Gp130-Dependent Release of Acute Phase Proteins Is Linked to the Activation of Innate Immune Signaling Pathways

Background: Elevated levels of acute phase proteins (APP) are often found in patients with cardiovascular diseases. In a previous study, we demonstrated the importance of the IL-6-gp130 axis-as a key regulator of inflammatory acute phase signaling in hepatocytes-for the development of atherosclerosis. Background/Principal Findings: Gp130-dependent gene expression was analyzed in a previously established hepatocytespecific gp130 knockout mouse model. We performed whole transcriptome analysis in isolated hepatocytes to measure tissue specific responses after proinflammatory stimulus with IL-6 across different time points. Our analyses revealed an unexpected small gene cluster that requires IL-6 stimulus for early activation. Several of the genes in this cluster are involved in different cell defense mechanisms. Thus, stressors that trigger both general stress and inflammatory responses lead to activation of a stereotypic innate cellular defense response. Furthermore, we identified a potential biomarker Lipocalin (LCN) 2 for the gp130 dependent early inflammatory response. Conclusions/Significance: Our findings suggest a complex network of tightly linked genes involved in the early activatio

The sex-specific factor SOA controls dosage compensation in <i>Anopheles</i> mosquitos

The Anopheles mosquito is one of thousands of species in which sex differences play a central role in their biology, as only females need a blood meal in order to produce eggs. Sex differentiation is regulated by sex chromosomes, but their presence creates a dosage imbalance between males (XY) and females (XX). Dosage compensation (DC) can re-equilibrate the expression of sex-chromosomal genes, but because DC mechanisms have only been fully characterized in a few model organisms, key questions about its evolutionary diversity and functional necessity remain unresolved 1. Here we report the discovery of a previously uncharacterized gene (SOA, for sex chromosome activation) as a master regulator of DC in the malaria mosquito Anopheles gambiae. Sex-specific alternative splicing prevents functional SOA protein expression in females. The male isoform encodes a DNA-binding protein that binds the promoters of active X chromosomal genes. Expressing male SOA is sufficient to induce DC in female cells. Male mosquitoes lacking SOA or female mosquitos ectopically expressing the male isoform exhibit X chromosome misregulation, which is compatible with viability but causes developmental delay. Thus, our molecular analysis of the first DC master regulator in a non-model organism elucidates the evolutionary steps leading to the establishment of a chromosome-specific fine-tuning mechanism

Systematic permutation testing in GWAS pathway analyses: identification of genetic networks in dilated cardiomyopathy and ulcerative colitis

Background: Genome wide association studies (GWAS) are applied to identify genetic loci, which are associated with complex traits and human diseases. Analogous to the evolution of gene expression analyses, pathway analyses have emerged as important tools to uncover functional networks of genome-wide association data. Usually, pathway analyses combine statistical methods with a priori available biological knowledge. To determine significance thresholds for associated pathways, correction for multiple testing and over-representation permutation testing is applied. Results: We systematically investigated the impact of three different permutation test approaches for over-representation analysis to detect false positive pathway candidates and evaluate them on genome-wide association data of Dilated Cardiomyopathy (DCM) and Ulcerative Colitis (UC). Our results provide evidence that the gold standard - permuting the case–control status – effectively improves specificity of GWAS pathway analysis. Although permutation of SNPs does not maintain linkage disequilibrium (LD), these permutations represent an alternative for GWAS data when case–control permutations are not possible. Gene permutations, however, did not add significantly to the specificity. Finally, we provide estimates on the required number of permutations for the investigated approaches. Conclusions: To discover potential false positive functional pathway candidates and to support the results from standard statistical tests such as the Hypergeometric test, permutation tests of case control data should be carried out. The most reasonable alternative was case–control permutation, if this is not possible, SNP permutations may be carried out. Our study also demonstrates that significance values converge rapidly with an increasing number of permutations. By applying the described statistical framework we were able to discover axon guidance, focal adhesion and calcium signaling as important DCM-related pathways and Intestinal immune network for IgA production as most significant UC pathway

Dichotomy between the transcriptomic landscape of naturally versus accelerated aged murine hearts

We investigated the transcriptomic landscape of the murine myocardium along the course of natural aging and in three distinct mouse models of premature aging with established aging-related cardiac dysfunction. Genome-wide total RNA-seq was performed and the expression patterns of protein-coding genes and non-coding RNAs were compared between hearts from naturally aging mice, mice with cardiac-specific deficiency of a component of the DNA repair machinery, mice with reduced mitochondrial antioxidant capacity and mice with reduced telomere length. Our results demonstrate that no dramatic changes are evident in the transcriptomes of naturally senescent murine hearts until two years of age, in contrast to the transcriptome of accelerated aged mice. Additionally, these mice displayed model-specific alterations of the expression levels of protein-coding and non-coding genes with hardly any overlap with age-related signatures. Our data demonstrate very limited similarities between the transcriptomes of all our murine aging models and question their reliability to study human cardiovascular senescence

Human atherosclerotic plaque transcriptomics reveals endothelial beta-2 spectrin as a potential regulator a leaky plaque microvasculature phenotype

The presence of atherosclerotic plaque vessels is a critical factor in plaque destabilization. This may be attributable to the leaky phenotype of these microvessels, although direct proof for this notion is lacking. In this study, we investigated molecular and cellular patterns of stable and hemorrhaged human plaque to identify novel drivers of intraplaque vessel dysfunction. From transcriptome data of a human atherosclerotic lesion cohort, we reconstructed a co-expression network, identifying a gene module strongly and selectively correlated with both plaque microvascular density and inflammation. Spectrin Beta Non-Erythrocytic 1 (sptbn1) was identified as one of the central hubs of this module (along with zeb1 and dock1) and was selected for further study based on its predominant endothelial expression. Silencing of sptbn1 enhanced leukocyte transmigration and vascular permeability in vitro, characterized by an increased number of focal adhesions and reduced junctional VE-cadherin. In vivo, sptbn1 knockdown in zebrafish impaired the development of the caudal vein plexus. Mechanistically, increased substrate stiffness was associated with sptbn1 downregulation in endothelial cells in vitro and in human vessels. Plaque SPTBN1 mRNA and protein expression were found to correlate with an enhanced presence of intraplaque hemorrhage and future cardiovascular disease (CVD) events during follow-up. In conclusion, we identify SPTBN1 as a central hub gene in a gene program correlating with plaque vascularisation. SPTBN1 was regulated by substrate stiffness in vitro while silencing blocked vascular development in vivo, and compromised barrier function in vitro. Together, SPTBN1 is identified as a new potential regulator of the leaky phenotype of atherosclerotic plaque microvessels

Examinations on the production and functionality of novel padlock probes for the detection of nucleic acids on microarrays

In this study a new approach for DNA-detection was developed, which combines DNA-microarrays with the principle of padlock probes. For this novel U-shaped capture molecules were developed, which ensure label-free and specific DNA detection on microarrays while simultaneously allowing a novel normalisation strategy of present signal intensities. This approach was implemented for and tested on the sequence detection of various pathogenic microorganisms. The specificity of the system could be demonstrated by differentiating single nucleotide variations of the target sequence. Moreover different variations of the normalisation strategy were tested to further improve the microarray's reproducibility. The reaction conditions of the enzymatic steps involved got optimised. Furthermore the realisability of a new concept for signal amplification was tested for this approach, which is based on in situ concatenation of special padlock probes

Untersuchungen zur Herstellung und Funktionalität neuartiger Padlock-Sonden für den Nukleinsäurenachweis auf Mikroarrays

In this study a new approach for DNA-detection was developed, which combines DNA-microarrays with the principle of padlock probes. For this novel U-shaped capture molecules were developed, which ensure label-free and specific DNA detection on microarrays while simultaneously allowing a novel normalisation strategy of present signal intensities. This approach was implemented for and tested on the sequence detection of various pathogenic microorganisms. The specificity of the system could be demonstrated by differentiating single nucleotide variations of the target sequence. Moreover different variations of the normalisation strategy were tested to further improve the microarray's reproducibility. The reaction conditions of the enzymatic steps involved got optimised. Furthermore the realisability of a new concept for signal amplification was tested for this approach, which is based on in situ concatenation of special padlock probes

Long non-coding RNA Databases in Cardiovascular Research

With the rising interest in the regulatory functions of long non-coding RNAs (lncRNAs) in complex human diseases such as cardiovascular diseases, there is an increasing need in public databases offering comprehensive and integrative data for all aspects of these versatile molecules. Recently, a variety of public data repositories that specialized in lncRNAs have been developed, which make use of huge high-throughput data particularly from next-generation sequencing (NGS) approaches. Here, we provide an overview of current lncRNA databases covering basic and functional annotation, lncRNA expression and regulation, interactions with other biomolecules, and genomic variants influencing the structure and function of lncRNAs. The prominent lncRNA antisense noncoding RNA in the INK4 locus (ANRIL), which has been unequivocally associated with coronary artery disease through genome-wide association studies (GWAS), serves as an example to demonstrate the features of each individual database