Two mice from each group and these are not shown in the figure. * denotes a significant effect on plaque progression in purchase (R)-K-13675 exposed mice vs. the control group.Discussion In this study we show that four repeated i.t. instillations of nTiO2 were associated with a modest increase in plaque progression in the aorta of ApoE-/- mice, whereas there was little pulmonary inflammation and only minor effects on vasodilatory function of endothelial and smooth muscle cells in segments after two instillations. Ex vivo addition of the SOD mimic agent, tempol,Mikkelsen et al. Particle and Fibre Toxicology 2011, 8:32 http://www.particleandfibretoxicology.com/content/8/1/Page 8 ofTable 4 Inflammatory markers measured in mice having received two intratracheal instillations of either control bronchoalveolar lavage (BAL) suspension or 0.5 mg/(kg bodyweight) of one of the particle suspensions; fTiO2, pTiO2, or nTiO2, separated by 24 hours (n = 10-11).Exposure i.t. instillation Mcp-1 (*10 )-mRNA expression level (Median (quartile 25-75 )) Mip-2 (*10-9) 84.5 (41.5-150.6) 71.9 (35.2-111.6) 77.8 (36.2-140.0) 49.1 (39.0-237.9) Icam-1 (*10-6) 40.8 (27.4-70.2) 32.8 (29.0-54.4) 30.3 (25.7-55.2) 38.9 (18.3-66.8) Vcam-1 (*10-6) 0.62 (0.32-0.88) 0.34 (0.19-0.60) 0.38 (0.19-1.04) 0.36 (0.27-0.63) Vegf (*10-6) 96.9 (59.3-271.2) 99.7 (74.2-109.4) 105.1 (90.3-168.0) 80.1 (47.0-144.1) 54.5 (27.7-89.3) 24.4 (9.9-38.9) 37.1 (24.3-52.8) 27.3 (20.7-64.9)Control fTiO2 pTiO2 nTiOExpression of mRNA is normalised to 18S rRNA.reduced the endothelium-dependent vasodilatory function in aorta segments from unexposed mice, whereas tempol increased this function in segments from mice exposed to pTiO 2 . The minimal effect of pulmonary TiO2 exposure on vasodilatory function was supported by minimal effects on NO production in HUVECs. The endothelium and its product NO are key regulators of vascular function. Reduced bioavailability of NO is involved in the initiation and progression of atherosclerosis. The particle exposure of HUVECs showed an increase in the level of NO when the cells were exposed to nTiO2. This increase was abolished after addition of DPI, which is a non-selective inhibitor of NO synthase activity in the concentration that we have used. The addition of DPI also blocked the accumulation of NO in HUVECs that were not exposed to particles, indicating that the basal NO production originated from NO-generating enzymes. DPI also inhibits NAD(P)H oxidase, but this probably increases the cellular NO level because of lower possibility of reaction with superoxide anion radicals. iNOS enzymes are expressed during inflammatory conditions, but the one-hour incubation time in our study is probably too short to be associated with upregulation of iNOS. PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27362935 Collectively, the results suggest that the particle-derived NO production originated from eNOS activity. We investigated the plaque progression in ApoE -/mice exposed to nTiO2 once a week for four weeks, followed by a period of five weeks before sacrifice. This particular sample was chosen because previous in vitro studies had indicated inflammatory response. Furthermore, Rossi et al. showed that a silica-coated TiO 2 material (similar to nTiO2) was the only among several TiO2 materials that gave pulmonary inflammation .The protocol used in the present study was similar to the one used for assessment of plaque progression by i.t. instillation of single-walled carbon nanotubes (SWCNT), which had been associated with slightly larger plaque.