RESUMO
The single nucleotide polymorphism (SNP) within the TCF7L2 gene, rs7903146, is, to date, the most significant genetic marker associated with Type 2 diabetes mellitus (T2DM) risk. Nonetheless, its functional role in disease pathology is poorly understood. The aim of the present study was to investigate, in vascular smooth muscle cells from 92 patients undergoing aortocoronary bypass surgery, the contribution of this SNP in T2DM using expression levels and expression correlation comparison approaches, which were visually represented as gene interaction networks. Initially, the expression levels of 41 genes (seven TCF7L2 splice forms and 40 other T2DM relevant genes) were compared between rs7903146 wild-type (CC) and T2DM-risk (CT + TT) genotype groups. Next, we compared the expression correlation patterns of these 41 genes between groups to observe if the relationships between genes were different. Five TCF7L2 splice forms and nine genes showed significant expression differences between groups. RXRα gene was pinpointed as showing the most different expression correlation pattern with other genes. Therefore, T2DM risk alleles appear to be influencing TCF7L2 splice form's expression in vascular smooth muscle cells, and RXRα gene is pointed out as a treatment target candidate for risk reduction in individuals with high risk of developing T2DM, especially individuals harboring TCF7L2 risk genotypes.
Assuntos
Doenças Cardiovasculares/genética , Diabetes Mellitus Tipo 2/genética , Redes Reguladoras de Genes/genética , Receptor X Retinoide alfa/genética , Proteína 2 Semelhante ao Fator 7 de Transcrição/genética , Brasil , Doenças Cardiovasculares/cirurgia , Ponte de Artéria Coronária , Primers do DNA/genética , Genótipo , Humanos , Músculo Liso Vascular/metabolismo , Polimorfismo de Nucleotídeo Único/genética , Isoformas de Proteínas/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de RiscoRESUMO
RATIONALE: Major coronary vessels derive from the proepicardium, the cellular progenitor of the epicardium, coronary endothelium, and coronary smooth muscle cells (CoSMCs). CoSMCs are delayed in their differentiation relative to coronary endothelial cells (CoEs), such that CoSMCs mature only after CoEs have assembled into tubes. The mechanisms underlying this sequential CoE/CoSMC differentiation are unknown. Retinoic acid (RA) is crucial for vascular development and the main RA-synthesizing enzyme is progressively lost from epicardially derived cells as they differentiate into blood vessel types. In parallel, myocardial vascular endothelial growth factor (VEGF) expression also decreases along coronary vessel muscularization. OBJECTIVE: We hypothesized that RA and VEGF act coordinately as physiological brakes to CoSMC differentiation. METHODS AND RESULTS: In vitro assays (proepicardial cultures, cocultures, and RALDH2 [retinaldehyde dehydrogenase-2]/VEGF adenoviral overexpression) and in vivo inhibition of RA synthesis show that RA and VEGF act as repressors of CoSMC differentiation, whereas VEGF biases epicardially derived cell differentiation toward the endothelial phenotype. CONCLUSION: Experiments support a model in which early high levels of RA and VEGF prevent CoSMC differentiation from epicardially derived cells before RA and VEGF levels decline as an extensive endothelial network is established. We suggest this physiological delay guarantees the formation of a complex, hierarchical, tree of coronary vessels.
Assuntos
Diferenciação Celular , Vasos Coronários/metabolismo , Células Endoteliais/metabolismo , Miócitos de Músculo Liso/metabolismo , Pericárdio/metabolismo , Transdução de Sinais , Tretinoína/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Aldeído Oxirredutases/genética , Aldeído Oxirredutases/metabolismo , Animais , Apoptose , Comunicação Autócrina , Diferenciação Celular/genética , Células Cultivadas , Embrião de Galinha , Técnicas de Cocultura , Vasos Coronários/embriologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Camundongos , Morfogênese , Miócitos Cardíacos/metabolismo , Comunicação Parácrina , Pericárdio/embriologia , Codorniz , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/genética , Técnicas de Cultura de Tecidos , Transdução Genética , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
There is a lack of standardization of a best practice protocol for Phosphatase and Tensin Homolog (PTEN) assessment by immunohistochemistry in anatomic pathology routine practice. We performed immunohistochemistry for 19 antibodies against PTEN, eleven of which were excluded during the standardization step. Immunohistochemistry of the remaining eight antibodies was performed on a Tissue Microarray containing 55 prostate and 40 renal carcinoma samples. Fluorescent in situ hybridization (FISH) was used as reference standard for immunohistochemistry specificity evaluation. Concerning nuclear staining, polyclonal (Cat#22034-1-AP); 6H2.1 mMAb (Cat#ABM-2052), Y184 RabMAb (Cat#NB110-57441) and 217702 mMAb antibodies presented the highest agreement with fluorescent in situ hybridization (p<0.001 for all) and with regard to cytoplasmic staining, Y184 RabMAb (Cat#NB110-57441); polyclonal (Cat#22034-1-AP) and 217702 mMAb presented the highest agreement (p<0.001 for all). Our results indicate that several commercially available antibodies do not show reliability of sensitivity and specificity for PTEN evaluation and we propose 6H2.1 mMAb (Cat#ABM-2052) as the antibody of choice for laboratory standardization and best practice in clinical routine, which demonstrated excellent sensitivity for both nuclear and cytoplasmic staining, specificity for PTEN by Western blot and good correlation with PTEN status by FISH with regard to nuclear staining.