Combination effects of cigarette smoke extract and ambient ultrafine particles on endothelial cells

Abstract

Previous studies have shown that ambient ultrafine particles with diameters less than 100nm (UFPs) can pass from the lungs to the circulation because of their very small diameter, and induce lung oxidative stress with a resultant dysfunction of lung endothelial cells. However, no studies have addressed the potential combined effects of UFPs and cigarette smoke on vascular endothelial cells. We hypothesized that co-exposure to UFPs and cigarette smoke extract (CSE) may cause combined effects on activation of endothelial cells and dysfunction of endothelium by oxidative stress through activation of NADPH oxidase. We determined the effects of UFPs with or without CSE on mouse pulmonary microvascular endothelial cells (MPMVEC) obtained from C57BL/6J (wild-type) and gp91(phox) knock-out mice (gp91(phox) is one of the key components of NADPH oxidase, one of ROS generators). Our results showed that exposure of MPMVEC from wild-type mice to UFPs or CSE, at a non-toxic dose, induced reactive oxygen species (ROS) generation, increased phosphorylation of p38 and Erk1/2, and up-regulated early growth response -1 (Egr-1) and IL-6 genes. These effects were significantly enhanced when cells were co-exposed to both UFPs and CSE. However, exposure of MPMVEC from gp91(phox) knock-out mice did not induce the above effects. Furthermore, UFPs- and/or CSE-induced Egr-1 mRNA upregulation was attenuated significantly when cells were pre-treated with p38 specific inhibitor, SB 203580, or MEK1/2 inhibitor, PD98059, and Egr-1 siRNA treatment abolished UFPs- and/or CSE-induced overexpression of IL-6. Our results suggest that UFPs and/or CSE caused activation of NADPH oxidase, resulting in ROS generation that led to activation of MAPKs through induced phosphorylation of p38 and ERK1/2 MAPKs and upregulation of Egr-1. Those effects may further result in endothelial dysfunction through production of cytokines such as IL-6. Our results suggest that co-exposure to UFPs and CSE causes enhanced injury to endothelial cells.

Figure 1. Cytotoxicity of UFPs (A), CSE (B) and UFPs with CSE (C) on MPMVEC from wild-type or gp91^phox KO mice

3 × 10³ cells were seeded into each well of 96-well plates. After overnight culture, cells were treated with UFPs, CSE or UFPs with CSE, respectively. Cytotoxicity was determined with MTS assay kit (Promega) after 24 h treatment. Cells without UFPs or CSE treatment were used as controls. Data are shown as mean ± SD of three experiments with six replicates in each experiment. * Significant difference as compared with the control, p < 0.05.

Figure 2. Effects of UFPs, CSE and UFPs with CSE on ROS generation in MPMVEC from wild-type or gp91^phox KO mice

1 × 10⁴ cells were seeded into each well of 96-well plates. After overnight culture, cells were pretreated with 5 μM H₂-DCFDA for 2 h prior to exposure to UFPs, CSE or UFPs with CSE for 4 h. Data are shown as mean ± SD of three experiments with six replicates in each experiment. DCF fluorescence in cells with 5 μM H₂-DCFDA pre-treatment but without UFPs and CSE treatment was used as control. * Significant difference as compared with the control, p < 0.05; # Significant difference as compared with the UFPs alone or CSE alone treated group, p < 0.05.

Figure 3. Increased phosphorylation of p38 and ERK1/2 in MPMVEC from wild-type mice exposed to UFPs, CSE and UFPs with CSE (A & B). In MPMVEC from gp91^phox KO mice, UFPs and CSE failed to induce such effects (C & D)

Cells were treated with UFPs, CSE or UFPs with CSE for one hour. Cells without UFPs and CSE treatment were used as control. 30 μg protein (phospho-p38 and p38) and 10 μg protein (phospho-ERK1/2 and ERK1/2) were loaded in each lane. For ERK, two different isoforms were identified, ERK1 (p44) and ERK2 (p42). P-p38, phospho-p38; P-ERK1/2, phospho-ERK1/2. A and C show the results of a single Western blot experiment. B and D represent normalized band densitometry readings averaged from 3 independent experiments ± SD of Western results. * Significant difference as compared with the control, p < 0.05; # Significant difference as compared with UFPs alone or CSE alone treated group, p < 0.05.

Figure 3. Increased phosphorylation of p38 and ERK1/2 in MPMVEC from wild-type mice exposed to UFPs, CSE and UFPs with CSE (A & B). In MPMVEC from gp91^phox KO mice, UFPs and CSE failed to induce such effects (C & D)

Cells were treated with UFPs, CSE or UFPs with CSE for one hour. Cells without UFPs and CSE treatment were used as control. 30 μg protein (phospho-p38 and p38) and 10 μg protein (phospho-ERK1/2 and ERK1/2) were loaded in each lane. For ERK, two different isoforms were identified, ERK1 (p44) and ERK2 (p42). P-p38, phospho-p38; P-ERK1/2, phospho-ERK1/2. A and C show the results of a single Western blot experiment. B and D represent normalized band densitometry readings averaged from 3 independent experiments ± SD of Western results. * Significant difference as compared with the control, p < 0.05; # Significant difference as compared with UFPs alone or CSE alone treated group, p < 0.05.

Figure 4. Egr-1 induction in MPMVEC from wild-type mice exposed to UFPs, CSE and UFPs with CSE (A & B), but not in MPMVEC from gp91^phox KO mice (C)

2 × 10⁵ cells were seeded into each well of 6-well plates. After overnight culture, cells were treated with UFPs, CSE, or UFPs with CSE. Cells were collected after one hour treatment for real-time PCR (A) or after 3 hours treatment for Western blot (B & C). (A) Data are shown as mean ± SD of three experiments with three replicates in each experiment. * Significant difference as compared with the control, p < 0.05; # Significant difference as compared with UFPs alone or CSE alone treated group, p < 0.05.

Figure 5. UFPs- and/or CSE- induced Egr-1 upregulation in MPMVEC from wild-type mice was abolished by pre-treatment of cells with SB203580 (A) and PD98059 (B)

2 × 10⁵ cells were seeded into each well of 6-well plates. After overnight culture, cells were pre-treated with 5 μM or 10 μM of SB203580 (A), or 20 μM of PD98059 (B) for 3 h, then treated with UFPs and/or CSE for one hour. Total RNA was extracted and used for real-time PCR. Data are shown as mean ± SD of three experiments with three replicates in each experiment. * Significant difference as compared with the control (without any treatment), p < 0.05; # Significant difference as compared with UFPs alone or CSE alone treated group, p < 0.05; $ Significant difference as compared with those with UFPs and/or CSE treatment but without SB203580 or PD98059 pre-treatment, p < 0.05.

Figure 6. Increased expression of IL-6 in MPMVEC from wild-type mice exposed to UFPs, CSE and UFPs with CSE

2 × 10⁵ cells were seeded into each well of 6-well plates. After overnight culture, cells were treated with UFPs, CSE, or UFPs with CSE for 12 h (A) or 24 h (B). Cells without any treatment were used as controls. (A) IL-6 mRNA was analyzed by real-time PCR. Data are shown as mean ± SD of three experiments with three replicates in each experiment. (B) IL-6 in the cell culture medium was determined by ELISA. Data are shown as mean ± SD of three experiments with duplicates in each experiment. * Significant difference as compared with the control, p < 0.05; # Significant difference as compared with UFPs alone or CSE alone treated group, p < 0.05.

Figure 7

Increased expression of IL-6 in MPMVEC from wild-type mice exposed to UFPs and/or CSE was attenuated by pre-treatment of cells with p38 inhibitor. 2 × 10⁵ cells were seeded into each well of 6-well plates. After overnight culture, cells were pre-treated with 10 μM SB203580 for 3 h, then treated with UFPs and/or CSE for 12 h. Cells without any treatment were used as controls. Real-time PCR was performed with iQ5 Cycler (Bio-Rad). Data are shown as mean ± SD of three experiments with three replicates in each experiment. * Significant difference as compared with the control, p < 0.05; # Significant difference as compared with UFPs alone or CSE alone treated group, p < 0.05; $ Significant difference as compared with those with UFPs and/or CSE treatment but without SB203580 pre-treatment, p < 0.05.

Figure 8. Egr-1 siRNA treatment inhibited Egr-1 expression (A) and abolished increased expression of IL-6 in MPMVEC from wild-type mice exposed to UFPs, CSE and UFPs with CSE (B)

(A) MPMVEC were collected for Western blot after transfected with 100 nM of Egr-1 or control siRNA. 40 μg (Egr-1) or 5 μg (β-actin) protein was loaded in each lane. Control, cells without transfection; Lipofectamine, cells treated with Lipofectamine 2000 Reagent (Invitrogen) but without any siRNA treatment; Control siRNA, cells transfected with control siRNA-A (Santa Cruz). (B) Cells were transfected with Egr-1 siRNA for 24 h following treatment with UFPs, CSE, or UFPs with CSE for 12h. Real-time PCR was performed with iQ5 Cycler (Bio-Rad). Data are shown as mean ± SD of three experiments with three replicates in each experiment. * Significant difference as compared with the control (without any treatment), p < 0.05; # Significant difference as compared with UFPs alone or CSE alone treated group, p < 0.05; $ Significant difference as compared with non-siRNA transfected UFPs- and/or CSE- treated group, p < 0.05.