Endothelial responses of the alveolar barrier in vitro in a dose-controlled exposure to diesel exhaust particulate matter

Abstract

Background: During the last 250 years, the level of exposure to combustion-derived particles raised dramatically in western countries, leading to increased particle loads in the ambient air. Among the environmental particles, diesel exhaust particulate matter (DEPM) plays a special role because of its omnipresence and reported effects on human health. During recent years, a possible link between air pollution and the progression of atherosclerosis is recognized. A central effect of DEPM is their impact on the endothelium, especially of the alveolar barrier. In the present study, a complex 3D tetraculture model of the alveolar barrier was used in a dose-controlled exposure scenario with realistic doses of DEPM to study the response of endothelial cells.

Results: Tetracultures were exposed to different doses of DEPM (SRM2975) at the air-liquid-interface. DEPM exposure did not lead to the mRNA expression of relevant markers for endothelial inflammation such as ICAM-1 or E-selectin. In addition, we observed neither a significant change in the expression levels of the genes relevant for antioxidant defense, such as HMOX1 or SOD1, nor the release of pro-inflammatory second messengers, such as IL-6 or IL-8. However, DEPM exposure led to strong nuclear translocation of the transcription factor Nrf2 and significantly altered expression of CYP1A1 mRNA in the endothelial cells of the tetraculture.

Conclusion: In the present study, we demonstrated the use of a complex 3D tetraculture system together with a state-of-the-art aerosol exposure equipment to study the effects of in vivo relevant doses of DEPM on endothelial cells in vitro. To the best of our knowledge, this study is the first that focuses on indirect effects of DEPM on endothelial cells of the alveolar barrier in vitro. Exposure to DEPM led to significant activation and nuclear translocation of the transcription factor Nrf2 in endothelial cells. The considerably low doses of DEPM had a low but measurable effect, which is in line with recent data from in vivo studies.

Keywords: Air-liquid-interface; Coculture; Diesel exhaust particles; in vitro model.

Fig. 1

The concept of the hierarchical oxidative stress response. In order to explain the observed effects in cells after exposure to DEPM, the concept of the hierarchical oxidative stress response was developed [23]. In this concept, the cellular response is divided in three different tiers, depending on the level of oxidative stress. In tier 1 the level of oxidative stress is moderate and cellular defense mechanisms are able to cope with the radicals. In tier 2 the level of oxidative stress causes already an inflammatory response and activation of cells. Finally, in tier 3 the oxidative stress leads to cytotoxicity and cellular damage (adapted and modified from [73])

Fig. 2

Size distribution of the DEPM aerosol that was produced with the PALAS powder aerosol generator and analyzed using particle counters (optical GRIMM Aerosol counter and SMPS + C counter by GRIMM Aerosol). Particle counts were registered for one minute with the rotating steel brush at 1200 rpm and the piston at 30 mm/h. Data represents the mean of three independent experiments ± SEM. a) measurement of the nano-range from 250 nm to 2 μm performed on an optical particle counter by GRIMM Aerosol b) measurement of the nano-range from 5 nm to 300 nm performed on a Sequential Mobility Particle Sizer and Counter (SMPS + C) by GRIMM Aerosol

Fig. 3

Scanning electron microscopy micrographs of empty Transwell™ inserts after exposure to DEPM aerosol using the PALAS steel-brush-generator. Transwell™ inserts were pre-wetted and then exposed to: a: 40 ng/cm²; b: 80 ng/cm²; c: 240 ng/cm²

Fig. 4

Distribution of metals on the outer shell of DEPM analyzed by NanoSIMS50. DEPM samples were analyzed for the presence of Ni, Cu, Cr and Zn. P was used as reference to draft an image of the outer shell of the particle. The relative amounts of the elements are presented as intensities with a logarithmic scale from black (lowest) to red (highest)

Fig. 5

Impact of the exposure to different amounts of DEPM on the cellular viability of the tetraculture at different time-points after exposure. Tetracultures were exposed to 40 ng/cm² (a), 80 ng/cm² (b) or 240 ng/cm² (c) of DEPM and the viability of the tetracultures was evaluated at 6, 24 and 48 h after exposure. DEPM were not significantly affecting the viability of the tetracultures compared to the control cells at any of the evaluated time-points. Tetracultures that without particles served as control (dotted line). Data represents the mean of three independent experiments ± SEM

Fig. 6

Potential of DEPM to induce nuclear translocation of Nrf2 in endothelial cells after different incubation times. Tetracultures were exposed to 240 ng/cm² of DEPM. Nrf2 translocation was evaluated for cells indirectly exposed to DEPM at 3, 4 and 5 h after exposure. Cells exposed for the same time but without particles in the air stream were used as controls for the fluorescence signals. Cells were fixed and stained for nucleus (Hoechst 33342) and anti-Nrf2 (green). Images were analyzed using a Zeiss LSM 510 META. At 3 h after exposure, first cells showed nuclear translocation. At 4 h after exposure, nuclear translocation was quite obvious in the majority of the cell population. At 5 h after exposure almost every endothelial cells showed strong colocalization of Nrf2 with the nucleus (counterstained in blue). In control EA.hy 926 cells, no nuclear translocation was observed at any time-point (compare with the representative control image)

Fig. 7

Differential gene expression profile of CYP1A1 in endothelial cells at different incubation time after DEPM exposure of the tetraculture system. The endothelial cells of the tetraculture showed a significant increase for CYP1A1 mRNA 6 h after the indirect exposure to 80 ng/cm² of DEPM (a). No significant change was observed for cells exposed to 240 ng/cm² of DEPM (b). Results were normalized to untreated control cells (dotted line). Data represents the mean of four independent experiments ± SEM. Asterisks indicate significant differences compared to untreated control cells (P < 0.05)