Marcelo F. Sampaio

Myocardial Chemokine Expression and Intensity of Myocarditis in Chagas Cardiomyopathy Are Controlled by Polymorphisms in CXCL9 and CXCL10

Background Chronic Chagas cardiomyopathy (CCC), a life-threatening inflammatory dilated cardiomyopathy, affects 30% of the approximately 8 million patients infected by Trypanosoma cruzi. Even though the Th1 T cell-rich myocarditis plays a pivotal role in CCC pathogenesis, little is known about the factors controlling inflammatory cell migration to CCC myocardium. Methods and Results Using confocal immunofluorescence and quantitative PCR, we studied cell surface staining and gene expression of the CXCR3, CCR4, CCR5, CCR7, CCR8 receptors and their chemokine ligands in myocardial samples from end-stage CCC patients. CCR5+, CXCR3+, CCR4+, CCL5+ and CXCL9+ mononuclear cells were observed in CCC myocardium. mRNA expression of the chemokines CCL5, CXCL9, CXCL10, CCL17, CCL19 and their receptors was upregulated in CCC myocardium. CXCL9 mRNA expression directly correlated with the intensity of myocarditis, as well as with mRNA expression of CXCR3, CCR4, CCR5, CCR7, CCR8 and their ligands. We also analyzed single-nucleotide polymorphisms for genes encoding the most highly expressed chemokines and receptors in a cohort of Chagas disease patients. CCC patients with ventricular dysfunction displayed reduced genotypic frequencies of CXCL9 rs10336 CC, CXCL10 rs3921 GG, and increased CCR5 rs1799988CC as compared to those without dysfunction. Significantly, myocardial samples from CCC patients carrying the CXCL9/CXCL10 genotypes associated to a lower risk displayed a 2–6 fold reduction in mRNA expression of CXCL9, CXCL10, and other chemokines and receptors, along with reduced intensity of myocarditis, as compared to those with other CXCL9/CXCL10 genotypes. Conclusions Results may indicate that genotypes associated to reduced risk in closely linked CXCL9 and CXCL10 genes may modulate local expression of the chemokines themselves, and simultaneously affect myocardial expression of other key chemokines as well as intensity of myocarditis. Taken together our results may suggest that CXCL9 and CXCL10 are master regulators of myocardial inflammatory cell migration, perhaps affecting clinical progression to the life-threatening form of CCC.

Leptin G-2548A promoter polymorphism is associated with increased plasma leptin and BMI in Brazilian women

Variants in leptin gene (LEP) have been implicated in the pathogenesis of obesity. The relationship between LEP G-2548A polymorphism and obesity-related traits was evaluated in a sample of Brazilian women (n = 228) who were randomly selected from two clinical centers in Sao Paulo city. Blood samples were collected for DNA extraction, plasma leptin and serum lipids measurements. LEP G-2548A genotypes were identified by a PCR- RFLP strategy using the endonuclease Alw44I. LEP G-2548A was associated with obesity after adjustment for covariates (age, hypertension, coronary artery disease, smoking and physical activity). Women carrying G allele had a four times higher risk of obesity than the A allele carriers (OR: 4.11, CI95%: 1.06-15.90, p = 0.041). G allele was also related to increased plasma leptin (p = 0.024) and body mass index (p = 0.027). Hypertension, hyperglycemia, dyslipidemia and coronary artery disease were associated with obesity. However LEP G-2548A polymorphism was not related to these variables. All together these data suggest that LEP G-2548A polymorphism has an important role in regulating plasma leptin levels and body mass index in women.

Genetic susceptibility to Chagas disease cardiomyopathy: involvement of several genes of the innate immunity and chemokine-dependent migration pathways

BackgroundChagas disease, caused by the protozoan Trypanosoma cruzi is endemic in Latin America. Thirty percent of infected individuals develop chronic Chagas cardiomyopathy (CCC), an inflammatory dilated cardiomyopathy that is, by far, the most important clinical consequence of T. cruzi infection. The others remain asymptomatic (ASY). A possible genetic component to disease progression was suggested by familial aggregation of cases and the association of markers of innate and adaptive immunity genes with CCC development. Migration of Th1-type T cells play a major role in myocardial damage.MethodsOur genetic analysis focused on CCR5, CCL2 and MAL/TIRAP genes. We used the Tag SNPs based approach, defined to catch all the genetic information from each gene. The study was conducted on a large Brazilian population including 315 CCC cases and 118 ASY subjects.ResultsThe CCL2rs2530797A/A and TIRAPrs8177376A/A were associated to an increase susceptibility whereas the CCR5rs3176763C/C genotype is associated to protection to CCC. These associations were confirmed when we restricted the analysis to severe CCC, characterized by a left ventricular ejection fraction under 40%.ConclusionsOur data show that polymorphisms affecting key molecules involved in several immune parameters (innate immunity signal transduction and T cell/monocyte migration) play a role in genetic susceptibility to CCC development. This also points out to the multigenic character of CCC, each polymorphism imparting a small contribution. The identification of genetic markers for CCC will provide information for pathogenesis as well as therapeutic targets.

Polymorphism in the Alpha Cardiac Muscle Actin 1 Gene Is Associated to Susceptibility to Chronic Inflammatory Cardiomyopathy

Aims Chagas disease, caused by the protozoan Trypanosoma cruzi is endemic in Latin America, and may lead to a life-threatening inflammatory dilated, chronic Chagas cardiomyopathy (CCC). One third of T. cruzi-infected individuals progress to CCC while the others remain asymptomatic (ASY). A possible genetic component to disease progression was suggested by familial aggregation of cases and the association of markers of innate and adaptive immunity genes with CCC development. Since mutations in multiple sarcomeric genes, including alpha-cardiac actin (ACTC1) have been involved in hereditary dilated cardiomyopathy, we investigated the involvement of the ACTC1 gene in CCC pathogenesis. Methods and Results We conducted a proteomic and genetic study on a Brazilian study population. The genetic study was done on a main cohort including 118 seropositive asymptomatic subjects and 315 cases and the replication was done on 36 asymptomatic and 102 CCC cases. ACTC1 protein and mRNA levels were lower in myocardial tissue from patients with end-stage CCC than those found in hearts from organ donors. Genotyping a case-control cohort of CCC and ASY subjects for all informative single nucleotide polymorphism (SNP) in the ACTC1 gene identified rs640249 SNP, located at the 5’ region, as associated to CCC. Associations are borderline after correction for multiple testing. Correlation and haplotype analysis led to the identification of a susceptibility haplotype. Functional assays have shown that the rs640249A/C polymorphism affects the binding of transcriptional factors in the promoter regions of the ACTC1 gene. Confirmation of the detected association on a larger independent replication cohort will be useful. Conclusions Genetic variations at the ACTC1 gene may contribute to progression to chronic Chagas Cardiomyopathy among T. cruzi-infected patients, possibly by modulating transcription factor binding to ACTC1 promoter regions.

Time course proteomic profiling of human myocardial infarction plasma samples: an approach to new biomarker discovery.

BACKGROUND The aim of this study was to identify novel candidate biomarker proteins differentially expressed in the plasma of patients with early stage acute myocardial infarction (AMI) using SELDI-TOF-MS as a high throughput screening technology. METHODS Ten individuals with recent acute ischemic-type chest pain (<12 h duration) and ST-segment elevation AMI (1STEMI) and after a second AMI (2STEMI) were selected. Blood samples were drawn at six times after STEMI diagnosis. The first stage (T0) was in Emergency Unit before receiving any medication, the second was just after primary angioplasty (T2), and the next four stages occurred at 12 h intervals after T0. Individuals (n=7) with similar risk factors for cardiovascular disease and normal ergometric test were selected as a control group (CG). Plasma proteomic profiling analysis was performed using the top-down (i.e. intact proteins) SELDI-TOF-MS, after processing in a Multiple Affinity Removal Spin Cartridge System (Agilent). RESULTS Compared with the CG, the 1STEMI group exhibited 510 differentially expressed protein peaks in the first 48 h after the AMI (p<0.05). The 2STEMI group, had ~85% fewer differently expressed protein peaks than those without previous history of AMI (76, p<0.05). Among the 16 differentially-regulated protein peaks common to both STEMI cohorts (compared with the CG at T0), 6 peaks were persistently down-regulated at more than one time-stage, and also were inversed correlated with serum protein markers (cTnI, CK and CKMB) during 48 h-period after IAM. CONCLUSIONS Proteomic analysis by SELDI-TOF-MS technology combined with bioinformatics tools demonstrated differential expression during a 48 h time course suggests a potential role of some of these proteins as biomarkers for the very early stages of AMI, as well as for monitoring early cardiac ischemic recovery.

Novel genes detected by transcriptional profiling from whole-blood cells in patients with early onset of acute coronary syndrome

BACKGROUND Genome-wide expression analysis using microarrays has been used as a research strategy to discovery new biomarkers and candidate genes for a number of diseases. We aim to find new biomarkers for the prediction of acute coronary syndrome (ACS) with a differentially expressed mRNA profiling approach using whole genomic expression analysis in a peripheral blood cell model from patients with early ACS. METHODS AND RESULTS This study was carried out in two phases. On phase 1 a restricted clinical criteria (ACS-Ph1, n=9 and CG-Ph1, n=6) was used in order to select potential mRNA biomarkers candidates. A subsequent phase 2 study was performed using selected phase 1 markers analyzed by RT-qPCR using a larger and independent casuistic (ACS-Ph2, n=74 and CG-Ph2, n=41). A total of 549 genes were found to be differentially expressed in the first 48 h after the ACS-Ph1. Technical and biological validation further confirmed that ALOX15, AREG, BCL2A1, BCL2L1, CA1, COX7B, ECHDC3, IL18R1, IRS2, KCNE1, MMP9, MYL4 and TREML4, are differentially expressed in both phases of this study. CONCLUSIONS Transcriptomic analysis by microarray technology demonstrated differential expression during a 48 h time course suggesting a potential use of some of these genes as biomarkers for very early stages of ACS, as well as for monitoring early cardiac ischemic recovery.

Functional IL18 polymorphism and susceptibility to Chronic Chagas Disease

BACKGROUND Chronic Chagas Disease cardiomyopathy (CCC), a life-threatening inflammatory dilated cardiomyopathy, affects 30% of the approximately 8 million patients infected by Trypanosoma cruzi, the rest of the infected subjects remaining asymptomatic (ASY). The Th1 T cell-rich myocarditis plays a pivotal role in CCC pathogenesis. Local expression of IL-18 in CCC myocardial tissue has recently been described. IL-18 could potentially amplify the process by inducing increased expression of IFN-γ which in turn can increase the production of IL-18, thereby creating a positive feedback mechanism. In order to assess the contribution of the IL-18 to susceptibility to Chronic Chagas Disease, we investigated the association between a single nucleotide polymorphism (SNP) located in the IL-18 gene with the risk of developing Chagas cardiomyopathy. METHODS AND RESULTS We analyzed the rs2043055 marker in the IL18 gene in a cohort of Chagas disease cardiomyopathy patients (n=849) and asymptomatic subjects (n=202). We found a significant difference in genotype frequencies among moderate and severe CCC patients with ventricular dysfunction. CONCLUSIONS Our analysis suggests that the IL18 rs2043055 polymorphism- or a SNP in tight linkage disequilibrium with it- may contribute to modulating the Chagas cardiomyopathy outcome.

Consequences of the Prolonged Waiting Time for Patients Candidates for Heart Surgery

OBJECTIVE To assess mortality and the psychological repercussions of the prolonged waiting time for candidates for heart surgery. METHODS From July 1999 to May 2000, using a standardized questionnaire, we carried out standardized interviews and semi-structured psychological interviews with 484 patients with coronary heart disease, 121 patients with valvular heart diseases, and 100 patients with congenital heart diseases. RESULTS The coefficients of mortality (deaths per 100 patients/year) were as follows: patients with coronary heart disease, 5.6; patients with valvular heart diseases, 12.8; and patients with congenital heart diseases, 3.1 (p<0.0001). The survival curve was lower in patients with valvular heart diseases than in patients with coronary heart disease and congenital heart diseases (p<0.001). The accumulated probability of not undergoing surgery was higher in patients with valvular heart diseases than in the other patients (p<0.001), and, among the patients with valvular heart diseases, this probability was higher in females than in males (p<0.01). Several patients experienced intense anxiety and attributed their adaptive problems in the scope of love, professional, and social lives, to not undergoing surgery. CONCLUSION Mortality was high, and even higher among the patients with valvular heart diseases, with negative psychological and social repercussions.

Polymorphisms in Genes Encoding Metalloproteinase 9 and Lymphotoxin- Alpha can Influence Warfarin Treatment

Objectives: Warfarin treatment is influenced by environmental and genetic factors. The influence of polymorphisms in genes encoding metalloproteinase 9 (MMP9), lymphotoxin-alpha (LTA) andTNFSF14 (LIGHT), related to the inflammatory process of coronary artery disease, on warfarin dose and time to reach target was investigated in this study. Methods: Outpatients on warfarin treatment (n=227), 20 to 92 years, were enrolled at the Institute Dante Pazzanese of Cardiology (IDPC). Genomic DNA was obtained from peripheral whole blood to evaluate MMP9 rs17576 (Gln279Arg, A>G), LTA rs1041981 (Thr60Asn, C>A) and rs909253 (c.252T>C) and TNFSF14rs2291668 (c.147C>T) and rs344560 (Lys214Glu, G>A) polymorphisms by pyrosequencing in Q24PyroMark. Results: The patients carrying MMP9 rs17576GG genotype were more likely to require a lower warfarin weekly dose (OR: 2.73, 95% CI: 1.01-7.41, p=0.048). Also, LTA rs909253 variant was associated with a longer time to reach the target international normalized ratio (INR) (OR: 1.98, 95% CI: 1.02-3.86, p=0.043). Age was inversely correlated with the target INR (r=-0.387, p<0.001), and dose was directly correlated with time to reach target INR (r=0.244, p<0.001). Conclusion: MMP9 rs17576 variant may have an important influence on warfarin weekly dose, and that LTA rs909253 polymorphism may also influence the time to reach the target INR.

Detection of the TLR4 1196C>T Polymorphism by Mismatched-Polymerase Chain Reaction Using Plasmid DNA as Internal Control in Restriction Fragment Length Polymorphism Assays

BACKGROUND Restriction fragment length polymorphism (RFLP) is a common molecular assay used for genotyping, and it requires validated quality control procedures to prevent mistyping caused by impaired endonuclease activity. We have evaluated the usefulness of a plasmid-based internal control in RFLP assays. RESULTS Blood samples were collected from 102 individuals with acute myocardial infarction (AMI) and 108 non-AMI individuals (controls) for DNA extraction and laboratory analyses. The 1196C > T polymorphism in the toll-like receptor 4 (TLR4) gene was amplified by mismatched-polymerase chain reaction (PCR). Amplicons and pBluescript II SK- plasmid were simultaneously digested with endonuclease HincII. Fragments were separated on 2% agarose gels. Plasmid was completely digested using up to 55.2 nmL/L DNA solutions and 1 microL PCR product. Nevertheless, plasmid DNA with 41.4 nM or higher concentrations was incompletely digested in the presence of 7 microL PCR product. In standardized conditions, TLR4 1196C>T variant was accurately genotyped. TLR4 1196T allele frequency was similar between AMI (3.1%) and controls (2.0%, p = 0.948). TLR4 SNP was not associated with AMI in this sample population. In conclusion, the plasmid-based control is a useful approach to prevent mistyping in RFLP assays, and it is validate for genetic association studies such as TLR4 1196C>T.