Ultrafine particles from diesel engines induce vascular oxidative stress via JNK activation

Abstract

Exposure to particulate air pollution is linked to increased incidences of cardiovascular diseases. Ambient ultrafine particles (UFP) from diesel vehicle engines have been shown to be proatherogenic in ApoE knockout mice and may constitute a major cardiovascular risk in humans. We posited that circulating nano-sized particles from traffic pollution sources induce vascular oxidative stress via JNK activation in endothelial cells. Diesel UFP were collected from a 1998 Kenworth truck. Intracellular superoxide assay revealed that these UFP dose-dependently induced superoxide (O(2)(-)) production in human aortic endothelial cells (HAEC). Flow cytometry showed that UFP increased MitoSOX red intensity specific for mitochondrial superoxide. Protein carbonyl content was increased by UFP as an indication of vascular oxidative stress. UFP also up-regulated heme oxygenase-1 (HO-1) and tissue factor (TF) mRNA expression, and pretreatment with the antioxidant N-acetylcysteine significantly decreased their expression. Furthermore, UFP transiently activated JNK in HAEC. Treatment with the JNK inhibitor SP600125 and silencing of both JNK1 and JNK2 with siRNA inhibited UFP-stimulated O(2)(-) production and mRNA expression of HO-1 and TF. Our findings suggest that JNK activation plays an important role in UFP-induced oxidative stress and stress response gene expression.

Figure 1

UFP Induces oxidative stress in HAEC. (A) UFP stimulated intracellular superoxide production. HAEC were treated with different concentration of UFP for 1 hour in the presence of NBT. Superoxide production was measured as absorbance at 700nm. (B) UFP stimulated mitochondrial superoxide production. HAEC were pretreated with mitochondrial superoxide specific dye MitoSOX Red for 10 minutes followed by treatment with 50ug/ml of UFP for 1 hour. The intensity of fluorescence was measured by FACS as quantification of mitochondrial superoxide. (C) UFP increased protein carbonyl contents. HAEC were treated with 50ug/ml of UFP for 6 hours. Cells were lysed and protein carbonyl content in equal amount of protein lysate was measured as described in methods.

Figure 2

UFP stimulated the expression of HO-1 and TF. HAEC were treated for 4 hours with indicated concentration of UFP. The expression of HO-1 (A) and TF (B) mRNA was measured by qRT- PCR.

Figure 3

Antioxidant, N-Acetyl-Cysteine (NAC), inhibited UFP stimulated gene expression. HAEC were pretreated with 5mM of NAC for 1 hour in M199/25mM Hepes(pH7.4)/0.1%FBS followed by co-treatment of NAC with or without 50ug/ml of UFP. The expression of HO-1 (A) and TF (B) mRNA was measured by qRT- PCR.

Figure 4

Transient activation of JNK by UFP. (A) HAEC were treated with 50 μg/ml of UFP for indicated time. Cells were lysed in RIPA buffer with protease and phosphatase inhibitors. 50μg of protein was loaded for SDS-PAGE in each lane. Activated JNK and total JNK were detected with anti-phospho-JNK and anti-total JNK antibodies. (B) Densitometry scan of (A). The relative activated JNK2 was expressed as ratio of phospho-JNK2 (p-JNK) to total JNK2.

Figure 5

JNK inhibitor SP600125 (SP) inhibited UFP stimulated superoxide production and gene expression. (A) HAEC in 96 wells were pretreated with 10uM of SP600125 for 1 hours followed by co-treatment of 25ug/ml of UFP and SP600125 for 1 hour in the presence of NBT. Superoxide production was measured as O.D. 700nm. (B) HAEC were pre-treated with 10uM of SP600125 for 1 hour followed by co-treatment of inhibitor and 50ug/ml of UFP for another 4 hours. HO-1 and TF expression were measured by qRT- PCR.

Figure 6

UFP stimulated superoxide production and gene expression is JNK-dependent. HAEC were transfected with scrambled control siRNA (Scr) or JNK1/JNK2 siRNA (siJNK). 48 hours after transfection, the cells were used for the following analysis. (A) Cells were lysed for measuring JNK2 mRNA expression by qRT-PCR. (B) Cell lysate was obtained to measure JNK1 and JNK2 protein expression by western blotting with anti-total JNK antibody. The relative levels of JNK1 and JNK2 were normalized to tubulin from densitometry scan data. (C) Cells in 96 well were used to measure superoxide production stimulated with or without 50ug/ml of UFP by NBT assay. (D) The expression of HO-1 and TF mRNA in cells treated with or without 50ug/ml of UFP was measured by qRT- PCR.

Figure 7

Mode of action of UFP stimulated effect in HAEC. UFP treatment activates JNK, which lead to increased intracellular superoxide production that eventually results in increased expression of stress response gene HO-1 and TF. Knockdown of JNK by siRNA or JNK inhibitor SP6000125 attenuate UFP stimulated superoxide production and gene expression. Scavenger of superoxide by Antioxidant NAC inhibits UF stimulated gene expression.