Biological, clinical and population relevance of 95 loci for blood lipids.

Plasma concentrations of total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol and triglycerides are among the most important risk factors for coronary artery disease (CAD) and are targets for therapeutic intervention. We screened the genome for common variants associated with plasma lipids in >100,000 individuals of European ancestry. Here we report 95 significantly associated loci (P < 5 x 10(-8)), with 59 showing genome-wide significant association with lipid traits for the first time. The newly reported associations include single nucleotide polymorphisms (SNPs) near known lipid regulators (for example, CYP7A1, NPC1L1 and SCARB1) as well as in scores of loci not previously implicated in lipoprotein metabolism. The 95 loci contribute not only to normal variation in lipid traits but also to extreme lipid phenotypes and have an impact on lipid traits in three non-European populations (East Asians, South Asians and African Americans). Our results identify several novel loci associated with plasma lipids that are also associated with CAD. Finally, we validated three of the novel genes-GALNT2, PPP1R3B and TTC39B-with experiments in mouse models. Taken together, our findings provide the foundation to develop a broader biological understanding of lipoprotein metabolism and to identify new therapeutic opportunities for the prevention of CAD

Genetic variants in novel pathways influence blood pressure and cardiovascular disease risk.

Blood pressure is a heritable trait influenced by several biological pathways and responsive to environmental stimuli. Over one billion people worldwide have hypertension (≥140 mm Hg systolic blood pressure or  ≥90 mm Hg diastolic blood pressure). Even small increments in blood pressure are associated with an increased risk of cardiovascular events. This genome-wide association study of systolic and diastolic blood pressure, which used a multi-stage design in 200,000 individuals of European descent, identified sixteen novel loci: six of these loci contain genes previously known or suspected to regulate blood pressure (GUCY1A3-GUCY1B3, NPR3-C5orf23, ADM, FURIN-FES, GOSR2, GNAS-EDN3); the other ten provide new clues to blood pressure physiology. A genetic risk score based on 29 genome-wide significant variants was associated with hypertension, left ventricular wall thickness, stroke and coronary artery disease, but not kidney disease or kidney function. We also observed associations with blood pressure in East Asian, South Asian and African ancestry individuals. Our findings provide new insights into the genetics and biology of blood pressure, and suggest potential novel therapeutic pathways for cardiovascular disease prevention

New genetic loci link adipose and insulin biology to body fat distribution.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms

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

Genetic associations at 53 loci highlight cell types and biological pathways relevant for kidney function.

Reduced glomerular filtration rate defines chronic kidney disease and is associated with cardiovascular and all-cause mortality. We conducted a meta-analysis of genome-wide association studies for estimated glomerular filtration rate (eGFR), combining data across 133,413 individuals with replication in up to 42,166 individuals. We identify 24 new and confirm 29 previously identified loci. Of these 53 loci, 19 associate with eGFR among individuals with diabetes. Using bioinformatics, we show that identified genes at eGFR loci are enriched for expression in kidney tissues and in pathways relevant for kidney development and transmembrane transporter activity, kidney structure, and regulation of glucose metabolism. Chromatin state mapping and DNase I hypersensitivity analyses across adult tissues demonstrate preferential mapping of associated variants to regulatory regions in kidney but not extra-renal tissues. These findings suggest that genetic determinants of eGFR are mediated largely through direct effects within the kidney and highlight important cell types and biological pathways

Functional Characterization of differential expressed Genes in a mouse model of cerebral ischemia

Der Schlaganfall ist eine der Haupttodesursachen in den Industrieländern und führt aufgrund resultierender körperlicher Behinderungen jährlich zu Kosten in Millionenhöhe im Gesundheitswesen. Es ist bereits bekannt, dass entzündliche Prozesse zu einer Verschlechterung des Gesundheitszustandes des Schlaganfallpatienten führen. Mit Hilfe globaler Genexpressionsanalysen wurde nicht nur die Hochregulierung der inflammatorischen Proteine TLR2 („Toll-Like Receptor 2“), TLR4 („Toll-Like Receptor 4“) und MRP14 („Myeloid Related Protein 14“), sondern auch die Hochregulierung TLR2- bzw. TLR4-Pathway zugehöriger Gene detektiert. Daher wurden im Rahmen dieser Dissertationsarbeit der Einfluss von TLR2 und das mit TLR4 interagierende Protein MRP14 in einem Mausmodell für Schlaganfall untersucht. Nach einem einstündigen Verschluss der mittleren Hirnarterie („middle cerebral artery occlusion“ bzw. MCAO) in C57Bl/6-Wildtyptieren wurde eine Hochregulierung der TLR2-mRNA zwischen 3 bis 48 Stunden Reperfusion sowie der MRP14-mRNA zwischen 12 und 48 Stunden Reperfusion gezeigt. TLR2-Protein ist fast ausschließlich in der ipsilateralen Hemisphäre, insbesondere im Infarktkern, zu finden. Die meisten TLR2-positiven Zellen ließen sich als Mikroglia identifizieren. Aber auch einige ausgewählte Endothelien, Neuronen und Astrozyten bilden TLR2-Protein aus. Es wurde ein ischämischer Infarkt (1 h MCAO/48 h Reperfusion) in C57Bl/6-Wildtypmäusen sowie in TLR2-defizienten Mäusen ausgelöst. Das Infarktvolumen, sowie die Anzahl an aktivierten Mikroglia bzw. eingewanderten Makrophagen waren in den TLR2-defizienten Tieren geringer als in den Wildtyptieren. Dementgegen war die Neuronenzahl größer. Dieses Ergebnis ließ auf eine wichtige Rolle des TLR2 hinsichtlich der Ausweitung des Gewebeschadens nach einem Schlaganfall schließen. MRP14 ist als endogener TLR4-Agonist bekannt und wurde hinsichtlich seiner Relevanz auf die Ausweitung des Gewebeschadens nach einem Schlaganfall untersucht. MRP14 wurde in der ipsilateralen Hemisphäre nachgewiesen und zwar ausschließlich in aktivierten Mikroglia und einwandernden Makrophagen. MRP14-positive Zellen lagern sich an Endothelien an. MRP14-defiziente Mäuse (die ebenfalls den Komplexpartner MRP8 nicht bilden können) zeigen nach 1 h MCAO und 48 h Reperfusion ein geringeres Infarktvolumen, ein kleineres Ödem sowie ein reduzierte Anzahl an aktivierten Mikroglia und einwandernden Makrophagen im Vergleich zu MRP14-Wurfgeschwister. MRP14 trägt somit zum schlaganfallinduzierten Gewebeschaden bei. Um eine mögliche Behandlung gegen eine Gewebeschadensausweitung zu entwickeln, wurden in C57Bl/6-Mäusen Schlaganfälle ausgelöst (45 min MCAO/48 h Reperfusion), die anschließend mit TLR2-blockierenden TLR2.5-Antikörper und nichtfunktionalen „isotype control”-Antikörper behandelt wurden. Es wurde ein verringertes Schlaganfallvolumen, eine hochsignifikante Reduzierung der Mikrogliaaktivierung bzw. Makrophageninfiltration, sowie eine signifikante Erhöhung des Neuronenerhalts festgestellt. Um eine mögliche Toxizität einer Antikörperapplikation ins Gehirn zu detektieren, wurden Wildtypmäuse einem Schlaganfall ausgesetzt und mit „isotype control”-Antikörper bzw. PBS-Solvent behandelt. Der so detektierte neurotoxische Effekt einer Antikörperapplikation wurde durch eine Blockade des TLR2 jedoch mehr als kompensiert. Zusammenfassend tragen beide Proteine (TLR2 und MRP14) zum sekundären Schaden nach einem ischämischen Schlaganfall bei. Ich vermute, diese Gewebe-schadensausweitung wird durch eine erhöhte Anzahl an Mikroglia und Makrophagen und damit durch eine Verschärfung der Inflammation verursacht. Des Weiteren hat eine Blockade des TLR2-Signalweges einen positiven Effekt auf den Neuronenerhalt und das klinische Ergebnis. Daraus ergibt sich die Möglichkeit zur Entwicklung medikamentöser Anwendungen.Stroke is a leading cause of death in industrialized countries and cost several million € each year because of disabilities. It is known that inflammation leads to exacerbation of ischemic infarction. Besides an up-regulation of TLR2 (toll-tike receptor 2), TLR4 and MRP14 (myeloid related protein 14) an up-regulation of TLR-pathway related genes was detected by global gene expression analysis. Therefore the influence of TLR2 and TLR4 interacting protein MRP14 were examined. A significant up-regulation of TLR2-mRNAs (3 h - 48 h of reperfusion) as well as MRP14-mRNA (12 h - 48 h of reperfusion) was shown after 1 h of transient focal cerebral ischemia in C57Bl/6 wildtype mice. TLR2-protein was up-regulated in ipsilateral brain hemisphere especially in infarction core. Most expression was shown in microglia, but also in selected endothelial cells, neurons and astrocytes. An ischemic infarction (1 h MCAO/48 h Reperfusion) was induced in C57Bl/6 wildtype mice as well as in TLR2-knockout mice. Infarct volume as well as number of activated microglia and infiltrating macrophages was significant decreased in TLR2 deficient mice compared to wildtype mice. However number of neurons was increased. It forces to conclude a major role of TLR2 in exacerbation of stroke. MRP14 is known for being an endogenous TLR4-activator. It was examined in respect of its relevance for exacerbation of ischemic infarction. MRP14 protein was detected in ipsilaterale brain hemisphere, namely exclusively in activated microglia and infiltrated macrophages. MRP14-positive cells attach to endothelia cells. Ischemic infarcts were induced in MRP14 knockout mice (which were also deficient its complex partner MRP8) as well as wild type littermates revealing a smaller infarct volume, less edema, and a reduced number of activated microglia and infiltrated macrophage in MRP14-/- mice compared to MRP+/+ mice. So MRP14 contributes to exacerbation of ischemic infarction. To find a possible treatment for injury increase, infracted C57Bl/6J mice (45 min MCAO/48 h Reperfusion) were treated with TLR2-blocking TLR2.5-antibody and nonfunctional “isotype control” antibody, respectively and revealed a decreased infarct volume, a highly significant decreased microglia activation and macrophage infiltration and a highly significant increased neuron preservation in the anti-TLR2.5 antibody treated mouse brain compared to “isotype control” antibody treated brain. For identification of a possible toxicity of antibody treatment, I tested “isotype control” antibody application vs. PBS control application and detected a detrimental effect of antibody in infarcted mouse brain what is compensated by blocking TLR2. In conclusion, both proteins (TLR2 and MRP14) contribute to a secondary brain damage after transient focal ischemia. I assume that exacerbation is due to increasing macrophage and microglia number and aggravation of inflammation. Furthermore I conclude, that turning off the TLR2 pathway by blocking its receptor limits microglia infiltration and preserves neurons in the infarct mouse brain and therefore leads to an improved outcome after transient focal cerebral ischemia. It therefore offers a possibility for development of future medication strategies

TLR2 has a detrimental role in mouse transient focal cerebral ischemia

A significant up-regulation of Toll-like-receptor (TLR) mRNAs between 3 and 48 h reperfusion time after induction of transient focal cerebral ischemia for 1 h was revealed by applying global gene expression profiling in postischemic mouse brains. Compared to TLR4 and TLR9, TLR2 proved to be the most significantly up-regulated TLR in the ipsilateral brain hemisphere. TLR2-protein was found to be expressed mainly in microglia in the postischemic brain tissue, but also in selected endothelial cells, neurons, and astrocytes. Additionally, TLR2-related genes with pro-inflammatory and pro-apoptotic capabilities were induced. Therefore we hypothesized that TLR2-signaling could exacerbate the primary brain damage after ischemia. Two days after induction of transient focal cerebral ischemia (1 h), we found a significant decrease of the infarct volume in TLR2 deficient mice compared to wild type mice (75 ± 5 vs. 42 ± 7 mm3). We conclude that TLR2 up-regulation and TLR2-signaling are important events in focal cerebral ischemia and contribute to the deterioration of ischemic damage

Blocking TLR2 in vivo protects against accumulation of inflammatory cells and neuronal injury in experimental stroke

Reduced infarct volume in TLR2-knockout mice compared with C57Bl/6 wild-type mice has recently been shown in experimental stroke and confirmed in this study. We now also show a significant decrease of CD11b-positive cell counts and decreased neuronal death in the ischemic hemispheres of TLR2-deficient mice compared with C57Bl/6wt mice 2 days after transient focal cerebral ischemia. To examine the potential benefit of intravascular TLR2 inhibition, C57Bl/6wt mice were treated intraarterially with TLR2-blocking anti-TLR2 antibody (clone T2.5) after 45 minutes of cerebral ischemia and compared with control antibody (isotype) treated wild-type mice. Whereas T2.5-treated mice had no reduction in infarct volumes at 48 hours after reperfusion, they did have decreased numbers of CD11b-positive inflammatory cells and decreased neuronal death compared with isotype-treated control mice. Comparison of the isotype antibody treatment to control (saline) treatment showed no effects on infarct volumes or neuronal survival. However, mice treated with the control isotype antibody had increased numbers of CD11b-positive inflammatory cells compared with saline-treated animals. Thus, antibody treatment itself (i.e., control isotype antibody, but potentially of any antibody) may have adverse effects and limit therapeutic benefit of anti-TLR2-antibody therapy. We conclude that TLR2 mediates leukocyte and microglial infiltration and neuronal death, which can be attenuated by TLR2 inhibition. The TLR2 inhibition in vivo improves neuronal survival and may represent a future stroke therapy