Meta-analysis of genome-wide association studies from the CHARGE consortium identifies common variants associated with carotid intima media thickness and plaque

Carotid intima media thickness (cIMT) and plaque determined by ultrasonography are established measures of subclinical atherosclerosis that each predicts future cardiovascular disease events. We conducted a meta-analysis of genome-wide association data in 31,211 participants of European ancestry from nine large studies in the setting of the Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) Consortium. We then sought additional evidence to support our findings among 11,273 individuals using data from seven additional studies. In the combined meta-analysis, we identified three genomic regions associated with common carotid intima media thickness and two different regions associated with the presence of carotid plaque (P < 5 × 10 -8). The associated SNPs mapped in or near genes related to cellular signaling, lipid metabolism and blood pressure homeostasis, and two of the regions were associated with coronary artery disease (P < 0.006) in the Coronary Artery Disease Genome-Wide Replication and Meta-Analysis (CARDIoGRAM) consortium. Our findings may provide new insight into pathways leading to subclinical atherosclerosis and subsequent cardiovascular events

Causal effect of plasminogen activator inhibitor type 1 on coronary heart disease

Background--Plasminogen activator inhibitor type 1 (PAI-1) plays an essential role in the fibrinolysis system and thrombosis. Population studies have reported that blood PAI-1 levels are associated with increased risk of coronary heart disease (CHD). However, it is unclear whether the association reflects a causal influence of PAI-1 on CHD risk. Methods and Results--To evaluate the association between PAI-1 and CHD, we applied a 3-step strategy. First, we investigated the observational association between PAI-1 and CHD incidence using a systematic review based on a literature search for PAI-1 and CHD studies. Second, we explored the causal association between PAI-1 and CHD using a Mendelian randomization approach using summary statistics from large genome-wide association studies. Finally, we explored the causal effect of PAI-1 on cardiovascular risk factors including metabolic and subclinical atherosclerosis measures. In the systematic meta-analysis, the highest quantile of blood PAI-1 level was associated with higher CHD risk comparing with the lowest quantile (odds ratio=2.17; 95% CI: 1.53, 3.07) in an age- and sex-adjusted model. The effect size was reduced in studies using a multivariable-adjusted model (odds ratio=1.46; 95% CI: 1.13, 1.88). The Mendelian randomization analyses suggested a causal effect of increased PAI-1 level on CHD risk (odds ratio=1.22 per unit increase of log-transformed PAI-1; 95% CI: 1.01, 1.47). In addition, we also detected a causal effect of PAI-1 on elevating blood glucose and high-density lipoprotein cholesterol. Conclusions--Our study indicates a causal effect of elevated PAI-1 level on CHD risk, which may be mediated by glucose dysfunction

Effects of irbesartan and dronedarone on in vivo gene expression signatures of left atrial tissue in a porcine model of acute rapid pacing

Zusammenfassend lässt sich aus der Irbesartan- und der Dronedaron-Studie schließen, dass akutes RAP für 7 h signifikante Änderungen in der linksatrialen Genexpression bewirkt, darunter solche, die indikativ sind für ANG II-vermittelten oxidativen Stress, atrialen Gewebeumbau (Remodeling) und zelluläre Energieverknappung. Darüberhinaus zeigen die Ergebnisse der Dronedaron-Studie, dass das Antiarrhythmikum eine große Zahl der RAP-induzierten Genexpressions-Änderungen, die durch oxidativen Stress bedingt sind, reduziert. In Übereinstimmung hiermit demonstrierten die haemodynamischen Parameter ebenfalls eine Verringerung RAP-induzierter mikrovaskulärer Durchblutungsstörungen durch Dronedaron. Das Antiarrhythmikum schwächte außerdem weitere RAP-abhängige Effekte ab, namentlich die Phosphorylierung von PKC und IκBα, die Induktion der Expression verschiedener NAPDH-Oxidasen, und die Erhöhung der Gewebekonzentration von F2-Isoprostanen. Diese Ergebnisse sind konsistent mit der Vorstellung, dass ein großer Teil der Änderungen der RAP-induzierten linksatrialen Genexpression sowie der damit verbundenen pathologischen Effekte durch ANG II-induzierten oxidativen Stress vermittelt werden. Konsistent hiermit schwächte Irbesartan, welches den ANG II-Typ 1-Rezeptor AT1R blockiert, seinerseits die meisten RAP-induzierten linksatrialen Genexpressionsänderungen ab. Ein weiteres wesentliches Ergebnis besteht darin, dass ET-1 zur VHF-bedingten atrialen Fibrose beiträgt, indem es nach ANG II-vermittelter transkriptioneller Aktivierung seines Strukturgens sowohl die Expression von SGK1 als auch die Phosphorylierung der davon kodierten Kinase stimuliert, die dann ihrerseits die Expression von CTGF induziert. Vom CTGF-Protein wird schon seit längerer Zeit angenommen, dass es pro-fibrotisch wirkt. An HL-1-Zellen durchgeführte in vitro-Analysen bestätigen diese Folgerungen: Hochfrequenz-Stimulation induziert die sequentielle transkriptionelle Induktion von ET-1, SGK1 und CTGF, und exogenes ET-1 führt zur Induktion der CTGF-Expression.In summary, the transcriptome data demonstrated that acute RAP for 7h induces significant changes in the expression of several left atrial genes, including those reflecting ANG II-mediated oxidative stress, tissue remodeling, and energy depletion. Furthermore, the results from the dronedarone study demonstrated that this drug is capable of attenuating most of RAP-induced changes in oxidative stress-related gene expression. Accordingly, the haemodynamic parameters also showed that dronedarone reduced RAP-induced microvascular flow abnormalities. This view is supported by the observation that in the used porcine model of acute AF, dronedarone decreased RAP-dependent PKC phosphorylation, NADPH isoform expression, F2-isoprostane release and IκBα phosphorylation. Additionally, the results of the irbesartan study indicate that ET-1 contributes to AF-dependent atrial fibrosis by synergistic activity with ANG-II to stimulate SGK1 expression and enhance phosphorylation of the SGK1 protein which, in turn, induces CTGF. The latter has been consistently associated with tissue fibrosis. In support of this view, in vitro analyses using HL-1 cells verified CTGF induction after short episodes of RAP and additionally in response to exogenous addition of ET-1. Accordingly, irbesartan was shown to attenuate most of the RAP-dependent changes in atrial or ventricular gene expression

Distinct Transcriptional Networks in Quiescent Myoblasts: A Role for Wnt Signaling in Reversible vs. Irreversible Arrest

<div><p>Most cells in adult mammals are non-dividing: differentiated cells exit the cell cycle permanently, but stem cells exist in a state of reversible arrest called quiescence. In damaged skeletal muscle, quiescent satellite stem cells re-enter the cell cycle, proliferate and subsequently execute divergent programs to regenerate both post-mitotic myofibers and quiescent stem cells. The molecular basis for these alternative programs of arrest is poorly understood. In this study, we used an established myogenic culture model (C2C12 myoblasts) to generate cells in alternative states of arrest and investigate their global transcriptional profiles. Using cDNA microarrays, we compared G₀ myoblasts with post-mitotic myotubes. Our findings define the transcriptional program of quiescent myoblasts in culture and establish that distinct gene expression profiles, especially of tumour suppressor genes and inhibitors of differentiation characterize reversible arrest, distinguishing this state from irreversibly arrested myotubes. We also reveal the existence of a tissue-specific quiescence program by comparing G₀ C2C12 myoblasts to isogenic G₀ fibroblasts (10T1/2). Intriguingly, in myoblasts but not fibroblasts, quiescence is associated with a signature of Wnt pathway genes. We provide evidence that different levels of signaling via the canonical Wnt pathway characterize distinct cellular states (proliferation vs. quiescence vs. differentiation). Moderate induction of Wnt signaling in quiescence is associated with critical properties such as clonogenic self-renewal. Exogenous Wnt treatment subverts the quiescence program and negatively affects clonogenicity. Finally, we identify two new quiescence-induced regulators of canonical Wnt signaling, Rgs2 and Dkk3, whose induction in G₀ is required for clonogenic self-renewal. These results support the concept that active signal-mediated regulation of quiescence contributes to stem cell properties, and have implications for pathological states such as cancer and degenerative disease.</p></div

C2C12 myoblasts enter alternate states of arrest.

<p>(<b>A</b>) Asynchronous proliferating myoblasts (MB) were held in suspension in mitogen-rich media to induce quiescence (arrest in an undifferentiated mono-nucleated state; G₀ MB), or shifted to mitogen-poor medium for 96 hrs to induce differentiation (arrest coupled with fusion into multinucleated myotubes; MT). Actin staining (Oregon Green-Phalloidin, top panel) reveals the distinct morphology of these 3 states. Nuclei are detected with Hoechst 33352. DNA synthesis was analyzed by detection of BrdU incorporated in a 15-minute pulse (middle panel)- ∼40% of cycling MB are labeled, while less than 1% of G₀ MB synthesize DNA. In differentiated cultures (MT), only residual mono-nucleated cycling cells (5–10%) incorporate label whereas myotube nuclei do not. Expression of determination factor MyoD can be detected in ∼50% of proliferating MB, is lost in G₀ but sustained in MT (lower panel). (<b>B</b>) FACS analysis of DNA content of cycling (Asynchronous MB) and 48-hr suspension-cultured populations confirms that >97% of cells in suspension (G₀ MB) possess a 2C DNA content while >40% of cells in an asynchronous culture have replicated their genomes (representative graphs from 4 independent experiments). (<b>C</b>) Muscle transcription factor expression distinguishes alternate states of arrest. Q-RT-PCR analysis of specification/survival factor Pax7, determination factor MyoD, and early differentiation marker Myogenin (MyoG) in asynchronous proliferating myoblasts (As), suspension-arrested MB (G₀) and differentiated MT (MT). Values represent normalized fold differences between GAPDH (control) mRNA and myogenic mRNAs in each sample (n = 3). (<b>D</b>) Cell cycle inhibitor expression distinguishes alternate states of arrest-G₀ MB and MT express different combinations of cyclin-dependent kinase inhibitors p21/p27 and Rb-related p130. Immuno-detection of p21, p130 or p27 in MT and G₀; p21 (green) co-localizes with MyoG (red) in all nuclei of differentiated MT but neither factor is expressed in G₀; p130 is specific to G₀ and p27 is expressed in both G₀ and MT. Data depicted is representative of three independent experiments. (<b>E</b>) G₀ arrest is reversible. DNA synthesis in asynchronous MB (Mb), 48-hour suspension cultures (G₀) and G₀ MB replated for 6 or 24 hours (R6, R24). BrdU detected as in Fig. 1A. Values represent mean<u>+</u>SEM (n = 3). (<b>F</b>) MyoD expression is suppressed in G₀ and restored during early reactivation. Asynchronous cultures (A) were arrested in G₀ (48 hrs) and reactivated by replating for 2 to 24 hrs (R2–R24). MyoD expression detected as in Fig. 1A. Values represent mean<u>+</u>SEM (n = 3).</p

Exogenous Wnt treatment compromises a G0-induced program that promotes clonogenic potential.

<p>(A) Wnt3A treatment of MB reduces clonogenic potential. Colony formation was measured after 48 hrs in control culture conditions (either in proliferating conditions-Mb, or in suspension culture-G₀), or in the presence of 50 ng/ml of rWnt3A. Cloning efficiency (a measure of self-renewal) was strongly reduced by Wnt3A supplementation and restored by simultaneous addition of 50ng/ml sFRP2. Values represent the mean±SEM from three independent experiments, <i>p</i><0.05 (denoted by asterisk *). (B) Knockdown of Rgs2 and Dkk3 transcripts using siRNAs. siRNAs were designed against the putative Wnt regulators Rgs2, Dkk3 or an irrelevant gene (GAPDH) or a control scrambled siRNA sequence and transfected into C2C12 myoblasts along with a GFP plasmid. GFP<b>⁺</b> transfected cells were enriched by FACS, RNA isolated and analysed by Q-RT-PCR and the relative mRNA levels calculated. In each pair, the mRNA level is depicted of cells transfected with scrambled siRNA (blue bars) and cells transfected with the targeting siRNA (pink bars). Values represent the mean and SEM of 3 independent experiments. In each case, modest but reproducible reduction of the target transcript level is observed. (C) Reduction of Rgs2 and Dkk3 protein expression by siRNA-mediated knockdown. Western blot analysis of total protein isolated from control and knockdown C2C12 muscle cells probed with antibodies against Rgs2 (top) and Dkk3 (bottom). GAPDH protein levels indicate equal loading. Data depicted is representative of 3 independent experiments. (D) Rgs2 and Dkk3 expression is necessary for Wnt signaling. Knockdown of either Rgs2 or Dkk3 in growing or quiescent MB leads to suppression of TOPflash activity. Cells were treated and enriched as described in (B) and luciferase activity measured. Despite modest reduction of protein levels, strong reduction in TOPflash activity are seen, indicating a critical role for Rgs2 and Dkk3 in Wnt-βcat signaling. Values represent the mean and SEM of 3 independent experiments. (E,F) Knockdown cells (‘Rgs sh’ and ‘Dkk sh’) were enriched as described in (B) cultured in quiescence-inducing conditions, recovered from suspension culture and plated at clonogenic density for assessment of self-renewal (colony formation). Controls include untransfected cells (‘UT’) and control shRNA transfected cells (‘Con sh’). Typical plates with colony assays are shown in (E) and data are quantified as CFU (colony forming units) in (F). Values represent the mean and SEM of 3 independent experiments.</p

Chromatin-IP analysis of Myf5 and MyoG promoters in different cellular states.

<p>Antibodies against β-cat or HBP were used to assess the association of these Wnt-regulated transcription factors with chromatin in different cellular states as described in Materials and Methods. Control pulldowns used IgG and all values shown represent fold enrichment of the specific transcription factor after normalization against control IgG values. (<b>A</b>)<b>.</b> The Wnt target transcription factor β-cat associates with the known Wnt-responsive site in Myf5 enhancer preferentially in G₀ (G₀) but shows low enrichment in either proliferating (MB) or differentiating muscle cells (MT) (Blue bars-MB; pink bars-G₀; green bars-MT). This observation is consistent with the hypothesis that Wnt signaling is active in quiescent myoblasts. Comparison of β-cat association on another myogenic promoter (Myogenin promoter) shows greater enrichment in MT. Taken together, these observations suggest that Wnt/β-cat regulates different genes in different cellular states. (<b>B</b>)<b>.</b> ChIP analysis shows that HBP1 (a Wnt-induced repressor) co-associates with the Myf5 enhancer only in G₀ (Blue bars-Mb; pink bars-G₀; green bars-MT) and does not associate with this element in either proliferating or differentiated muscle cells. This observation suggests that fine-tuning of Myf5 expression by both activating and repressive mechanisms may occur in quiescent cells by association of two types of Wnt-responsive transcription factors. Taken together, this observation would account for the absence of induction of Myf5 mRNA in quiescent myoblasts despite the association of the transcriptional activator β-cat.</p

Involvement of surface proteins in Hla mediated cytotoxicity.

<p><b>A.</b> Western blot analysis of ADAM10 expression in 16HBE14o- and S9 cells. Signals corresponding to mature and processed ADAM10 are indicated. B. Quantification of surface E-cadherin (left panel) and ADAM10 (right panel) in 16HBE14o- (red), A549 (blue) and S9 (orange) cells as analyzed by flow cytometry. Corresponding isotype controls are indicated as dashed shapes. C. Flow cytometry analysis of surface expression of E-cadherin (left panels) and ADAM10 (right panels) in 16HBE14o- (upper panels) and S9 (lower panels) cells after 2 h under control conditions (blue) or following 2 h treatment with rHla (red). The corresponding isotype control is indicated by a dashed line. MFI: mean fluorescence intensity.</p

Functional annotation analysis of proteins with altered phosphorylation in rHla-treated 16HBE14o- and S9 cells.

<p>A. KEGG pathways with statistically noticeable number of proteins with altered phosphorylation following rHla-treatment for 2 h. The dotted vertical line indicates the cut-off for significant enrichment (Benjamini-Hochberg corrected p-value ≤0.01). Numbers next to the horizontal bars provide the number of assigned proteins. B. Matrix for cross-comparison of recurrent proteins with altered phosphorylation in both cell types between KEGG pathways from panel A. Numbers greater than the half of the maximum possible are highlighted. C. Ranking of phosphoproteins with altered phosphorylation that appear in at least three enriched KEGG pathways. D. Network visualization of proteins from the enriched KEGG pathways that have been identified with altered phosphorylation in both cell lines. Grey lines indicate experimentally validated protein-protein-interactions and red arrows kinase-substrate relationship taken from STRING and PhosphoSitePlus, respectively. rHla-induced changes are indicated in ruby (increased p-sites); cyan (decreased) or purple (both) for kinases (diamond shape) and substrates (circles) for 16HBE14o- cells (left half of shape) and S9 cells (right).</p

rHla-induced EGFR activation mediates resistance of S9 cells towards Hla.

<p>A. Cell counts of rHla-treated S9 cells co-treated with or without the EGFR-selective inhibitor tyrphostin AG1478. Graphs represent means ± SEM (n = 3). B. Quantification of EGFR surface expression in 16HBE14o- (red), A549 (blue) and S9 (orange) epithelial cells as analyzed by flow cytometry. The corresponding isotype control is indicated by a dashed line. MFI: mean fluorescence intensity.</p