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1.  The periodontal pathogen Porphyromonas gingivalis changes the gene expression in vascular smooth muscle cells involving the TGFbeta/Notch signalling pathway and increased cell proliferation 
BMC Genomics  2013;14:770.
Porphyromonas gingivalis is a gram-negative bacterium that causes destructive chronic periodontitis. In addition, this bacterium is also involved in the development of cardiovascular disease. The aim of this study was to investigate the effects of P. gingivalis infection on gene and protein expression in human aortic smooth muscle cells (AoSMCs) and its relation to cellular function.
AoSMCs were exposed to viable P. gingivalis for 24 h, whereafter confocal fluorescence microscopy was used to study P. gingivalis invasion of AoSMCs. AoSMCs proliferation was evaluated by neutral red assay. Human genome microarray, western blot and ELISA were used to investigate how P. gingivalis changes the gene and protein expression of AoSMCs. We found that viable P. gingivalis invades AoSMCs, disrupts stress fiber structures and significantly increases cell proliferation. Microarray results showed that, a total of 982 genes were identified as differentially expressed with the threshold log2 fold change > |1| (adjust p-value <0.05). Using bioinformatic data mining, we demonstrated that up-regulated genes are enriched in gene ontology function of positive control of cell proliferation and down-regulated genes are enriched in the function of negative control of cell proliferation. The results from pathway analysis revealed that all the genes belonging to these two categories induced by P. gingivalis were enriched in 25 pathways, including genes of Notch and TGF-beta pathways.
This study demonstrates that P. gingivalis is able to invade AoSMCs and stimulate their proliferation. The activation of TGF-beta and Notch signaling pathways may be involved in the bacteria-mediated proliferation of AoSMCs. These findings further support the association between periodontitis and cardiovascular diseases.
PMCID: PMC3827841  PMID: 24209892
Porphyromonas gingivalis; Aortic smooth muscle cells; Proliferation; Gene expression profiling
2.  Polymorphism in the Retinoic Acid Metabolizing Enzyme CYP26B1 and the Development of Crohn’s Disease 
PLoS ONE  2013;8(8):e72739.
Several studies suggest that Vitamin A may be involved in the pathogenesis of inflammatory bowel disease (IBD), but the mechanism is still unknown. Cytochrome P450 26 B1 (CYP26B1) is involved in the degradation of retinoic acid and the polymorphism rs2241057 has an elevated catabolic function of retinoic acid, why we hypothesized that the rs2241057 polymorphism may affect the risk of Crohn’s disease (CD) and Ulcerative Colitis (UC). DNA from 1378 IBD patients, divided into 871 patients with CD and 507 with UC, and 1205 healthy controls collected at Örebro University Hospital and Karolinska University Hospital were analyzed for the CYP26B1 rs2241057 polymorphism with TaqMan® SNP Genotyping Assay followed by allelic discrimination analysis. A higher frequency of patients homozygous for the major (T) allele was associated with CD but not UC compared to the frequency found in healthy controls. A significant association between the major allele and non-stricturing, non-penetrating phenotype was evident for CD. However, the observed associations reached borderline significance only, after correcting for multiple testing. We suggest that homozygous carriers of the major (T) allele, relative to homozygous carriers of the minor (C) allele, of the CYP26B1 polymorphism rs2241057 may have an increased risk for the development of CD, which possibly may be due to elevated levels of retinoic acid. Our data may support the role of Vitamin A in the pathophysiology of CD, but the exact mechanisms remain to be elucidated.
PMCID: PMC3747106  PMID: 23977348
3.  Q705K variant in NLRP3 gene confers protection against myocardial infarction in female individuals 
Biomedical Reports  2013;1(6):879-882.
Inflammation is a multifaceted process that underlies the pathophysiology of acute myocardial infarction (MI). Variations in the inflammasome-related NLRP3 gene have been associated with risk for a number of different inflammatory diseases. Therefore, Q705K polymorphism in NLRP3 gene likely confers susceptibility to risk for MI. A First-ever myocardial Infarction study in Ac-county (FIA) cohort comprising 555 MI patients and 1,016 controls was used to genotype rs35829419 in the NLRP3 gene by TaqMan single-nucleotide polymorphism assay. C-reactive protein (CRP) was measured in the study participants by ELISA. The results showed no significant association between the variant rs35829419 and MI. However, the minor A allele of the rs35829419 polymorphism conferred a protective effect against the risk of developing MI in females. The minor A allele of rs35829419 polymorphism was also associated with increased CRP levels in males. Results of the study suggested a gender-specific deregulation of NLRP3 gene mediated by rs35829419 polymorphism that confers protection against MI in females but has no effect on MI susceptibility in males. However, the rs35829419 polymorphism was associated with increased CRP levels among the male subjects, thereby demonstrating the possible effect of the Q705K polymorphism in elevating the basal active state of innate immune response.
PMCID: PMC3917090  PMID: 24649046
myocardial infarction; Q705K polymorphism; NLRP3; inflammasome; rs35829419; cytokine
4.  Application of an Integrative Computational Framework in Trancriptomic Data of Atherosclerotic Mice Suggests Numerous Molecular Players 
Advances in Bioinformatics  2012;2012:453513.
Atherosclerosis is a multifactorial disease involving a lot of genes and proteins recruited throughout its manifestation. The present study aims to exploit bioinformatic tools in order to analyze microarray data of atherosclerotic aortic lesions of ApoE knockout mice, a model widely used in atherosclerosis research. In particular, a dynamic analysis was performed among young and aged animals, resulting in a list of 852 significantly altered genes. Pathway analysis indicated alterations in critical cellular processes related to cell communication and signal transduction, immune response, lipid transport, and metabolism. Cluster analysis partitioned the significantly differentiated genes in three major clusters of similar expression profile. Promoter analysis applied to functional related groups of the same cluster revealed shared putative cis-elements potentially contributing to a common regulatory mechanism. Finally, by reverse engineering the functional relevance of differentially expressed genes with specific cellular pathways, putative genes acting as hubs, were identified, linking functionally disparate cellular processes in the context of traditional molecular description.
PMCID: PMC3502768  PMID: 23193398
5.  Retinoic Acid Signalling Is Activated in the Postischemic Heart and May Influence Remodelling 
PLoS ONE  2012;7(9):e44740.
All-trans retinoic acid (atRA), an active derivative of vitamin A, regulates cell differentiation, proliferation and cardiac morphogenesis via transcriptional activation of retinoic acid receptors (RARs) acting on retinoic acid response elements (RARE).We hypothesized that the retinoic acid (RA) signalling pathway is activated in myocardial ischemia and postischemic remodelling.
Methods and Findings
Myocardial infarction was induced through ligating the left coronary artery in mice. In vivo cardiac activation of the RARs was measured by imaging RARE-luciferase reporter mice, and analysing expression of RAR target genes and proteins by real time RT-PCR and western blot. Endogenous retinoids in postinfarcted hearts were analysed by triple-stage liquid chromatography/tandem mass spectrometry. Cardiomyocytes (CM) and cardiofibroblasts (CF) were isolated from infarcted and sham operated RARE luciferase reporter hearts and monitored for RAR activity and expression of target genes. The effect of atRA on CF proliferation was evaluated by EdU incorporation. Myocardial infarction increased thoracic RAR activity in vivo (p<0.001), which was ascribed to the heart through ex vivo imaging (p = 0.002) with the largest signal 1 week postinfarct. This was accompanied by increased cardiac gene and protein expression of the RAR target genes retinol binding protein 1 (p = 0.01 for RNA, p = 0,006 for protein) and aldehyde dehydrogenase 1A2 (p = 0.04 for RNA, p = 0,014 for protein), while gene expression of cytochrome P450 26B1 was downregulated (p = 0.007). Concomitantly, retinol accumulated in the infarcted zone (p = 0.02). CM and CF isolated from infarcted hearts had higher luminescence than those from sham operated hearts (p = 0.02 and p = 0.008). AtRA inhibited CF proliferation in vitro (p = 0.02).
The RA signalling pathway is activated in postischemic hearts and may play a role in regulation of damage and repair during remodelling.
PMCID: PMC3460971  PMID: 23028599
6.  Cloning and Functional Studies of a Splice Variant of CYP26B1 Expressed in Vascular Cells 
PLoS ONE  2012;7(5):e36839.
All-trans retinoic acid (atRA) plays an essential role in the regulation of gene expression, cell growth and differentiation and is also important for normal cardiovascular development but may in turn be involved in cardiovascular diseases, i.e. atherosclerosis and restenosis. The cellular atRA levels are under strict control involving several cytochromes P450 isoforms (CYPs). CYP26 may be the most important regulator of atRA catabolism in vascular cells. The present study describes the molecular cloning, characterization and function of atRA-induced expression of a spliced variant of the CYP26B1 gene.
Methodology/Principal Findings
The coding region of the spliced CYP26B1 lacking exon 2 was amplified from cDNA synthesized from atRA-treated human aortic smooth muscle cells and sequenced. Both the spliced variant and full length CYP26B1 was found to be expressed in cultured human endothelial and smooth muscle cells, and in normal and atherosclerotic vessel. atRA induced both variants of CYP26B1 in cultured vascular cells. Furthermore, the levels of spliced mRNA transcript were 4.5 times higher in the atherosclerotic lesion compared to normal arteries and the expression in the lesions was increased 20-fold upon atRA treatment. The spliced CYP26B1 still has the capability to degrade atRA, but at an initial rate one-third that of the corresponding full length enzyme. Transfection of COS-1 and THP-1 cells with the CYP26B1 spliced variant indicated either an increase or a decrease in the catabolism of atRA, probably depending on the expression of other atRA catabolizing enzymes in the cells.
Vascular cells express the spliced variant of CYP26B1 lacking exon 2 and it is also increased in atherosclerotic lesions. The spliced variant displays a slower and reduced degradation of atRA as compared to the full-length enzyme. Further studies are needed, however, to clarify the substrate specificity and role of the CYP26B1 splice variant in health and disease.
PMCID: PMC3362586  PMID: 22666329
7.  A CYP26B1 Polymorphism Enhances Retinoic Acid Catabolism and May Aggravate Atherosclerosis 
Molecular Medicine  2012;18(1):712-718.
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.
PMCID: PMC3388133  PMID: 22415012

Results 1-7 (7)