Let-7a Downregulation Accompanied by KRAS Mutation Is Predictive of Lung Cancer Onset in Cigarette Smoke-Exposed Mice.

BACKGROUND: Let-7 is a tumor suppressor microRNA targeting the KRAS lung oncogene. Let-7a downregulation is reversible during the early stages of lung carcinogenesis but is irreversible in cancer cells. The aim of this study is to shed light on the relationship between oncogene (KRAS) mutation and let-7a downregulation in cigarette smoke (CS)-induced lung carcinogenesis. METHODS: A total of 184 strain H Swiss albino mice were either unexposed (control) or exposed to CS for 2 weeks (short CS) or 8 months (long CS). After 8 months, the lungs were individually collected. The following end points have been evaluated: (a) DNA methylation of the let-7a gene promoter by bisulphite-PCR and pyrosequencing; (b) let-7a expression by qPCR; (c) KRAS mutation by DNA pyrosequencing; (d) cancer incidence by histopathological examination. RESULTS: let-7a expression decreased by 8.3% in the mice exposed to CS for two weeks (CS short) and by 33.4% (p ≤ 0.01) in the mice exposed to CS for 8 months (CS long). No significant difference was detected in the rate of let-7a-promoter methylation between the Sham-exposed mice (55.1%) and the CS short-(53%) or CS long (51%)-exposed mice. The percentage of G/T transversions in KRAS codons 12 and 13 increased from 2.3% (Sham) to 6.4% in CS short- and to 11.5% in CS long-exposed mice. Cancer incidence increased significantly in the CS long-exposed mice (11%) as compared to both the Sham (4%) and the CS short-exposed (2%) mice. In the CS long-exposed mice, the correlation between let-7a expression and the number of KRAS mutations was positive (R = +0.5506) in the cancer-free mice and negative (R = -0.5568) in the cancer-bearing mice. CONCLUSIONS: The effects of CS-induced mutations in KRAS are neutralized by the high expression of let-7a in cancer-free mice (positive correlation) but not in cancer-bearing mice where an irreversible let-7a downregulation occurs (negative correlation). This result provides evidence that both genetic (high load of KRAS mutation) and epigenetic alterations (let-7a irreversible downregulation) are required to produce lung cancer in CS-exposed organisms.

Air pollution and human endogenous retrovirus methylation in the school inner-city asthma intervention study.

Human endogenous retroviruses (HERVs) are transposable genomic elements generally repressed through DNA methylation. HERVs can be demethylated and expressed in response to environmental stimuli. Therefore, more research is needed to understand the influence of environmental exposures on HERV methylation. Air pollutants are commonly linked with global hypomethylation, and as HERVs comprise of nearly 8% of repetitive elements in the human genome, our objective was to examine the association between air pollutant exposure and HERV methylation. We investigated 180 students with asthma participating in the School Inner-City Asthma Intervention Study, which evaluated the efficacy of classroom air filters and school-wide pest management on air pollutant/allergen exposure and asthma. Both air pollutants measured in classrooms and asthma outcomes assessed by surveys were collected pre- and post-intervention. Buccal swabs were also collected pre- and post-intervention, and methylation levels from 9 transposable genomic elements (HERV-E, -FRD, -K, -L, -R, -W, -9, and HRES and LINE1) were measured. Adjusting for relevant covariates, the overall air pollutant mixture was cross-sectionally associated with higher HERV-W and lower HERV-L and LINE1 methylation. Coarse PM was cross-sectionally associated with higher HERV-K methylation and CO2 with lower LINE1 methylation. These results suggest that exposure to air pollutants is associated with HERV-W and HERV-K hypermethylation and HERV-L and LINE1 hypomethylation in children with asthma. Future studies are needed to characterize the links between HERV methylation and possible adverse outcomes.

Association between night shift work and methylation of a subset of immune-related genes.

Introduction: Night shift (NS) work has been associated with an increased risk of different conditions characterized by altered inflammatory and immune responses, such as cardio-metabolic and infectious diseases, cancer, and obesity. Epigenetic modifications, such as DNA methylation, might mirror alterations in biological processes that are influenced by NS work. Methods: The present study was conducted on 94 healthy female workers with different working schedules and aimed at identifying whether NS was associated with plasmatic concentrations of the inflammatory proteins NLRP3 and TNF-alpha, as well as with DNA methylation levels of ten human endogenous retroviral (HERV) sequences, and nine genes selected for their role in immune and inflammatory processes. We also explored the possible role of the body mass index (BMI) as an additional susceptibility factor that might influence the effects of NS work on the tested epigenetic modifications. Results and discussion: We observed a positive association between NS and NLRP3 levels (p-value 0.0379). Moreover, NS workers retained different methylation levels for ERVFRD-1 (p-value = 0.0274), HERV-L (p-value = 0.0377), and HERV-P (p-value = 0.0140) elements, and for BIRC2 (p-value = 0.0460), FLRT3 (p-value = 0.0422), MIG6 (p-value = 0.0085), and SIRT1 (p-value = 0.0497) genes. We also observed that the BMI modified the relationship between NS and the methylation of ERVE, HERV-L, and ERVW-1 elements. Overall, our results suggest that HERV methylation could pose as a promising biomolecular sensor to monitor not only the effect of NS work but also the cumulative effect of multiple stressors.

The Methylation of Clock Genes in Perinatal Depression: Which Role for Oxytocin?

Background: Perinatal Depression (PD) is a widespread disabling condition that is hypothesized to be associated with abnormalities in circadian rhythms and neuropeptide release including oxytocin (OXT). Methods: Fourty-four pregnant women (28 with PD, and 16 controls) were evaluated through the Edinburgh Postnatal Depression Scale (EPDS), the State/Trait Anxiety Inventory Form Y (STAI-Y), and the Prenatal Attachment Inventory (PAI). A blood sample was collected from all participants, and OXT plasma levels, DNA methylation of clock genes, as well as of FOXp3 and HERV-W were measured. Linear regression analyses were performed to assess the effect of oxytocin on the methylation of selected genes. Continuous ordinal regression models was further applied to see if the score of rating scales was associated to gene methylation, adjusting for oxytocin-methylation interaction. Results: OXT plasma levels were positively associated with CRY1 methylation. Women with higher OXT plasma levels showed an association between higher degree of CRY2 methylation (thus, reduced expression) and lower EPDS (OR = 0.21; P = 0.043) and STAI-S scores (OR = 6.96; P = 0.019). Finally, with high OXT levels, hypermethylation of CRY1 was associated to higher scores on the PAI (OR = 2.74; P = 0.029) while higher methylation of HERV-W related to lower PAI scores (OR = 0.273; P = 0.019). Conclusion: Our results suggest a possible protective role played by oxytocin in the development of PD by promoting a favorable methylation profile characterized by reduced expression of CRY1 and CRY2. Moreover, oxytocin strengthens the association between maternal prenatal attachment with a favorable pattern of methylation of clock genes and HERV-W, which is essential for pregnancy outcomes.

Stress Modifies the Expression of Glucocorticoid-Responsive Genes by Acting at Epigenetic Levels in the Rat Prefrontal Cortex: Modulatory Activity of Lurasidone.

Epigenetics is one of the mechanisms by which environmental factors can alter brain function and may contribute to central nervous system disorders. Alterations of DNA methylation and miRNA expression can induce long-lasting changes in neurobiological processes. Hence, we investigated the effect of chronic stress, by employing the chronic mild stress (CMS) and the chronic restraint stress protocol, in adult male rats, on the glucocorticoid receptor (GR) function. We focused on DNA methylation specifically in the proximity of the glucocorticoid responsive element (GRE) of the GR responsive genes Gadd45ß, Sgk1, and Gilz and on selected miRNA targeting these genes. Moreover, we assessed the role of the antipsychotic lurasidone in modulating these alterations. Chronic stress downregulated Gadd45ß and Gilz gene expression and lurasidone normalized the Gadd45ß modification. At the epigenetic level, CMS induced hypermethylation of the GRE of Gadd45ß gene, an effect prevented by lurasidone treatment. These stress-induced alterations were still present even after a period of rest from stress, indicating the enduring nature of such changes. However, the contribution of miRNA to the alterations in gene expression was moderate in our experimental conditions. Our results demonstrated that chronic stress mainly affects Gadd45ß expression and methylation, effects that are prolonged over time, suggesting that stress leads to changes in DNA methylation that last also after the cessation of stress procedure, and that lurasidone is a modifier of such mechanisms.

The Independent Role of Body Mass Index (BMI) and Severity of Depressive Symptoms on Biological Changes of Women Affected by Overweight/Obesity.

Background: Both obesity and depression are medical conditions associated with severe disability and biological abnormalities. Our aim was to study associations between Body Mass Index (BMI), depression and biological changes in women affected by overweight or obesity. Methods: Depressive symptoms were evaluated by the Beck Depression Inventory II (BDI-II) questionnaire in 200 women affected by overweight/obesity (mean age of the sample 52.7 ± 12.9 years, BMI 33.8 ± 5.5 kg/m2). A blood sample was obtained for evaluation of biochemical (oxytocin and vitamin D), inflammatory and epigenetic (methylation of clock genes) parameters. Multivariable linear regression models were used to study the association between BMI or severity of depressive symptoms (BDI-II scores) with different biomarkers. Results: BMI was found to be associated with severity of depressive symptoms (p = 0.050). Severity of obesity resulted to be associated with lower plasma levels of oxytocin (p = 0.053), vitamin D deficiency (p = 0.006) and higher plasma levels of IFN-γ (p = 0.004), IL-6 (p = 0.013), IL-7 (p = 0.013), TNF-alpha (p = 0.036) and chemokine ligand 3 (CCL3) (p = 0.013, R2 = 0.03). Severity of depression was significantly associated with more methylation of clock genes CRY1 (p = 0.034, R2 = 0.16) and CRY2 (p = 0.019, R2 = 0.47). More severe depression together with higher levels of IL-8 strongly predicted lower methylation of CLOCK gene (p = 0.009); Conclusions: Different biological abnormalities have been found to be independently associated with BMI and severity of depressive symptoms in women affected by overweight/obesity. The complex interplay between overweight, depression and biological changes will have to be better clarified by future studies.

Effects of PM Exposure on the Methylation of Clock Genes in a Population of Subjects with Overweight or Obesity.

The expression of clock genes, regulating the synchronization of metabolic and behavioral processes with environmental light/dark cycles, is regulated by methylation and might be influenced by short-term exposure to airborne particulate matter (PM), especially in individuals that are hypersensitive to proinflammatory cues. The present study aimed to evaluate the effects of PM2.5 and PM10 on the methylation profile of the clock genes ARNTL, CLOCK, CRY1, CRY2, PER1, PER2, and PER3 in a population of 200 women with obesity. A significant association between PM10 exposure and the methylation of clock genes was found, namely, this was negative for PER2 gene and positive for the CLOCK, CRY1, CRY2, and PER3 genes. PM2.5 was negatively associated with methylation of PER2 gene and positively with methylation of CRY2 gene. Evidence was observed for effect modification from body mass index (BMI) regarding the PER1 gene: as PM2.5/10 increases, DNA methylation increases significantly for relatively low BMI values (BMI = 25), while it decreases in participants with severe obesity (BMI = 51). PM may therefore alter the epigenetic regulation of clock genes, possibly affecting circadian rhythms. Future studies are needed to clarify how alterations in clock gene methylation are predictive of disease development and how obesity can modulate the adverse health effects of PM.

Human Endogenous Retroviruses Long Terminal Repeat Methylation, Transcription, and Protein Expression in Human Colon Cancer.

Colon cancer is the fourth most common malignancy in both incidence and mortality in developed countries. Infectious agents are among the risk factors for colon cancer. Variations in human endogenous retrovirus (HERV) transcript and protein levels are associated with several types of cancers, but few studies address HERV expression in colon cancer. Fifty-eight patients with advanced-stage colon cancer were enrolled in this study. HERV-H, -K (HML-2), -P LTRs, Alu, and LINE-1 methylation levels and transcription of HERV-H, -K (HML-2), and -P env and HERV-K pol genes in normal adjacent and tumor tissues were investigated by pyrosequencing and RT-qPCR, respectively. Expression of the HERV-K (HML-2) Pol and Env proteins in selected tissues was examined by Western blotting. Associations between HERV transcript expression and methylation levels and between clinical characteristics and HERV expression were evaluated. Compared to adjacent normal tissues, LINE-1 was hypomethylated in tumor tissues (p < 0.05), whereas Alu, HERV-K (HML-2), and -H LTRs showed a decreasing trend in tumor tissue compared to normal tissue, though without a significant difference. The transcription levels of HERV env and pol genes were similar. However, the HERV-K (HML-2) Pol protein was more highly expressed (p < 0.01) in surrounding normal tissues, but the HERV-K (HML-2) Env protein was only expressed in tumor tissues. Although HERV LTR methylation and gene expression did not show significant differences between tumor and normal tissues, HERV protein expression differed greatly. Pol protein expression in normal cells may induce reverse transcription and subsequent integration into the host genome, likely favoring cell transformation; in contrast, the Env protein in tumor tissue may contribute to cancer progression through cell-to-cell fusion.

DNA methylation level in blood and relations to breast cancer, risk factors and environmental exposure in Greenlandic Inuit women.

Several studies have found aberrant DNA methylation levels in breast cancer cases, but factors influencing DNA methylation patterns and the mechanisms are not well understood. This case-control study evaluated blood methylation level of two repetitive elements and selected breast cancer-related genes in relation to breast cancer risk, and the associations with serum level of persistent organic pollutants (POPs) and breast cancer risk factors in Greenlandic Inuit. DNA methylation was determined using bisulphite pyrosequencing in blood from 74 breast cancer cases and 80 controls. Using first tertile as reference, the following was observed. Positive associations for ATM in second tertile (OR: 2.33, 95% CI: 1.04; 5.23) and ESR2 in third tertile (OR: 2.22, 95% CI: 0.97; 5.05) suggest an increased breast cancer risk with high DNA methylation. LINE-1 methylation was lower in cases than controls. In third tertile (OR: 0.42, 95% CI: 0.18; 0.98), associations suggest in accordance with the literature an increased risk of breast cancer with LINE-1 hypomethylation. Among controls, significant associations between methylation levels and serum level of POPs and breast cancer risk factors (age, body mass index, cotinine level) were found. Thus, breast cancer risk factors and POPs may alter the risk through changes in methylation levels; further studies are needed to elucidate the mechanisms.

Human endogenous retroviruses env gene expression and long terminal repeat methylation in colorectal cancer patients.

Human endogenous retroviruses (HERV) are remnants of exogenous retroviral infections, representing 8% of the human genome. Their regulation is based on the DNA methylation of promoters, the long terminal repeats (LTRs). Transcripts from HERV have been associated with cancers, but reports concerning HERV expression in colorectal cancer remain sporadic. Sixty-three patients with advanced stages of colorectal cancer were enrolled in this study. The expressions of HERV env gene, and HERV-H, -K, -R and -P LTRs and Alu, LINE-1 methylation levels, were investigated in the tumor, normal adjacent tissues, and, where possible, blood and plasmatic extracellular vesicles (EVs). Associations among HERV env expression, methylation status and clinical characteristics were evaluated. No differences were observed in HERV env gene expression levels among the clinical specimens, while Alu, LINE-1, HERV-H and -K LTRs were demethylated in the tumor compared to the normal adjacent tissues (p < 0.05).The HERV env gene was expressed in the EVs at of 54% (-H), 38% (-K), 31% (-R) patients. Association was not found between HERV env expression and LTR methylation, but significant higher expression of HERV-P and -R env was found in tumor tissues arising from the right colon. Our findings do not demonstrate significant overexpression of the studied HERV in colorectal cancer, but their association with tumor localization and specificity of the changes in DNA methylation of retroelements are shown. HERV sequences were packaged in the EVs and might be transferred from one cell to another.

Targeted resequencing of FECH locus reveals that a novel deep intronic pathogenic variant and eQTLs may cause erythropoietic protoporphyria (EPP) through a methylation-dependent mechanism.

PURPOSE: Existing data do not explain the reason why some individuals homozygous for the hypomorphic FECH allele develop erythropoietic protoporphyria (EPP) while the majority are completely asymptomatic. This study aims to identify novel possible genetic variants contributing to this variable phenotype. METHODS: High-throughput resequencing of the FECH gene, qualitative analysis of RNA, and quantitative DNA methylation examination were performed on a cohort of 72 subjects. RESULTS: A novel deep intronic variant was found in four homozygous carriers developing a clinically overt disease. We demonstrate that this genetic variant leads to the insertion of a pseudo-exon containing a stop codon in the mature FECH transcript by the abolition of an exonic splicing silencer site and the concurrent institution of a new methylated CpG dinucleotide. Moreover, we show that the hypomorphic FECH allele is linked to a single haplotype of about 20 kb in size that encompasses three noncoding variants that were previously associated with expression quantitative trait loci (eQTLs). CONCLUSION: This study confirms that intronic variants could explain the variability in the clinical manifestations of EPP. Moreover, it supports the hypothesis that the control of the FECH gene expression can be mediated through a methylation-dependent modulation of the precursor messenger RNA (pre-mRNA) splicing pattern.

Effects of Physical Exercise on Endothelial Function and DNA Methylation.

Essential hypertension is the leading preventable cause of death in the world. Epidemiological studies have shown that physical training can reduce blood pressure (BP), both in hypertensive and healthy individuals. Increasing evidence is emerging that DNA methylation is involved in alteration of the phenotype and of vascular function in response to environmental stimuli. We evaluated repetitive element and gene-specific DNA methylation in peripheral blood leukocytes of 68 volunteers, taken before (T0) and after (T1) a three-month intervention protocol of continuative aerobic physical exercise. DNA methylation was assessed by bisulfite-PCR and pyrosequencing. Comparing T0 and T1 measurements, we found an increase in oxygen consumption at peak of exercise (VO2peak) and a decrease in diastolic BP at rest. Exercise increased the levels of ALU and Long Interspersed Nuclear Element 1 (LINE-1) repetitive elements methylation, and of Endothelin-1 (EDN1), Inducible Nitric Oxide Synthase (NOS2), and Tumour Necrosis Factor Alpha (TNF) gene-specific methylation. VO2peak was positively associated with methylation of ALU, EDN1, NOS2, and TNF; systolic BP at rest was inversely associated with LINE-1, EDN1, and NOS2 methylation; diastolic BP was inversely associated with EDN1 and NOS2 methylation. Our findings suggest a possible role of DNA methylation for lowering systemic BP induced by the continuative aerobic physical training program.

Extracellular Vesicles Released by Colorectal Cancer Cell Lines Modulate Innate Immune Response in Zebrafish Model: The Possible Role of Human Endogenous Retroviruses.

Extracellular vesicles (EVs) are important components of the metastatic niche and are crucial in infiltration, metastasis, and immune tolerance processes during tumorigenesis. We hypothesized that human endogenous retroviruses (HERV) positive EVs derived from tumor cellsmay have a role in modulating the innate immune response. The study was conducted in two different colorectal cancer cell lines, representing different stages of cancer development: Caco-2, derived from a non-metastatic colorectal adenocarcinoma, and SK-CO-1, derived from metastatic colorectal adenocarcinoma (ascites). Both cell lines were treated with decitabine to induce global hypomethylation and to reactivate HERV expression. EVs were quantified by nanoparticle tracking analysis, and HERV-positive EV concentrations were measured by flow cytometry. The effect of EVs isolated from both untreated and decitabine-treated cells on the innate immune response was evaluated by injecting them in zebrafish embryos and then assessing Interleukin 1ß (IL1-ß), Interleukin 10 (IL-10), and the myeloperoxidase (mpx) expression levels by real-time qPCR. Interestingly, HERV-K positive EVs concentrations were significantly associated with a reduced expression of IL1-ß and mpx, supporting our hypothesis that HERV-positive EVs may act as immunomodulators in tumor progression. The obtained results open new perspectives about the modulation of the immune response in cancer therapy.

Night Shift Work, DNA Methylation and Telomere Length: An Investigation on Hospital Female Nurses.

Increased breast cancer risk has been reported in some night shift (NS) workers but underlying biological mechanisms are still unclear. We assessed the association between NS work and DNA methylation of tumor suppressor (TP53, CDKN2A, BRCA1, BRCA2) and estrogen receptor (ESR1, ESR2) genes, methylation of repetitive elements (LINE-1, Alu), and telomere length (TL). Forty six female nurses employed in NS for at least two years were matched by age (30-45 years) and length of service (≥1 year) with 51 female colleagues not working in NS. Each subject underwent a semi-structured interview and gave a blood sample. We applied linear regression and spline models adjusted for age, BMI, smoking habit, oral contraceptive use, parity and marital status/age at marriage. Currently working in NS was associated with ESR1 hypomethylation (ß: -1.85 (95%CI: -3.03; -0.67), p = 0.003). In current and former NS workers we observed TP53 (-0.93 (-1.73; -0.12), p = 0.03) and BRCA1 (-1.14 (-1.71; -0.58), p <0.001) hypomethylation. We found an increase between TL and number of years in NS in subjects employed in NS <12 years (0.06 (0.03; 0.09), p <0.001), while a decrease if employed in NS ≥12 years (-0.07 -0.10; -0.04), p <0.001). Our findings show NS-associated markers potentially involved in cellular aging, genomic instability, and cancer development.

50-Hz MF does not affect global DNA methylation of SH-SY5Y cells treated with the neurotoxin MPP.

Exposure to extremely low frequency magnetic fields (ELF-MFs) has been associated with an increased risk of neurodegenerative disorders. The underlying mechanisms, however, are still debated. Since epigenetics play a key role in the neurodegenerative process, we investigated whether exposure to ELF-MF (50 Hz, 1 mT) might affect global DNA methylation of SH-SY5Y dopaminergic-like neuroblastoma cells. We assessed the percentage of 5-methylcytosine (5-mC) of three repetitive interspersed sequences (ALU, LINE-1, or SATα), through pyrosequencing analysis. We demonstrated that ELF exposure (up to 72 h) does not induce any change in the methylation pattern of ALU, LINE-1, and SATα in both proliferating and differentiated SH-SY5Y cells. Furthermore, when administered in combination with 1-methyl-4-phenylpyridinium (MPP+ ), a neurotoxin mimicking the Parkinson's Disease (PD) phenotype, ELF-MF exposure does not trigger any modulation in the percentage of 5-mC of the repetitive elements. Our findings demonstrate that exposure to 50-Hz MF does not affect global DNA methylation in proliferating and dopaminergic differentiated SH-SY5Y cells, either under basal culture conditions or under neurotoxic stress. Bioelectromagnetics. 40:33-41, 2019. © 2018 Bioelectromagnetics Society.

Placental circadian pathway methylation and in utero exposure to fine particle air pollution.

In mammals, a central clock maintains the daily rhythm in accordance with the external environment. At the molecular level, the circadian rhythm is maintained by epigenetic regulation of the Circadian pathway. Here, we tested the role of particulate matter with an aerodynamic diameter ≤ 2.5 µm (PM2.5) exposure during gestational life on human placental Circadian pathway methylation, as an important molecular target for healthy development. In 407 newborns, we quantified placental methylation of CpG sites within the promoter regions of the following genes: CLOCK, BMAL1, NPAS2, CRY1-2 and PER1-3 using bisulfite-PCR-pyrosequencing. Daily PM2.5 exposure levels were estimated for each mother's residence, using a spatiotemporal interpolation model. We applied mixed-effects models to study the methylation status of the Circadian pathway genes and in utero PM2.5 exposure, while adjusting for a priori chosen covariates. In a multi-gene model, placental Circadian pathway methylation was positively and significantly (p < 0.0001) associated with 3rd trimester PM2.5 exposure. Consequently, the single-gene models showed relative methylation differences [Log(fold change)] in placental NPAS2 (+0.16; p = 0.001), CRY1 (+0.59; p = 0.0023), PER2 (+0.36; p = 0.0005), and PER3 (+0.42; p = 0.0008) for an IQR increase (8.9 µg/m3) in 3rd trimester PM2.5 exposure. PM2.5 air pollution, an environmental risk factor leading to a pro-inflammatory state of the mother and foetus, is associated with the methylation pattern of genes in the Circadian pathway. The observed alterations in the placental CLOCK epigenetic signature might form a relevant molecular mechanism through which fine particle air pollution exposure might affect placental processes and foetal development.

Extracellular vesicle-packaged miRNA release after short-term exposure to particulate matter is associated with increased coagulation.

BACKGROUND: Exposure to particulate matter (PM) is associated with increased incidence of cardiovascular disease and increased coagulation, but the molecular mechanisms underlying these associations remain unknown. Obesity may increase susceptibility to the adverse effects of PM exposure, exacerbating the effects on cardiovascular diseases. Extracellular vesicles (EVs), which travel in body fluids and transfer microRNAs (miRNAs) between tissues, might play an important role in PM-induced cardiovascular risk. We sought to determine whether the levels of PM with an aerodynamic diameter ≤ 10 µm (PM10) are associated with changes in fibrinogen levels, EV release, and the miRNA content of EVs (EV-miRNAs), investigating 1630 overweight/obese subjects from the SPHERE Study. RESULTS: Short-term exposure to PM10 (Day before blood drawing) was associated with an increased release of EVs quantified by nanoparticle tracking analysis, especially EVs derived from monocyte/macrophage components (CD14+) and platelets (CD61+) which were characterized by flow cytometry. We first profiled miRNAs of 883 subjects by the QuantStudio™ 12 K Flex Real Time PCR System and the top 40 EV-miRNAs were validated through custom miRNA plates. Nine EV-miRNAs (let-7c-5p; miR-106a-5p; miR-143-3p; miR-185-5p; miR-218-5p; miR-331-3p; miR-642-5p; miR-652-3p; miR-99b-5p) were downregulated in response to PM10 exposure and exhibited putative roles in cardiovascular disease, as highlighted by integrated network analysis. PM10 exposure was significantly associated with elevated fibrinogen levels, and five of the nine downregulated EV-miRNAs were mediators between PM10 exposure and fibrinogen levels. CONCLUSIONS: Research on EVs opens a new path to the investigation of the adverse health effects of air pollution exposure. EVs have the potential to act both as markers of PM susceptibility and as potential molecular mechanism in the chain of events connecting PM exposure to increased coagulation, which is frequently linked to exposure and CVD development.

Evaluation of DNA methylation of inflammatory genes following treatment of chronic periodontitis: A pilot case-control study.

OBJECTIVE: To evaluate the influence of periodontal therapy on DNA methylation in patients with chronic periodontitis as compared to healthy individuals. MATERIAL AND METHODS: Twenty patients were enrolled into two groups: (i) 10 diagnosed as clinically healthy; and (ii) 10 diagnosed with chronic periodontitis. Clinical measures were recorded and gingival biopsies were harvested at baseline (both patient groups) and at 2 and 8 weeks post-baseline for diseased individuals. Molecular DNA methylation analysis was performed by pyrosequencing for the putative inflammation-associated genes LINE-1, COX-2, IFN-γ and TNF-α. Random-intercept linear regression models were applied to evaluate methylation levels across groups at baseline and the methylation changes over time in the diseased and normal tissues. RESULTS: Periodontal therapy did not influence gene expression methylation of TNF-α, IFN-γ and LINE-1 levels at normal and periodontitis sites over time. However, it significantly reduced COX-2 methylation levels comparable to healthy individuals at both 2 and 8 weeks post-treatment (p < .05). CONCLUSIONS: Periodontal therapy resets the DNA methylation status of inflammatory gene for COX-2 in patients with periodontal disease. DNA methylation levels of TNF-α, IFN-γ and LINE-1 were sustained in periodontitis sites despite therapy. Future studies should consider an expanded panel of inflammatory genes over time. (ClinicalTrials.gov NCT02835898).

Particulate matter exposure is associated with inflammatory gene methylation in obese subjects.

BACKGROUND: Overweight and obesity are becoming more widespread with alarming projections for the coming years. Obesity may increase susceptibility to the adverse effects of PM exposure, exacerbating the effects on cardiovascular diseases and altering the biomarkers of vascular inflammation. The associated biological mechanisms have not been fully understood yet; the common denominator in the pathogenesis of the co-morbidities of obesity is the presence of an active, low-grade inflammatory process. DNA methylation has been shown to regulate inflammatory pathways that are responsible for the development of cardiovascular diseases. OBJECTIVES: The aim of the study was to investigate, in a population of overweight/obese subjects, the effects of PM on blood DNA methylation in genes associated to inflammatory response. METHODS: Using bisulfite pyrosequencing, we measured DNA methylation in peripheral blood mononuclear cells from 186 overweighted/obese subjects. In particular, we quantified DNA methylation in a set of 3 candidate genes, including CD14, TLR4 and TNF-α, because of the important roles that these genes play in the inflammatory pathway. Personal exposure to PM10 was estimated for each subject based on the local PM10 concentrations, measured by monitoring stations at residential address. Repeated measure models were used to evaluate the association of PM10 with each genes, accounting for possible correlations among the genes that regulate the same inflammatory pathway. RESULTS: We found an inverse association between the daily PM10 exposure and the DNA methylation of inflammatory genes, measured in peripheral blood of healthy overweight/obese subjects. Considering different exposure time-windows, the effect on CD14 and TLR4 methylation was observed, respectively, in days 4-5-6, and days 6-7-8. TNF-α methylation was not associated to PM10. CONCLUSIONS: Our findings support a picture in which PM10 exposure and transcriptional regulation of inflammatory gene pathway in obese subjects are associated.