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.