Effects of FW2 Nanoparticles Toxicity in a New In Vitro Pulmonary Vascular Cells Model Mimicking Endothelial Dysfunction.

Several epidemiological studies have revealed the involvement of nanoparticles (NPs) in respiratory and cardiovascular mortality. In this work, the focus will be on the effect of manufactured carbon black NPs for risk assessment of consumers and workers, as human exposure is likely to increase. Since the pulmonary circulation could be one of the primary targets of inhaled NPs, patients suffering from pulmonary hypertension (PH) could be a population at risk. To compare the toxic effect of carbon black NPs in the pulmonary circulation under physiologic and pathological conditions, we developed a new in vitro model mimicking the endothelial dysfunction and vascular dynamics observed in vascular pathology such as PH. Human pulmonary artery endothelial cells were cultured under physiological conditions (static and normoxia 21% O2) or under pathological conditions (20% cycle stretch and hypoxia 1% O2). Then, cells were treated for 4 or 6 h with carbon black FW2 NPs from 5 to 10 µg/cm2. Different endpoints were studied: (i) NPs internalization by transmission electronic microscopy; (ii) oxidative stress by CM-H2DCFDA probe and electron paramagnetic resonance; (iii) NO (nitrites and nitrates) production by Griess reaction; (iv) inflammation by ELISA assay; and (v) calcium signaling by confocal microscopy. The present study characterizes the in vitro model mimicking endothelial dysfunction in PH and indicates that, under such pathological conditions, oxidative stress and inflammation are increased along with calcium signaling alterations, as compared to the physiological conditions. Human exposure to carbon black NPs could produce greater deleterious effects in vulnerable patients suffering from cardiovascular diseases.

In vitro study of carbon black nanoparticles on human pulmonary artery endothelial cells: effects on calcium signaling and mitochondrial alterations.

Human exposure to manufactured nanoparticles (NPs) is a public health concern. Endothelial cells lining the inner surface of arteries could be one of the primary targets for inhaled nanoparticles. Moreover, it is well known that alteration in calcium signaling is a critical event involved in the physiopathology of cardiovascular diseases. The objective of this study was to assess the role of oxidative stress in carbon black FW2 NPs-induced alteration in calcium signaling and mitochondria in human pulmonary artery endothelial cells. To this end, cells were exposed for 4 or 24 h to FW2 NPs (1-10 µg/cm2) and the following endpoints were studied: (i) production of ROS by fluorimetry and electron paramagnetic resonance, (ii) variation in intracellular calcium concentration by confocal microscopy, and (iii) mitochondrial alteration and apoptosis by confocal microscopy and transmission electronic microscopy. Exposure to FW2 NPs concentration-dependently increases oxidative stress, evidenced by the production of superoxide anion leading to an alteration in calcium content of intracellular organelles, such as endoplasmic reticulum and mitochondria activating, in turn, intrinsic apoptosis. This study provides evidence that FW2 NPs exposure impairs calcium signaling and mitochondria triggered by oxidative stress, and, thus, could act as a cardiovascular disease risk owing to the key role of calcium homeostasis in the control of vascular tone.

Involvement of oxidative stress and calcium signaling in airborne particulate matter - induced damages in human pulmonary artery endothelial cells.

Recent studies have revealed that particulate matter (PM) exert deleterious effects on vascular function. Pulmonary artery endothelial cells (HPAEC), which are involved in the vasomotricity regulation, can be a direct target of inhaled particles. Modifications in calcium homeostasis and oxidative stress are critical events involved in the physiopathology of vascular diseases. The objectives of this study were to assess the effects of PM2.5 on oxidative stress and calcium signaling in HPAEC. Different endpoints were studied, (i) intrinsic and intracellular production of reactive oxygen species (ROS) by the H2DCF-DA probe, (ii) intrinsic, intracellular and mitochondrial production of superoxide anion (O2-) by electronic paramagnetic resonance spectroscopy and MitoSOX probe, (iii) reactive nitrosative species (RNS) production by Griess reaction, and (vi) calcium signaling by the Fluo-4 probe. In acellular conditions, PM2.5 leads to an intrinsic free radical production (ROS, O2-) and a 4h-exposure to PM2.5 (5-15µg/cm2), induced, in HPAEC, an increase of RNS, of global ROS and of cytoplasmic and mitochondrial O2- levels. The basal intracellular calcium ion level [Ca2+]i was also increased after 4h-exposure to PM2.5 and a pre-treatment with superoxide dismutase and catalase significantly reduced this response. This study provides evidence that the alteration of intracellular calcium homeostasis induced by PM2.5 is closely correlated to an increase of oxidative stress.