From genotype to phenotype in human atherosclerosis - recent findings

Purpose of reviewSince 2007, genome-wide association studies (GWAS) have led to the identification of numerous loci of atherosclerotic cardiovascular disease. The majority of these loci harbor genes previously not known to be involved in atherogenesis. In this review, we summarize the recent progress in understanding the pathophysiology of genetic variants in atherosclerosis.Recent findingsFifty-eight loci with P<10(-7) have been identified in GWAS for coronary heart disease and myocardial infarction. Of these, 23 loci (40%) overlap with GWAS loci of classical risk factors such as lipids, blood pressure, and diabetes mellitus, suggesting a potential causal relation. The vast majority of the remaining 35 loci (60%) are at genomic regions where the mechanism in atherogenesis is unclear. Loci most frequently found in independent GWAS were at Chr9p21.3 (ANRIL/CDKN2B-AS1), Chr6p24.1 (PHACTR1), and Chr1p13.3 (CELSR2, PSRC1, MYBPHL, SORT1). Recent work suggests that Chr9p21.3 exerts its effects through epigenetic regulation of target genes, whereas mechanisms at Chr6p24.1 remain obscure, and Chr1p13.3 affects plasma LDL cholesterol.SummaryNovel GWAS loci indicate that our understanding of atherosclerosis is limited and implicate a role of hitherto unknown mechanisms, such as epigenetic gene regulation in atherogenesis

Long Noncoding RNA ANRIL: Lnc-ing Genetic Variation at the Chromosome 9p21 Locus to Molecular Mechanisms of Atherosclerosis

Ever since the first genome-wide association studies (GWAS) on coronary artery disease (CAD), the Chr9p21 risk locus has emerged as a top signal in GWAS of atherosclerotic cardiovascular disease, including stroke and peripheral artery disease. The CAD risk SNPs on Chr9p21 lie within a stretch of 58 kilobases of non-protein-coding DNA, containing the gene body of the long noncoding RNA (lncRNA) antisense non coding RNA in the INK4 locus (ANRIL). How risk is affected by the Chr9p21 locus in molecular detail is a matter of ongoing research. Here we will review recent advances in the understanding that ANRIL serves as a key risk effector molecule of atherogenesis at the locus. One focus of this review is the shift in understanding that genetic variation at Chr9p21 not only affects the abundance of ANRIL, and in some cases expression of the adjacent CDKN2A/B tumor suppressors, but also impacts ANRIL splicing, such that 3′-5′-linked circular noncoding ANRIL RNA species are produced. We describe how the balance of linear and circular ANRIL RNA, determined by the Chr9p21 genotype, regulates molecular pathways and cellular functions involved in atherogenesis. We end with an outlook on how manipulating circular ANRIL abundance may be exploited for therapeutic purposes

Circular RNAs as Therapeutic Agents and Targets

It has recently been reported that thousands of covalently linked circular RNAs (circRNAs) are expressed from human genomes. circRNAs emerge during RNA splicing. circRNAs are circularized in a reaction termed “backsplicing,” whereby the spliceosome fuses a splice donor site in a downstream exon to a splice acceptor site in an upstream exon. Although a young field of research, first studies indicate that backsplicing is not an erroneous reaction of the spliceosome. Instead, circRNAs are produced in cells with high cell-type specificity and can exert biologically meaningful and specific functions. These observations and the finding that circRNAs are stable against exonucleolytic decay are raising the question whether circRNAs may be relevant as therapeutic agents and targets. In this review, we start out with a short introduction into classification, biogenesis and general molecular mechanisms of circRNAs. We then describe reports, where manipulating circRNA abundance has been shown to have therapeutic value in animal disease models in vivo, with a focus on cardiovascular disease (CVD). Starting from existing approaches, we outline particular challenges and opportunities for future circRNA-based therapeutic approaches that exploit stability and molecular effector functions of native circRNAs. We end with considerations which designer functions could be engineered into artificial therapeutic circular RNAs

Comparison of Whole Blood RNA Preservation Tubes and Novel Generation RNA Extraction Kits for Analysis of mRNA and MiRNA Profiles

Background: Whole blood expression profiling is frequently performed using PAXgene (Qiagen) or Tempus (Life Technologies) tubes. Here, we compare 6 novel generation RNA isolation protocols with respect to RNA quantity, quality and recovery of mRNA and miRNA. Methods: 3 PAXgene and 3 Tempus Tubes were collected from participants of the LIFE study with (n=12) and without (n=35) acute myocardial infarction (AMI). RNA was extracted with 4 manual protocols from Qiagen (PAXgene Blood miRNA Kit), Life Technologies (MagMAX for Stabilized Blood Tubes RNA Isolation Kit), and Norgen Biotek (Norgen Preserved Blood RNA Purification Kit I and Kit II), and 2 (semi-) automated protocols on the QIAsymphony (Qiagen) and MagMAX Express-96 Magnetic Particle Processor (Life Technologies). RNA quantity and quality was determined. For biological validation, RNA from 12 representative probands, extracted with all 6 kits (n=72), was reverse transcribed and mRNAs (matrix metalloproteinase 9, arginase 1) and miRNAs (miR133a, miR1), shown to be altered by AMI, were analyzed. Results: RNA yields were highest using the Norgen Kit I with Tempus Tubes and lowest using the Norgen Kit II with PAXgene. The disease status was the second major determinant of RNA yields (LIFE-AMI 11.2 vs. LIFE 6.7 mu g, p < 0.001) followed by the choice of blood collection tube. (Semi-) automation reduced overall RNA extraction time but did not generally reduce hands-on-time. RNA yields and quality were comparable between manual and automated extraction protocols. mRNA expression was not affected by collection tubes and RNA extraction kits but by RT/qPCR reagents with exception of the Norgen Kit II, which led to mRNA depletion. For miRNAs, expression differences related to collection tubes (miR30b), RNA isolation (Norgen Kit II), and RT/qRT reagents (miR133a) were observed. Conclusion: We demonstrate that novel generation RNA isolation kits significantly differed with respect to RNA recovery and affected miRNA but not mRNA expression profiles

Identification of a novel SERPINA-1 mutation causing alpha-1 antitrypsin deficiency in a patient with severe bronchiectasis and pulmonary embolism

Deficiency in the serine protease inhibitor, alpha-1 antitrypsin (AAT), is known to cause emphysema and liver disease. Other manifestations, including airway disease or skin disorders, have also been described. A 44-year-old woman presented to our emergency department with dyspnea and respiratory insufficiency. She had never smoked, and had been diagnosed with COPD 9 years earlier. Three months previously, she had suffered a pulmonary embolism. Chest computed tomography scan revealed severe cystic bronchiectasis with destruction of the lung parenchyma. The sweat test was normal and there was no evidence of the cystic fibrosis transmembrane conductance regulator (CFTR) mutation. Capillary zone electrophoresis showed a decrease of alpha-1 globin band and AAT levels were below the quantification limit (<25 mg/dL). No S or Z mutation was identified, but sequencing analysis found a homozygous cytosine and adenine (CA) insertion in exon 2 of the SERPINA-1 gene, probably leading to a dysfunctional protein (PI Null/Null). This mutation has not been previously identified. The atypical presentation of the patient, with severe cystic bronchiectasis, highlights AAT deficiency as a differential diagnosis in bronchiectasis. Further, awareness should be raised regarding a possible increased risk of thromboembolism associated with AAT deficiency

Severe Atherosclerosis and Hypercholesterolemia in Mice Lacking Both the Melanocortin Type 4 Receptor and Low Density Lipoprotein Receptor

Dysfunction of the melanocortin system can result in severe obesity accompanied with dyslipidemia and symptoms of the metabolic syndrome but the effect on vascular atherogenesis is not known. To study the impact of obesity and dyslipidemia on the cardiovascular system, we generated mice double-deficient for the melanocortin type 4 receptor (Mc4r(mut) mice) and the LDL receptor (Ldlr(-/-) mice). Mc4r(mut) mice develop obesity due to hyperphagia. Double-mutant mice (Mc4r(mut);Ldlr(-/-)) exhibited massive increases in body weight, plasma cholesterol and triacylglycerol levels and developed atherosclerosis. Atherosclerotic lesion size was affected throughout the aortic root and brachiocephalic artery not only under semisynthetic, cholesterol-containing diet but also under cholesterol-free standard chow. The Mc4r(mut) mice developed a hepatic steatosis which contributes to increased plasma cholesterol levels even under cholesterol-free standard chow. Transcripts of cholesterol biosynthesis components and liver cholesterol levels did not significantly differ between wild-type and all mutant mouse strains but RNA sequencing data and biochemical measurements point to an altered bile acid elimination in Mc4r(mut);Ldlr(-/-). Therefore, the unchanged endogenous cholesterol biosynthesis together with a reduced hepatic VLDL and LDL-cholesterol clearance most likely led to increased plasma lipid levels and consequently to atherosclerosis in this animal model. Our data indicate that dysfunction of the melanocortin-regulated food intake and the resulting obesity significantly add to the proatherogenic lipoprotein profile caused by LDL receptor deficiency and, therefore, can be regarded as relevant risk factor for atherosclerosis

Plasma Proteome Profiling to Assess Human Health and Disease

SummaryProteins in the circulatory system mirror an individual’s physiology. In daily clinical practice, protein levels are generally determined using single-protein immunoassays. High-throughput, quantitative analysis using mass-spectrometry-based proteomics of blood, plasma, and serum would be advantageous but is challenging because of the high dynamic range of protein abundances. Here, we introduce a rapid and robust “plasma proteome profiling” pipeline. This single-run shotgun proteomic workflow does not require protein depletion and enables quantitative analysis of hundreds of plasma proteomes from 1 μl single finger pricks with 20 min gradients. The apolipoprotein family, inflammatory markers such as C-reactive protein, gender-related proteins, and >40 FDA-approved biomarkers are reproducibly quantified (CV <20% with label-free quantification). Furthermore, we functionally interpret a 1,000-protein, quantitative plasma proteome obtained by simple peptide pre-fractionation. Plasma proteome profiling delivers an informative portrait of a person’s health state, and we envision its large-scale use in biomedicine

Sex-specific associations between serum lipids and hemostatic factors: the cross-sectional population-based KORA-fit study

Background Studies on the associations between lipid parameters and different hemostatic factors in men and women from the general population are scarce. It was therefore examined whether there are possible relationships between routinely measured serum lipids (total cholesterol, HDL-cholesterol, non-HDL-cholesterol, LDL-cholesterol, and triglycerides) and different hemostatic factors (activated partial thromboplastin time (aPTT), fibrinogen, factor VIII, antithrombin III (AT III), protein C, protein S, and D-dimer). Methods The analysis was based on data from the Cooperative Health Research in the Region of Augsburg (KORA)-Fit study, which included 805 participants (378 men, 427 women) with a mean age of 63.1 years. Sex-specific associations between serum lipids and coagulation factors were investigated using multivariable linear regression models. Results In men, total cholesterol was inversely related to aPTT but positively associated with protein C activity. HDL cholesterol was inversely related to aPTT and fibrinogen. LDL cholesterol, non-HDL cholesterol, and triglycerides showed a positive association with protein C and protein S activity. In women, LDL-cholesterol, total cholesterol, and non-HDL-cholesterol were positively related to AT III concentrations and protein C and S activity. Additionally, non-HDL-cholesterol was positively associated with factor VIII activity. HDL cholesterol was inversely related to fibrinogen. Triglycerides showed a positive relationship with protein C activity. Conclusions There seem to be sex differences regarding various associations between blood lipid levels and hemostatic factors. Further studies are needed to address the possible impact of these associations on cardiovascular risk and the underlying mechanisms

Rationale and Design of the Leipzig (LIFE) Heart Study: Phenotyping and Cardiovascular Characteristics of Patients with Coronary Artery Disease

We established the Leipzig (LIFE) Heart Study, a biobank and database of patients with different stages of coronary artery disease (CAD) for studies of clinical, metabolic, cellular and genetic factors of cardiovascular diseases.The Leipzig (LIFE) Heart Study (NCT00497887) is an ongoing observational angiographic study including subjects with different entities of CAD. Cohort 1, patients undergoing first-time diagnostic coronary angiography due to suspected stable CAD with previously untreated coronary arteries. Cohort 2, patients with acute myocardial infarction (MI) requiring percutaneous revascularization. Cohort 3, patients with known left main coronary artery disease (LMCAD).We present preliminary results of demographics and phenotyping based on a 4-years analysis of a total of 3,165 subjects. Cohort 1 (n=2,274) shows the typical distribution of elective coronary angiography cohorts with 43% cases with obstructive CAD and 37% normal angiograms. Cohorts 2 and 3 consist of 590 and 301 subjects, respectively, adding patients with severe forms of CAD. The suitability of the database and biobank to perform association studies was confirmed by replication of the CAD susceptibility locus on chromosome 9p21 (OR per allele: 1.55 (any CAD), 1.54 (MI), 1.74 (LMCAD), p<10(-6), respectively). A novel finding was that patients with LMCAD had a stronger association with 9p21 than patients with obstructive CAD without LMCAD (OR 1.22, p=0.042). In contrast, 9p21 did not associate with myocardial infarction in excess of stable CAD.The Leipzig (LIFE) Heart Study provides a basis to identify molecular targets related to atherogenesis and associated metabolic disorders. The study may contribute to an improvement of individual prediction, prevention, and treatment of CAD