SUSCEPTIBILITY TO PARTICULATE MATTER AND HEALTH EFFECTS MEDIATED BY MICRORNAS CARRIED IN PLASMA EXTRACELLULAR VESICLES

Air pollution exposure is a major problem worldwide and has been linked to many diseases. PM10 is one of the components of air pollution and it includes a mixture of compounds. Several studies suggest that PM produces significant effects on the cardiovascular system, in relation to acute as well as chronic exposure. This process has been extensively studied, but to date it has not yet been fully understood. Ambient particles have been shown to produce a strong inflammatory reaction, and beside pro-inflammatory mediators, cell-derived membrane Extracellular Vesicles (EVs) are also released. EVs (particularly microvesicles) might be the ideal candidate to mediate the effects of air pollution, since potentially they could transfer miRNAs, after internalization within target cells through surface-expressed ligands, enabling intercellular communication in the body. Our hypothesis is that, after air pollution exposure, the respiratory system can release extracellular vesicles that could reach the systemic circulation and lead to the development of endothelial disfunction. The aim of the study is to determine whether exposure to air particles and PM-associated metals can modify EVs in plasma in term of: - miRNAs content - Quantity of MVs subpopulations Our study population represents a subgroup of the SPHERE projects that includes patients enrolled from Center for Obesity and Weight Control of the Department of Environmental and Occupational Health, University of Milan and IRCCS Fondazione Ca’Granda – Ospedale Maggiore Policlinico. In particular we recruited 883 overweight/obese subjects for miRNAs study (recruited from 2010 till 2012) and 266 (recruited from January till November 2013) subjects for MVs characterization. We chose overweight/obese people because some evidence shows that obesity may bring greater susceptibility to the adverse cardiovascular effects of PM exposure. Exposure is defined using a multifaceted approach. In particular, PM10 is assigned to each subjects following two approaches: (1) use of daily PM10 concentration series from air quality monitors; (2) use of daily PM10 concentration estimates by the FARM model (the flexible air quality regional model) supplied by ARPA Lombardy. In the first part of the study, we analyzed the variation, in term of concentration, of plasma EVs in association with PM10 exposure. In particular we decided to investigate 5 subpopulations of microvesicles (MVs) by flow cytometry (MACS Quant Analyzers-Miltenyi Biotech): MVs from monocytes (CD14+), MVs from platelets (CD61+), MVs from neutrophils (CD66+), MVs from endothelium (CD105+), MVs from epithelium (EpCAM+). The second phase of the study represents a discovery stage that involves a miRNAs expression profiling using the OpenArray technology (QuantStudioTM 12K FlexOpenArray-Life Technologies). The results of MVs subpopulation analysis in association with PM10 exposure show an increase of 6.6% in the concentration of MVs CD61+ (from platelets); 3.4% for MVs CD66+ (neutrophils); 3.9% MVs for CD105+ (endothelium); 7% for MVs CD14+ (monocytes); while there is no significant association for MVs EpCAM+ (from pulmonary epithelium). These results indicate the percentage increase of MVs concentration with an increase of 1μg / m3 of PM10. The total concentration of extracellular vesicles was analyzed by Nanoparticle Tracking Analysis (NTA), allowing to separate microvesicles from exosomes. The total amount of microvesicles is associated with PM10 exposure, while the total amount of exosomes is not significant. From miRNAs analysis, performed through TaqMan Open Array Real Time PCR, we test the association between PM10 exposure and the different levels of expression of 733 human miRNAs. This phase of the SPHERE study has the aim of screening the entire miRNome on a population of 833 subjects. From the results of this analysis have emerged 52 miRNAs as strongly associated with PM10 exposure. We performed a Mediation Analysis to investigate the potential role of the top 10 miRNAs as mediators of the effect of PM10 exposure on cardiac outcomes, as Systolic Blood Pressure and Diastolic Blood Pressure. We can estimate that 36.8% of the effect of PM10 on Systolic Blood Pressure is mediated by miR-106a. Interestingly miR-106a seems to play a crucial regulatory role in regulate macrophage inflammatory responses, and is well known that macrophage infiltration is involved in the processes of formation of atherosclerotic plaques. The identified expression profile may provide a useful starting point for the understanding of the key mechanism that links PM10 exposure to the onset of cardiovascular diseases.