Nathalie Gaudreault

Molecular Signature of Smoking in Human Lung Tissues

Cigarette smoking is the leading risk factor for lung cancer. To identify genes deregulated by smoking and to distinguish gene expression changes that are reversible and persistent following smoking cessation, we carried out genome-wide gene expression profiling on nontumor lung tissue from 853 patients with lung cancer. Gene expression levels were compared between never and current smokers, and time-dependent changes in gene expression were studied in former smokers. A total of 3,223 transcripts were differentially expressed between smoking groups in the discovery set (n = 344, P < 1.29 × 10(-6)). A substantial number of smoking-induced genes also were validated in two replication sets (n = 285 and 224), and a gene expression signature of 599 transcripts consistently segregated never from current smokers across all three sets. The expression of the majority of these genes reverted to never-smoker levels following smoking cessation, although the time course of normalization differed widely among transcripts. Moreover, some genes showed very slow or no reversibility in expression, including SERPIND1, which was found to be the most consistent gene permanently altered by smoking in the three sets. Our findings therefore indicate that smoking deregulates many genes, many of which reverse to normal following smoking cessation. However, a subset of genes remains altered even decades following smoking cessation and may account, at least in part, for the residual risk of lung cancer among former smokers. Cancer Res; 72(15); 3753-63. ©2012 AACR.

ATP acts as a survival signal and prevents the mineralization of aortic valve

Calcific aortic valve disease (CAVD) is a disorder related to progressive mineralization of valvular tissue that is a leading cause of heart disease. Thus far, there is no medical treatment to prevent the mineralization of aortic valves. It is generally thought that pathologic mineralization is linked to apoptosis of vascular cells. However, the role of apoptosis during mineralization as well as the survival signals for valvular interstitial cells (VICs), the main cellular component of aortic valves, remains to be identified. Here, through several lines of evidence, we show that bioavailability of extracellular ATP is a signal which determines survival or apoptosis of VICs and, in doing so, plays a major role in the development of CAVD. Specifically, in CAVD and in VIC cultures undergoing mineralization, we found a high level of the ectonucleotidase ENPP1. In addition, a genetic polymorphism in the intron 9 of the ENPP1 gene was associated with CAVD in a case-control cohort as well as with mRNA expression levels of ENPP1 in aortic valves. A high level of ENPP1 in CAVD promoted apoptosis-mediated mineralization of VICs by depleting the extracellular pool of ATP. We then documented that release of ATP by VICs promoted cell survival via the P2Y(2) receptor and the PI3K/Akt signaling pathway. Hence, our results show that level of ENPP1 modulates extracellular concentration of ATP, which is an important survival signal for VICs. These findings may help to develop novel pharmacological treatment for CAVD.

ApoE Suppresses Atherosclerosis by Reducing Lipid Accumulation in Circulating Monocytes and the Expression of Inflammatory Molecules on Monocytes and Vascular Endothelium

Objective—We investigated atheroprotective properties of apolipoprotein (apo) E beyond its ability to lower plasma cholesterol. We hypothesized that apoE reduces atherosclerosis by decreasing lipid accumulation in circulating monocytes and the inflammatory state of monocytes and the vascular endothelium. Methods and Results—We developed mice with spontaneous hyperlipidemia with and without plasma apoE. Hypomorphic apoE mice deficient in low-density lipoprotein receptor (Apoeh/hLdlr−/−) were compared to Apoe−/−Ldlr−/− mice. Despite 4-fold more plasma apoE than WT mice, Apoeh/hLdlr−/− mice displayed similar plasma cholesterol as Apoe−/− Ldlr−/− mice but developed 4-fold less atherosclerotic lesions by 5 months of age. The aortic arch of Apoeh/hLdlr−/− mice showed decreased endothelial expression of ICAM-1, PECAM-1, and JAM-A. In addition, Apoeh/hLdlr−/− mice had less circulating leukocytes and proinflammatory Ly6Chigh monocytes. These monocytes had decreased neutral lipid content and reduced surface expression of ICAM-1, VLA-4, and L-Selectin. Apoeh/hLdlr−/− mice displayed increased levels of apoA1-rich HDL that were potent in promoting cellular cholesterol efflux. Conclusions—Our findings suggest that apoE reduces atherosclerosis in the setting of hyperlipidemia by increasing plasma apoA1-HDL that likely contribute to reduce intracellular lipid accumulation and thereby the activation of circulating leukocytes and the vascular endothelium.

Altered DNA methylation of long noncoding RNA H19 in calcific aortic valve disease promotes mineralization by silencing NOTCH1

Background: Calcific aortic valve disease is characterized by an abnormal mineralization of the aortic valve. Osteogenic activity in the aortic valve is under the control of NOTCH1, which regulates the expression of key pro-osteogenic genes such as RUNX2 and BMP2. Long noncoding RNAs (lncRNAs) may reprogram cells by altering the gene expression pattern. Methods: Multidimensional genomic profiling was performed in human aortic valves to document the expression of lncRNAs and the DNA methylation pattern in calcific aortic valve disease. In-depth functional assays were carried out to document the impact of lncRNA on the mineralization of the aortic valve. Results: We documented that lncRNA H19 (H19) was increased in calcific aortic valve disease. Hypomethylation of the promoter region was observed in mineralized aortic valves and was inversely associated with H19 expression. Knockdown and overexpression experiments showed that H19 induces a strong osteogenic phenotype by altering the NOTCH1 pathway. Gene promoter analyses showed that H19 silenced NOTCH1 by preventing the recruitment of p53 to its promoter. A knockdown of H19 in valve interstitial cells (VICs) increased the expression of NOTCH1 and decreased the level of RUNX2 and BMP2, 2 downstream targets repressed by NOTCH1. In rescue experiments, the transfection of a vector encoding for the active Notch intracellular domain prevented H19-induced mineralization of valve interstitial cells. Conclusions: These findings indicate that a dysregulation of DNA methylation in the promoter of H19 during calcific aortic valve disease is associated with a higher expression of this lncRNA, which promotes an osteogenic program by interfering with the expression of NOTCH1.

Replication of Genetic Association Studies in Aortic Stenosis in Adults

Only a handful of studies have attempted to unravel the genetic architecture of calcific aortic valve stenosis (AS). The goal of this study was to validate genes previously associated with AS. Seven genes were assessed: APOB, APOE, CTGF, IL10, PTH, TGFB1, and VDR. Each gene was tested for a comprehensive set of single-nucleotide polymorphisms (SNPs). SNPs were genotyped in 457 patients who underwent surgical aortic valve replacement, and allele frequencies were compared to 3,294 controls. A missense mutation in the APOB gene was significantly associated with AS (rs1042031, E4181K, p = 0.00001). A second SNP located 5.6 kilobases downstream of the APOB stop codon was also associated with the disease (rs6725189, p = 0.000013). Six SNPs surrounding the IL10 locus were strongly associated with AS (0.02 > p > 6.2 × 10⁻¹¹). The most compelling association for IL10 was found with a promoter polymorphism (rs1800872) well known to regulate the production of the encoded anti-inflammatory cytokine. The frequency of the low-producing allele was greater in cases compared to controls (30% vs 20%, p = 6.2 × 10⁻¹¹). SNPs in PTH, TGFB1, and VDR had nominal p values <0.05 but did not resist Bonferroni correction. In conclusion, this study suggests that subjects carrying specific polymorphisms in the IL10 and APOB genes are at higher risk for developing AS.

Characterisation of glucose transporters in the intact coronary artery endothelium in rats: GLUT-2 upregulated by long-term hyperglycaemia

Aims/hypothesisWe have examined the effects of streptozotocin-induced type 1 diabetes on the expression and subcellular distribution of the classic sugar transporters (GLUT-1 to 5 and sodium-dependent glucose transporter-1 [SGLT-1]) in the endothelial cells of an en face preparation of septal coronary artery from Wistar rats.MethodsThe presence of the GLUT isoforms and SGLT-1 in the endothelial cell layer was determined by immunohistochemistry using wide-field fluorescence microscopy coupled to deconvolution, and was quantified by digital image analysis.ResultsWe found that all of the transporters were expressed within these cells and that all except SGLT-1 were preferentially located on the abluminal side. The heaviest labelling was adjacent to the cell-to-cell junctions where the luminal and abluminal membranes are in close proximity, which may reflect a spatial organisation specialised for vectorial glucose transport across the thinnest part of the cytoplasm. Long-term hyperglycaemia, induced by streptozotocin, significantly downregulated GLUT-1, 3, 4 and 5 and dramatically upregulated GLUT-2, leaving SGLT-1 unchanged.Conclusions/interpretationWe conclude that the high susceptibility of endothelial cells to glucose toxicity may be the result of the subcellular organisation of their GLUTs and the increased expression of GLUT-2.

The transcriptome of human epicardial, mediastinal and subcutaneous adipose tissues in men with coronary artery disease.

Background The biological functions of epicardial adipose tissue (EAT) remain largely unknown. However, the proximity of EAT to the coronary arteries suggests a role in the pathogenesis of coronary artery disease (CAD). The objectives of this study were to identify genes differentially regulated among three adipose tissues, namely EAT, mediastinal (MAT) and subcutaneous (SAT) and to study their possible relationships with the development of cardiovascular diseases. Methods and Results Samples were collected from subjects undergoing coronary artery bypass grafting surgeries. Gene expression was evaluated in the three adipose depots of six men using the Illumina® HumanWG-6 v3.0 expression BeadChips. Twenty-three and 73 genes were differentially up-regulated in EAT compared to MAT and SAT, respectively. Ninety-four genes were down-regulated in EAT compared to SAT. However, none were significantly down-regulated in EAT compared to MAT. More specifically, the expression of the adenosine A1 receptor (ADORA1), involved in myocardial ischemia, was significantly up-regulated in EAT. Levels of the prostaglandin D2 synthase (PTGDS) gene, recently associated with the progression of atherosclerosis, were significantly different in the three pairwise comparisons (EAT>MAT>SAT). The results of ADORA1 and PTGDS were confirmed by quantitative real-time PCR in 25 independent subjects. Conclusions Overall, the transcriptional profiles of EAT and MAT were similar compared to the SAT. Despite this similarity, two genes involved in cardiovascular diseases, ADORA1 and PTGDS, were differentially up-regulated in EAT. These results provide insights about the biology of EAT and its potential implication in CAD.

Identification of Gender-Specific Genetic Variants in Patients With Bicuspid Aortic Valve

Bicuspid aortic valve (BAV) is the most frequent congenital heart defect and has a male predominance of 3 to 1. A large proportion of patients develop valvular and aortic complications. Despite the high prevalence of BAV, its cause and genetic origins remain elusive. The goal of this study was to identify genetic variants associated with BAV. Nine genes previously associated with BAV (NOTCH1, AXIN1, EGFR, ENG, GATA5, NKX2-5, NOS3, PDIA2, and TGFBR2) were sequenced in 48 patients with BAV using the Ion Torrent Personal Genome Machine. Pathogenicity of genetic variants was evaluated with the Combined Annotation Dependent Depletion framework. A selection of 89 variants identified by sequencing or in previous BAV genetic studies was genotyped, and allele frequencies were compared in 323 patients with BAV confirmed at surgery and 584 controls. Analyses were also performed by gender. Nine novel and 19 potentially pathogenic variants were identified by next-generation sequencing and confirmed by Sanger sequencing, but they were not associated with BAV in the case-control population. A significant association was observed between an in silico-predicted benign EGFR intronic variant (rs17290301) and BAV. Analyses performed by gender revealed different variants associated with BAV in men (EGFR rs533525993 and TEX26 rs12857479) and women (NOTCH1 rs61751489, TGFBR2 rs1155705, and NKX2-5 rs2277923). In conclusion, these results constitute the first association between EGFR genetic variants and BAV in humans and support a possible role of gender-specific polymorphisms in the development of BAV.

Calcium Signaling Pathway Genes RUNX2 and CACNA1C Are Associated With Calcific Aortic Valve Disease

Background—Calcific aortic valve stenosis (AS) is a life-threatening disease with no medical therapy. The genetic architecture of AS remains elusive. This study combines genome-wide association studies, gene expression, and expression quantitative trait loci mapping in human valve tissues to identify susceptibility genes of AS. Methods and Results—A meta-analysis was performed combining the results of 2 genome-wide association studies in 474 and 486 cases from Quebec City (Canada) and Paris (France), respectively. Corresponding controls consisted of 2988 and 1864 individuals with European ancestry from the database of genotypes and phenotypes. mRNA expression levels were evaluated in 9 calcified and 8 normal aortic valves by RNA sequencing. The results were integrated with valve expression quantitative trait loci data obtained from 22 AS patients. Twenty-five single-nucleotide polymorphisms had P<5×10−6 in the genome-wide association studies meta-analysis. The calcium signaling pathway was the top gene set enriched for genes mapped to moderately AS-associated single-nucleotide polymorphisms. Genes in this pathway were found differentially expressed in valves with and without AS. Two single-nucleotide polymorphisms located in RUNX2 (runt-related transcription factor 2), encoding an osteogenic transcription factor, demonstrated some association with AS (genome-wide association studies P=5.33×10−5). The mRNA expression levels of RUNX2 were upregulated in calcified valves and associated with eQTL-SNPs. CACNA1C encoding a subunit of a voltage-dependent calcium channel was upregulated in calcified valves. The eQTL-SNP with the most significant association with AS located in CACNA1C was associated with higher expression of the gene. Conclusions—This integrative genomic study confirmed the role of RUNX2 as a potential driver of AS and identified a new AS susceptibility gene, CACNA1C, belonging to the calcium signaling pathway.

Isradipine and insulin sensitivity in hypertensive rats

The present study was designed to investigate the effect of a reduction in blood pressure, by using the calcium channel antagonist isradipine, on insulin sensitivity and vascular responses to insulin in conscious spontaneously hypertensive male rats (SHR). The rats were instrumented with intravascular catheters and pulsed Doppler flow probes to measure blood pressure, heart rate, and blood flows. Insulin sensitivity was assessed by the euglycemic-hyperinsulinemic clamp technique. Two groups of rats received isradipine at a dose of 0.05 or 0.15 mg ⋅ kg-1 ⋅ h-1, whereas a third group received a continuous infusion of vehicle (15% DMSO). Both doses of isradipine were found to decrease mean blood pressure (-25 ± 4 mmHg at the dose of 0.05 mg ⋅ kg-1 ⋅ h-1and -20 ± 2 mmHg at the dose of 0.15 mg ⋅ kg-1 ⋅ h-1) and to improve insulin sensitivity. Moreover, in the rats treated with the low dose of isradipine, we observed vasodilations in renal, superior mesenteric, and hindquarter vascular beds. In the untreated group, the euglycemic infusion of insulin (4 mU ⋅ kg-1 ⋅ min-1) was found to cause vasoconstrictions in superior mesenteric and hindquarter vascular beds, but no changes in mean blood pressure, heart rate, or renal vascular conductance were found. In contrast, in the isradipine-treated groups, the same dose of insulin was found to produce vasodilations in the renal vascular bed and to abolish the vasoconstrictor responses previously observed. We concluded that short-term treatment with isradipine in SHR can lower blood pressure and improve insulin sensitivity, mainly through hemodynamic factors, as supported by experiments with hydralazine as a positive vasodilator control.The present study was designed to investigate the effect of a reduction in blood pressure, by using the calcium channel antagonist isradipine, on insulin sensitivity and vascular responses to insulin in conscious spontaneously hypertensive male rats (SHR). The rats were instrumented with intravascular catheters and pulsed Doppler flow probes to measure blood pressure, heart rate, and blood flows. Insulin sensitivity was assessed by the euglycemic-hyperinsulinemic clamp technique. Two groups of rats received isradipine at a dose of 0.05 or 0.15 mg. kg-1. h-1, whereas a third group received a continuous infusion of vehicle (15% DMSO). Both doses of isradipine were found to decrease mean blood pressure (-25 +/- 4 mmHg at the dose of 0.05 mg. kg-1. h-1 and -20 +/- 2 mmHg at the dose of 0.15 mg. kg-1. h-1) and to improve insulin sensitivity. Moreover, in the rats treated with the low dose of isradipine, we observed vasodilations in renal, superior mesenteric, and hindquarter vascular beds. In the untreated group, the euglycemic infusion of insulin (4 mU. kg-1. min-1) was found to cause vasoconstrictions in superior mesenteric and hindquarter vascular beds, but no changes in mean blood pressure, heart rate, or renal vascular conductance were found. In contrast, in the isradipine-treated groups, the same dose of insulin was found to produce vasodilations in the renal vascular bed and to abolish the vasoconstrictor responses previously observed. We concluded that short-term treatment with isradipine in SHR can lower blood pressure and improve insulin sensitivity, mainly through hemodynamic factors, as supported by experiments with hydralazine as a positive vasodilator control.