous veins were cultured either under venous (flow: 5 ml/min) or arterial hemodynamic conditions (flow: 50 ml/min, pressure: 80 mmHg) for 1-, 2- and 4-day periods. Cell viability, cell density and apoptosis were compared before and after these intervals using MTT, Hoeschst 33258 stain, and TUNEL assays, respectively.
Fresh excised tissue segments were well preserved prior to the study. Hoechst 33258 and MTT stains showed progressive losses in cell density and cell viability in veins cultured under arterial hemodynamic conditions from 1 to 4 days, while no alterations were observed in veins cultured under venous conditions. Although the cell density from 1-day cultured veins under arterial conditions was similar to that of freshly excised veins, the TUNEL assay indicated that most of these cells were undergoing apoptosis.
The results observed resemble the events taking place during early in vivo arterial-vein grafting and provide evidence that an ex vivo perfusion system may be useful for the identification of new therapeutic targets that ameliorate vein graft remodeling and increase graft patency over time.
PMCID: PMC2664728  PMID: 18925330
Saphenous vein graft; Ex vivo organ culture; Vascular biology

Results 1-7 (7)