Loci cg06256735 and cg15815843 in the MFAP5 gene regulatory regions are hypomethylated in varicose veins apparently due to active demethylation.

Varicose vein disease (VVD) is a common health problem worldwide. Microfibril Associated Protein 5 (MFAP5) is one of the potential key players in its pathogenesis. Our previous microarray analysis revealed the cg06256735 and cg15815843 loci in the regulatory regions of the MFAP5 gene as hypomethylated in varicose veins which correlated with its up-regulation. The aim of this work was to validate preliminary microarray data, estimate the level of 5-hydroxymethylcytosine (5hmC) at these loci, and determine the methylation status of one of them in different layers of the venous wall. For this, methyl- and hydroxymethyl-sensitive restriction techniques were used followed by real-time PCR and droplet digital PCR, correspondingly, as well as bisulfite pyrosequencing of +/- oxidized DNA. Our microarray data on hypomethylation at the cg06256735 and cg15815843 loci in whole varicose vein segments were confirmed and it was also demonstrated that the level of 5hmC at these loci is increased in VVD. Specifically, among other layers of the venous wall, t. intima is the main contributor to hypomethylation at the cg06256735 locus in varicose veins. Thus, it was shown that hypomethylation at the cg06256735 and cg15815843 loci takes place in VVD, with evidence to suggest that it happens through their active demethylation leading to up-regulation of the MFAP5 gene, and t. intima is most involved in this biochemical process.

Epigenome-Wide Changes in the Cell Layers of the Vein Wall When Exposing the Venous Endothelium to Oscillatory Shear Stress.

Epigenomic changes in the venous cells exerted by oscillatory shear stress towards the endothelium may result in consolidation of gene expression alterations upon vein wall remodeling during varicose transformation. We aimed to reveal such epigenome-wide methylation changes. Primary culture cells were obtained from non-varicose vein segments left after surgery of 3 patients by growing the cells in selective media after magnetic immunosorting. Endothelial cells were either exposed to oscillatory shear stress or left at the static condition. Then, other cell types were treated with preconditioned media from the adjacent layer's cells. DNA isolated from the harvested cells was subjected to epigenome-wide study using Illumina microarrays followed by data analysis with GenomeStudio (Illumina), Excel (Microsoft), and Genome Enhancer (geneXplain) software packages. Differential (hypo-/hyper-) methylation was revealed for each cell layer's DNA. The most targetable master regulators controlling the activity of certain transcription factors regulating the genes near the differentially methylated sites appeared to be the following: (1) HGS, PDGFB, and AR for endothelial cells; (2) HGS, CDH2, SPRY2, SMAD2, ZFYVE9, and P2RY1 for smooth muscle cells; and (3) WWOX, F8, IGF2R, NFKB1, RELA, SOCS1, and FXN for fibroblasts. Some of the identified master regulators may serve as promising druggable targets for treating varicose veins in the future.

Quantitative and structural characteristics of mitochondrial DNA in varicose veins.

OBJECTIVE: We examined quantitative (in terms of mtDNA/nuclear DNA) and structural (in terms of common deletions in the MT-ND4 gene region) characteristics of mitochondrial DNA (mtDNA) in varicose veins (VVs) and venous wall layers by comparing mitochondrial genome parameters, as well as mitochondrial function (in terms of mitochondrial membrane potential (MtMP)), in varicose vein (VV) vs. non-varicose vein (NV) tissue samples. METHODS: We analyzed paired great saphenous vein samples (VV vs. NV segments from each patient left after venous surgery) harvested from patients with VVs. Relative mtDNA level and the proportion of no-deletion mtDNA were determined by a multiplex quantitative PCR (qPCR), confirming the latter with a more sensitive method - droplet digital PCR (ddPCR). Mitochondria's functional state in VVs was assessed using fluorescent (dependent on MtMP) live-staining of mitochondria in venous tissues. RESULTS: Total mtDNA level was lower in VV than in NV samples (predominantly in the t. media layer). ddPCR analysis showed lower proportion of no-deletion mtDNA in VVs. Because of the decrease in relative MtMP in VVs, our results suggest a possible reduction of mitochondrial function in VVs. CONCLUSION: Quantitative and structural changes (copy number and integrity) of mtDNA are plausibly involved in VV pathogenesis. Future clinical studies implementing the mitochondrial targeting may be eventually fostered after auxiliary mechanistic studies.

DNA methylation and gene expression profiling reveal MFAP5 as a regulatory driver of extracellular matrix remodeling in varicose vein disease.

AIM: To integrate transcriptomic and DNA-methylomic measurements on varicose versus normal veins using a systems biological analysis to shed light on the interplay between genetic and epigenetic factors. MATERIALS & METHODS: Differential expression and methylation were measured using microarrays, supported by real-time quantitative PCR and immunohistochemistry confirmation for relevant gene products. A systems biological 'upstream analysis' was further applied. RESULTS: We identified several potential key players contributing to extracellular matrix remodeling in varicose veins. Specifically, our analysis suggests MFAP5 acting as a master regulator, upstream of integrins, of the cellular network affecting the varicose vein condition. Possible mechanism and pathogenic model were outlined. CONCLUSION: A coherent model proposed incorporates the relevant signaling networks and will hopefully aid further studies on varicose vein pathogenesis.

Mycoplasma hyorhinis reduces sensitivity of human lung carcinoma cells to Nutlin-3 and promotes their malignant phenotype.

PURPOSE: MDM2 inhibitors are promising anticancer agents that induce cell cycle arrest and tumor cells death via p53 reactivation. We examined the influence of Mycoplasma hyorhinis infection on sensitivity of human lung carcinoma cells NCI-H292 to MDM2 inhibitor Nutlin-3. In order to unveil possible mechanisms underlying the revealed effect, we investigated gene expression changes and signal transduction networks activated in NCI-H292 cells in response to mycoplasma infection. METHODS: Sensitivity of NCI-Н292 cells to Nutlin-3 was estimated by resazurin-based cell viability assay. Genome-wide transcriptional profiles of NCI-H292 and NCI-Н292Myc.h cell lines were determined using Illumina Human HT-12 v3 Expression BeadChip. Search for key transcription factors and key node molecules was performed using the geneXplain platform. Ability for anchorage-independent growth was tested by soft agar colony formation assay. RESULTS: NCI-Н292Myc.h cells were shown to be 1.5- and 5.2-fold more resistant to killing by Nutlin-3 at concentrations of 15 and 30 µM than uninfected NCI-Н292 cells (P < 0.05 and P < 0.001, respectively). Transcriptome analysis revealed differential expression of multiple genes involved in cancer progression and metastasis as well as epithelial-mesenchymal transition (EMT). Moreover, we have shown experimentally that NCI-Н292Myc.h cells were more capable of growing and dividing without binding to a substrate. The most likely mechanism explaining the observed changes was found to be TLR4- and IL-1b-mediated activation of NF-κB pathway. CONCLUSIONS: Our results provide evidence that mycoplasma infection is an important factor modulating the effect of MDM2 inhibitors on cancer cells and is able to induce EMT-related changes.

Genome-wide association study in ethnic Russians suggests an association of the MHC class III genomic region with the risk of primary varicose veins.

Heredity is a well-known risk factor for varicose veins, but genetic basis of this condition remains poorly studied. Our aim was to conduct a large-scale genetic association study for primary varicose veins (PVVs) in the population of ethnic Russians. An initial scan using Illumina HumanExome-12 v1.0 BeadChip was performed for 273 patients with PVVs and 250 controls without a history of chronic venous disease and other venous disorders. After quality control and removal of monomorphic markers, 25,424 common and 48,232 rare variants were included in the analysis. 42 single nucleotide polymorphisms (SNPs) were genotyped in the independent replication cohort of 447 PVVs patients and 443 controls. Association of common variants with PVVs was investigated by logistic regression, and the impact of rare variants was analyzed using sequence kernel association test. No effect of low frequency alleles has been revealed in our study. Common variant analysis identified a promising signal at chromosome 6 within classical major histocompatibility complex (MHC) class III subregion. The most strongly associated SNP in a combined analysis that reached a suggestive significance level of 3.2e-05 was polymorphism rs4151657 in the complement factor B gene. Testing for potential pleiotropy with other traits indicated that the same causal variant in this region increases the risk of rheumatoid arthritis and has a negative impact on human height. Our results provide suggestive evidence for the involvement of the MHC class III genes in the pathogenesis of PVVs. Further independent studies are needed to confirm our pilot findings.

Allele rs2010963 C of the VEGFA gene is associated with the decreased risk of primary varicose veins in ethnic Russians.

Objective To study the association of polymorphisms rs699947, rs2010963, rs3025039 in the VEGFA gene region and rs1870377, rs2305949, rs2071559 in the VEGFR2 gene region with the risk of primary varicose veins in ethnic Russians. Methods Genotypes were determined by real-time PCR allelic discrimination. The case group consisted of 448 patients with primary varicose veins and the control group comprised 609 individuals without a history of chronic venous disease. Association was studied by logistic regression analysis. Results Allele rs2010963 C was associated with the decreased risk of varicose veins (additive model of inheritance: odds ratio = 0.73, 95% confidence interval = 0.59-0.91, P = 0.004). Conclusions Our results provide evidence that polymorphism rs2010963 located in the 5' untranslated region of the VEGFA gene can influence genetic susceptibility to primary varicose veins in Russians. Otherwise, it can be in linkage disequilibrium with another functional single nucleotide polymorphism that can alter the level of vascular endothelial growth factor A protein.

Polymorphisms in inflammation-related genes and the risk of primary varicose veins in ethnic Russians.

Inflammation was shown to be activated in varicose veins, although its role in the development of vein wall transformation remains inconclusive. We aimed to investigate the influence of 13 inflammation-related single nucleotide polymorphisms (SNPs) TNF rs1800629 and rs3093661, IL1A rs1800587, IL1RN rs4251961, IL6 rs1800795 and rs1800796, IFNG rs2430561, IL10 rs1800896, TGFB1 rs1800469, HIF1A rs11549465, NFKB1 rs28362491, and rs4648068 on the risk of primary varicose veins (PVVs) in ethnic Russians. We genotyped 709 patients with PVVs and 278 individuals without a history of chronic venous disease and performed a single SNP and a haplotype analysis. Several associations with P < 0.05 were revealed in our study. Variant allele HIF1A rs11549465 T, TNF rs3093661 A, and NFKB1 rs28362491 ATTG deletion showed the reverse association with PVV risk, and allele IL6 rs1800795 C was associated with the increased risk of the studied pathology. Haplotype analysis revealed associations of TNF haplotypes rs3093661 A-rs1800629 G and IL6 rs1800795 C-rs1800796 G with the decreased and the increased risk of PVVs, correspondingly. However, all the observed associations failed to reach statistical significance after the correction for multiple testing, which was set at a level of 10-3 due to many tests performed. Our study therefore provides evidence that investigated polymorphisms do not play a major role in susceptibility to PVVs.

Functional polymorphism rs1024611 in the MCP1 gene is associated with the risk of varicose veins of lower extremities.

OBJECTIVE: Monocyte chemoattractant protein 1 (MCP-1) is a chemokine responsible for monocyte, basophil, and T-lymphocyte attraction. Polymorphism rs1024611 located in the regulatory region of the MCP1 gene has previously been shown to be associated with increased MCP-1 production. In our study, we aimed to examine the association of rs1024611 with the risk of primary varicose veins (PVVs) of lower extremities. METHODS: The case group comprised 470 patients with PVVs, and the control group included 269 individuals without a history of chronic venous disease. All cases and controls were ethnic Russians. Genotypes were determined by real-time polymerase chain reaction allelic discrimination. Association was studied by logistic regression analysis. RESULTS: We revealed the association of genotype G/G with the increased risk of PVVs (odds ratio [OR], 1.87; 95% confidence interval [CI], 1.02-3.44; P = .04). In the subgroup analysis, association was revealed only in patients with C2 Clinical, Etiology, Anatomy, and Pathophysiology class (allele G: OR, 1.62 [95% CI, 1.13-2.33; P = .008]; genotype G/G: OR, 3.22 [95% CI, 1.43-7.27; P = .005]), in patients with age at onset of PVVs before 30 years (allele G: OR, 1.41 [95% CI, 1.08-1.85; P = .01]; genotype G/G: OR, 2.35 [95% CI, 1.22-4.55; P = .01]), and in patients who declared no family history (allele G: OR, 1.46 [95% CI, 1.02-2.09; P = .04]; genotype G/G: OR, 2.50 [95% CI, 1.11-5.63; P = .03]). CONCLUSIONS: Our results provide evidence for MCP-1 involvement in the development of PVVs and indicate that inflammation could be implicated in the pathogenesis of this condition.

Polymorphisms in the MTHFR and MTR genes and the risk of varicose veins in ethnical Russians.

OBJECTIVES: The objective of this study was to study the association of polymorphisms MTHFR C677T (rs1801133) and MTR A2756G (rs1805087) with the risk of varicose veins in ethnical Russians. METHODS: We genotyped 475 patients with varicose veins, 168 individual without chronic venous disease, and the population-based group of 896 subjects. Association was studied using logistic regression analysis adopting co-dominant, additive, recessive, and dominant models of inheritance. RESULTS: None of the polymorphisms showed a statistically significant association with the risk of varicose veins. CONCLUSIONS: Our results provide evidence that the studied polymorphisms do not contribute to genetic susceptibility to varicose veins in ethnical Russians.

HFE p.C282Y gene variant is associated with varicose veins in Russian population.

Recently, the association of polymorphism rs1800562 (p.C282Y) in the hemochromatosis (HFE) gene with the increased risk of venous ulceration was shown. We hypothesized that HFE gene polymorphism might be involved not only in ulceration process, but also in susceptibility to primary varicose veins. We genotyped HFE p.C282Y (rs1800562) and p.H63D (rs1799945) variants in patients with primary varicose veins (n = 463) and in the control group (n = 754). In our study, p.282Y variant (rs1800562 A allele) was significantly associated with the risk of varicose veins (OR 1.79, 95 % CI = 1.11-2.89, P = 0.02). A borderline significant reverse association of p.63D variant (rs1799945 G allele) with venous leg ulcer development was revealed in Russians (OR 0.25, 95 % CI = 0.06-1.00, P = 0.05), but not in the meta-analysis (P = 0.56). We conclude that the HFE gene polymorphism can affect the risk of developing primary varicose veins.

Association of polymorphisms near the FOXC2 gene with the risk of varicose veins in ethnic Russians.

OBJECTIVE: To investigate the association of polymorphisms located near the FOXC2 gene with the risk of varicose veins in ethnic Russians. METHODS: Allele, genotype, and haplotype frequencies were determined in the sample of 474 patients with primary varicose veins and in the control group of 478 individuals without a history of chronic venous disease. RESULTS: Polymorphisms rs7189489, rs4633732, and rs1035550 showed the association with the increased risk of varicose veins, but none of the observed associations remained significant after correction for multiple testing. Haplotype analysis revealed the association of haplotype rs7189489 C-rs4633732 T-rs34221221 C-rs1035550 C-rs34152738 T-rs12711457 G with the increased risk of varicose veins (OR = 2.67, P = 0.01). CONCLUSIONS: Our results provide evidence that the studied polymorphisms do not play a major role in susceptibility to varicose veins development in the Russian population.

Ortho-aminoazotoluene activates mouse constitutive androstane receptor (mCAR) and increases expression of mCAR target genes.

2'-3-dimethyl-4-aminoazobenzene (ortho-aminoazotoluene, OAT) is an azo dye and a rodent carcinogen that has been evaluated by the International Agency for Research on Cancer (IARC) as a possible (class 2B) human carcinogen. Its mechanism of action remains unclear. We examined the role of the xenobiotic receptor Constitutive Androstane Receptor (CAR, NR1I3) as a mediator of the effects of OAT. We found that OAT increases mouse CAR (mCAR) transactivation in a dose-dependent manner. This effect is specific because another closely related azo dye, 3'-methyl-4-dimethyl-aminoazobenzene (3'MeDAB), did not activate mCAR. Real-time Q-PCR analysis in wild-type C57BL/6 mice revealed that OAT induces the hepatic mRNA expression of the following CAR target genes: Cyp2b10, Cyp2c29, Cyp3a11, Ugt1a1, Mrp4, Mrp2 and c-Myc. CAR-null (Car(-/-)) mice showed no increased expression of these genes following OAT treatment, demonstrating that CAR is required for their OAT dependent induction. The OAT-induced CAR-dependent increase of Cyp2b10 and c-Myc expression was confirmed by Western blotting. Immunohistochemistry analysis of wild-type and Car(-/-) livers showed that OAT did not acutely induce hepatocyte proliferation, but at much later time points showed an unexpected CAR-dependent proliferative response. These studies demonstrate that mCAR is an OAT xenosensor, and indicate that at least some of the biological effects of this compound are mediated by this nuclear receptor.

The increased CAR-dependent metabolism of thyroid hormones in mice with high cancer susceptibility.

AIM: our aim was to compare activation of the constitutive androstane receptor (CAR), hepatic expression of its target genes, and the serum thyroid hormone levels in C3H/He, C57BL/6J, and CC57BR/Mv mice following phenobarbital treatment. These differences, if present, could help to explain the different susceptibility to phenobarbital-induced liver tumor promotion among these strains of mice. MAIN METHODS: CAR DNA-binding activity and CAR content in nuclear protein extracts from mouse livers were assessed using the electrophoretic mobility shift assay and immunoblotting. Serum thyroid hormone concentrations were determined by radioimmunoassay. Real-time PCR was used to measure the hepatic expression level of CAR target genes. KEY FINDINGS: we found a 2.3-fold increase of CAR DNA-binding activity in response to phenobarbital in the sensitive C3H/He mice, but no change in the relatively resistant C57BL/6J and CC57BR/Mv mice. Phenobarbital treatment caused a significant decrease in triiodothyronine and free thyroxine concentrations (17% and 40%, respectively) in the sensitive C3H/He mice by the end of 60-day treatment, while in the resistant mice, these changes were not observed. In the sensitive C3H/He mice only, the expression of a CAR target gene encoding sulfotransferase Sult2a1, the thyroid hormone inactivation enzyme, increased by 260-fold after phenobarbital administration. The expression of another CAR target gene, Mdm2, was also increased by phenobarbital treatment in C3H/He mice. SIGNIFICANCE: we have shown that phenobarbital activates CAR and increases the expression of its target genes thereby accelerating the metabolism of thyroid hormones only in mice susceptible to liver tumor promotion by phenobarbital, but not in relatively resistant animals.