Carbon Black Nanoparticles Promote Endothelial Activation and Lipid Accumulation in Macrophages Independently of Intracellular ROS Production

Loft S (2014) Carbon Black Nanoparticles Promote Endothelial Activation and Lipid Accumulation in Macrophages Independently of Intracellular ROS Production. PLoS ONE 9(9): e106711. doi:10.1371/journal.pone.0106711 Carbon Black Nanoparticles Promote Endothelial Activation and Lipid Accumulation in Macrophages Independently of Intracellular ROS Production Yi Cao 0 Martin Roursgaard 0 Pernille Hgh Danielsen 0 Peter Mller 0 Steffen Loft 0 Clarissa Menezes Maya-Monteiro, Fundacao Oswaldo Cruz, Brazil 0 Section of Environmental Health, Department of Public Health, University of Copenhagen , Copenhagen , Denmark Exposure to nanoparticles (NPs) may cause vascular effects including endothelial dysfunction and foam cell formation, with oxidative stress and inflammation as supposed central mechanisms. We investigated oxidative stress, endothelial dysfunction and lipid accumulation caused by nano-sized carbon black (CB) exposure in cultured human umbilical vein endothelial cells (HUVECs), THP-1 (monocytes) and THP-1 derived macrophages (THP-1a). The proliferation of HUVECs or cocultures of HUVECs and THP-1 cells were unaffected by CB exposure, whereas there was increased cytotoxicity, assessed by the LDH and WST-1 assays, especially in THP-1 and THP-1a cells. The CB exposure decreased the glutathione (GSH) content in THP-1 and THP-1a cells, whereas GSH was increased in HUVECs. The reactive oxygen species (ROS) production was increased in all cell types after CB exposure. A reduction of the intracellular GSH concentration by buthionine sulfoximine (BSO) pre-treatment further increased the CB-induced ROS production in THP-1 cells and HUVECs. The expression of adhesion molecules ICAM-1 and VCAM-1, but not adhesion of THP-1 to HUVECs or culture dishes, was elevated by CB exposure, whereas these effects were unaffected by BSO pre-treatment. qRT-PCR showed increased VCAM1 expression, but no change in GCLM and HMOX1 expression in CB-exposed HUVECs. Pre-exposure to CB induced lipid accumulation in THP1a cells, which was not affected by the presence of the antioxidant N-acetylcysteine. In addition, the concentrations of CB to induce lipid accumulation were lower than the concentrations to promote intracellular ROS production in THP-1a cells. In conclusion, exposure to nano-sized CB induced endothelial dysfunction and foam cell formation, which was not dependent on intracellular ROS production. - Funding: This work was supported by the Centre for Pharmaceutical Nanoscience and Nanotoxicology financed by the Danish Strategic Research Council and by the Lundbeck Foundation Center for Biomembranes in Nanomedicine (CBN). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Exposure to nanoparticles (NPs) has been suggested to cause vascular health effects with oxidative stress and inflammation as central mechanisms [1]. The NP-mediated vascular effects include expression of endothelial cell adhesion molecules such as intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), vasomotor dysfunction and accelerated progression of atherosclerosis [1]. The expression of ICAM-1 and VCAM-1 promotes the firm adhesion of monocytes onto the endothelium and the monocytes can subsequently differentiate into macrophages, migrate to the intima and transform to foam cells [2]. It has been shown that exposure of endothelial cells to NPs promotes the expression of ICAM-1 and VCAM-1 as well as adhesion of monocytes onto the endothelial cells [3,4]. Furthermore, it has also been shown that NP exposure induces intracellular lipid accumulation [57]. The process of endothelial activation might not require oxidative stress, as suggested by increased adhesion molecule expression by NP exposure in a manner not associated with generation of ROS [8,9]. In addition, it has been shown that addition of the antioxidant ascorbic acid to the cell culture medium did not alleviate particle-induced ICAM-1 and VCAM-1 expression on human umbilical vein endothelial cells (HUVECs) [10]. On the other hand, NP induced lipid accumulation in rat cells was inhibited by pre-treatment with the antioxidant N-acetylcysteine (NAC) [11]. We hypothesized that oxidatively stressed endothelial cells would be more readily activated and interact more strongly with monocytes or macrophages, and that oxidative stress could further promote the lipid accumulation in macrophages by exposure to NPs. To this end we investigated the effect of exposure to nanosized carbon black (CB) on the activation of endothelial cells by ICAM-1 and VCAM-1 expression on HUVECs and adhesion of THP-1 monocytes onto HUVECs as well as lipid accumulation in THP-1 macrophages. We used nano-sized CB because it generates high levels of intracellular ROS [12]. In addition, we have previously shown that HUVECs express increased levels of ICAM1 and VCAM-1 after exposure to nano-sized CB [9,10,13]. CB is widely used as black pigment in rubber, paints and inks as well as being a widely used type of particle in toxicological studies including studies on ROS production [14], endothelial-dependent vasomotor function [15] and atherosclerosis [16]. The intracellular ROS generation and GSH concentration were used as markers of oxidative stress, whereas the mRNA expression of adhesion molecule VCAM-1 as well as the oxidative stress response genes in the NRF-2 signaling pathway, glutamate-cysteine ligase, modifier subunit (GCLM), the rate limiting enzyme in GSH synthesis, and heme oxygenase 1 (HMOX1), one of the essential enzymes in heme catabolism [17], was assessed in HUVECs by qRT-PCR. Materials and Methods Cell lines The HUVECs and culture medium were purchased from Cell Applications (San Diego, CA, USA). The cells were cultured in Endothelial Cell Growth Medium Kit with 2% serum at 37uC in an incubator with 5% CO2. The medium was changed 2436 h after seeding and the cells were cultured until they were 90% confluent. The cells were used between passages 25 because they maintain their morphologic and phenotypic characteristics within these passages [9,13]. The THP-1 monocytes were obtained from the American Type Culture Collection (Manassas, VA, USA) and was cultured in RPMI with 10% serum as previously described [18]. The THP-1 cells were differentiated into adherent macrophages (denoted THP-1a) by treatment with 10 ng/ml phorbol 12-myristate 13-acetate (PMA, Sigma, St. Louis, MO, USA) overnight [19]. The THP-1a cells attach to the surface of the culture flasks, whereas THP-1 cells stay in suspension and were removed with the supernatant. Particles CB particles (Printex 90) were obtained from Evonik Industries, Frankfurt, Germany (primary particle size 14 nm; surface area 300 m2/g). Printex 90 is an extensively studied model NP and has been characterized elsewhere [20,21]. The mean size of the (...truncated)