RESUMO
AIMS: Lipid phosphate phosphatase 3; type 2 phosphatidic acid phosphatase ß (LPP3; PPAP2B) is a transmembrane protein dephosphorylating and thereby terminating signalling of lipid substrates including lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P). Human LPP3 possesses a cell adhesion motif that allows interaction with integrins. A polymorphism (rs17114036) in PPAP2B is associated with coronary artery disease, which prompted us to investigate the possible role of LPP3 in human endothelial dysfunction, a condition promoting atherosclerosis. METHODS AND RESULTS: To study the role of LPP3 in endothelial cells we used human primary aortic endothelial cells (HAECs) in which LPP3 was silenced or overexpressed using either wild type or mutated cDNA constructs. LPP3 silencing in HAECs enhanced secretion of inflammatory cytokines, leucocyte adhesion, cell survival, and migration and impaired angiogenesis, whereas wild-type LPP3 overexpression reversed these effects and induced apoptosis. We also demonstrated that LPP3 expression was negatively correlated with vascular endothelial growth factor expression. Mutations in either the catalytic or the arginine-glycine-aspartate (RGD) domains impaired endothelial cell function and pharmacological inhibition of S1P or LPA restored it. LPA was not secreted in HAECs under silencing or overexpressing LPP3. However, the intra- and extra-cellular levels of S1P tended to be correlated with LPP3 expression, indicating that S1P is probably degraded by LPP3. CONCLUSIONS: We demonstrated that LPP3 is a negative regulator of inflammatory cytokines, leucocyte adhesion, cell survival, and migration in HAECs, suggesting a protective role of LPP3 against endothelial dysfunction in humans. Both the catalytic and the RGD functional domains were involved and S1P, but not LPA, might be the endogenous substrate of LPP3.
Assuntos
Aorta/enzimologia , Células Endoteliais/enzimologia , Neovascularização Fisiológica , Fosfatidato Fosfatase/metabolismo , Apoptose , Domínio Catalítico , Adesão Celular , Movimento Celular , Células Cultivadas , Citocinas/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Lisofosfolipídeos/metabolismo , Mutação , Fosfatidato Fosfatase/química , Fosfatidato Fosfatase/genética , Cultura Primária de Células , Domínios Proteicos , Interferência de RNA , Transdução de Sinais , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Especificidade por Substrato , Transfecção , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Macrophages are key players involved in numerous pathophysiological pathways and an in-depth characterization of their gene regulatory networks can help in better understanding how their dysfunction may impact on human diseases. We here conducted a cross-species network analysis of macrophage gene expression data between human and mouse to identify conserved networks across both species, and assessed whether such networks could reveal new disease-associated regulatory mechanisms. From a sample of 684 individuals processed for genome-wide macrophage gene expression profiling, we identified 27 groups of coexpressed genes (modules). Six modules were found preserved (P < 10-4) in macrophages from 86 mice of the Hybrid Mouse Diversity Panel. One of these modules was significantly [false discovery rate (FDR) = 8.9 × 10-11] enriched for genes belonging to the oxidative phosphorylation (OXPHOS) pathway. This pathway was also found significantly (FDR < 10-4) enriched in susceptibility genes for Alzheimer, Parkinson, and Huntington diseases. We further conducted an expression quantitative trait loci analysis to identify SNP that could regulate macrophage OXPHOS gene expression in humans. This analysis identified the PARK2 rs192804963 as a trans-acting variant influencing (minimal P-value = 4.3 × 10-8) the expression of most OXPHOS genes in humans. Further experimental work demonstrated that PARK2 knockdown expression was associated with increased OXPHOS gene expression in THP1 human macrophages. This work provided strong new evidence that PARK2 participates to the regulatory networks associated with oxidative phosphorylation and suggested that PARK2 genetic variations could act as a trans regulator of OXPHOS gene macrophage expression in humans.
Assuntos
Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Macrófagos/metabolismo , Fosforilação Oxidativa , Transcriptoma , Ubiquitina-Proteína Ligases/genética , Animais , Biologia Computacional/métodos , Evolução Molecular , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Estudo de Associação Genômica Ampla , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Camundongos , Locos de Características Quantitativas , Especificidade da Espécie , Ubiquitina-Proteína Ligases/metabolismo , Fluxo de TrabalhoRESUMO
OBJECTIVE: We have previously reported that SASH1 expression is increased in circulating human monocytes from smokers and was positively correlated with the number of carotid atherosclerotic plaques. The aim of this study was to further validate the link between smoking, SASH1 and atherosclerosis within the vascular wall and to assess the impact of SASH1 expression on endothelial cell functions. METHOD: Human carotids with atherosclerotic plaques were obtained from 58 patients (45 of them with known smoking status: smoker, non-smoker, ex-smokers), and were processed for gene expression analyses and immunostaining. To investigate its function, SASH1 was silenced in human aortic endothelial cells (HAECs) using two different siRNA and subcellular localization of SASH1 was determined by immunostaining and subcellular fractionation. Subsequently the transcriptomic analyses and functional experiments (wound healing, WST-1 proliferation or Matrigel assays) were performed to characterize SASH1 function. RESULTS: SASH1 was expressed in human vascular cells (HAECs, smooth muscle cells) and in monocytes/macrophages. Its tissue expression was significantly higher in the atherosclerotic carotids of smokers compared to non-smokers (p < 0.01). In HAECs, SASH1 was expressed mostly in the cytoplasm and SASH1 knockdown resulted in an increased cell migration, proliferation and angiogenesis. Transcriptomic and pathway analyses showed that SASH1 silencing results in a decreased CYP1A1 expression possibly through the inhibition of TP53 activity. CONCLUSION: We showed that SASH1 expression is increased in atherosclerotic carotids in smokers and its silencing affects endothelial angiogenic functions; therefore we provide a potential link between smoking and atherosclerosis through SASH1 expression.
Assuntos
Aterosclerose/metabolismo , Regulação da Expressão Gênica , Fumar/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Idoso , Idoso de 80 Anos ou mais , Aorta/metabolismo , Aterosclerose/genética , Aterosclerose/fisiopatologia , Ciclo Celular , Linhagem Celular , Movimento Celular , Proliferação de Células , Ciclina D1/metabolismo , Ciclina D3/metabolismo , Citocromo P-450 CYP1A1/metabolismo , Células Endoteliais/metabolismo , Feminino , Inativação Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Neovascularização Patológica , RNA Interferente Pequeno/metabolismo , Transcriptoma , Proteína Supressora de Tumor p53/metabolismoRESUMO
Runs of homozygosity (ROHs) are recognized signature of recessive inheritance. Contributions of ROHs to the genetic architecture of coronary artery disease and regulation of gene expression in cells relevant to atherosclerosis are not known. Our combined analysis of 24,320 individuals from 11 populations of white European ethnicity showed an association between coronary artery disease and both the count and the size of ROHs. Individuals with coronary artery disease had approximately 0.63 (95% CI: 0.4-0.8) excess of ROHs when compared to coronary-artery-disease-free control subjects (p = 1.49 × 10(-9)). The average total length of ROHs was approximately 1,046.92 (95% CI: 634.4-1,459.5) kb greater in individuals with coronary artery disease than control subjects (p = 6.61 × 10(-7)). None of the identified individual ROHs was associated with coronary artery disease after correction for multiple testing. However, in aggregate burden analysis, ROHs favoring increased risk of coronary artery disease were much more common than those showing the opposite direction of association with coronary artery disease (p = 2.69 × 10(-33)). Individual ROHs showed significant associations with monocyte and macrophage expression of genes in their close proximity-subjects with several individual ROHs showed significant differences in the expression of 44 mRNAs in monocytes and 17 mRNAs in macrophages when compared to subjects without those ROHs. This study provides evidence for an excess of homozygosity in coronary artery disease in outbred populations and suggest the potential biological relevance of ROHs in cells of importance to the pathogenesis of atherosclerosis.
Assuntos
Doença da Artéria Coronariana/genética , Regulação da Expressão Gênica/genética , Genes Recessivos/genética , Homozigoto , Macrófagos/metabolismo , Monócitos/metabolismo , Fatores Etários , Humanos , RNA Mensageiro/metabolismo , População Branca/genéticaRESUMO
Smoking is a major risk factor in many diseases. Genome wide association studies have linked genes for nicotine dependence and smoking behavior to increased risk of cardiovascular, pulmonary, and malignant diseases. We conducted an epigenome wide association study in peripheral-blood DNA in 464 individuals (22 current smokers and 263 ex-smokers), using the Human Methylation 450 K array. Upon replication in an independent sample of 356 twins (41 current and 104 ex-smokers), we identified 30 probes in 15 distinct loci, all of which reached genome-wide significance in the combined analysis P < 5 × 10(-8). All but one probe (cg17024919) remained significant after adjusting for blood cell counts. We replicated all 9 known loci and found an independent signal at CPOX near GPR15. In addition, we found 6 new loci at PRSS23, AVPR1B, PSEN2, LINC00299, RPS6KA2, and KIAA0087. Most of the lead probes (13 out of 15) associated with cigarette smoking, overlapped regions of open chromatin (FAIRE and DNaseI hypersensitive sites) or/and H3K27Ac peaks (ENCODE data set), which mark regulatory elements. The effect of smoking on DNA methylation was partially reversible upon smoking cessation for longer than 3 months. We report the first statistically significant interaction between a SNP (rs2697768) and cigarette smoking on DNA methylation (cg03329539). We provide evidence that the metSNP for cg03329539 regulates expression of the CHRND gene located circa 95 Kb downstream of the methylation site. Our findings suggest the existence of dynamic, reversible site-specific methylation changes in response to cigarette smoking , which may contribute to the extended health risks associated with cigarette smoking.
Assuntos
Metilação de DNA , Polimorfismo de Nucleotídeo Único , Fumar/efeitos adversos , Adulto , Idoso , Idoso de 80 Anos ou mais , Ilhas de CpG , Epigênese Genética , Feminino , Estudo de Associação Genômica Ampla , Humanos , Modelos Lineares , Masculino , Pessoa de Meia-Idade , Locos de Características Quantitativas , Análise de Sequência de RNARESUMO
The nature of an inherited platelet disorder was investigated in three siblings affected by severe bleeding. Using whole-exome sequencing, we identified the culprit mutation (cG742T) in the RAS guanyl-releasing protein-2 (RASGRP2) gene coding for calcium- and DAG-regulated guanine exchange factor-1 (CalDAG-GEFI). Platelets from individuals carrying the mutation present a reduced ability to activate Rap1 and to perform proper αIIbß3 integrin inside-out signaling. Expression of CalDAG-GEFI mutant in HEK293T cells abolished Rap1 activation upon stimulation. Nevertheless, the PKC- and ADP-dependent pathways allow residual platelet activation in the absence of functional CalDAG-GEFI. The mutation impairs the platelet's ability to form thrombi under flow and spread normally as a consequence of reduced Rac1 GTP-binding. Functional deficiencies were confined to platelets and megakaryocytes with no leukocyte alteration. This contrasts with the phenotype seen in type III leukocyte adhesion deficiency caused by the absence of kindlin-3. Heterozygous did not suffer from bleeding and have normal platelet aggregation; however, their platelets mimicked homozygous ones by failing to undergo normal adhesion under flow and spreading. Rescue experiments on cultured patient megakaryocytes corrected the functional deficiency after transfection with wild-type RASGRP2. Remarkably, the presence of a single normal allele is sufficient to prevent bleeding, making CalDAG-GEFI a novel and potentially safe therapeutic target to prevent thrombosis.
Assuntos
Transtornos Herdados da Coagulação Sanguínea , Plaquetas , Fatores de Troca do Nucleotídeo Guanina , Hemorragia , Mutação , Agregação Plaquetária/genética , Difosfato de Adenosina/genética , Difosfato de Adenosina/metabolismo , Transtornos Herdados da Coagulação Sanguínea/genética , Transtornos Herdados da Coagulação Sanguínea/metabolismo , Transtornos Herdados da Coagulação Sanguínea/patologia , Plaquetas/metabolismo , Plaquetas/patologia , Linhagem Celular , Feminino , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Guanosina Trifosfato/genética , Guanosina Trifosfato/metabolismo , Hemorragia/genética , Hemorragia/metabolismo , Hemorragia/patologia , Heterozigoto , Homozigoto , Humanos , Masculino , Megacariócitos/metabolismo , Megacariócitos/patologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Complexo Shelterina , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismoRESUMO
Smoking is a risk factor for atherosclerosis with reported widespread effects on gene expression in circulating blood cells. We hypothesized that a molecular signature mediating the relation between smoking and atherosclerosis may be found in the transcriptome of circulating monocytes. Genome-wide expression profiles and counts of atherosclerotic plaques in carotid arteries were collected in 248 smokers and 688 non-smokers from the general population. Patterns of co-expressed genes were identified by Independent Component Analysis (ICA) and network structure of the pattern-specific gene modules was inferred by the PC-algorithm. A likelihood-based causality test was implemented to select patterns that fit models containing a path "smokingâgene expressionâplaques". Robustness of the causal inference was assessed by bootstrapping. At a FDR ≤0.10, 3,368 genes were associated to smoking or plaques, of which 93% were associated to smoking only. SASH1 showed the strongest association to smoking and PPARG the strongest association to plaques. Twenty-nine gene patterns were identified by ICA. Modules containing SASH1 and PPARG did not show evidence for the "smokingâgene expressionâplaques" causality model. Conversely, three modules had good support for causal effects and exhibited a network topology consistent with gene expression mediating the relation between smoking and plaques. The network with the strongest support for causal effects was connected to plaques through SLC39A8, a gene with known association to HDL-cholesterol and cellular uptake of cadmium from tobacco, while smoking was directly connected to GAS6, a gene reported to have anti-inflammatory effects in atherosclerosis and to be up-regulated in the placenta of women smoking during pregnancy. Our analysis of the transcriptome of monocytes recovered genes relevant for association to smoking and atherosclerosis, and connected genes that before, were only studied in separate contexts. Inspection of correlation structure revealed candidates that would be missed by expression-phenotype association analysis alone.
Assuntos
Aterosclerose/genética , Artérias Carótidas/metabolismo , Expressão Gênica , Modelos Genéticos , Monócitos/metabolismo , Placa Aterosclerótica/genética , Fumar/genética , Algoritmos , Aterosclerose/etiologia , Aterosclerose/metabolismo , Aterosclerose/patologia , Artérias Carótidas/patologia , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Funções Verossimilhança , Masculino , Pessoa de Meia-Idade , Monócitos/química , Monócitos/patologia , Família Multigênica , PPAR gama/genética , PPAR gama/metabolismo , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patologia , Gravidez , Fatores de Risco , Fumar/efeitos adversos , Fumar/metabolismo , Fumar/patologia , Transcriptoma , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
BACKGROUND: Cardiac surgery modulates pro- and anti-inflammatory cytokine balance involving plasma tumour necrosis factor alpha (TNFα) and interleukin-10 (IL-10) together with urinary transforming growth factor beta-1 (TGFß1), interleukin-1 receptor antagonist (IL1ra) and tumour necrosis factor soluble receptor-2 (TNFsr2). Effects on post-operative renal function are unclear. We investigated if following cardiac surgery there is a relationship between cytokine (a) phenotype and renal outcome; (b) genotype and phenotype and (c) genotype and renal outcome. Since angiotensin-2 (AG2), modulates TGFß1 production, we determined whether angiotensin converting enzyme insertion/deletion (ACE I/D) genotype affects urinary TGFß1 phenotype as well as renal outcome. METHODS: In 408 elective cardiac surgery patients we measured pre- and 24 h post-operative urinary TGFß-1, IL1ra and TNFsr2 and pre- and 2 h post-operative plasma TNFα and IL-10. Post-operative responses were compared for each cytokine in patients grouped according to presence or absence of renal dysfunction defined as a drop from baseline eGFR of greater than 25% (as calculated by the method of modification of diet in renal disease (MDRD)) occurring (1) within the first 24 and (2) 48 postoperative hours (early renal dysfunction), (3) on the fifth postoperative day (late renal dysfunction) or (4) at any time throughout the 5 day postoperative period (early and late combined). Patient genotype was determined for TNF/G-308A, TGFß1-509 C/T, IL10/G-1082A and ACE I/D. RESULTS: Post-operative plasma IL-10 and urinary TGFß1 responses were significantly higher in patients who developed early renal dysfunction. IL1ra and TNFsr2 responses were significantly lower 24h post-operatively in patients who developed late renal dysfunction. Genotype did not alter cytokine phenotype or outcome. CONCLUSIONS/INFERENCES: Cytokine profiling may help predict early and late renal dysfunction. Genotypes studied did not alter phenotype or outcome.
Assuntos
Injúria Renal Aguda/etiologia , Procedimentos Cirúrgicos Cardíacos/efeitos adversos , Citocinas/sangue , Nefropatias/etiologia , Injúria Renal Aguda/genética , Idoso , Enzima de Conversão de Angiotensina 2 , Citocinas/metabolismo , Feminino , Genótipo , Humanos , Proteína Antagonista do Receptor de Interleucina 1/sangue , Interleucina-10/sangue , Nefropatias/genética , Masculino , Pessoa de Meia-Idade , Peptidil Dipeptidase A/genética , Fenótipo , Fator de Crescimento Transformador beta1/sangue , Resultado do Tratamento , Fator de Necrose Tumoral alfa/sangueRESUMO
BACKGROUND: A sexual dimorphism exists in the incidence and prevalence of coronary artery disease--men are more commonly affected than are age-matched women. We explored the role of the Y chromosome in coronary artery disease in the context of this sexual inequity. METHODS: We genotyped 11 markers of the male-specific region of the Y chromosome in 3233 biologically unrelated British men from three cohorts: the British Heart Foundation Family Heart Study (BHF-FHS), West of Scotland Coronary Prevention Study (WOSCOPS), and Cardiogenics Study. On the basis of this information, each Y chromosome was tracked back into one of 13 ancient lineages defined as haplogroups. We then examined associations between common Y chromosome haplogroups and the risk of coronary artery disease in cross-sectional BHF-FHS and prospective WOSCOPS. Finally, we undertook functional analysis of Y chromosome effects on monocyte and macrophage transcriptome in British men from the Cardiogenics Study. FINDINGS: Of nine haplogroups identified, two (R1b1b2 and I) accounted for roughly 90% of the Y chromosome variants among British men. Carriers of haplogroup I had about a 50% higher age-adjusted risk of coronary artery disease than did men with other Y chromosome lineages in BHF-FHS (odds ratio 1·75, 95% CI 1·20-2·54, p=0·004), WOSCOPS (1·45, 1·08-1·95, p=0·012), and joint analysis of both populations (1·56, 1·24-1·97, p=0·0002). The association between haplogroup I and increased risk of coronary artery disease was independent of traditional cardiovascular and socioeconomic risk factors. Analysis of macrophage transcriptome in the Cardiogenics Study revealed that 19 molecular pathways showing strong differential expression between men with haplogroup I and other lineages of the Y chromosome were interconnected by common genes related to inflammation and immunity, and that some of them have a strong relevance to atherosclerosis. INTERPRETATION: The human Y chromosome is associated with risk of coronary artery disease in men of European ancestry, possibly through interactions of immunity and inflammation. FUNDING: British Heart Foundation; UK National Institute for Health Research; LEW Carty Charitable Fund; National Health and Medical Research Council of Australia; European Union 6th Framework Programme; Wellcome Trust.
Assuntos
Cromossomos Humanos Y/genética , Doença da Artéria Coronariana/genética , Haplótipos , Estudos de Casos e Controles , Feminino , Genótipo , Humanos , Macrófagos/metabolismo , Masculino , Pessoa de Meia-Idade , Monócitos/metabolismo , Polimorfismo de Nucleotídeo Único , Fatores de Risco , Distribuição por Sexo , TranscriptomaRESUMO
One major expectation from the transcriptome in humans is to characterize the biological basis of associations identified by genome-wide association studies. So far, few cis expression quantitative trait loci (eQTLs) have been reliably related to disease susceptibility. Trans-regulating mechanisms may play a more prominent role in disease susceptibility. We analyzed 12,808 genes detected in at least 5% of circulating monocyte samples from a population-based sample of 1,490 European unrelated subjects. We applied a method of extraction of expression patterns-independent component analysis-to identify sets of co-regulated genes. These patterns were then related to 675,350 SNPs to identify major trans-acting regulators. We detected three genomic regions significantly associated with co-regulated gene modules. Association of these loci with multiple expression traits was replicated in Cardiogenics, an independent study in which expression profiles of monocytes were available in 758 subjects. The locus 12q13 (lead SNP rs11171739), previously identified as a type 1 diabetes locus, was associated with a pattern including two cis eQTLs, RPS26 and SUOX, and 5 trans eQTLs, one of which (MADCAM1) is a potential candidate for mediating T1D susceptibility. The locus 12q24 (lead SNP rs653178), which has demonstrated extensive disease pleiotropy, including type 1 diabetes, hypertension, and celiac disease, was associated to a pattern strongly correlating to blood pressure level. The strongest trans eQTL in this pattern was CRIP1, a known marker of cellular proliferation in cancer. The locus 12q15 (lead SNP rs11177644) was associated with a pattern driven by two cis eQTLs, LYZ and YEATS4, and including 34 trans eQTLs, several of them tumor-related genes. This study shows that a method exploiting the structure of co-expressions among genes can help identify genomic regions involved in trans regulation of sets of genes and can provide clues for understanding the mechanisms linking genome-wide association loci to disease.
Assuntos
Doença Celíaca/genética , Diabetes Mellitus Tipo 1/genética , Regulação da Expressão Gênica/genética , Variação Genética/genética , Hipertensão/genética , Monócitos/metabolismo , Locos de Características Quantitativas/genética , Proteínas Adaptadoras de Transdução de Sinal , Adulto , Idoso , Feminino , Perfilação da Expressão Gênica , Predisposição Genética para Doença , Genoma Humano , Estudo de Associação Genômica Ampla , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Pessoa de Meia-Idade , Muramidase/genética , Polimorfismo de Nucleotídeo Único , Proteínas/genética , Proteínas Ribossômicas/genética , Fatores de Transcrição/genéticaRESUMO
Combined analyses of gene networks and DNA sequence variation can provide new insights into the aetiology of common diseases that may not be apparent from genome-wide association studies alone. Recent advances in rat genomics are facilitating systems-genetics approaches. Here we report the use of integrated genome-wide approaches across seven rat tissues to identify gene networks and the loci underlying their regulation. We defined an interferon regulatory factor 7 (IRF7)-driven inflammatory network (IDIN) enriched for viral response genes, which represents a molecular biomarker for macrophages and which was regulated in multiple tissues by a locus on rat chromosome 15q25. We show that Epstein-Barr virus induced gene 2 (Ebi2, also known as Gpr183), which lies at this locus and controls B lymphocyte migration, is expressed in macrophages and regulates the IDIN. The human orthologous locus on chromosome 13q32 controlled the human equivalent of the IDIN, which was conserved in monocytes. IDIN genes were more likely to associate with susceptibility to type 1 diabetes (T1D)-a macrophage-associated autoimmune disease-than randomly selected immune response genes (P = 8.85 × 10(-6)). The human locus controlling the IDIN was associated with the risk of T1D at single nucleotide polymorphism rs9585056 (P = 7.0 × 10(-10); odds ratio, 1.15), which was one of five single nucleotide polymorphisms in this region associated with EBI2 (GPR183) expression. These data implicate IRF7 network genes and their regulatory locus in the pathogenesis of T1D.
Assuntos
Diabetes Mellitus Tipo 1/genética , Loci Gênicos/genética , Predisposição Genética para Doença/genética , Imunidade Inata/genética , Vírus/imunologia , Animais , Sequência de Bases , Cromossomos Humanos Par 13/genética , Cromossomos de Mamíferos/genética , Diabetes Mellitus Tipo 1/imunologia , Redes Reguladoras de Genes/genética , Estudo de Associação Genômica Ampla , Humanos , Inflamação/genética , Inflamação/imunologia , Fator Regulador 7 de Interferon/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Especificidade de Órgãos , Polimorfismo de Nucleotídeo Único/genética , Locos de Características Quantitativas/genética , Ratos , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMO
AIMS: multimarker approaches for risk prediction in coronary artery disease have remained inconsistent. We assessed multiple biomarkers representing distinct pathophysiological pathways in relation to cardiovascular events in stable angina. METHODS AND RESULTS: we investigated 12 biomarkers reflecting inflammation [C-reactive protein, growth-differentiation factor (GDF)-15, neopterin], lipid metabolism (apolipoproteins AI, B100), renal function (cystatin C, serum creatinine), and cardiovascular function and remodelling [copeptin, C-terminal-pro-endothelin-1, mid-regional-pro-adrenomedullin (MR-proADM), mid-regional-pro-atrial natriuretic peptide (MR-proANP), N-terminal-pro-B-type natriuretic peptide (Nt-proBNP)] in 1781 stable angina patients in relation to non-fatal myocardial infarction and cardiovascular death (n = 137) over 3.6 years. Using Cox proportional hazards models and C-indices, the strongest association with outcome for log-transformed biomarkers in multivariable-adjusted analyses was observed for Nt-proBNP [hazard ratio (HR) for one standard deviation increase 1.65, 95% confidence interval (CI) 1.28-2.13, C-index 0.686], GDF-15 (HR 1.59, 95% CI 1.25-2.02, C-index 0.681), MR-proANP (HR 1.46, 95% CI 1.14-1.87, C-index 0.673), cystatin C (HR 1.39, 95% CI 1.10-1.75, C-index 0.671), and MR-proADM (HR 1.63, 95% CI 1.21-2.20, C-index 0.668). Each of these top single markers and their combination (C-index 0.690) added predictive information beyond the baseline model consisting of the classical risk factors assessed by C-index and led to substantial reclassification (P-integrated discrimination improvement <0.05). CONCLUSION: comparative analysis of 12 biomarkers revealed Nt-proBNP, GDF-15, MR-proANP, cystatin C, and MR-proADM as the strongest predictors of cardiovascular outcome in stable angina. All five biomarkers taken separately offered incremental predictive ability over established risk factors. Combination of the single markers slightly improved model fit but did not enhance risk prediction from a clinical perspective.
Assuntos
Biomarcadores/metabolismo , Doença da Artéria Coronariana/prevenção & controle , Idoso , Angina Estável/sangue , Angina Estável/mortalidade , Angina Estável/prevenção & controle , Doença da Artéria Coronariana/sangue , Doença da Artéria Coronariana/mortalidade , Feminino , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Infarto do Miocárdio/sangue , Infarto do Miocárdio/mortalidade , Infarto do Miocárdio/prevenção & controle , Prognóstico , Estudos Prospectivos , Medição de RiscoRESUMO
Secreted phospholipases A2 (sPLA2s) are present in atherosclerotic plaques and are now considered novel attractive therapeutic targets and potential biomarkers as they contribute to the development of atherosclerosis through lipoprotein-dependent and independent mechanisms. We have previously shown that hGX-sPLA2-phospholipolyzed LDL (LDL-X) induces proinflammatory responses in human umbilical endothelial cells (HUVECs); here we explore the molecular mechanisms involved. Global transcriptional gene expression profiling of the response of endothelial cells exposed to either LDL or LDL-X revealed that LDL-X activates multiple distinct cellular pathways including the unfolded protein response (UPR). Mechanistic insight showed that LDL-X activates UPR through calcium depletion of intracellular stores, which in turn disturbs cytoskeleton organization. Treatment of HUVECs and aortic endothelial cells (HAECs) with LDL-X led to activation of all 3 proximal initiators of UPR: eIF-2alpha, IRE1alpha, and ATF6. In parallel, we observed a sustained phosphorylation of the p38 pathway resulting in the phosphorylation of AP-1 downstream targets. This was accompanied by significant production of the proinflammatory cytokines IL-6 and IL-8. Our study demonstrates that phospholipolyzed LDL uses a range of molecular pathways including UPR to initiate endothelial cell perturbation and thus provides an LDL oxidation-independent mechanism for the initiation of vascular inflammation in atherosclerosis.
Assuntos
Retículo Endoplasmático/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Lipoproteínas LDL/farmacologia , Fator 6 Ativador da Transcrição/metabolismo , Western Blotting , Células Cultivadas , Endorribonucleases/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Imunofluorescência , Inativação Gênica , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Lipoproteínas LDL/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Fosfolipases A2 Secretórias , Proteínas Serina-Treonina Quinases/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Veias Umbilicais/citologia , Resposta a Proteínas não DobradasRESUMO
BACKGROUND: Variability of gene expression in human may link gene sequence variability and phenotypes; however, non-genetic variations, alone or in combination with genetics, may also influence expression traits and have a critical role in physiological and disease processes. METHODOLOGY/PRINCIPAL FINDINGS: To get better insight into the overall variability of gene expression, we assessed the transcriptome of circulating monocytes, a key cell involved in immunity-related diseases and atherosclerosis, in 1,490 unrelated individuals and investigated its association with >675,000 SNPs and 10 common cardiovascular risk factors. Out of 12,808 expressed genes, 2,745 expression quantitative trait loci were detected (P<5.78x10(-12)), most of them (90%) being cis-modulated. Extensive analyses showed that associations identified by genome-wide association studies of lipids, body mass index or blood pressure were rarely compatible with a mediation by monocyte expression level at the locus. At a study-wide level (P<3.9x10(-7)), 1,662 expression traits (13.0%) were significantly associated with at least one risk factor. Genome-wide interaction analyses suggested that genetic variability and risk factors mostly acted additively on gene expression. Because of the structure of correlation among expression traits, the variability of risk factors could be characterized by a limited set of independent gene expressions which may have biological and clinical relevance. For example expression traits associated with cigarette smoking were more strongly associated with carotid atherosclerosis than smoking itself. CONCLUSIONS/SIGNIFICANCE: This study demonstrates that the monocyte transcriptome is a potent integrator of genetic and non-genetic influences of relevance for disease pathophysiology and risk assessment.
Assuntos
Perfilação da Expressão Gênica , Predisposição Genética para Doença , Monócitos/metabolismo , Adulto , Idoso , Aterosclerose/genética , Sequência de Bases , Movimento Celular/genética , Cromossomos Humanos Par 21/genética , Sondas de DNA/metabolismo , Feminino , Regulação da Expressão Gênica , Variação Genética , Genoma Humano/genética , Estudo de Associação Genômica Ampla , Humanos , Imunidade/genética , Masculino , Pessoa de Meia-Idade , Monócitos/citologia , Fenótipo , Polimorfismo de Nucleotídeo Único/genética , Locos de Características Quantitativas/genética , Característica Quantitativa Herdável , Fatores de Risco , Fumar/efeitos adversos , Fumar/genéticaRESUMO
OBJECTIVE: Osteoprotegerin (OPG) has been reported to be involved in the development of atherosclerotic disease, and OPG gene variation has been associated with plasma OPG levels and different cardiovascular disease phenotypes. However, the genetic architecture of the OPG promoter and its transcriptional regulation are poorly characterized. METHODS AND RESULTS: We identified 1008 bp of the OPG 5'-flanking region to be sufficiently transcriptionally active in osteosarcoma cell lines and generated serial promoter deletion constructs. Individual subcloning revealed the existence of 3 molecular haplotypes (MolHaps): [T(-960)-A(-946)-G(-900)-T(-864); MolHap1, wild type], [T(-960)-G(-946)-G(-900)-T(-864); MolHap2], [C(-960)-G(-946)-A(-900)-G(-864); MolHap4]. Compared to MolHap1, transcriptional activities of MolHaps 2 and 4 were significantly reduced (P=0.0018). Whereas introduction of the -159C allele reduced transcriptional activities of the full-length constructs (P=0.0014), it significantly increased activities of the deletion constructs (P=0.0005). Electrophoretic mobility shift, competition, and chromatin immunoprecipitation assays revealed specific DNA:protein interactions for the MolHaps with Sp1 and NF-1, and identified Egr1 interacting exclusively with the -159T allele. CONCLUSIONS: We propose new structural and transcriptional features within the OPG promoter region and identified MolHaps being differentially transcriptionally active and allele-dependently interacting with a proximal polymorphic site.
Assuntos
Haplótipos , Osteoprotegerina/genética , Regiões Promotoras Genéticas/fisiologia , Sítios de Ligação , Linhagem Celular Tumoral , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Neurofibromina 1/metabolismo , RNA Mensageiro/análise , Fator de Transcrição Sp1/metabolismo , Transcrição GênicaRESUMO
BACKGROUND: Interleukin-18 is a pro-inflammatory cytokine suspected to be associated with atherosclerosis and its complications. We had previously shown that one single nucleotide polymorphism (SNP) of the IL18 gene was associated with cardiovascular disease (CVD) through an interaction with smoking. As a further step for elucidating the contribution of the IL-18 pathway to the etiology of CVD, we here investigated the association between the genetic variability of two IL-18 receptor genes, IL18R1 and IL18RAP, with the risk of developing CVD. METHODS: Eleven tagging SNPs, 5 in IL18R1 and 6 in IL18RAP, characterizing the haplotypic variability of the corresponding genes; were genotyped in 5 European prospective CVD cohorts including 1416 cases and 1772 non-cases, as part of the MORGAM project. Both single-locus and haplotypes analyses were carried out to investigate the association of these SNPs with CVD. RESULTS: We did not find any significant differences in allele, genotype and haplotype frequencies between cases and non-cases for either of the two genes. Moreover, the search for interactions between SNPs located in different genes, including 5 IL18 SNPs previously studied in the MORGAM project, and between SNPs and environmental factors remained unfruitful. CONCLUSION: Our analysis suggests that the variability of IL18R1 and IL18RAP genes are unlikely to contribute to modulate the risk of CVD.
Assuntos
Doenças Cardiovasculares/genética , Subunidade alfa de Receptor de Interleucina-18/genética , Subunidade beta de Receptor de Interleucina-18/genética , Polimorfismo de Nucleotídeo Único , Estudos de Coortes , Finlândia , França , Frequência do Gene , Estudo de Associação Genômica Ampla , Genótipo , Haplótipos , Humanos , Desequilíbrio de Ligação , Irlanda do Norte , Razão de Chances , Fatores de Risco , População BrancaRESUMO
We aimed at associating common osteopontin (OPN) gene variants with cardiovascular disease phenotypes.We scanned the OPN gene in 190 chromosomes from myocardial infarction (MI) patients and identified five variants in the promoter, three synonymous and one non-synonymous variant. All variants were investigated in case-control studies for MI (ECTIM: 990 cases, 900 controls) and brain infarction (BI) (GENIC: 466 cases, 444 controls). Promoter variants were functionally analyzed by bandshift assays, the coding D147D [T/C] by Western blot. Allele D147D C was independently and significantly associated with lower apoB levels (P=0.044 [ECTIM] P=0.03 [GENIC]), its allele frequency was significantly lower in patients with BI compared to controls (OR [95% CI] 0.39 [0.20-0.74], P=0.004), and C allele carriers had a significantly lower frequency of presence of carotid plaques (P=0.02). Bandshifts with HepG2 and Ea.hy926 nuclear proteins did not reveal any functionality of promoter variants, whereas the OPN-441C-containing construct resulted in reduced OPN protein expression in Western blots, complying with its potential protective effect on the phenotypes studied.We here provide evidence that a portion of the OPN locus is likely to associate with cardiovascular disease-related phenotypes. However, further experiments are warranted to clarify the functional role of OPN variants.
Assuntos
Transtornos Cerebrovasculares/genética , Infarto do Miocárdio/genética , Osteopontina/genética , Idoso , Infarto Encefálico/genética , Artérias Carótidas/diagnóstico por imagem , Proteínas de Transporte/genética , Linhagem Celular , Linhagem Celular Tumoral , Feminino , Humanos , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Fenótipo , Polimorfismo Genético , UltrassonografiaRESUMO
Insulin-like growth factor 1 (IGF1) exerts important endocrine and paracrine functions in the cardiovascular system. We identified the common variant -1411C>T in the IGF1 upstream promoter P1, located within several overlapping transcription factor binding sites. Using transient transfection assays, we identified this site as a functional enhancer. The T allele-carrying enhancer, compared with the C allelic portion, exerts significantly reduced or even abrogated activity, respectively, in SaOs-2 and HepG2 (all P<0.0001) as well as in differentiated THP-1 macrophages. Electrophoretic mobility shift assay and subsequent supershift experiments in HepG2 identified c-Jun as the binding partner exclusively to the T allele, whereas CCAAT/enhancer-binding protein delta and interferon consensus site-binding protein/interferon-regulating factor 8 interacted only with the C allelic promoter portion. Furthermore, genotyping in a case-control study for essential hypertension (n=745 hypertensive patients; n=769 normotensive control subjects) for this variant revealed an odds ratio for hypertension of 0.73 (95% confidence interval 0.58-0.91, P=0.006) associated with the T allele, and normotensive subjects carrying the protective T allele displayed a significant decrease in diastolic (P=0.036) and systolic (P=0.024) blood pressure levels. We here report detection of a functional enhancer module in the upstream IGF1 promoter region, which might play a key role in local IGF1 bioavailability. Whether -1411C>T is also associated with other IGF1-related disease phenotypes should be evaluated further in population studies.
Assuntos
Fator de Crescimento Insulin-Like I/genética , Regiões Promotoras Genéticas , Sequência de Bases , Proteína delta de Ligação ao Facilitador CCAAT/genética , Linhagem Celular Tumoral , Células Cultivadas , Biologia Computacional , Sequência Consenso , Elementos Facilitadores Genéticos/genética , Feminino , Humanos , Hipertensão/genética , Fatores Reguladores de Interferon/genética , Masculino , Pessoa de Meia-Idade , Transcrição Gênica/fisiologiaRESUMO
Interleukin-18 (IL-18) is a key inflammatory molecule suspected of being involved in the etiology of cardiovascular diseases (CVD). Five single nucleotide polymorphisms (SNPs) capturing the common genetic variation of the IL-18 gene (tag SNPs) were genotyped in five European prospective CVD cohorts including 1933 cases and 1938 non-cases as part of the MORGAM Project. Not a single SNP was found associated with CVD. However, a significant (P=0.002) gene-smoking interaction was observed. In smokers, the -105T allele was more frequent in cases than in non-cases (0.29 vs 0.25) and associated with an increased risk of disease (odds ratio (OR)=1.25 (1.07-1.45), P=0.005), whereas the inverse relationship tended to be observed in non-smokers (OR=0.90 (0.78-1.02), P=0.131). The gene-smoking interaction was broadly homogenous across the cohorts and was also observed through haplotype analyses. In conclusion, using the concerted effort of several European prospective CVD cohorts, we are able to show that one IL-18 tag SNP interacts with smoking to modulate the risk of developing CVD.
Assuntos
Doenças Cardiovasculares/genética , Interleucina-18/genética , Polimorfismo de Nucleotídeo Único , Doenças Cardiovasculares/epidemiologia , Estudos de Casos e Controles , Estudos de Coortes , Simulação por Computador , Genética Populacional/métodos , Estudo de Associação Genômica Ampla , Haplótipos , Humanos , Análise de Regressão , Sitios de Sequências Rotuladas , Fumar/epidemiologia , Fumar/genéticaRESUMO
OBJECTIVE: Genetic and environmental factors modulate the susceptibility to diabetic nephropathy, as initiating and/or progression factors. The objective of the European Rational Approach for the Genetics of Diabetic Complications (EURAGEDIC) study is to identify nephropathy susceptibility genes. We report molecular genetic studies for 127 candidate genes for nephropathy. RESEARCH DESIGN AND METHODS: Polymorphisms were identified through sequencing of promoter, exon, and flanking intron gene regions and a database search. A total of 344 nonredundant SNPs and nonsynonymous variants were tested for association with diabetic nephropathy (persistent albuminuria >/=300 mg/24 h) in a large type 1 diabetes case/control (1,176/1,323) study from three European populations. RESULTS: Only one SNP, rs2281999, located in the UNC13B gene, was significantly associated with nephropathy after correction for multiple testing. Analyses of 21 additional markers fully characterizing the haplotypic variability of the UNC13B gene showed consistent association of SNP rs13293564 (G/T) located in intron 1 of the gene with nephropathy in the three populations. The odds ratio (OR) for nephropathy associated with the TT genotype was 1.68 (95% CI 1.29-2.19) (P = 1.0 x 10(-4)). This association was replicated in an independent population of 412 case subjects and 614 control subjects (combined OR of 1.63 [95% CI 1.30-2.05], P = 2.3 x 10(-5)). CONCLUSIONS: We identified a polymorphism in the UNC13B gene associated with nephropathy. UNC13B mediates apopotosis in glomerular cells in the presence of hyperglycemia, an event occurring early in the development of nephropathy. We propose that this polymorphism could be a marker for the initiation of nephropathy. However, further studies are needed to clarify the role of UNC13B in nephropathy.