Cigarette smoke and α,β-unsaturated aldehydes elicit VEGF release through the p38 MAPK pathway in human airway smooth muscle cells and lung fibroblasts

Abstract

Background and purpose: Vascular endothelial growth factor (VEGF) is an angiogenic factor known to be elevated in the sputum of asymptomatic smokers as well as smokers with bronchitis type of chronic obstructive pulmonary disease. The aim of this study was to investigate whether acute exposure to cigarette smoke extract altered VEGF production in lung parenchymal cells.

Experimental approach: We exposed human airway smooth muscle cells (ASMC), normal human lung fibroblasts (NHLF) and small airways epithelial cells (SAEC) to aqueous cigarette smoke extract (CSE) in order to investigate the effect of cigarette smoke on VEGF expression and release.

Key results: Vascular endothelial growth factor release was elevated by sub-toxic concentrations of CSE in both ASMC and NHLF, but not in SAEC. CSE-evoked VEGF release was mimicked by its component acrolein at concentrations (10-100 µM) found in CSE, and prevented by the antioxidant and α,β-unsaturated aldehyde scavenger, N-acetylcysteine (NAC). Both CSE and acrolein (30 µM) induced VEGF mRNA expression in ASMC cultures, suggesting an effect at transcriptional level. Crotonaldehyde and 4-hydroxy-2-nonenal, an endogenous α,β-unsaturated aldehyde, stimulated VEGF release, as did H(2)O(2). CSE-evoked VEGF release was accompanied by rapid and lasting phosphorylation of p38 MAPK (mitogen-activated protein kinase), which was abolished by NAC and mimicked by acrolein. Both CSE- and acrolein-evoked VEGF release were blocked by selective inhibition of p38 MAPK signalling.

Conclusions and implications: α,β-Unsaturated aldehydes and possibly reactive oxygen species contained in cigarette smoke stimulate VEGF expression and release from pulmonary cells through p38 MAPK signalling.

Figure 1

Cigarette smoke extract (CSE) enhances vascular endothelial growth factor (VEGF) release from airway smooth muscle cell (ASMC) and normal human lung fibroblast (NHLF) cells. Effects of increasing concentrations [expressed as optical density (OD) at 320 nm] of CSE on VEGF release in ASMC (A) and in NHLF (B) cultures. CSE effect on cell viability (MTT test) in ASMC (C) and NHLF (D) cultures. Each histogram is the mean ± SD of three independent experiments performed in quadruplicate. n.d., not detectable. Statistically different from basal (vehicle-treated), Dunnett's test after anova, *P < 0.05, **P < 0.01.

Figure 2

Cigarette smoke extract (CSE) does not stimulate vascular endothelial growth factor (VEGF) release from small airways epithelial cell (SAEC). Effects of increasing concentrations (expressed as optical density, OD) of CSE (A) and acrolein (B) on VEGF release in SAEC cultures. Effects on cell viability (MTT test) of CSE (C) and acrolein (D). Each histogram is the mean ± SD of three independent experiments performed in quadruplicate. Statistically different from basal (vehicle-treated), Dunnett's test after anova, **P < 0.01.

Figure 3

α,β-Unsaturated aldehydes stimulate vascular endothelial growth factor (VEGF) release. Effects of increasing concentrations of acrolein on VEGF release in airway smooth muscle cell (ASMC) (A) and normal human lung fibroblast (NHLF) cultures (B). Acrolein effects on cell viability (MTT test) in ASMC (C) and NHLF (D) cultures. Effects of increasing concentrations of 4-hydroxy-2-nonenal (4-HNE) (E) and crotonaldehyde (F) on the secretion of VEGF in ASMC cultures. Effects of 4-HNE (G) and crotonaldehyde (H) on cell viability (MTT test) in ASMC cultures. Each histogram is the mean ± SD of three independent experiments performed in quadruplicate. n.d., not detectable. Statistically different from basal (vehicle-treated), Dunnett's test after anova, *P < 0.05, **P < 0.01.

Figure 4

Cigarette smoke extract (CSE) and acrolein increase vascular endothelial growth factor (VEGF) mRNA expression. Airway smooth muscle cell cultures were treated with CSE (optical density = 0.075) (A) or acrolein (30 µM) (B) for the indicated periods. Total RNAs were extracted and relative VEGF mRNA amounts were measured by TaqMan real-time PCR. mRNA VEGF levels are expressed as fold change over the basal after normalizing to β-actin. Each bar is the mean ± SD of three independent experiments. Statistically different from basal (vehicle-treated), Dunnett's test after anova, **P < 0.01.

Figure 5

Effects of nicotinic and transient receptor potential type A1 (TRPA1) receptor antagonists and of various kinase inhibitors against cigarette smoke extract (CSE)-evoked vascular endothelial growth factor (VEGF) release. The nicotinic receptor antagonist mecamylamine (mecamyl; 10 µM) does not alter VEGF release evoked by CSE (optical density, OD = 0.075) in normal human lung fibroblast (NHLF) cultures (A). Nicotine (10 µM) did not affect constitutive VEGF release in NHLF cultures (A). Lack of effect of the EGF receptor inhibitor gefitinib (1 µM) and the TRPA1 antagonist AP-18 (10 µM) on VEGF release stimulated by CSE (OD = 0.075) in NHLF cultures (B). Effect on CSE (OD = 0.075)-evoked VEGF release of 10 µM of the following compounds: the ERK1/2 inhibitor FR180204 (ERKi), the HIF-1α inhibitor 2-methoxy-estradiol (2-ME) and the PI3K-γ inhibitor II (PI3Ki) in airway smooth muscle cell (ASMC) cultures (C). Each histogram is the mean ± SD of three independent experiments performed in quadruplicate. Dunnett's test (A) and Bonferroni's test after anova, *P < 0.05, **P < 0.01. n.s. not significant.

Figure 6

Effects of pharmacological inhibition of p38 MAPK (mitogen-activated protein kinase) and N-acetylcysteine (NAC) on vascular endothelial growth factor (VEGF) release. Effects of NAC (0.3 mM) on VEGF release elicited by cigarette smoke extract (CSE) (optical density, OD = 0.075) and acrolein (30 µM) in airway smooth muscle cell (ASMC) cultures (A). Effects of NAC (0.3 mM) on VEGF release stimulated by CSE (OD = 0.075) in normal human lung fibroblast (NHLF) cultures (B). Inhibitory effects of increasing concentrations of the p38 MAPK inhibitors SB203580 (C) and SB202190 (D) on VEGF release induced by CSE (OD = 0.075) in ASMC cultures. Effects of 1 µM of SB203580 (E) and 1 µM of SB202190 (F) on VEGF release induced by acrolein (30 µM) in ASMC cultures. Effects of H₂O₂ (1 mM) on VEGF release in ASMC cultures in the presence or absence of the p38 MAPK inhibitors SB203580 or SB202190 (G). Each histogram is the mean ± SD of three independent experiments performed in quadruplicate. n.d., not detectable. Bonferroni's test after anova in A–D and Dunnett's test after anova in E–G, **P < 0.01.

Figure 7

Effects of cigarette smoke extract (CSE) and acrolein on p38 MAPK (mitogen-activated protein kinase) and ERK1/2 phosphorylation. Representative blots of p38 MAPK immunoreactivity in airway smooth muscle cell cultures treated with CSE (optical density, OD = 0.1) or acrolein (60 µM) and harvested at the indicated time points (A and B, respectively). Representative blots of ERK1/2 (ERK42/44) immunoreactivity in cells treated with CSE (OD = 0.1) or acrolein (60 µM) and harvested at the indicated time points (C and D, respectively). Effects of 1 µM of the p38 MAPK inhibitors SB203580 and SB202190 (E) and of 0.3 mM of N-acetylcysteine (NAC) (F) on p38 MAPK phosphorylation induced by CSE or acrolein. Quantitative densitometry values (fold changes over basal of phospho-p38 MAPK/total p38 MAPK) are reported below each lane.