Laura Pergoli

DNA methylation in Repetitive Elements and Alzheimer disease

Epigenetics is believed to play a role in Alzheimers disease (AD). DNA methylation, the most investigated epigenetic hallmark, is a reversible mechanism that modifies genome function and chromosomal stability through the addition of methyl groups to cytosine located in CpG dinucleotides to form 5 methylcytosine (5mC). Methylation status of repetitive elements (i.e. Alu, LINE-1 and SAT-α) is a major contributor of global DNA methylation patterns and has been investigated in relation to a variety of human diseases. However, the role of methylation of repetitive elements in blood of AD patients has never been investigated so far. In the present study, a quantitative bisulfite-PCR pyrosequencing method was used to evaluate methylation of Alu, LINE-1 and SAT-α sequences in 43 AD patients and 38 healthy donors. In multivariate analysis adjusting for age and gender, LINE-1 was increased in AD patients compared with healthy volunteers (ADs: 83.6%5mC, volunteers: 83.1%5mC, p-value: 0.05). The group with best performances in mini mental state examination (MMSE) showed higher levels of LINE-1 methylation compared to the group with worst performances (MMSE>22: 83.9%5mC; MMSE≤22: 83.2%5mC; p=0.05). Our data suggest that LINE-1 methylation may lead to a better understanding of AD pathogenesis and course, and may contribute to identify novel markers useful to assess risk stratification. Further prospective investigations are warranted to evaluate the dynamics of DNA methylation from early-stage AD to advanced phases of the disease.

Microvesicle-associated microRNA expression is altered upon particulate matter exposure in healthy workers and in A549 cells

Cardiovascular disease risk has been consistently linked with particulate matter (PM) exposure. Cell‐derived microvesicles (MVs) are released into plasma and transfer microRNAs (miRNAs) between tissues. MVs can be produced by the respiratory system in response to proinflammatory triggers, enter the circulatory system and remotely modify gene expression in cardiovascular tissues. However, whether PM affects MV signaling has never been investigated. In this study, we evaluated expression of microRNAs contained within plasma MVs upon PM exposure both in vivo and in vitro. In the in vivo study, we isolated plasma MVs from healthy steel plant workers before and after workplace PM exposure. We measured the expression of 88 MV‐associated miRNAs by real‐time polymerase chain reaction. To assess a possible source of the MV miRNAs identified in vivo, we measured their miRNA expression in PM‐treated A549 pulmonary cell lines in vitro. MiRNA profiling of plasma MVs showed 5.62‐ and 13.95‐fold increased expression of miR‐128 and miR‐302c, respectively, after 3 days of workplace PM exposure (P < 0.001). According to Ingenuity Pathway Analysis, miR‐128 is part of coronary artery disease pathways, and miR‐302c is part of coronary artery disease, cardiac hypertrophy and heart failure pathways. In vitro experiments confirmed a dose‐dependent expression of miR‐128 in MVs released from A549 cells after 6 h of PM treatment (P = 0.030). MiR‐302c was expressed neither from A549 cells nor in reference lung RNA. These results suggest novel PM‐activated molecular mechanisms that may mediate the effects of air pollution and could lead to the identification of new diagnostic and therapeutic interventions. Copyright

Particulate matter induces prothrombotic microparticle shedding by human mononuclear and endothelial cells.

Particulate airborne pollution is associated with increased cardiopulmonary morbidity. Microparticles are extracellular vesicles shed by cells upon activation or apoptosis involved in physiological processes such as coagulation and inflammation, including airway inflammation. We investigated the hypothesis that particulate matter causes the shedding of microparticles by human mononuclear and endothelial cells. Cells, isolated from the blood and the umbilical cords of normal donors, were cultured in the presence of particulate from a standard reference. Microparticles were assessed in the supernatant as phosphatidylserine concentration. Microparticle-associated tissue factor was assessed by an one-stage clotting assay. Nanosight technology was used to evaluate microparticle size distribution. Particulate matter induces a dose- and time- dependent, rapid (1h) increase in microparticle generation in both cells. These microparticles express functional tissue factor. Particulate matter increases intracellular calcium concentration and phospholipase C inhibition reduces microparticle generation. Nanosight analysis confirmed that upon exposure to particulate matter both cells express particles with a size range consistent with the definition of microparticles (50-1000 nm). Exposure of mononuclear and endothelial cells to particulate matter upregulates the generation of microparticles at least partially mediated by calcium mobilization. This observation might provide a further link between airborne pollution and cardiopulmonary morbidity.

Blood DNA methylation, nevi number, and the risk of melanoma

Germline mutations determining increased cutaneous malignant melanoma (CMM) risk have been identified in familial and sporadic CMM cases, but they account only for a small proportion of CMM cases. Recent evidence suggests that germline epimutations (e.g. DNA methylation alterations), which can be inherited similarly to genomic mutations and can be detected in normal body cells (including blood), might increase susceptibility to cancer. The aim of the study was to identify germline epimutations of genes that were found to be mutated in familial CMM (p16INK4a, p14ARF, CDK4, MC1R, hTERT), immune and inflammatory genes (ICAM-1, TNF&agr;), DNA mismatch repair gene (MLH1), and repetitive elements (ALU, LINE-1, HERV-w). We measured DNA methylation using bisulfite pyrosequencing in peripheral blood mononuclear cells from 167 CMM cases and 164 sex-matched and age-matched controls. We used multivariable logistic regression models to evaluate the association between methylation levels and CMM status or presence of dysplastic nevi. We found an association between the risk of CMM and peripheral blood mononuclear cell methylation levels of TNF&agr; [odds ratio (OR)=1.11, 95% confidence interval (CI)=1.03–1.18], CDK4 (OR=0.76, 95% CI=0.64–0.91), and MLH1 (OR=1.12, 95% CI=1.02–1.22). In control participants, the risk of developing dysplastic nevi was associated with methylation levels of TNF&agr; (OR=0.81, 95% CI=0.69–0.95), hTERT (OR=0.90, 95% CI=0.82–0.99), and ALU (OR=1.56, 95% CI=1.02–2.39). Epimutations in CMM susceptibility genes and in genes involved in response to oxidative damage are associated with the risk of developing CMM or dysplastic nevi. Further studies measuring methylation levels of these genes in prospectively collected samples are warranted to further elucidate their role in the development and progression of CMM.

Short-term particulate matter exposure induces extracellular vesicle release in overweight subjects

Background: Extracellular vesicles (EVs) represent a plausible molecular mechanism linking particulate matter (PM) inhalation to its systemic effects. Microvesicles (MVs) are released from many cell types in response to various stimuli. Increased body mass index (BMI) could modify the response to PM exposure due to enhanced PM uptake and/or an underlying pro‐oxidative state. We investigated the relationship between EV release and PM10/PM2.5 exposure in a cohort of 51 volunteers. Subjects were stratified based on their BMI to evaluate whether overweight BMI is a determinant of hypersusceptibility to PM effects. Results: Exposure to PM10/PM2.5 was assessed with a personal sampler worn for 24 hours before plasma collection and confirmed with monitoring station data. Size and cellular origin of plasma EVs were characterized by Nanosight analysis and flow cytometry, respectively. Multivariate regression models were run after log‐transformation, stratifying subjects based on BMI (≥ or <25 kg/m2). PM exposure resulted in increased release of EVs, with the maximum observed effect for endothelial MVs. For PM10 and PM2.5, the adjusted geometric mean ratio and 95% confidence interval were 3.47 (1.30, 9.27) and 3.14 (1.23, 8.02), respectively. Compared to those in normal subjects, PM‐induced EV alterations in overweight subjects were more pronounced, with visibly effect in all MV subtypes, particularly endothelial MVs. Conclusions: Our findings emphasize the role of EV release after PM exposure and the susceptibility of overweight subjects. Larger studies with accurate exposure assessment and complete EVs characterization/content analysis, could further clarify the molecular mechanism responsible for PM effects and of hypersusceptibility of overweight subjects. HighlightsWe investigated the association between EV release and personal exposure to PM.Increased PM exposure was associated with increased release of EVs.The maximum effect was found for endothelial EVs.PM‐induced EV alterations were more pronounced in overweight subjects.

Extracellular vesicle-driven information mediates the long-term effects of particulate matter exposure on coagulation and inflammation pathways

BACKGROUND Continuous exposure to particulate air pollution (PM) is a serious worldwide threat to public health as it coherently links with increased morbidity and mortality of cardiorespiratory diseases (CRD), and of type 2 diabetes (T2D). Extracellular vesicles (EVs) are circular plasma membrane fragments released from human cells that transfer microRNAs between tissues. In the present work it was explored the hypothesis that EVs with their encapsulated microRNAs (EVmiRNAs) contents might mediate PM effects by triggering key pathways in CRD and T2D. METHODS Expression of EVmiRNAs analyzed by real-time PCR was correlated with oxidative stress, coagulation and inflammation markers, from healthy steel plant workers (n=55) with a well-characterized exposure to PM and PM-associated metals. All p-values were adjusted for multiple comparisons. In-silico Ingenuity Pathway Analysis (IPA) was performed to identify biological pathways regulated by PM-associated EVmiRNAs. RESULTS Increased expression in 17 EVmiRNAs is associated with PM and metal exposure (p<0.01). Mir-196b that tops the list, being related to 9 different metals, is fundamental in insulin biosynthesis, however three (miR-302b, miR-200c, miR-30d) out of these 17 EVmiRNAs are in turn also related to disruptions (p<0.01) in inflammatory and coagulation markers. CONCLUSIONS The studys findings support the hypothesis that adverse cardiovascular and metabolic effects stemming from inhalation exposures in particular to PM metallic component may be mediated by EVmiRNAs that target key factors in the inflammation, coagulation and glucose homeostasis pathways.

1281 Mirnas in extracellular vesiclesmediate the effect of particulate matter exposure on coagulation in a large sample of overweight/obese adults

Introduction In Italy about 45% of people aged ≥18 years are overweight/obese and might thus be more susceptible to the adverse health effects of air pollution exposure. Particulate matter ≤10 µm (PM10) represents a common pollutant of living and working environments and has been associated with increased risk of cardiovascular diseases (CVD) and hypercoagulability. Extracellular vesicles (EV) might play an important role in PM-related CVD, as they can travel in body fluids and transfer miRNAs between cells. We investigated whether PM10 exposure is associated with changes in fibrinogen levels, EV release, and EV-miRNA content in a large sample of overweight/obese adults. Methods EV concentrations were quantified by nanoparticle tracking analysis and flow cytometry. To identify altered levels of EV-miRNAs, we profiled miRNAs of 883 subjects by the QuantStudio 12K Flex Real Time PCR System. The top 40 EV-miRNAs were validated through custom miRNA plates. Statistical analyses included multiple linear regressions, mediation analysis and bioinformatics analysis. Results In a sample of 1630 overweight/obese subjects from the SPHERE ( S usceptibility to P article H ealth E ffects, mi R NAs and E xosomes) study, short-term exposure to PM10 was associated with increased release of EVs, especially those from monocyte/macrophage components (CD14+) and platelets (CD61+). Nine EV-miRNAs (let-7c-5p; miR-106a-5p; miR-143–3 p; miR-185–5 p; miR-218–5 p; miR-331–3 p; miR-642–5 p; miR-652–3 p; miR-99b-5p) were downregulated in response to PM10 exposure and exhibited putative roles in CVD. Five of these nine EV-miRNAs were mediators in the positive association between PM10 exposure and fibrinogen levels. Conclusions Our study sheds some light on the potential mechanisms underlying the adverse cardiovascular health effects of air pollution exposure. Our results were obtained in a hypersusceptible population and thus strengthen the relevance of health promotion interventions for both the general public and the working population, as they might be particularly feasible in the workplace.

PICALM gene methylation in blood of Alzheimer's disease patients is associated with cognitive decline

Epigenetic mechanisms might be involved in Alzheimers disease (AD). Genetic polymorphisms in several genes, including APOE (Apolipoprotein E), PSEN1 (Presenilin 1), CR1 (Complement receptor 1), and PICALM (Phosphatidylinositol binding clathrin assembly protein), have been associated to an increased AD risk. However, data regarding methylation of these specific genes are lacking. We evaluated DNA methylation measured by quantitative bisulfite-PCR pyrosequencing in 43 AD patients and 38 healthy subjects (HS). In a multivariate age- and gender-adjusted model, PICALM methylation was decreased in AD compared to HS (mean = 3.54 and 4.63, respectively, p = 0.007). In AD, PICALM methylation level was also positively associated to Mini-Mental Scale Examination (MMSE) score (percent change 3.48%, p = 0.008). Moreover, a negative association between PICALM methylation and age was observed only in HS (percent change - 2.29%, p = 0.002). In conclusion, our data suggest a possible role of PICALM methylation in AD, particularly related to cognitive function. Given the small study sample and the associative nature of our study, further prospective investigations are required to assess the dynamics of DNA methylation in the early stages of AD development.

Is there a link between air pollution and mental disorders

Several studies have demonstrated the association between air pollution and different medical conditions including respiratory and cardiovascular diseases. Air pollutants might have a role also in the etiology of mental disorders in the light of their toxicity on central nervous system. Purpose of the present manuscript was to review and summarize available data about an association between psychiatric disorders and air pollution. A research in the main database sources has been conducted to identify relevant papers about the topic. Different air pollutants and in particular PM and nitric oxides have been associated with poor mental health; long exposition to PM2.5 has been associated with an increased risk of new onset of depressive symptoms (Cohens effect size d: 0.05-0.81), while increased concentration of nitric dioxide in summer with worsening of existing depressive conditions (Cohens effect size d: 0.05-1.77). However, the interpretation of these finding should take into account the retrospective design of most of studies, different periods of observations, confounding factors such as advanced age or medical comorbidity. Further studies with rigorous methodology are needed to confirm the results of available literature about this topic.

O18-2 Extracellular vesicles are associated with particulate matter exposure

Background Exposure to Particulate Matter (PM) has been associated with increased risk for cardiovascular diseases. In the lungs PM triggers the release of extracellular vesicles (EVs) that travel through the bloodstream and reach target organs. EVs might represent an essential component of the effect of PM on human health. Aim to characterise plasma EVs and assess their association with exposure to PM with diameter ≤10 µm (PM10) and ≤2.5 µm (PM2.5), considering the role of Body Mass Index (BMI). Methods we recruited 20 male and 30 female healthy volunteers (Nov 2014–Mar 2015) in Milan, Italy, which provided personal information and a blood sample. After centrifugation, EV membrane determinants were characterised by Flow Cytometry (to assess cellular origin from platelets, monocytes, epithelium, endothelium, neutrophils), EV size and counted by Nanosight. Each subject wore a personal air sampler for 24 h, retrieving individual daily mean PM10 and PM2.5 concentrations. Same-day PM10 and PM2.5 data from air quality monitoring stations were also obtained. Associations between PM and log-EV were assessed applying multivariate linear models adjusted for age, sex, BMI and smoking. Results Personal sampler and monitor data showed a Spearman’s ρ of 0.59 (p < 0.001) for PM10 and of 0.68 (p < 0.001) for PM2.5. Regression analysis showed a variation of 10.5% (95% Confidence Interval [CI]: 1.8; 19.8, p = 0.018) in endothelium-derived EV count (CD105+) per 10 µg/m3 increase in PM10, and of 11.4% (95% CI: 2.0; 21.6, p = 0.017) per an equal increase in PM2.5. When stratifying by BMI, all EV types showed a positive increase in overweight subjects only (BMI ≥ 25) and a null-negative variation in normal weight subjects. Variation in EV-CD105+ was 13.4% (95% CI: 3.3; 24.4, p = 0.011) for PM10 and 14.0% (95% CI: 2.6; 26.7, p = 0.018) for PM2.5 in overweight. Conclusions Our findings suggest an association between PM exposure and EV, particularly in hypersusceptible subjects.