Comparative analysis of genome-wide association studies signals for lipids, diabetes, and coronary heart disease: Cardiovascular Biomarker Genetics Collaboration

To evaluate the associations of emergent genome-wide-association study-derived coronary heart disease (CHD)-associated single nucleotide polymorphisms (SNPs) with established and emerging risk factors, and the association of genome-wide-association study-derived lipid-associated SNPs with other risk factors and CHD events

Involvement of long non-coding RNAs in beta cell failure at the onset of type 1 diabetes in NOD mice

Aims/hypothesis: Exposure of pancreatic beta cells to cytokines released by islet-infiltrating immune cells induces alterations in gene expression, leading to impaired insulin secretion and apoptosis in the initial phases of type 1 diabetes. Long non-coding RNAs (lncRNAs) are a new class of transcripts participating in the development of many diseases. As little is known about their role in insulin-secreting cells, this study aimed to evaluate their contribution to beta cell dysfunction. Methods: The expression of lncRNAs was determined by microarray in the MIN6 beta cell line exposed to proinflammatory cytokines. The changes induced by cytokines were further assessed by real-time PCR in islets of control and NOD mice. The involvement of selected lncRNAs modified by cytokines was assessed after their overexpression in MIN6 cells and primary islet cells. Results: MIN6 cells were found to express a large number of lncRNAs, many of which were modified by cytokine treatment. The changes in the level of selected lncRNAs were confirmed in mouse islets and an increase in these lncRNAs was also seen in prediabetic NOD mice. Overexpression of these lncRNAs in MIN6 and mouse islet cells, either alone or in combination with cytokines, favoured beta cell apoptosis without affecting insulin production or secretion. Furthermore, overexpression of lncRNA-1 promoted nuclear translocation of nuclear factor of κ light polypeptide gene enhancer in B cells 1 (NF-κB). Conclusions/interpretation: Our study shows that lncRNAs are modulated during the development of type 1 diabetes in NOD mice, and that their overexpression sensitises beta cells to apoptosis, probably contributing to their failure during the initial phases of the disease

Influence of matrix metalloproteinase- 12 on fibrinogen level

In vitro studies have shown that matrixmetalloproteinase-12 (MMP12) can degrade fibrinogen, a clotting factor whose level predicts risk of advanced atherosclerosis and myocardial infarction. In this study, we found that mean plasma fibrinogenlevel was approximately 10-fold higher in MMP12 knockout mice than wildtype mice (p = 0.0006). Differential allelic expression analysis of human MMP12 gene polymorphism rs17368582 in human vascular tissues showed an allele-specific effect on MMP12 expression, with one allele (T) having 1.6 fold higher expression level than the other allele (C) (p = 0.0006). In a population cohort, we found that individuals homozygous for the MMP12 low expression allele had higher plasma fibrinogenlevels (2.95 mg/mL compared with 2.61 mg/mL in other individuals, p = 0.029) and increased risk of advanced atherosclerosis [odds ratio 6.3 (95% CI 1.9–20.8), p = 0.003] and myocardial infarction [hazard ratio 5.6 (95% CI 1.7–18.3), p = 0.005]. In summary, our study in mouse and humans provides in vivo evidence of an effect of MMP12 on fibrinogenleve

Allele-specific regulation of matrix metalloproteinase-3 gene by transcription factor NF?B

Background: matrix metalloproteinase-3 (MMP3) is implicated in the pathogenesis and progression of atherosclerotic lesions. Previous studies suggested that MMP3 expression is influenced by a polymorphism (known as the 5A/6A polymorphism) in the promoter of the MMP3 gene and that this polymorphism is located within a cis-element that interacts with the transcription factor NF?B. In the present study, we sought to investigate whether MMP3 and NF?B were co-localized in atherosclerotic lesions and whether NF?B had differential effects on the two alleles of the MMP3 5A/6A polymorphism.Methodology/Principal Findings: immunohistochemical examination showed that MMP3 and both the NF?B p50 and p65 subunits were expressed abundantly in macrophages in atherosclerotic lesions and that MMP3 expression was co-localized with p50 and p65. Chromatin immunoprecipitation experiments showed interaction of p50 and p65 with the MMP3 promoter in macrophages, with greater binding to the 5A allele than to the 6A allele. Reporter gene assays in transiently transfected macrophages showed that the 5A allele had greater transcriptional activity than the 6A allele, and that this allele-specific effect was augmented when the cells were treated with the NF?B activator lipopolysaccharides or co-transfected with p50 and/or p65 expressing plasmids, but was reduced when the cells were treated with the NF?B inhibitor 6-Amino-4-(4-phenoxyphenylethylamino)-qu?inazoline or transfected with a dominant negative mutant of IkB kinase-?.Conclusion: these results corroborate an effect of the 5A/6A polymorphism on MMP3 transcription and indicate that NF?B has differential effects on the 5A and 6A alleles.<br/

Identification of islet-enriched long non-coding RNAs contributing to β-cell failure in type 2 diabetes

Objective: Non-coding RNAs constitute a major fraction of the β-cell transcriptome. While the involvement of microRNAs is well established, the contribution of long non-coding RNAs (lncRNAs) in the regulation of β-cell functions and in diabetes development remains poorly understood. The aim of this study was to identify novel islet lncRNAs differently expressed in type 2 diabetes models and to investigate their role in β-cell failure and in the development of the disease. Methods: Novel transcripts dysregulated in the islets of diet-induced obese mice were identified by high throughput RNA-sequencing coupled with de novo annotation. Changes in the level of the lncRNAs were assessed by real-time PCR. The functional role of the selected lncRNAs was determined by modifying their expression in MIN6 cells and primary islet cells. Results: We identified about 1500 novel lncRNAs, a number of which were differentially expressed in obese mice. The expression of two lncRNAs highly enriched in β-cells, βlinc2, and βlinc3, correlated to body weight gain and glycemia levels in obese mice and was also modified in diabetic db/. db mice. The expression of both lncRNAs was also modulated in vitro in isolated islet cells by glucolipotoxic conditions. Moreover, the expression of the human orthologue of βlinc3 was altered in the islets of type 2 diabetic patients and was associated to the BMI of the donors. Modulation of the level of βlinc2 and βlinc3 by overexpression or downregulation in MIN6 and mouse islet cells did not affect insulin secretion but increased β-cell apoptosis. Conclusions: Taken together, the data show that lncRNAs are modulated in a model of obesity-associated type 2 diabetes and that variations in the expression of some of them may contribute to β-cell failure during the development of the disease

The MMP3 gene 5A allele more readily interacts with p50 and p65, than the 6A allele.

<p>Chromatin proteins and DNA in THP1 cells were cross-linked in1% formaldehyde for 10 minutes. Cells were then lysed, and the lysate sonicated to reduce DNA length to 200–1000 bp. The sonicated chromatin was subjected to immunoprecipitation using a p50 or p65 antibody, or incubated with a rabbit IgG, followed by PCR amplification of 179 bp and 180 bp DNA fragments surrounding the <i>MMP3</i> gene 5A/6A site using fluorescence labelled primers. The PCR amplicons were analyzed using an ABI 3730xl analyzer and Genemapper software. Three independent experiments were performed. (A). Representative output from the Genemapper programme. The panels from top to bottom represent input chromatin DNA, chromatin immunoprecipitated with the p50 antibody, and chromatin immunoprecipitated with the p65 antibody. (B). Chart shows mean (± standard error of mean) of the relative peak heights of the 5A and 6A alleles in the p50 and p65 antibody precipitates, standardized against the relative peak heights of the 5A and 6A alleles in the input DNA, in three independent experiments. * denotes p<0.05 comparing 5A versus 6A alleles.</p

Macrophages and smooth muscle cells in atherosclerotic plaques express MMP3 and NFκB.

<p>Sections (4 µm) of paraffin embedded tissue blocks of atherosclerotic plaques were subjected to double immunostaining of MMP3 and the macrophage marker CD68 (A and B), MMP3 and the smooth muscle cell marker SMA (C and D), p50 and CD68 (E and F), p50 and SMA (G and H), p65 and CD68 (I and J), p65 and SMA (K and M), using rabbit polyclonal NFκB p50 and MMP3 antibodies (Abcam), a rabbit monoclonal NFκB p65 antibody (Abcam), and mouse monoclonal CD68 and smooth muscle actin (SMA) antibodies (DAKO), respectively, and goat anti-rabbit or anti-mouse secondary antibodies (Thermo/LabVision). The double immunostaining was visualized with Liquid Permanent Red chromogen (Dako) and Vector Blue chromogen (Vector Labs). Pink colour indicates expression of MMP3 (A, B, C and D), p50 (E, F, G and H) or p65 (I, J, K and M). Blue colour indicates expression of CD68 (A, B, E, F, I and J) or SMA (C, D, G, H, K and M). Arrow indicates cell co-expressing MMP3 with CD68 (B) or SMA (D), p50 with CD68 (F) or SMA (H), and p65 with CD68 (J) or SMA (M). 200X magnification in A, C, E, G, I and K; 400X magnification in B, D, F, H, J and M.</p