Till Örebro universitet

Coronary atherosclerosis can lead to myocardial infarction, and secondarily to post-infarct remodelling and heart failure. Retinoic acid (RA) influences cell proliferation. We hypothesized that RA could influence gene expression and proliferation of cardiovascular cells. Left ventricular biopsies from patients with end-stage heart failure due to coronary artery disease (CAD) or dilated cardiomyopathy were investigated for the content of RA metabolites using liquid chromatography mass spectrometry (LC-MS/MS), and compared with healthy donors. All-trans retinoic acid (ATRA) was increased in the hearts of CAD patients. Gene expression (quantitative PCR) of RA target genes was not influenced in failing hearts, but was increased in the hearts of patients with CAD undergoing open heart surgery. The expression of RA target genes was increased in atherosclerotic lesions from carotid arteries compared to healthy arteries. Stimulation of cardiomyocytes, cardiofibroblasts, smooth muscle cells and endothelial cells with ATRA increased the gene expression of the key enzymes. Cardiofibroblast and smooth muscle cell proliferation were reduced by ATRA, which increased endothelial cell proliferation. Coronary artery disease leads to increased expression of RA target genes. ATRA accumulated in the failing human heart. All investigated cell types present in the heart had induced expression of RA target genes when stimulated with ATRA, which also influenced cell proliferation.

Inflammation is a key factor in the development of atherosclerotic coronary artery disease. It is promoted through the inflammasome, a molecular machine that produces IL (interleukin)-1 beta in response to cholesterol crystal accumulation in macrophages. The CARD8 (caspase recruitment domain 8) protein modulates this process by suppressing caspase 1 and the transcription factor NF-kappa B (nuclear factor kappa B). The expression of CARD8 mRNA was examined in atherosclerotic vascular tissue and the impact on MI (myocardial infarction) of a polymorphism in the CARD8 gene determined. CARD8 mRNA was analysed by microarray of human atherosclerotic tissue and compared with transplant donor arterial tissue. Microarray analysis was performed for proximal genes associated with the rs2043211 locus in plaque. The CARD8 rs2043211 polymorphism was analysed by genotyping of two Swedish MI cohorts, FIA (First Myocardial Infarction in Northern Sweden) and SCARF (Stockholm Coronary Atherosclerosis Risk Factor). The CRP (C-reactive protein) level was measured in both cohorts, but the levels of the pro-inflammatory cytokines IL-1 beta, IL-18, TNF (tumour necrosis factor) and MCP-1 (monocyte chemoattractant protein) were measured in sera available from the SCARF cohort. CARD8 mRNA was highly expressed in atherosclerotic plaques compared with the expression in transplant donor vessel (P < 0.00001). The minor allele was associated with lower expression of CARD8 in the plaques, suggesting that CARD8 may promote inflammation. Carriers of the minor allele of the rs2043211 polymorphism also displayed lower circulating CRP and lower levels of the pro-atherosclerotic chemokine MCP-1. However, no significant association could be detected between this polymorphism and MI in the two cohorts. Genetic alterations in the CARD8 gene therefore seem to be of limited importance for the development of MI.

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.

Background: 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.

Results: 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.

Conclusions: 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.

All-trans retinoic acid, controlled by CYP26 enzymes, potentially has beneficial effects in atherosclerosis treatment. This study investigates CYP26B1 in atherosclerosis and 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 co-localized in human atherosclerotic lesions. In mice, Cyp26B1 mRNA was higher in atherosclerotic than normal arteries. Databases were queried for non-synonymous CYP26B1 SNPs and rs2241057 selected for further studies. Constructs of the CYP26B1 variants were created and used for production of purified proteins and transfection of macrophage-like 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.

Background: 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.

Conclusions/Significance: 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 fulllength enzyme. Further studies are needed, however, to clarify the substrate specificity and role of the CYP26B1 splice variant in health and disease.

Cardiovascular disease (CVD), currently the most common cause of morbidity and mortality worldwide, is caused mainly by atherosclerosis. Atherosclerosis is a chronic multifocal, immunoinflammatory, fibroproliferative disease of medium and large arteries. Atherosclerotic lesions and vascular cells express different genes, among these are genes regulated by retinoic acid. Retinoids have pleiotropic effects and are able to modulate gene expression involved in growth, function and adaptation. During atherosclerosis development, there is endothelial perturbation, lipid accumulation, attraction of immune cells, smooth muscle cell migration and extracellular matrix remodeling and eventually fibrous cap formation which results in plaques. Retinoids have been demonstrated to either inhibit or modulate the above processes, resulting in amelioration of atherosclerosis. So far, retinoids are known to have impact on cellular processes in SMC, vascular injury and atherosclerosis. However, little is known about catabolism of retinoids in vascular cells and lesions and the effects of alteration of retinoic catabolizing enzymes on retinoids’ status. Therefore, we investigated the expression of Cytochrome P450 26 (CYP26) which is thought to be dedicated to retinoid catabolism. In vascular SMCs and atherosclerotic lesions, we found that CYP26B1 was the only member of the CYP26 family expressed, and it was highly inducible by atRA. Our data revealed that blocking CYP26B1 by chemical inhibition, or by targeted siRNA knock-down, resulted in significantly increased cellular retinoid levels. This indicates that CYP26B1 is an important modulator of endogenous retinoic acid levels. Therefore, we studied the effect of the CYP26B1 nonsynonymous polymorphism rs224105 on retinoic acid availability and found that the minor allele was associated with an enhanced retinoic acid catabolism rate and also with a slightly larger area of atherosclerotic lesions. The expression of CYP26B1 in human atherosclerotic lesions was localized to macrophage rich areas, suggesting retinoic acid activity in macrophages. Furthermore, we demonstrated that a CYP26B1 splice variant, that lack exon two, is expressed in vascular cells and in vessels walls. It is functional, with a reduced catabolic activity to around 70%, inducible by atRA in vascular cells and expressed 4.5 times more in atherosclerotic lesions compared to normal arteries. Moreover, the statins simvastatin and rosuvastatin reduced CYP26B1 mediated atRA catabolism in a concentration-dependent manner, and in vascular cells increased the mRNA expression of the atRA-responsive genes CYP26B1 and RARβ. This could lead to statins indirectly augmenting retinoic acid action in vascular cells which mimic statins roles. In conclusion, CYP26B1 is a major retinoic acid modulator in vascular cells and atherosclerotic lesions. Blocking of CYP26B1 could provide an advantageous therapeutic alternative to exogenous retinoid administration for treatment of vascular disorders.

Cigarette smoke (CS) is a well established risk factor in the development of chronic obstructive pulmonary disease (COPD). In contrast, the extent to which CS exposure contributes to the development of the systemic manifestations of COPD, such as skeletal muscle dysfunction and wasting remains largely unknown. Decreased skeletal muscle capillarization has been previously reported in early stages of COPD and might play an important role in the development of COPD-associated skeletal muscle abnormalities. To investigate the effects of chronic CS exposure on skeletal muscle capillarization and exercise tolerance a mouse model of CS exposure was used. The129/SvJ mice were exposed to CS for 6 months, and the expression of putative elements of the hypoxia-angiogenic signaling cascade as well as muscle capillarization were studied. Additionally, functional tests assessing exercise tolerance/endurance were performed in mice. Compared to controls, skeletal muscles from CS-exposed mice exhibited significantly enhanced expression of von Hippel-Lindau tumor suppressor (VHL), ubiquitin-conjugating enzyme E2D1 (UBE2D1) and prolyl hydroxylase-2 (PHD2). In contrast, hypoxia-inducible factor-1 (HIF1-α) and vascular endothelial growth factor (VEGF) expression was reduced. Furthermore, reduced muscle fiber cross-sectional area, decreased skeletal muscle capillarization, and reduced exercise tolerance were also observed in CS-exposed animals. Taken together, the current results provide evidence linking chronic CS exposure and induction of VHL expression in skeletal muscles leading towards impaired hypoxia-angiogenesis signal transduction, reduced muscle fiber cross-sectional area and decreased exercise tolerance.

Homology models of CYP26B1 (cytochrome P450RAI2) and CYP26B1 spliced-variant were derived using the crystal structure of cyanobacterial CYP120A1 as template for the model building. The quality of the homology models generated were carefully evaluated, and the natural substrate all-trans-retinoic acid (atRA), several tetralone-derived retinoic acid metabolizing blocking agents (RAMBAs) and a well known potent inhibitor of CYP26B1 (R115866) were docked into the homology model of full-length cytochrome P450 26B1. The results show that in the model of the full length CYP26B1, the protein is capable of distinguishing between the natural substrate (atRA), R115866 and the tetralone derivatives. The spliced-variant of CYP26B1 model displays a reduced affinity for atRA compared to the full length enzyme, in accordance with recently described experimental information.

Interleukin (IL)-1β is known to be activated by the inflammasome. Inflammasome activities depend on a plethora of moieties including NLRP3 and CARD8, which have been reported to be associated with several inflammatory diseases. Aortic smooth muscle cells (AOSMCs) were transfected with siRNA targeting the NLRP3 and CARD8 genes, followed by tumor necrosis factor-α (TNF-α) treatment. We found that TNF-α induces IL-1β, IL-1Ra and NLRP3 genes but not CARD8. Silencing of the NLRP3 gene significantly decreased IL-1β expression and release, the IL-1Ra expression showed a borderline non-significant increment, while CARD8 knockdown did not affect the IL-1β and IL-1Ra mRNA expression or IL-1β protein release. Our results suggest that mainly NLRP3 plays a role in the regulation of IL-1β expression and release in AOSMC and could be a potential future target for the treatment of atherosclerosis and other inflammatory diseases.