FINE PARTICULATE MATTER EXACERBATES FOAM CELLS FORMATION BY MACROPHAGES, BY ENHANCING THE LDL OXIDATION IN AN OXIDATIVE MEDIUM Rafael Kazmirczak de Moraes Bioquímica - UFRGS The exposure to fine particulate material (PM2.5), an atmospheric pollutant, is a risk factor for cardiovascular diseases, especially atherosclerosis. The reduced diameter of PM2.5 (0.1 to 2.5 µm) allows the pollutant to go deeper in the respiratory tract and trigger systemic effects. In this context, macrophages are the first immune cells to phagocyte these particles and to activate the immune defense, by releasing inflammatory cytokines. In an inflammatory milieu, circulating monocytes can switch their phenotype to an oxidative one, and enhance the risk of vascular damage. Within the vessels, the oxidated LDL can be phagocyted by macrophages, and lead to the formation of foam cells. Moreover, macrophages apoptosis can aggravate thrombus formation. However, if the cardiovascular effects of PM2.5 are related to changes in macrophages phenotype, and the consequent foam cells formation remains unclear until now. To verify if the exposure of macrophages to the PM2.5 can exacerbate the formation of foam cells by creating a pro-oxidative medium. PM2.5 retained in fiberglass filters was partially extracted with PBS and further centrifuged at 1000g for 15min. This solution (1g filter/125 mL PBS) was diluted in DMEM 10% FBS ten times. The LDL was extracted from human plasma (CEP 4.557.728). A murine macrophage cell line (RAW264.7) was exposed to PM2.5 for 48h and PBS was used as a Control. To verify foam cell formation, non-oxidized human LDL (50µg/mL) was added in the last 24h. Triglycerides intracellular accumulation was verified by AdipoRed staining; Cell death by Annexin and PI kit, all in flow cytometry. Nitrite levels were measured by Griess method, and ROS production by DCF fluorescence. The LDL oxidation was assessed by conjugated diene formation, spectrophotometrically. The data are expressed in mean ± SEM, and analyzed by Student`s t-test, except for triglycerides accumulation that was compared through Two-way ANOVA followed by Tukey, P˂0.05). Exposing the macrophages to the non-oxidized human LDL induced the intracellular accumulation of lipids, and subsequently the formation of foam cells, which was exacerbated by the pollutant (Ctrl: 1.00±0.02; LDL: 1.27±0.06*; PM: 1.26±0.11; LDL+PM: 1.39±0.06*; Two-way ANOVA: LDL: P=0.009; PM: P=0.01; Interaction: P=0.34). Thus, we investigated if it could be derived from an effect on macrophages activated phenotype. The DCF showed that PM2.5 increased ROS production after 48h (Ctrl: 0.97±0.04; PM: 1.55±0.13; t-test P=0.0003). The nitrite levels also increased (Ctrl: 11.99±1.11; PM: 18.45±1.09; P=0.0002), indicating a pro-oxidative medium. This pro-oxidative medium was able to oxidate the LDL, evidenced by the increase in conjugated dienes formation (Ctrl: 0.02±0.005; PM: 0.08±0.025; P=0.05). The cell viability was reduced in the group exposed to PM2.5 as shown by the Annexin test (Ctrl: 8.58 ± 0.95; PM: 12.75±1.10; P=0.017). These results shows that the foam cell formation was increased when macrophages were exposed to PM2.5 and human LDL. This could be happening because the pollutant exacerbates the production of ROS and NO, that in turn oxidates the LDL, increasing its phagocytosis by macrophages.