Clinical characteristics of the study population.

<p>Data expressed as mean±SD or N. Laboratory and diagnostic assays were from fasted blood samples.</p><p>HbA_1c = glycated hemoglobin;</p><p>HDL = high-denisty lipoprotein;</p><p>LDL = low-density lipoprotein;</p><p>GOT = glutamic-oxaloacetic transaminase;</p><p>GPT = glutamic-pyruvic transaminase;</p><p>BMI = body mass index;</p><p>SBP = systolic blood pressure;</p><p>DBP = diastolic blood pressure.</p><p>Cigarette smoking refers to current cigarette smoker.</p

Pioglitazone effect on EPC pro-inflammatory molecule expression.

<p>Effect of pioglitazone on PPARγ, adhesion molecule and TNFα expression in EPCs. PPARγ gene expression in early and late-outgrowth EPC exposed to pioglitazone (10 µM) and pioglitazone + GW9662 (1 µM) (A) Effect of pioglitazone in modulating ICAM-1 and VCAM-1 expression by early and late-outgrowth EPCs (cytofluorimetric analyses). Results are reported as delta % of MFI (mean fluorescence intensity) with vehicle condition (B). Effects of pioglitazone on TNFα gene (C) and protein (D) expression in early and late-outgrowth EPC. (*p<0.05 vs vehicle; **p<0.01 vs vehicle). Real time PCR data are expressed as −ΔΔCt and represent the relative gene expression of EPC cultured in the presence of pioglitazone 10 µM (with or without GW9662 1 µM) in relation to vehicle, normalized for the endogenous control GAPDH. Protein expression is measured with ELISA assay from culture supernatants. Results from five independent experiments performed in duplicate are shown (*p<0.05 vs vehicle) (Pio = pioglitazone).</p

Characterization of late-outgrowth EPCs.

<p>Morphology and phenotype of late-outgrowth EPC derived from human peripheral blood mononuclear cells. Colonies of late-outgrowth EPCs with a cobblestone-like morphology (A). Representative flow cytometry analysis of late-outgrowth EPCs (red) compared to early EPCs (purple) for the expression of CD31, KDR, CD146 and CD14 (B). Immunofluorescent staining of late-outgrowth EPCs for CD34, Ve-cad (vascular endothelial cadherin) and vWF (von Willebrand factor) (C), immunofluorescence staining of late-outgrowth EPC for DiI-LDL (red), lectin (green) and merge (bar = 50 µm) (D). eNOS and GAPDH gene expression in late EPCs (E) (bp = base pairs; eNOS = endothelial nitric oxide synthase; GAPDH = Glyceraldehyde 3-phosphate dehydrogenase).</p

Effect of pioglitazone on EPC function.

<p>Effect of pioglitazone (10 µM), pioglitazone + GW9662 (1 µM) and vehicle culture conditions on early and late-outgrowth EPC tube formation capacity expressed as total tube length (A) and as number of closed circles formed by tube-like structures (B); representation of early and late-outgrowth EPC tube formation assay showing the network formed by EPCs plus HUVEC on Matrigel (EPCs are red stained with Dil) in the presence of pioglitazone and vehicle (C); (*p<0.05 vs vehicle) (Pio = pioglitazone).</p

Effect of pioglitazone on EPC viability.

<p>The effects of pioglitazone (10 µM), pioglitazone (10 µM) + GW9662 (1 µM) and 0.2% DMSO vehicle culture conditions were examined on early (A) and late-outgrowth EPC viability alone and in the presence of H₂O₂ (500 µM, 24 h) (B); (*p<0.05 vs vehicle; **p<0.01 vs vehicle; §p<0.01 vs vehicle+H₂O₂).</p

NP effects on CAC viability.

<p>Effect of TiO₂ and Co₃O₄ NPs in affecting CAC viability following 24 and 48h of exposure (A) function assessed by adhesion assay on fibronectin (B) in CACs following 24h of exposure (NP = nanoparticle; BSA = bovine serum albumin; SE = standard error) (*p<0.05; **p<0.01 vs control).</p

Effects of TiO₂ and Co₃O₄ Nanoparticles on Circulating Angiogenic Cells

<div><p>Background and Aim</p><p>Sparse evidence suggests a possible link between exposure to airborne nanoparticles (NPs) and cardiovascular (CV) risk, perhaps through mechanisms involving oxidative stress and inflammation. We assessed the effects of TiO₂ and Co₃O₄ NPs in human circulating angiogenic cells (CACs), which take part in vascular endothelium repair/replacement.</p><p>Methods</p><p>CACs were isolated from healthy donors’ buffy coats after culturing lymphomonocytes on fibronectin-coated dishes in endothelial medium for 7 days. CACs were pre-incubated with increasing concentration of TiO₂ and Co₃O₄ (from 1 to 100 μg/ml) to test the effects of NP – characterized by Transmission Electron Microscopy – on CAC viability, apoptosis (caspase 3/7 activation), function (fibronectin adhesion assay), oxidative stress and inflammatory cytokine gene expression.</p><p>Results</p><p>Neither oxidative stress nor cell death were associated with exposure to TiO₂ NP (except at the highest concentration tested), which, however, induced a higher pro-inflammatory effect compared to Co₃O₄ NPs (p<0.01). Exposure to Co₃O₄ NPs significantly reduced cell viability (p<0.01) and increased caspase activity (p<0.01), lipid peroxidation end-products (p<0.05) and pro-inflammatory cytokine gene expression (p<0.05 or lower). Notably, CAC functional activity was impaired after exposure to both TiO₂ (p<0.05 or lower) and Co₃O₄ (p<0.01) NPs.</p><p>Conclusions</p><p>In vitro exposure to TiO₂ and Co₃O₄ NPs exerts detrimental effects on CAC viability and function, possibly mediated by accelerated apoptosis, increased oxidant stress (Co₃O₄ NPs only) and enhancement of inflammatory pathways (both TiO₂ and Co₃O₄ NPs). Such adverse effects may be relevant for a potential role of exposure to TiO₂ and Co₃O₄ NPs in enhancing CV risk in humans.</p></div

Cellular morphology.

<p>Morphology of CACs cells untreated (A) and following incubation with different concentration of TiO₂ (B) and Co₃O₄ NPs (C). (CAC = circulating angiogenic cells).</p

Effects of NPs on CAC inflammation.

<p>Effects of TiO₂ and Co₃O₄ NPs on IL-1β (A), TNF-α (B) and MCP-1(C) gene expression in CACs following 6h and 24h (D-F) of exposure. Data are expressed as -ΔΔCt and represent the relative gene expression of CACs cultured in the presence of NPs in relation to control, normalized for the endogenous control GAPDH. (<i>N = 7</i>; *p<0.05 vs control;** p<0.01 vs control) (IL-1β = interleukin-1β; TNF-α = tumor necrosis factor-α; MCP-1 = monocyte chemoattractant protein-1; BSA = bovine serum albumin).</p

Effects of NPs on CAC apoptosis and oxidative stress.

<p>Effect of TiO₂ and Co₃O₄ NPs on caspase 3/7 activation (A) and on oxidant stress formation (TBARS) (B) in CACs following 24 h of exposure (MDA = Malondialdehyde; NP = nanoparticle; BSA = bovine serum albumin) (*p<0.05 vs control; **p<0.01 vs control).</p