Elsa Bronze-da-Rocha

MicroRNAs Expression Profiles in Cardiovascular Diseases

The current search for new markers of cardiovascular diseases (CVDs) is explained by the high morbidity and mortality still observed in developed and developing countries due to cardiovascular events. Recently, microRNAs (miRNAs or miRs) have emerged as potential new biomarkers and are small sequences of RNAs that regulate gene expression at posttranscriptional level by inhibiting translation or inducing degradation of the target mRNAs. Circulating miRNAs are involved in the regulation of signaling pathways associated to aging and can be used as novel diagnostic markers for acute and chronic diseases such as cardiovascular pathologies. This review summarizes the biogenesis, maturation, and stability of miRNAs and their use as potential biomarkers for coronary artery disease (CAD), myocardial infarction (MI), and heart failure (HF).

LAMA2 gene analysis in a cohort of 26 congenital muscular dystrophy patients

Congenital muscular dystrophy type 1A (MDC1A) is caused by mutations in the LAMA2 gene encoding laminin‐α2. We describe the molecular study of 26 patients with clinical presentation, magnetic resonance imaging and/or laminin‐α2 expression in muscle, compatible with MDC1A. The combination of full genomic sequencing and complementary DNA analysis led to the particularly high mutation detection rate of 96% (50/52 disease alleles). Besides 22 undocumented polymorphisms, 18 different mutations were identified in the course of this work, 14 of which were novel. In particular, we describe the first fully characterized gross deletion in the LAMA2 gene, encompassing exon 56 (c.7750‐1713_7899‐2153del), detected in 31% of the patients. The only two missense mutations detected were found in heterozygosity with nonsense or truncating mutations in the two patients with the milder clinical presentation and a partial reduction in muscle laminin‐α2. Our results corroborate the previous few genotype/phenotype correlations in MDC1A and illustrate the importance of screening for gross rearrangements in the LAMA2 gene, which may be underestimated in the literature.

Iron-Hepcidin Dysmetabolism, Anemia and Renal Hypoxia, Inflammation and Fibrosis in the Remnant Kidney Rat Model

Anemia is a common complication of chronic kidney disease (CKD) that develops early and its severity increases as renal function declines. It is mainly due to a reduced production of erythropoietin (EPO) by the kidneys; however, there are evidences that iron metabolism disturbances increase as CKD progresses. Our aim was to study the mechanisms underlying the development of anemia of CKD, as well as renal damage, in the remnant kidney rat model of CKD induced by 5/6 nephrectomy. This model of CKD presented a sustained degree of renal dysfunction, with mild and advanced glomerular and tubulointerstitial lesions. Anemia developed 3 weeks after nephrectomy and persisted throughout the protocol. The remnant kidney was still able to produce EPO and the liver showed an increased EPO gene expression. In spite of the increased EPO blood levels, anemia persisted and was linked to low serum iron and transferrin levels, while serum interleukin (IL)-6 and high sensitivity C-reactive protein (hs-CRP) levels showed the absence of systemic inflammation. The increased expression of duodenal ferroportin favours iron absorption; however, serum iron is reduced which might be due to iron leakage through advanced kidney lesions, as showed by tubular iron accumulation. Our data suggest that the persistence of anemia may result from disturbances in iron metabolism and by an altered activity/function of EPO as a result of kidney cell damage and a local inflammatory milieu, as showed by the increased gene expression of different inflammatory proteins in the remnant kidney. In addition, this anemia and the associated kidney hypoxia favour the development of fibrosis, angiogenesis and inflammation that may underlie a resistance to EPO stimuli and reduced iron availability. These findings might contribute to open new windows to identify putative therapeutic targets for this condition, as well as for recombinant human EPO (rHuEPO) resistance, which occurs in a considerable percentage of CKD patients.

Risk Factors for Mortality in Hemodialysis Patients: Two-Year Follow-Up Study

Background. End-stage renal disease (ESRD) patients under hemodialysis (HD) have high mortality rate. Inflammation, dyslipidemia, disturbances in erythropoiesis, iron metabolism, endothelial function, and nutritional status have been reported in these patients. Our aim was to identify any significant association of death with these disturbances, by performing a two-year follow-up study. Methods and Results. A large set of data was obtained from 189 HD patients (55.0% male; 66.4 ± 13.9 years old), including hematological data, lipid profile, iron metabolism, nutritional, inflammatory, and endothelial (dys)function markers, and dialysis adequacy. Results. 35 patients (18.5%) died along the follow-up period. Our data showed that the type of vascular access, C-reactive protein (CRP), and triglycerides (TG) are significant predictors of death. The risk of death was higher in patients using central venous catheter (CVC) (Hazard ratio [HR] =3.03, 95% CI = 1.49–6.13), with higher CRP levels (fourth quartile), compared with those with lower levels (first quartile) (HR = 17.3, 95% CI = 2.40–124.9). Patients with higher TG levels (fourth quartile) presented a lower risk of death, compared with those with the lower TG levels (first quartile) (HR = 0.18, 95% CI = 0.05–0.58). Conclusions. The use of CVC, high CRP, and low TG values seem to be independent risk factors for mortality in HD patients.

Impact of UGT1A1 gene variants on total bilirubin levels in Gilbert syndrome patients and in healthy subjects

The Gilbert syndrome is a benign form of unconjugated hyperbilirubinemia, mainly associated with alterations in UGT1A1 gene. This work investigated the effect of UGT1A1 variants on total bilirubin levels in Gilbert patients (n=45) and healthy controls (n=161). Total bilirubin levels were determined using a colorimetric method; molecular analysis of exons 1-5 and two UGT1A1 promoter regions were performed by direct sequencing and automatic analysis of fragments. Five in silico methods predicted the effect of new identified variants. A significant different allelic distribution, in Gilbert patients and in controls, was found for two promoter polymorphisms. Among patients, 82.2% were homozygous and 17.8% heterozygous for the c.-41_-40dupTA allele; in control group, 9.9% were homozygous and 43.5% heterozygous for this promoter variant, while 46.6% (n=75) presented the [A(TA)6TAA]. For the T>G transition at c.-3279 promoter region, in patients, 86.7% were homozygous and 13.3% heterozygous; in control group, 33.5% were homozygous for the wild type allele, 44.1% were heterozygous and 22.4% homozygous for the mutated allele. The two polymorphisms were in Hardy-Weinberg equilibrium in both groups. Sequencing of UGT1A1 coding region identified nine novel variants, five in patients and four in controls. In silico analysis of these amino acids replacements predicted four of them as benign and three as damaging. In conclusion, we demonstrated that total bilirubin levels are mainly determined by the TA duplication in the TATA-box promoter and by the c.-3279T>G variant. Alterations in the UGT1A1 coding region seem to be associated with increased bilirubin levels, and, therefore, with Gilbert syndrome.

Atypical phenotype in two patients with LAMA2 mutations

Congenital muscular dystrophy type 1A is caused by mutations in the LAMA2 gene, which encodes the α2-chain of laminin. We report two patients with partial laminin-α2 deficiency and atypical phenotypes, one with almost exclusive central nervous system involvement (cognitive impairment and refractory epilepsy) and the second with marked cardiac dysfunction, rigid spine syndrome and limb-girdle weakness. Patients underwent clinical, histopathological, imaging and genetic studies. Both cases have two heterozygous LAMA2 variants sharing a potentially pathogenic missense mutation c.2461A>C (p.Thr821Pro) located in exon 18. Brain MRI was instrumental for the diagnosis, since muscular examination and motor achievements were normal in the first patient and there was a severe cardiac involvement in the second. The clinical phenotype of the patients is markedly different which could in part be explained by the different combination of mutations types (two missense versus a missense and a truncating mutation).

Bilirubin dependence on UGT1A1 polymorphisms, hemoglobin, fasting time and body mass index.

Abstract:In humans, bilirubin levels are influenced by different factors. This study evaluates how several nongenetic causes and the genetic UGT1A1 polymorphisms contribute for bilirubin levels, in a cohort of 146 young Caucasian females. Hematological data, bilirubin, screening of TA duplication in the UGT1A1 gene, body mass index (BMI) and body fat were determined. A questionnaire about fasting time, smoking habits, oral contraceptive therapy, caloric intake and physical activity was done. Participants were divided according to the tertiles of bilirubin. Subjects from the second and third tertile had significant rises in hemoglobin (Hb), hematocrit, mean cell Hb and mean cell Hb concentration, and a significant increased frequency for the c.−41_−40dupTA allele in homozygosity, when compared to the first tertile. Red blood cell count was significantly increased in the third tertile. Results showed that the c.−41_−40dupTA allele (genetic), Hb, BMI and fasting time (nongenetic) were the main factors associated bilirubin levels.

Cytotoxicity and genotoxicity of chitooligosaccharides upon lymphocytes

Two COS mixtures and a low molecular weight chitosan (LMWC) were tested for potential cytotoxicity and genotoxicity upon human lymphocytes. Genotoxicity was evaluated in vitro by cytokinesis-blocked micronucleus and alkaline comet assays, while cytotoxicity was assessed by flow cytometry analysis. Our results suggest that COS do not exhibit any genotoxicity upon human lymphocytes, independently of MW or concentration. However, above 0.07mg/mL COS induced strong cytotoxic effects. According to the concentration used, such cytotoxicity will induce cell death, essentially by necrosis (>0.10mg/mL) and/or apoptosis (<0.10mg/mL). The level of necrosis/apoptosis induced by high COS concentrations, suggests a promising use as apoptosis inducers in specific cancer situations.

Peroxiredoxin 2, glutathione peroxidase, and catalase in the cytosol and membrane of erythrocytes under H2O2-induced oxidative stress

Abstract Erythrocytes are continuously exposed to risk of oxidative injury due to oxidant oxygen species. To prevent damage, they have antioxidant agents namely, catalase (Cat), glutathione peroxidase (GPx), and peroxiredoxin 2 (Prx2). Our aim was to contribute to a better understanding of the interplay between Prx2, Cat, and GPx under H2O2-induced oxidative stress, by studying their changes in the red blood cell cytosol and membrane, in different conditions. These three enzymes were quantified by immunoblotting. Malondialdehyde, that is, lipoperoxidation (LPO) in the erythrocyte membrane, and membrane-bound hemoglobin (MBH) were evaluated, as markers of oxidative stress. We also studied the erythrocyte membrane protein profile, to estimate how oxidative stress affects the membrane protein structure. We showed that under increasing H2O2 concentrations, inhibition of the three enzymes with or without metHb formation lead to the binding of Prx2 and GPx (but not Cat) to the erythrocyte membrane. Prx2 was detected mainly in its oxidized form and the linkage of metHb to the membrane seems to compete with the binding of Prx2. Catalase played a major role in protecting erythrocytes from high exogenous flux of H2O2, since whenever Cat was active there were no significant changes in any of the studied parameters. When only Cat was inhibited, Prx2 and GPx were unable to prevent H2O2-induced oxidative stress resulting in increasing MBH and membrane LPO. Additionally, the inhibition of one or more of these enzymes induced changes in the anchor/linker proteins of the junctional complexes of the membrane cytoskeleton–lipid bilayer, which might lead to membrane destabilization.

Body fat percentage is a major determinant of total bilirubin independently of UGT1A1*28 polymorphism in young obese.

Objectives Bilirubin has potential antioxidant and anti-inflammatory properties. The UGT1A1*28 polymorphism (TA repeats in the promoter region) is a major determinant of bilirubin levels and recent evidence suggests that raised adiposity may also be a contributing factor. We aimed to study the interaction between UGT1A1 polymorphism, hematological and anthropometric variables with total bilirubin levels in young individuals. Methods 350 obese (mean age of 11.6 years; 52% females) and 79 controls (mean age of 10.5 years; 59% females) were included. Total bilirubin and C-reactive protein (CRP) plasma levels, hemogram, anthropometric data and UGT1A1 polymorphism were determined. In a subgroup of 74 obese and 40 controls body composition was analyzed by dual-energy X-ray absorptiometry. Results The UGT1A1 genotype frequencies were 49.9%, 42.7% and 7.5% for 6/6, 6/7 and 7/7 genotypes, respectively. Patients with 7/7 genotype presented the highest total bilirubin levels, followed by 6/7 and 6/6 genotypes. Compared to controls, obese patients presented higher erythrocyte count, hematocrit, hemoglobin and CRP levels, but no differences in bilirubin or in UGT1A1 genotype distribution. Body fat percentage was inversely correlated with bilirubin in obese patients but not in controls. This inverse association was observed either in 6/7 or 6/6 genotype obese patients. UGT1A1 polymorphism and body fat percentage were the main factors affecting bilirubin levels within obese patients (linear regression analysis). Conclusion In obese children and adolescents, body fat composition and UGT1A1 polymorphism are independent determinants of total bilirubin levels. Obese individuals with 6/6 UGT1A1 genotype and higher body fat mass may benefit from a closer clinical follow-up.