Activated factor XI (FXIa) inhibitors are anticipated to combine anticoagulant and profibrinolytic effects with a low bleeding risk. This motivated a structure aided fragment based lead generation campaign to create novel FXIa inhibitor leads. A virtual screen, based on docking experiments, was performed to generate a FXIa targeted fragment library for an NMR screen that resulted in the identification of fragments binding in the FXIa S1 binding pocket. The neutral 6-chloro-3,4-dihydro-1H-quinolin-2-one and the weakly basic quinolin-2-amine structures are novel FXIa P1 fragments. The expansion of these fragments towards the FXIa prime side binding sites was aided by solving the X-ray structures of reported FXIa inhibitors that we found to bind in the S1-S1’-S2’ FXIa binding pockets. Combining the X-ray structure information from the identified S1 binding 6-chloro-3,4-dihydro-1H-quinolin-2-one fragment and the S1-S1’-S2’ binding reference compounds enabled structure guided linking and expansion work to achieve one of the most potent and selective FXIa inhibitors reported to date, compound 13, with a FXIa IC50 of 1.0 nM. The hydrophilicity and large polar surface area of the potent S1-S1’-S2’ binding FXIa inhibitors compromised permeability. Initial work to expand the 6-chloro-3,4-dihydro-1H-quinolin-2-one fragment towards the prime side to yield molecules with less hydrophilicity shows promise to afford potent, selective and orally bioavailable compounds.
Background/purpose of the study
Epidemiological evidence suggests that low doses of ionising radiation (≤1.0 Gy) produce persistent alterations in cognition if the exposure occurs at a young age. The mechanisms underlying such alterations are unknown. We investigated the long-term effects of low doses of total body gamma radiation on neonatally exposed NMRI mice on the molecular and cellular level to elucidate neurodegeneration.
Significant alterations in spontaneous behaviour were observed at 2 and 4 months following a single 0.5 or 1.0 Gy exposure. Alterations in the brain proteome, transcriptome, and several miRNAs were analysed 6–7 months post-irradiation in the hippocampus, dentate gyrus (DG) and cortex. Signalling pathways related to synaptic actin remodelling such as the Rac1-Cofilin pathway were altered in the cortex and hippocampus. Further, synaptic proteins MAP-2 and PSD-95 were increased in the DG and hippocampus (1.0 Gy). The expression of synaptic plasticity genes Arc, c-Fos and CREB was persistently reduced at 1.0 Gy in the hippocampus and cortex. These changes were coupled to epigenetic modulation via increased levels of microRNAs (miR-132/miR-212, miR-134). Astrogliosis, activation of insulin-growth factor/insulin signalling and increased level of microglial cytokine TNFα indicated radiation-induced neuroinflammation. In addition, adult neurogenesis within the DG was persistently negatively affected after irradiation, particularly at 1.0 Gy.
These data suggest that neurocognitive disorders may be induced in adults when exposed at a young age to low and moderate cranial doses of radiation. This raises concerns about radiation safety standards and regulatory practices.
Electronic supplementary material
The online version of this article (doi:10.1186/1750-1326-9-57) contains supplementary material, which is available to authorized users.
Dendritic spines; Hippocampus; Cortex; CREB; miR-132; Ionising radiation; Proteomics; Rac1; Cofilin; Alzheimer
Primary Sjögren’s syndrome (pSS) is a chronic autoimmune disease characterized by a lymphocytic exocrinopathy. However, patients often have evidence of systemic autoimmunity and they are at markedly increased risk for the development of non- Hodgkin’s lymphoma. Similar to other autoimmune disorders, a strong interferon (IFN) signature is present among subsets of pSS patients, though the precise etiology remains uncertain. NCR3/NKp30 is a NK-specific activating receptor regulating the cross-talk between NK and dendritic cells and type II IFN secretion. We performed a case-control study of genetic polymorphisms of the NCR3/NKp30 gene and found that rs11575837 (G>A) residing in the promoter was associated with reduced gene transcription and function as well as protection to pSS. We also demonstrated that circulating levels of NCR3/NKp30 were markedly increased among pSS patients compared with controls and correlated with higher NCR3/NKp30 but not CD16-dependent IFN-γ secretion by NK cells. Excess accumulation of NK cells in minor salivary glands correlated with the severity of the exocrinopathy. B7H6, the ligand of NKp30, was expressed by salivary epithelial cells. These findings suggest that NK cells may promote an NKp30-dependent inflammatory state in salivary glands, and that blockade of the B7H6/NKp30 axis could be clinically relevant in pSS.
Sjögren’s syndrome; autoimmunity; NK cells; innate immunity; NKp30/NCR3
Identification and treatment of abdominal aortic aneurysm (AAA) remain among the most prominent challenges in vascular medicine. MicroRNAs (miRNAs) are crucial regulators of cardiovascular pathology and represent intriguing targets to limit AAA expansion. Here we show, by using two established murine models of AAA disease along with human aortic tissue and plasma analysis, that miR-24 is a key regulator of vascular inflammation and AAA pathology. In vivo and in vitro studies reveal chitinase 3-like 1 (Chi3l1) to be a major target and effector under the control of miR-24, regulating cytokine synthesis in macrophages as well as their survival, promoting aortic smooth muscle cell migration and cytokine production, and stimulating adhesion molecule expression in vascular endothelial cells. We further show that modulation of miR-24 alters AAA progression in animal models, and that miR-24 and CHI3L1 represent novel plasma biomarkers of AAA disease progression in humans.
Abdominal aortic aneurysm (AAA) is a potentially fatal and often asymptomatic disease whose causes remain unclear. Here the authors show that a microRNA, miR-24, and its target, the glycoprotein chitinase 3-like 1, represent key regulators of AAA development.
Patients suffering from brain malignancies are treated with high-dose ionising radiation. However, this may lead to severe learning and memory impairment. Preventive treatments to minimise these side effects have not been possible due to the lack of knowledge of the involved signalling pathways and molecular targets. Mouse hippocampal neuronal HT22 cells were irradiated with acute gamma doses of 0.5 Gy, 1.0 Gy and 4.0 Gy. Changes in the cellular proteome were investigated by isotope-coded protein label technology and tandem mass spectrometry after 4 and 24 hours. To compare the findings with the in vivo response, male NMRI mice were irradiated on postnatal day 10 with a gamma dose of 1.0 Gy, followed by evaluation of the cellular proteome of hippocampus and cortex 24 hours post-irradiation. Analysis of the in vitro proteome showed that signalling pathways related to synaptic actin-remodelling were significantly affected at 1.0 Gy and 4.0 Gy but not at 0.5 Gy after 4 and 24 hours. We observed radiation-induced reduction of the miR-132 and Rac1 levels; miR-132 is known to regulate Rac1 activity by blocking the GTPase-activating protein p250GAP. In the irradiated hippocampus and cortex we observed alterations in the signalling pathways similar to those in vitro. The decreased expression of miR-132 and Rac1 was associated with an increase in hippocampal cofilin and phospho-cofilin. The Rac1-Cofilin pathway is involved in the modulation of synaptic actin filament formation that is necessary for correct spine and synapse morphology to enable processes of learning and memory. We suggest that acute radiation exposure leads to rapid dendritic spine and synapse morphology alterations via aberrant cytoskeletal signalling and processing and that this is associated with the immediate neurocognitive side effects observed in patients treated with ionising radiation.
The current study was carried out to test the potential of a new nanomaterial (Spago Pix) as a macromolecular magnetic MR contrast agent for tumor detection and to verify the presence of nanomaterial in tumor tissue. Spago Pix, synthesized by Spago Nanomedical AB, is a nanomaterial with a globular shape, an average hydrodynamic diameter of 5 nm, and a relaxivity (r1) of approximately 30 (mM Mn)−1 s−1 (60 MHz). The material consists of an organophosphosilane hydrogel with strongly chelated manganese (II) ions and a covalently attached PEG surface layer. In vivo MRI of the MMTV-PyMT breast cancer model was performed on a 3 T clinical scanner. Tissues were thereafter analyzed for manganese and silicon content using inductively coupled plasma-atomic emission spectroscopy (ICP-AES). The presence of nanomaterial in tumor and muscle tissue was assessed using an anti-PEG monoclonal antibody. MR imaging of tumor-bearing mice (n = 7) showed a contrast enhancement factor of 1.8 (tumor versus muscle) at 30 minutes post-administration. Contrast was retained and further increased 2–4 hours after administration. ICP-AES and immunohistochemistry confirmed selective accumulation of nanomaterial in tumor tissue. A blood pharmacokinetics analysis showed that the concentration of Spago Pix gradually decreased over the first hour, which was in good agreement with the time frame in which the accumulation in tumor occurred. In summary, we demonstrate that Spago Pix selectively enhances MR tumor contrast in a clinically relevant animal model. Based on the generally higher vascular leakiness in malignant compared to benign tissue lesions, Spago Pix has the potential to significantly improve cancer diagnosis and characterization by MRI.
Estimates of the heritability of plasma fibrinogen concentration, an established predictor of cardiovascular disease (CVD), range from 34 to 50%. Genetic variants so far identified by genome-wide association (GWA) studies only explain a small proportion (< 2%) of its variation.
Methods and Results
We conducted a meta-analysis of 28 GWA studies, including more than 90,000 subjects of European ancestry, the first GWA meta-analysis of fibrinogen levels in 7 African Americans studies totaling 8,289 samples, and a GWA study in Hispanic-Americans totaling 1,366 samples. Evaluation for association of SNPs with clinical outcomes included a total of 40,695 cases and 85,582 controls for coronary artery disease (CAD), 4,752 cases and 24,030 controls for stroke, and 3,208 cases and 46,167 controls for venous thromboembolism (VTE). Overall, we identified 24 genome-wide significant (P<5×10−8) independent signals in 23 loci, including 15 novel associations, together accounting for 3.7% of plasma fibrinogen variation. Gene-set enrichment analysis highlighted key roles in fibrinogen regulation for the three structural fibrinogen genes and pathways related to inflammation, adipocytokines and thyrotrophin-releasing hormone signaling. Whereas lead SNPs in a few loci were significantly associated with CAD, the combined effect of all 24 fibrinogen-associated lead SNPs was not significant for CAD, stroke or VTE.
We identify 23 robustly associated fibrinogen loci, 15 of which are new. Clinical outcome analysis of these loci does not support a causal relationship between circulating levels of fibrinogen and CAD, stroke or VTE.
Fibrinogen; cardiovascular disease; genome-wide association study
We performed a genome-wide association study on 1,292 individuals with abdominal aortic aneurysms (AAAs) and 30,503 controls from Iceland and The Netherlands, with a follow-up of top markers in up to 3,267 individuals with AAAs and 7,451 controls. The A allele of rs7025486 on 9q33 was found to associate with AAA, with an odds ratio (OR) of 1.21 and P = 4.6 × 10−10. In tests for association with other vascular diseases, we found that rs7025486[A] is associated with early onset myocardial infarction (OR = 1.18, P = 3.1 × 10−5), peripheral arterial disease (OR = 1.14, P = 3.9 × 10−5) and pulmonary embolism (OR = 1.20, P = 0.00030), but not with intracranial aneurysm or ischemic stroke. No association was observed between rs7025486[A] and common risk factors for arterial and venous diseases—that is, smoking, lipid levels, obesity, type 2 diabetes and hypertension. Rs7025486 is located within DAB2IP, which encodes an inhibitor of cell growth and survival.
Chronic obstructive pulmonary disease (COPD) independently associates with an increased risk of coronary artery disease (CAD), but it has not been fully investigated whether this co-morbidity involves shared pathophysiological mechanisms. To identify potential common pathways across the two diseases, we tested all recently published single nucleotide polymorphisms (SNPs) associated with human lung function (spirometry) for association with carotid intima-media thickness (cIMT) in 3,378 subjects with multiple CAD risk factors, and for association with CAD in a case-control study of 5,775 CAD cases and 7,265 controls. SNPs rs2865531, located in the CFDP1 gene, and rs9978142, located in the KCNE2 gene, were significantly associated with CAD. In addition, SNP rs9978142 and SNP rs3995090 located in the HTR4 gene, were associated with average and maximal cIMT measures. Genetic risk scores combining the most robustly spirometry–associated SNPs from the literature were modestly associated with CAD, (odds ratio (OR) (95% confidence interval (CI95) = 1.06 (1.03, 1.09); P-value = 1.5×10−4, per allele). In conclusion, our study suggests that some genetic loci implicated in determining human lung function also influence cIMT and susceptibility to CAD. The present results should help elucidate the molecular underpinnings of the co-morbidity observed across COPD and CAD.
The study hypothesis was that thoracic aortic disease (TAD) is associated with a higher‐than‐expected prevalence of inguinal hernia. Such an association has been reported for abdominal aortic aneurysm (AAA) and hernia. Unlike AAA, TAD is not necessarily detectable with clinical examination or ultrasound, and there are no population‐based screening programs for TAD. Therefore, conditions associated with TAD, such as inguinal hernia, are of particular clinical relevance.
Methods and Results
The prevalence of inguinal hernia in subjects with TAD was determined from nation‐wide register data and compared to a non‐TAD group (patients with isolated aortic stenosis). Groups were balanced using propensity score matching. Multivariable statistical analysis (logistic regression) was performed to identify variables independently associated with hernia. Hernia prevalence was 110 of 750 (15%) in subjects with TAD versus 29 of 301 (9.6%) in non‐TAD, P=0.03. This statistically significant difference remained after propensity score matching: 21 of 159 (13%) in TAD versus 14 of 159 (8.9%) in non‐TAD, P<0.001. Variables independently associated with hernia in multivariable analysis were male sex (odds ratio [OR] with 95% confidence interval [95% CI]) 3.4 (2.1 to 5.4), P<0.001; increased age, OR 1.02/year (1.004 to 1.04), P=0.014; and TAD, OR 1.8 (1.1 to 2.8), P=0.015.
The prevalence of inguinal hernia (15%) in TAD is higher than expected in a general population and higher in TAD, compared to non‐TAD. TAD is independently associated with hernia in multivariable analysis. Presence or history of hernia may be of importance in detecting TAD, and the association warrants further study.
aneurysm; aorta; risk factors; surgery
Sjögren’s syndrome is a common autoimmune disease (~0.7% of European Americans) typically presenting as keratoconjunctivitis sicca and xerostomia. In addition to strong association within the HLA region at 6p21 (Pmeta=7.65×10−114), we establish associations with IRF5-TNPO3 (Pmeta=2.73×10−19), STAT4 (Pmeta=6.80×10−15), IL12A (Pmeta =1.17×10−10), FAM167A-BLK (Pmeta=4.97×10−10), DDX6-CXCR5 (Pmeta=1.10×10−8), and TNIP1 (Pmeta=3.30×10−8). Suggestive associations with Pmeta<5×10−5 were observed with 29 regions including TNFAIP3, PTTG1, PRDM1, DGKQ, FCGR2A, IRAK1BP1, ITSN2, and PHIP amongst others. These results highlight the importance of genes involved in both innate and adaptive immunity in Sjögren’s syndrome.
Cardiac left ventricle hypertrophy (LVH) constitutes a major risk factor for heart failure. Although LVH is most commonly caused by chronic elevation in arterial blood pressure, reduction of blood pressure to normal levels does not always result in regression of LVH, suggesting that additional factors contribute to the development of this pathology. We tested whether genetic preconditions associated with the imbalance in sodium homeostasis could trigger the development of LVH without concomitant increases in blood pressure. The results showed that the presence of a hypertensive variant of α-adducin gene in Milan rats (before they become hypertensive) resulted in elevated expression of genes associated with LVH, and of salt-inducible kinase 2 (SIK2) in the left ventricle (LV). Moreover, the mRNA expression levels of SIK2, α-adducin, and several markers of cardiac hypertrophy were positively correlated in tissue biopsies obtained from human hearts. In addition, we found in cardiac myocytes that α-adducin regulates the expression of SIK2, which in turn mediates the effects of adducin on hypertrophy markers gene activation. Furthermore, evidence that SIK2 is critical for the development of LVH in response to chronic high salt diet (HS) was obtained in mice with ablation of the sik2 gene. Increases in the expression of genes associated with LVH, as well as increases in LV wall thickness upon HS, occurred only in sik2+/+ but not in sik2−/− mice. Thus LVH triggered by HS or the presence of a genetic variant of α-adducin requires SIK2 and is independent of elevated blood pressure. Inhibitors of SIK2 may constitute part of a novel therapeutic regimen aimed at prevention/regression of LVH.
To replicate the associations of leukocyte telomere length (LTL) with variants at four loci and to investigate their associations with coronary heart disease (CHD) and type II diabetes (T2D), in order to examine possible causal effects of telomere maintenance machinery on disease aetiology.
Four SNPs at three loci BICD1 (rs2630578 GγC), 18q12.2 (rs2162440 GγT), and OBFC1 (rs10786775 CγG, rs11591710 AγC) were genotyped in four studies comprised of 2353 subjects out of which 1148 had CHD and 566 T2D. Three SNPs (rs12696304 CγG, rs10936601G>T and rs16847897 GγC) at the TERC locus were genotyped in these four studies, in addition to an offspring study of 765 healthy students. For all samples, LTL had been measured using a real-time PCR-based method.
Only one SNP was associated with a significant effect on LTL, with the minor allele G of OBFC1 rs10786775 SNP being associated with longer LTL (β=0.029, P=0.04). No SNPs were significantly associated with CHD or T2D. For OBFC1 the haplotype carrying both rare alleles (rs10786775G and rs11591710C, haplotype frequency 0.089) was associated with lower CHD prevalence (OR: 0.77; 95% CI: 0.61–0.97; P= 0.03). The TERC haplotype GTC (rs12696304G, rs10936601T and rs16847897C, haplotype frequency 0.210) was associated with lower risk for both CHD (OR: 0.86; 95% CI: 0.75-0.99; P=0.04) and T2D (OR: 0.74; 95% CI: 0.61–0.91; P= 0.004), with no effect on LTL. Only the last association remained after adjusting for multiple testing.
Of reported associations, only that between the OBFC1 rs10786775 SNP and LTL was confirmed, although our study has a limited power to detect modest effects. A 2-SNP OBFC1 haplotype was associated with higher risk of CHD, and a 3-SNP TERC haplotype was associated with both higher risk of CHD and T2D. Further work is required to confirm these results and explore the mechanisms of these effects.
Nonfasting (postprandial) triglyceride concentrations have emerged as a clinically significant cardiovascular disease risk factor that results from accumulation of remnant triglyceride-rich lipoproteins (TRLs) in the circulation. The remnant TRLs are cleared from the circulation by hepatic uptake, but the specific mechanisms involved are unclear. The syndecan-1 heparan sulfate proteoglycan (HSPG) pathway is important for the hepatic clearance of remnant TRLs in mice, but its relevance in humans is unclear.
We sought to determine whether polymorphisms of the genes responsible for HSPG assembly and disassembly contribute to atherogenic dyslipoproteinemias in humans.
Patients And Design
We performed an oral fat load in 68 healthy subjects. Lipoproteins (chylomicrons and very low density lipoproteins 1 and 2) were isolated from blood, and the area under curve and incremental area under curve for postprandial variables were calculated. Single nucleotide polymorphisms in genes encoding syndecan-1 and enzymes involved in the synthesis or degradation of HSPG were genotyped in the study subjects.
Our results indicate that the genetic variation rs2281279 in SULF2 associates with postprandial clearance of remnant TRLs and triglyceride levels in healthy subjects. Furthermore, the SNP rs2281279 in SULF2 associates with hepatic SULF2 mRNA levels.
In humans, mild but clinically relevant postprandial hyperlipidemia due to reduced hepatic clearance of remnant TRLs may result from genetic polymorphisms that affect hepatic HSPG.
This study sought to investigate the role of secretory phospholipase A2 (sPLA2)-IIA in cardiovascular disease.
Higher circulating levels of sPLA2-IIA mass or sPLA2 enzyme activity have been associated with increased risk of cardiovascular events. However, it is not clear if this association is causal. A recent phase III clinical trial of an sPLA2 inhibitor (varespladib) was stopped prematurely for lack of efficacy.
We conducted a Mendelian randomization meta-analysis of 19 general population studies (8,021 incident, 7,513 prevalent major vascular events [MVE] in 74,683 individuals) and 10 acute coronary syndrome (ACS) cohorts (2,520 recurrent MVE in 18,355 individuals) using rs11573156, a variant in PLA2G2A encoding the sPLA2-IIA isoenzyme, as an instrumental variable.
PLA2G2A rs11573156 C allele associated with lower circulating sPLA2-IIA mass (38% to 44%) and sPLA2 enzyme activity (3% to 23%) per C allele. The odds ratio (OR) for MVE per rs11573156 C allele was 1.02 (95% confidence interval [CI]: 0.98 to 1.06) in general populations and 0.96 (95% CI: 0.90 to 1.03) in ACS cohorts. In the general population studies, the OR derived from the genetic instrumental variable analysis for MVE for a 1-log unit lower sPLA2-IIA mass was 1.04 (95% CI: 0.96 to 1.13), and differed from the non-genetic observational estimate (OR: 0.69; 95% CI: 0.61 to 0.79). In the ACS cohorts, both the genetic instrumental variable and observational ORs showed a null association with MVE. Instrumental variable analysis failed to show associations between sPLA2 enzyme activity and MVE.
Reducing sPLA2-IIA mass is unlikely to be a useful therapeutic goal for preventing cardiovascular events.
cardiovascular diseases; drug development; epidemiology; genetics; Mendelian randomization; ACS, acute coronary syndrome(s); CI, confidence interval; LDL-C, low-density lipoprotein cholesterol; MI, myocardial infarction; MVE, major vascular events; OR, odds ratio; RCT, randomized clinical trial; SNP, single-nucleotide polymorphism; sPLA2, secretory phospholipase A2
The NLRP3 inflammasome has been recognized as one of the key components of the innate immunity by sensing a diversity of insults. Inflammasome activation results in the maturation of the pro-inflammatory cytokines interleukin (IL)-1β and IL-18. Increased production of IL-1β is found in patients with gain-of-function polymorphisms in genes encoding the NLRP3 inflammasome. Since approximately 5% of the Swedish population are heterozygote carriers of these combined gene variants, their impact on inflammasome status and a relationship on disease development is therefore highly relevant to study. The present study investigates levels of inflammasome-produced cytokines as a measure of inflammasome activation in healthy individuals carrying Q705K polymorphism in the NLRP3 gene combined with C10X in the CARD8 gene.
Materials and Methods
Genotyping of 1006 healthy blood donors was performed for the polymorphisms Q705K in the NLRP3 and C10X in the CARD8 genes. IL-1β, IL-18, IL-33, as well as a number of other pro-inflammatory cytokines, were analyzed by Luminex or ELISA in plasma from individuals carrying the polymorphisms and in age and gender matched non-carrier controls.
Results & Discussion
The prevalence of the polymorphisms was in line with previous studies. Plasma levels of IL-1β and IL-33 were elevated among carriers of combined Q705K+C10X polymorphisms compared to controls, whereas no difference was found for IL-18 and the other cytokines measured. Moreover, carriers of C10X or Q705K per
se had similar plasma levels of IL-1β as non-carriers. These data suggest that the combined polymorphisms create inflammasomes with increased basal activation state, which might provide a more favourable innate immune response. In spite of this, it could also represent the mechanisms by which the inflammatory loop is triggered into a long-term inflammatory phenotype.
Exploring genetic pleiotropy can provide clues to a mechanism underlying the observed epidemiological association between type 2 diabetes and heightened fracture risk. We examined genetic variants associated with bone mineral density (BMD) for association with type 2 diabetes and glycemic traits in large well-phenotyped and -genotyped consortia. We undertook follow-up analysis in ∼19,000 individuals and assessed gene expression. We queried single nucleotide polymorphisms (SNPs) associated with BMD at levels of genome-wide significance, variants in linkage disequilibrium (r2 > 0.5), and BMD candidate genes. SNP rs6867040, at the ITGA1 locus, was associated with a 0.0166 mmol/L (0.004) increase in fasting glucose per C allele in the combined analysis. Genetic variants in the ITGA1 locus were associated with its expression in the liver but not in adipose tissue. ITGA1 variants appeared among the top loci associated with type 2 diabetes, fasting insulin, β-cell function by homeostasis model assessment, and 2-h post–oral glucose tolerance test glucose and insulin levels. ITGA1 has demonstrated genetic pleiotropy in prior studies, and its suggested role in liver fibrosis, insulin secretion, and bone healing lends credence to its contribution to both osteoporosis and type 2 diabetes. These findings further underscore the link between skeletal and glucose metabolism and highlight a locus to direct future investigations.
Expansive remodelling is the process of compensatory arterial enlargement in response to atherosclerotic stimuli. The genetic determinants of this process are poorly characterized.
Genetic association analyses of inter-adventitial common carotid artery diameter (ICCAD) in the IMPROVE study (n = 3427) using the Illumina 200k Metabochip was performed. Single nucleotide polymorphisms (SNPs) that met array-wide significance were taken forward for analysis in three further studies (n = 5704), and tested for association with Abdominal Aortic Aneurysm (AAA).
rs3768445 on Chromosome 1q24.3, in a cluster of protein coding genes (DNM3, PIGC, C1orf105) was associated with larger ICCAD in the IMPROVE study. For each copy of the rare allele carried, ICCAD was on average 0.13 mm greater (95% CI 0.08–0.18 mm, P = 8.2 × 10−8). A proxy SNP (rs4916251, R2 = 0.99) did not, however, show association with ICCAD in three follow-up studies (P for replication = 0.29). There was evidence of interaction between carotid intima-media thickness (CIMT) and rs4916251 on ICCAD in two of the cohorts studies suggesting that it plays a role in the remodelling response to atherosclerosis. In meta-analysis of 5 case–control studies pooling data from 5007 cases and 43,630 controls, rs4916251 was associated with presence of AAA 1.10, 95% CI 1.03–1.17, p = 2.8 × 10−3, I2 = 18.8, Q = 0.30). A proxy SNP, rs4916251 was also associated with increased expression of PIGC in aortic tissue, suggesting that this may the mechanism by which this locus affects vascular remodelling.
Common variation at 1q24.3 is associated with expansive vascular remodelling and risk of AAA. These findings support a hypothesis that pathways involved in systemic vascular remodelling play a role in AAA development.
► In the IMPROVE study (n > 3000) variants at 1q24.3 were strongly associated with larger carotid diameters. ► The lead variant was associated with Abdominal Aortic Aneurysm (AAA) in meta-analysis of 5 studies (n > 50,000). ► Variants at 1q24.3 appear to be associated with vascular remodelling and risk of AAA.
Abdominal aortic aneurysm; Genome-wide association studies; Vascular remodelling; Carotid artery
Patients with bicuspid aortic valve (BAV) have an increased risk of developing ascending aortic aneurysm. In the present study, collagen homeostasis in nondilated and dilated aorta segments from patients with BAV was studied, with normal and dilated aortas from tricuspid aortic valve (TAV) patients as reference.
Methods and Results
Ascending aortas from 56 patients were used for biochemical and morphological analyses of collagen. mRNA expression was analyzed in 109 patients. Collagen turnover rates were similar in nondilated and dilated aortas of BAV patients, showing that aneurysmal formation in BAV is, in contrast to TAV, not associated with an increased collagen turnover. However, BAV in general was associated with an increased aortic collagen turnover compared with nondilated aortas of TAV patients. Importantly, the ratio of hydroxylysyl pyridinoline (HP) to lysyl pyridinoline (LP), 2 distinct forms of collagen cross‐linking, was lower in dilated aortas from patients with BAV, which suggests that BAV is associated with a defect in the posttranslational collagen modification. This suggests a deficiency at the level of lysyl hydroxylase (PLOD1), which was confirmed by mRNA and protein analyses that showed reduced PLOD1 expression but normal lysyl oxidase expression in dilated aortas from patients with BAV. This suggests that impaired collagen cross‐linking in BAV patients may be attributed to changes in the expression and/or activity of PLOD1.
Our results demonstrate an impaired biosynthesis and posttranslational modification of collagen in aortas of patients with BAV, which may explain the increased aortic aneurysm formation in BAV patients.
aneurysm; aorta; bicuspid; collagen; valve
Shear forces play a key role in the maintenance of vessel wall integrity. Current understanding regarding shear-dependent gene expression is mainly based on in vitro or in vivo observations with experimentally deranged shear, hence reflecting acute molecular events in relation to flow. Our objective was to combine computational fluid dynamic (CFD) simulations with global microarray analysis to study flow-dependent vessel wall biology in the aortic wall under physiological conditions.
Methods and Results
Male Wistar rats were used. Animal-specific wall shear stress (WSS) magnitude and vector direction were estimated using CFD based on aortic geometry and flow information acquired by magnetic resonance imaging. Two distinct flow pattern regions were identified in the normal rat aortic arch; the distal part of the lesser curvature being exposed to low WSS and a non-uniform vector direction, and a region along the greater curvature being subjected to markedly higher levels of WSS and a uniform vector direction. Microarray analysis identified numerous novel mechanosensitive genes, including Trpc4 and Fgf12, and confirmed well-known ones, e.g. Klf2 and Nrf2. Gene ontology analysis revealed an over-representation of genes involved in transcriptional regulation. The most differentially expressed gene, Hand2, is a transcription factor previously shown to be involved in extracellular matrix remodeling. HAND2 protein was endothelial specific and showed higher expression in the regions exposed to low WSS with disturbed flow.
Microarray analysis validated the CFD-defined WSS regions in the rat aortic arch, and identified numerous novel shear-sensitive genes. Defining the functional importance of these genes in relation to atherosusceptibility may provide important insight into the understanding of vascular pathology.
We conducted a systematic review and meta-analysis of studies reporting circulating IL-6 in AAA, and new investigations of the association between a common non-synonymous functional variant (Asp358Ala) in the IL-6R gene (IL6R) and AAA, followed the analysis of the variant both in vitro and in vivo.
Inflammation may play a role in the development of abdominal aortic aneurysms (AAA). Interleukin-6 (IL-6) signalling through its receptor (IL-6R) is one pathway that could be exploited pharmacologically. We investigated this using a Mendelian randomization approach.
Up to October 2011, we identified seven studies (869 cases, 851 controls). Meta-analysis demonstrated that AAA cases had higher levels of IL-6 than controls [standardized mean difference (SMD) = 0.46 SD, 95% CI = 0.25–0.66, I2 = 70%, P = 1.1 × 10–5 random effects]. Meta-analysis of five studies (4524 cases/15 710 controls) demonstrated that rs7529229 (which tags the non-synonymous variant Asp358Ala, rs2228145) was associated with a lower risk of AAA, per Ala358 allele odds ratio 0.84, 95% CI: 0.80–0.89, I2 = 0%, P = 2.7 × 10–11). In vitro analyses in lymphoblastoid cell lines demonstrated a reduction in the expression of downstream targets (STAT3, MYC and ICAM1) in response to IL-6 stimulation in Ala358 carriers.
A Mendelian randomization approach provides robust evidence that signalling via the IL-6R is likely to be a causal pathway in AAA. Drugs that inhibit IL-6R may play a role in AAA management.
Abdominal aortic aneurysm; Mendelian randomization; Interleukin-6; Polymorphism
Proinsulin is a precursor of mature insulin and C-peptide. Higher circulating proinsulin levels are associated with impaired β-cell function, raised glucose levels, insulin resistance, and type 2 diabetes (T2D). Studies of the insulin processing pathway could provide new insights about T2D pathophysiology.
RESEARCH DESIGN AND METHODS
We have conducted a meta-analysis of genome-wide association tests of ∼2.5 million genotyped or imputed single nucleotide polymorphisms (SNPs) and fasting proinsulin levels in 10,701 nondiabetic adults of European ancestry, with follow-up of 23 loci in up to 16,378 individuals, using additive genetic models adjusted for age, sex, fasting insulin, and study-specific covariates.
Nine SNPs at eight loci were associated with proinsulin levels (P < 5 × 10−8). Two loci (LARP6 and SGSM2) have not been previously related to metabolic traits, one (MADD) has been associated with fasting glucose, one (PCSK1) has been implicated in obesity, and four (TCF7L2, SLC30A8, VPS13C/C2CD4A/B, and ARAP1, formerly CENTD2) increase T2D risk. The proinsulin-raising allele of ARAP1 was associated with a lower fasting glucose (P = 1.7 × 10−4), improved β-cell function (P = 1.1 × 10−5), and lower risk of T2D (odds ratio 0.88; P = 7.8 × 10−6). Notably, PCSK1 encodes the protein prohormone convertase 1/3, the first enzyme in the insulin processing pathway. A genotype score composed of the nine proinsulin-raising alleles was not associated with coronary disease in two large case-control datasets.
We have identified nine genetic variants associated with fasting proinsulin. Our findings illuminate the biology underlying glucose homeostasis and T2D development in humans and argue against a direct role of proinsulin in coronary artery disease pathogenesis.
Inflammation and increased ceramide concentrations characterise adipose tissue of obese women with high liver fat content compared to equally obese women with normal liver fat content. The present study characterises enzymes involved in ceramide metabolism in subcutaneous and intra-abdominal adipose tissue.
Pathways leading to increased ceramide concentrations in inflamed versus non-inflamed adipose tissue were investigated by quantifying expression levels of key enzymes involved in ceramide metabolism. Sphingomyelinases (sphingomyelin phosphodiesterases SMPD1-3) were investigated further using immunohistochemistry to establish their location within adipose tissue, and their mRNA expression levels were determined in subcutaneous and intra-abdominal adipose tissue from both non-obese and obese subject.
Gene expression levels of sphingomyelinases, enzymes that hydrolyse sphingomyelin to ceramide, rather than enzymes involved in de novo ceramide synthesis, were higher in inflamed compared to non-inflamed adipose tissue of obese women (with high and normal liver fat contents respectively). Sphingomyelinases were localised to both macrophages and adipocytes, but also to blood vessels and to extracellular regions surrounding vessels within adipose tissue. Expression levels of SMPD3 mRNA correlated significantly with concentrations of different ceramides and sphingomyelins. In both non-obese and obese subjects SMPD3 mRNA levels were higher in the more inflamed intra-abdominal compared to the subcutaneous adipose tissue depot.
Generation of ceramides within adipose tissue as a result of sphingomyelinase action may contribute to inflammation in human adipose tissue.
Adipose tissue; Ceramide; Human; Inflammation; Sphingomyelinase
Individuals with bicuspid aortic valve (BAV) are at significantly higher risk of developing serious aortic complications than individuals with tricuspid aortic valves (TAV). Studies have indicated an altered aortic blood flow in patients with BAV; however, the extent to which altered flow influences the pathological state of BAV aorta is unclear. In the present study, we dissected flow-mediated aortic gene expression in patients undergoing elective open heart surgery. A large collection of public microarray data sets were firstly screened for consistent co-expression with five well-characterized flow-regulated genes (query genes). Genes with co-expression probability of >0.5 were selected and further analysed in expression profiles (127 arrays) from ascending aorta of BAV and TAV patients. Forty-four genes satisfied two filtering criteria: a significant correlation with one or more of the query genes (R > 0.40) and differential expression between patients with BAV and TAV. No gene fulfilled the criteria in mammary artery (88 arrays), an artery not in direct contact with the valve. Fifty-five percent of the genes significantly altered between BAV and TAV patients showed differential expression between two identified flow regions in the rat aorta. A large proportion of the identified genes were related to angiogenesis and/or wound healing, with pro-angiogenesis genes downregulated and inhibitory genes upregulated in patients with BAV. Moreover, differential expression of ZFP36, GRP116 and PKD2 was confirmed using immunohistochemistry. Implementing a new strategy, we have demonstrated an angiostatic gene expression signature in patients with BAV, indicating impaired wound healing in these patients, potentially involved in BAV-associated aortopathy.
Electronic supplementary material
The online version of this article (doi:10.1007/s00109-012-0942-8) contains supplementary material, which is available to authorized users.
Aortic aneurysm; Transcriptome; Wound healing; Aorta; Thoracic; Congenital heart defects
Following the widespread use of genome-wide association studies (GWAS), focus is turning towards identification of causal variants rather than simply genetic markers of diseases and traits. As a step towards a high-throughput method to identify genome-wide, non-coding, functional regulatory variants, we describe the technique of allele-specific FAIRE, utilising large-scale genotyping technology (FAIRE-gen) to determine allelic effects on chromatin accessibility and regulatory potential. FAIRE-gen was explored using lymphoblastoid cells and the 50,000 SNP Illumina CVD BeadChip. The technique identified an allele-specific regulatory polymorphism within NR1H3 (coding for LXR-α), rs7120118, coinciding with a previously GWAS-identified SNP for HDL-C levels. This finding was confirmed using FAIRE-gen with the 200,000 SNP Illumina Metabochip and verified with the established method of TaqMan allelic discrimination. Examination of this SNP in two prospective Caucasian cohorts comprising 15,000 individuals confirmed the association with HDL-C levels (combined beta = 0.016; p = 0.0006), and analysis of gene expression identified an allelic association with LXR-α expression in heart tissue. Using increasingly comprehensive genotyping chips and distinct tissues for examination, FAIRE-gen has the potential to aid the identification of many causal SNPs associated with disease from GWAS.
The identification of genetic variants associated with complex diseases has rapidly grown through lowering costs of genome sequencing and the use of large-scale genotyping chips based on this sequencing data. There have not been corresponding advances in the identification of causal genetic variants compared to variants simply associated with diseases or traits. Most of these causal variants are thought to be located not within regions coding for proteins, but within genomic regions that regulate the level of protein. We have combined the use of large-scale gene chips with functional analysis, to determine regions of the genome that confer a greater potential for controlling gene regulation dependent on the genotype of that individual. Combining this data with population data and gene expression data, we identify a potential causal variant that alters regulation of LXR-α, a key mediator in lipid metabolism, and show that this variant is associated with HDL-C levels. This methodology provides a model for future analyses to identify further causal variants for disease.