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

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

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

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 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