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.
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.
Secretory phospholipase A2 group IIA (sPLA2-IIA) has been identified as a biomarker of atherosclerosis in observational and animal studies. The protein is encoded by the PLA2G2A gene and the aim of this study was to test the functionality of two PLA2G2A non-coding SNPs, rs11573156 C>G and rs3767221 T>G where the rare alleles have been previously associated with higher and lower sPLA2-IIA levels respectively.
Luciferase assays, electrophoretic mobility shift assays (EMSA), and RNA expression by RT-PCR were used to examine allelic differences. For rs3767221 the G allele showed ∼55% lower luciferase activity compared to the T allele (T = 62.1 (95% CI 59.1 to 65.1) G = 27.8 (95% CI 25.0 to 30.6), p = 1.22×10−35, and stronger EMSA binding of a nuclear protein compared to the T-allele. For rs11573156 C >G there were no luciferase or EMSA allelic differences seen. In lymphocyte cell RNA, from individuals of known rs11573156 genotype, there was no allelic RNA expression difference for exons 5 and 6, but G allele carriers (n = 7) showed a trend to lower exon 1–2 expression compared to CC individuals. To take this further, in the ASAP study (n = 223), an rs11573156 proxy (r2 = 0.91) showed ∼25% higher liver expression of PLA2G2A (1.67×10−17) associated with the G allele. However, considering exon specific expression, the association was greatly reduced for exon 2 (4.5×10−5) compared to exons 3–6 (10−10 to 10−20), suggesting rs11573156 G allele-specific exon 2 skipping.
Both SNPs are functional and provide useful tools for Mendelian Randomisation to determine whether the relationship between sPLA2-IIA and coronary heart disease is causal.
Combination antiretroviral therapy (cART) is associated with lipodystrophy, i.e., loss of subcutaneous adipose tissue in the abdomen, limbs, and face and its accumulation intra-abdominally. No fat is lost dorsocervically and it can even accumulate in this region (buffalo hump). It is unknown how preserved dorsocervical fat differs from abdominal subcutaneous fat in HIV-1–infected cART-treated patients with (cART+LD+) and without (cART+LD−) lipodystrophy.
RESEARCH DESIGN AND METHODS
We used histology, microarray, PCR, and magnetic resonance imaging to compare dorsocervical and abdominal subcutaneous adipose tissue in cART+LD+ (n = 21) and cART+LD− (n = 11).
Albeit dorsocervical adipose tissue in cART+LD+ seems spared from lipoatrophy, its mitochondrial DNA (mtDNA; copies/cell) content was significantly lower (by 62%) than that of the corresponding tissue in cART+LD−. Expression of CD68 mRNA, a marker of macrophages, and numerous inflammatory genes in microarray were significantly lower in dorsocervical versus abdominal subcutaneous adipose tissue. Genes with the greatest difference in expression between the two depots were those involved in regulation of transcription and regionalization (homeobox genes), irrespective of lipodystrophy status. There was negligible mRNA expression of uncoupling protein 1, a gene characteristic of brown adipose tissue, in either depot.
Because mtDNA is depleted even in the nonatrophic dorsocervical adipose tissue, it is unlikely that the cause of lipoatrophy is loss of mtDNA. Dorsocervical adipose tissue is less inflamed than lipoatrophic adipose tissue. It does not resemble brown adipose tissue. The greatest difference in gene expression between dorsocervical and abdominal subcutaneous adipose tissue is in expression of homeobox genes.
Genome-wide association studies have identified a coronary artery disease (CAD) risk locus in a non-coding region at 9p21.3, the nearest genes being CDKN2A and CDKN2B. To understand the pathways by which this locus might influence CAD susceptibility, we investigated associations between the 9p21.3 risk genotype and global gene expression in heart tissue from donors with no diagnosed heart disease (n = 108, predominant cause of death, cerebral vascular accident) and in carotid plaque (n = 106), aorta (n = 104) and mammary artery (n = 88) tissues from heart valve and carotid endarterectomy patients. Genotyping was performed with Taqman assays and Illumina arrays, and gene expression profiles generated with Affymetrix microarrays. Associations were analyzed with an additive genetic model. In heart tissue, 46 genes were putatively altered in association with the 9p21.3 risk allele (70% down-regulated, fold-change >1.1 per allele, p<0.05 adjusted for age, gender, ethnicity, cause of death). These genes were enriched for biomarkers of myocardial infarction (p = 1.53×10−9), response to wounding (p = 2.65×10−10) and inflammatory processes (p<1.97×10−7). Among the top 10 most down-regulated genes, 7 genes shared a set of transcription factor binding sites within conserved promoter regions (p<1.14×10−5), suggesting they may be co-regulated. Canonical pathway modelling of the most differentially expressed transcripts across all tissues (154 genes, 60% down-regulated, fold-change >1.1 per allele, p<0.01) showed that 75% of the genes could be transcriptionally regulated through the cell cycle G1 phase progression pathway (p<1.08×10−258), in which CDKN2A and CDKN2B play a regulatory role. These data suggest that the cell cycle G1 phase progression pathway is activated in individuals with the 9p21.3 risk allele. This may contribute to a proliferative phenotype that promotes adverse cardiac hypertrophy and vascular remodeling, leading to an increased CAD risk.
The Q705K polymorphism in NLRP3 has been implicated in several chronic inflammatory diseases. In this study we determine the functional role of this commonly occurring polymorphism using an in-vitro system.
NLRP3-WT and NLRP3-Q705K were retrovirally transduced into the human monocytic cell line THP-1, followed by the assessment of IL-1β and IL-18 levels in the cell culture supernatant. THP-1 cells expressing the above NLRP3 variants were sorted based upon Green Fluorescent Protein (GFP) expression. Cytokine response to alum (one of the most widely used adjuvants in vaccines) in the cells stably expressing NLRP3-WT and NLRP3-Q705K were determined. IL-1β and IL-18 levels were found to be elevated in THP-1 cells transduced with NLRP3-Q705K compared to the NLRP3-WT. Upon exposure to alum, THP-1 cells stably expressing NLRP3-Q705K displayed an increased release of IL-1β, IL-18 and TNF-α, in a caspase-1 and IL-1 receptor-dependent manner.
Collectively, these findings show that the Q705K polymorphism in NLRP3 is a gain-of-function alteration leading to an overactive NLRP3 inflammasome. The option of IL-1β blockade may be considered in patients with chronic inflammatory disorders that are unresponsive to conventional treatments.
All-trans retinoic acid, controlled by cytochrome P450, family 26 (CYP26) enzymes, potentially has beneficial effects in atherosclerosis treatment. This study investigates CYP26 subfamily B, polypeptide 1 (CYP26B1) in atherosclerosis and the effects of a genetic polymorphism in CYP26B1 on retinoid catabolism. We found that CYP26B1 mRNA was induced by retinoic acid in human atherosclerotic arteries, and CYP26B1 and the macrophage marker CD68 were colocalized in human atherosclerotic lesions. In mice, Cyp26B1 mRNA was higher in atherosclerotic arteries than in normal arteries. Databases were queried for nonsynonymous CYP26B1 single nucleotide polymorphisms (SNPs) and rs2241057 selected for further studies. Constructs of the CYP26B1 variants were created and used for production of purified proteins and transfection of macrophagelike cells. The minor variant catabolized retinoic acid with significantly higher efficiency, indicating that rs2241057 is functional and suggesting reduced retinoid availability in tissues with the minor variant. rs2241057 was investigated in a Stockholm Coronary Atherosclerosis Risk Factor (SCARF) subgroup. The minor allele was associated with slightly larger lesions, as determined by angiography. In summary, this study identifies the first CYP26B1 polymorphism that alters CYP26B1 capacity to metabolize retinoic acid. CYP26B1 was expressed in macrophage-rich areas of human atherosclerotic lesions, induced by retinoic acid and increased in murine atherosclerosis. Taken together, the results indicate that CYP26B1 capacity is genetically regulated and suggest that local CYP26B1 activity may influence atherosclerosis.
Neutrophils are key-players in the innate host defense and their programmed cell death and removal are essential for efficient resolution of inflammation. These cells recognize a variety of pathogens, and the NOD-like receptors (NLRs) have been suggested as intracellular sensors of microbial components and cell injury/stress. Some NLR will upon activation form multi-protein complexes termed inflammasomes that result in IL-1β production. NLR mutations are associated with auto-inflammatory syndromes, and our previous data propose NLRP3 (Q705K)/CARD-8 (C10X) polymorphisms to contribute to increased risk and severity of inflammatory disease by acting as genetic susceptibility factors. These gene products are components of the NALP3 inflammasome, and approximately 6.5% of the Swedish population are heterozygote carriers of these combined gene variants. Since patients carrying the Q705K/C10X polymorphisms display leukocytosis, the aim of the present study was to find out whether the inflammatory phenotype was related to dysfunctional apoptosis and impaired clearance of neutrophils by macrophages.
Methods and Findings
Patients carrying the Q705K/C10X polymorphisms displayed significantly delayed spontaneous as well as microbe-induced apoptosis compared to matched controls. Western blotting revealed increased levels and phosphorylation of Akt and Mcl-1 in the patients' neutrophils. In contrast to macrophages from healthy controls, macrophages from the patients produced lower amounts of TNF; suggesting impaired macrophage clearance response.
The Q705K/C10X polymorphisms are associated with delayed apoptosis of neutrophils. These findings are explained by altered involvement of different regulators of apoptosis, resulting in an anti-apoptotic profile. Moreover, the macrophage response to ingestion of microbe-induced apoptotic neutrophils is altered in the patients. Taken together, the patients display impaired turnover and clearance of apoptotic neutrophils, pointing towards a dysregulated innate immune response that influences the resolution of inflammation. The future challenge is to understand how microbes affect the activation of inflammasomes, and why this interaction will develop into severe inflammatory disease in certain individuals.
Macrophages play a critical role in innate immunity, and the expression of early response genes orchestrate much of the initial response of the immune system. Macrophages undergo extensive transcriptional reprogramming in response to inflammatory stimuli such as Lipopolysaccharide (LPS).
To identify gene transcription regulation patterns involved in early innate immune responses, we used two genome-wide approaches - gene expression profiling and chromatin immunoprecipitation-sequencing (ChIP-seq) analysis. We examined the effect of 2 hrs LPS stimulation on early gene expression and its relation to chromatin remodeling (H3 acetylation; H3Ac) and promoter binding of Sp1 and RNA polymerase II phosphorylated at serine 5 (S5P RNAPII), which is a marker for transcriptional initiation. Our results indicate novel and alternative gene regulatory mechanisms for certain proinflammatory genes. We identified two groups of up-regulated inflammatory genes with respect to chromatin modification and promoter features. One group, including highly up-regulated genes such as tumor necrosis factor (TNF), was characterized by H3Ac, high CpG content and lack of TATA boxes. The second group, containing inflammatory mediators (interleukins and CCL chemokines), was up-regulated upon LPS stimulation despite lacking H3Ac in their annotated promoters, which were low in CpG content but did contain TATA boxes. Genome-wide analysis showed that few H3Ac peaks were unique to either +/−LPS condition. However, within these, an unpacking/expansion of already existing H3Ac peaks was observed upon LPS stimulation. In contrast, a significant proportion of S5P RNAPII peaks (approx 40%) was unique to either condition. Furthermore, data indicated a large portion of previously unannotated TSSs, particularly in LPS-stimulated macrophages, where only 28% of unique S5P RNAPII peaks overlap annotated promoters. The regulation of the inflammatory response appears to occur in a very specific manner at the chromatin level for specific genes and this study highlights the level of fine-tuning that occurs in the immune response.
Thoracic aortic aneurysm (TAA) is a common complication in patients with a bicuspid aortic valve (BAV), the most frequent congenital heart disorder. For unknown reasons TAA occurs at a younger age, with a higher frequency in BAV patients than in patients with a tricuspid aortic valve (TAV), resulting in an increased risk for aortic dissection and rupture. To investigate the increased TAA incidence in BAV patients, we obtained tissue biopsy samples from nondilated and dilated aortas of 131 BAV and TAV patients. Global gene expression profiles were analyzed from controls and from aortic intima-media and adventitia of patients (in total 345 samples). Of the genes found to be differentially expressed with dilation, only a few (<4%) were differentially expressed in both BAV and TAV patients. With the use of gene set enrichment analysis, the cell adhesion and extracellular region gene ontology sets were identified as common features of TAA in both BAV and TAV patients. Immune response genes were observed to be particularly overexpressed in the aortic media of dilated TAV samples. The divergent gene expression profiles indicate that there are fundamental differences in TAA etiology in BAV and TAV patients. Immune response activation solely in the aortic media of TAV patients suggests that inflammation is involved in TAA formation in TAV but not in BAV patients. Conversely, genes were identified that were only differentially expressed with dilation in BAV patients. The result has bearing on future clinical studies in which separate analysis of BAV and TAV patients is recommended.
Impaired regulation of the transforming growth factor-β (TGFβ) signaling pathway has been linked to thoracic aortic aneurysm (TAA). Previous work has indicated that differential splicing is a common phenomenon, potentially influencing the function of proteins. In the present study we investigated the occurrence of differential splicing in the TGFβ pathway associated with TAA in patients with bicuspid aortic valve (BAV) and tricuspid aortic valve (TAV). Affymetrix human exon arrays were applied to 81 intima/media tissue samples from dilated (n = 51) and nondilated (n = 30) aortas of TAV and BAV patients. To analyze the occurrence of alternative splicing in the TGFβ pathway, multivariate techniques, including principal component analysis and OPLS-DA (orthogonal partial least squares to latent structures discriminant analysis), were applied on all exons (n = 614) of the TGFβ pathway. The scores plot, based on the splice index of individual exons, showed separate clusters of patients with both dilated and nondilated aorta, thereby illustrating the potential importance of alternative splicing in TAA. In total, differential splicing was detected in 187 exons. Furthermore, the pattern of alternative splicing is clearly differs between TAV and BAV patients. Differential splicing was specific for BAV and TAV patients in 40 and 86 exons, respectively, and splicings of 61 exons were shared between the two phenotypes. The occurrence of differential splicing was demonstrated in selected genes by reverse transcription–polymerase chain reaction. In summary, alternative splicing is a common feature of TAA formation. Our results suggest that dilatation in TAV and BAV patients has different alternative splicing fingerprints in the TGFβ pathway.
The TNFSF4/TNFRSF4 system, along with several other receptor-ligand pairs, is involved in the recruitment and activation of T-cells and is therefore tentatively implicated in atherosclerosis and acute coronary syndromes. We have previously shown that genetic variants in TNFSF4 are associated with myocardial infarction (MI) in women. This prompted functional studies of TNFSF4 expression.
Methods and Results
Based on a screening of the TNFSF4 genomic region, a promoter polymorphism (rs45454293) and a haplotype were identified, conceivably involved in gene regulation. The rs45454293T-allele, in agreement with the linked rs3850641G-allele, proved to be associated with increased risk of MI in women. Haplotype-specific chromatin immunoprecipitation of activated polymerase II, as a measure of transcriptional activity in vivo, suggested that the haplotype including the rs45454293 and rs3850641 polymorphisms is functionally important, the rs45454293T- and rs3850641G-alleles being associated with lower transcriptional activity in cells heterozygous for both polymorphisms. The functional role of rs45454293 on transcriptional levels of TNFSF4 was clarified by luciferase reporter assays, where the rs45454293T-allele decreased gene expression when compared with the rs45454293C-allele, while the rs3850641 SNP did not have any effect on TNFSF4 promoter activity. Electromobility shift assay showed that the rs45454293 polymorphism, but not rs3850641, affects the binding of nuclear factors, thus suggesting that the lower transcriptional activity is attributed to binding of one or more transcriptional repressor(s) to the T-allele.
Our data indicate that the TNFSF4 rs45454293T-allele is associated with lower TNFSF4 expression and increased risk of MI.
Several genome-wide association studies have recently linked a group of single nucleotide polymorphisms in the 9p21 region with cardiovascular disease. The molecular mechanisms of this link are not fully understood. We investigated five different expression microarray datasets in order to determine if the genotype had effect on expression of any gene transcript in aorta, mammary artery, carotid plaque and lymphoblastoid cells.
After multiple testing correction, no genes were found to have relation to the rs2891168 risk genotype, either on a genome-wide scale or on a regional (8 MB) scale. The neighbouring ANRIL gene was found to have eight novel transcript variants not previously known from literature and these varied by tissue type. We therefore performed a detailed probe-level analysis and found small stretches of significant relation to genotype but no consistent associations. In all investigated tissues we found an inverse correlation between ANRIL and the MTAP gene and a positive correlation between ANRIL and CDKN2A and CDKN2B.
Investigation of relation of the risk genotype to gene expression is complicated by the transcript complexity of the locus. With our investigation of a range of relevant tissue we wish to underscore the need for careful attention to the complexity of the alternative splicing issues in the region and its implications to the design of future gene expression studies.
Human cytomegalovirus (HCMV) has been suggested to contribute to the development of vascular diseases. Since matrix metalloproteinases (MMPs) have been implicated in atherosclerosis and plaque rupture, we investigated the effect of HCMV infection on MMP expression in human macrophages. We used quantitative real-time PCR, Western blotting, and gelatin zymography to study the expression and activity of MMP-2, -3, -7, -9, -12, -13, and -14 and of tissue inhibitor of metalloproteinase 1 (TIMP-1), -2, -3, and -4. HCMV infection reduced MMP-9 mRNA, protein, and activity levels but increased TIMP-1 mRNA and protein levels. Furthermore, a decrease in MMP-12, MMP-14, TIMP-2, and TIMP-3 mRNA levels could be detected. The MMP-9 and TIMP-1 mRNA alterations required viral replication. MMP-9 mRNA expression was affected by an immediate-early or early viral gene product, whereas TIMP-1 mRNA expression was affected by late viral gene products. We conclude that HCMV infection specifically alters the MMP-9/TIMP-1 balance in human macrophages, which in turn reduces MMP-9 activity in infected cells. Since MMP-9 prevents atherosclerotic plaque development in mice, these results suggest that HCMV may contribute to atherogenesis through specific effects on MMP-9 activity.
Summary: Whole-genome microarrays allow us to interrogate the entire transcriptome of a cell. Affymetrix microarrays are constructed using several probes that match to different regions of a gene and a summarization step reduces this complexity into a single value, representing the expression level of the gene or the expression level of an exon in the case of exon arrays. However, this simplification eliminates information that might be useful when focusing on specific genes of interest. To address these limitations, we present a software package for the R platform that allows detailed analysis of expression at the probe level. The package matches the probe sequences against a target gene sequence (either mRNA or DNA) and shows the expression levels of each probe along the gene. It also features functions to fit a linear regression based on several genetic models that enables study of the relationship between gene expression and genotype.
Availability and implementation: The software is implemented as a platform-independent R package available through the Bioconductor repository at http://www.bioconductor.org/. It is licensed as GPL 2.0.
Supplementary Information: Supplementary data are available at Bioinformatics online.
Identification of functional genetic variation associated with increased susceptibility to complex diseases can elucidate genes and underlying biochemical mechanisms linked to disease onset and progression. For genes linked to genetic diseases, most identified causal mutations alter an encoded protein sequence. Technological advances for measuring RNA abundance suggest that a significant number of undiscovered causal mutations may alter the regulation of gene transcription. However, it remains a challenge to separate causal genetic variations from linked neutral variations. Here we present an in silico driven approach to identify possible genetic variation in regulatory sequences. The approach combines phylogenetic footprinting and transcription factor binding site prediction to identify variation in candidate cis-regulatory elements. The bioinformatics approach has been tested on a set of SNPs that are reported to have a regulatory function, as well as background SNPs. In the absence of additional information about an analyzed gene, the poor specificity of binding site prediction is prohibitive to its application. However, when additional data is available that can give guidance on which transcription factor is involved in the regulation of the gene, the in silico binding site prediction improves the selection of candidate regulatory polymorphisms for further analyses. The bioinformatics software generated for the analysis has been implemented as a Web-based application system entitled RAVEN (regulatory analysis of variation in enhancers). The RAVEN system is available at http://www.cisreg.ca for all researchers interested in the detection and characterization of regulatory sequence variation.
DNA sequence variations (polymorphisms) that affect the expression levels of genes play important roles in the pathogenesis of many complex diseases. Compared with genetic variations that alter the amino acid sequences of encoded proteins, which are relatively easy to identify, sequence variants that affect the regulation of genes are difficult to pinpoint among the large amount of nonfunctional polymorphisms located in the vicinity of genes. Computational methods to distinguish functional from neutral variations could therefore prove useful to direct limited laboratory resources to sites most likely to exhibit a phenotypic effect. In this paper we present a Web-based tool for the identification of genetic variation in potential transcription factor binding sites. This tool can be used by any scientist interested in the characterization of regulatory polymorphisms. Using experimentally verified regulatory polymorphisms and background data collected from the literature, we evaluate the method's capacity to identify regulatory genetic variation, and we discuss the limitations of its application.
CD36 is a membrane glycoprotein involved in a variety of cellular processes such as lipid transport, immune regulation, hemostasis, adhesion, angiogenesis and atherosclerosis. It is expressed in many tissues and cell types, with a tissue specific expression pattern that is a result of a complex regulation for which the molecular mechanisms are not yet fully understood. There are several alternative mRNA isoforms described for the gene. We have investigated the expression patterns of five alternative first exons of the CD36 gene in several human tissues and cell types, to better understand the molecular details behind its regulation.
We have identified one novel alternative first exon of the CD36 gene, and confirmed the expression of four previously known alternative first exons of the gene. The alternative transcripts are all expressed in more than one human tissue and their expression patterns vary highly in skeletal muscle, heart, liver, adipose tissue, placenta, spinal cord, cerebrum and monocytes. All alternative first exons are upregulated in THP-1 macrophages in response to oxidized low density lipoproteins. The alternative promoters lack TATA-boxes and CpG islands. The upstream region of exon 1b contains several features common for house keeping gene and monocyte specific gene promoters.
Tissue-specific expression patterns of the alternative first exons of CD36 suggest that the alternative first exons of the gene are regulated individually and tissue specifically. At the same time, the fact that all first exons are upregulated in THP-1 macrophages in response to oxidized low density lipoproteins may suggest that the alternative first exons are coregulated in this cell type and environmental condition. The molecular mechanisms regulating CD36 thus appear to be unusually complex, which might reflect the multifunctional role of the gene in different tissues and cellular conditions.
Exposure to the brominated flame retardant 2,2′,4,4′,5-pentabromodiphenyl ether (PBDE-99) during the brain growth spurt disrupts normal brain development in mice and results in disturbed spontaneous behavior in adulthood. The neurodevelopmental toxicity of PBDE-99 has been reported to affect the cholinergic and catecholaminergic systems. In this study we use a proteomics approach to study the early effect of PBDE-99 in two distinct regions of the neonatal mouse brain, the striatum and the hippocampus. A single oral dose of PBDE-99 (12 mg/kg body weight) or vehicle was administered to male NMRI mice on neonatal day 10, and the striatum and the hippocampus were isolated. Using two-dimensional fluorescence difference gel electrophoresis (2D-DIGE), we found 40 and 56 protein spots with significantly (p < 0.01) altered levels in the striatum and the hippocampus, respectively. We used matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI–ToF–MS) to determine the protein identity of 11 spots from the striatum and 10 from the hippocampus. We found that the levels of proteins involved in neurodegeneration and neuroplasticity (e.g., Gap-43/neuromodulin, stathmin) were typically altered in the striatum, and proteins involved in metabolism and energy production [e.g., α-enolase; γ-enolase; ATP synthase, H+ transporting, mitochondrial F1 complex, β subunit (Atp5b); and α-synuclein] were typically altered in the hippocampus. Interestingly, many of the identified proteins have been linked to protein kinase C signaling. In conclusion, we identify responses to early exposure to PBDE-99 that could contribute to persistent neurotoxic effects. This study also shows the usefulness of proteomics to identify potential biomarkers of developmental neurotoxicity of organohalogen compounds.
2D-GE; brain development; brain growth spurt; MALDI–ToF–MS; neonatal; neurodegeneration; PBDE-99; PKC; proteomics
Aortic valve calcification (AVC), even without haemodynamic significance, may be prognostically import as an expression of generalized atherosclerosis, but techniques for echocardiographic assessment are essentially unexplored.
Two-dimensional (2D) echocardiographic recordings (Philips IE33) of the aortic valve in short-axis and long-axis views were performed in 185 consecutive patients within 1 week before surgery for aortic stenosis (n = 109, AS), aortic regurgitation (n = 61, AR), their combination (n = 8) or dilation of the ascending aorta (n = 7). The grey scale mean (GSMn) of the aortic valve in an end-diastolic short-axis still frame was measured. The same frame was scored visually 1–5 as indicating that the aortic valve was normal, thick, or had mild, moderate or severe calcification. The visual echodensity of each leaflet was determined real time applying the same 5-grade scoring system for each leaflet, and the average for the whole valve was calculated. Finally, a similar calcification score for the whole valve based on inspection and palpation by the surgeon was noted.
Visual assessment of real-time images using the proposed scoring system showed better correlation with the surgical evaluation of the degree of valve calcification (r = 0·83, P<0·001) compared to evaluation of stop frames by visual assessment (r = 0·66, P<0·001) or the GSMn score (r = 0·64, P<0·001). High inter- and intra-observer correlations were observed for real-time visual score (both intraclass correlation coefficient = 0·93).
Real-time evaluation of the level of AVC is superior to using stop frames assessed either visually or by dedicated computer grey scale measurement software.
aortic regurgitation; aortic stenosis; grey scale; sclerosis; ultrasound