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Protective Effects of N-Acetyl Cysteine against Diesel Exhaust Particles-Induced Intracellular ROS Generates Pro-Inflammatory Cytokines to Mediate the Vascular Permeability of Capillary-Like Endothelial Tubes.

Tseng CY, Chang JF, Wang JS, Chang YJ, Gordon MK, Chao MW - PLoS ONE (2015)

Bottom Line: Previous studies using in vitro endothelial tubes as a simplified model of capillaries have found that DEP-induced ROS increase vascular permeability with rearrangement or internalization of adherens junctional VE-cadherin away from the plasma membrane.Our data suggests that DEP-induced intracellular ROS and release of the pro-inflammatory cytokines TNF- α and IL-6, which would contribute to VEGF-A secretion and disrupt cell-cell borders and increase vasculature permeability.Addition of NAC suppresses DEP-induced ROS efficiently and reduces subsequent damages by increasing endogenous glutathione.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, College of Engineering, Chung Yuan Christian University, Zhongli district, Taoyuan city, Taiwan; Center of Nanotechnology, Chung Yuan Christian University, Zhongli district, Taoyuan city, Taiwan.

ABSTRACT
Exposure to diesel exhaust particles (DEP) is associated with pulmonary and cardiovascular diseases. Previous studies using in vitro endothelial tubes as a simplified model of capillaries have found that DEP-induced ROS increase vascular permeability with rearrangement or internalization of adherens junctional VE-cadherin away from the plasma membrane. This allows DEPs to penetrate into the cell and capillary lumen. In addition, pro-inflammatory cytokines are up-regulated and mediate vascular permeability in response to DEP. However, the mechanisms through which these DEP-induced pro-inflammatory cytokines increase vascular permeability remain unknown. Hence, we examined the ability of DEP to induce permeability of human umbilical vein endothelial cell tube cells to investigate these mechanisms. Furthermore, supplementation with NAC reduces ROS production following exposure to DEP. HUVEC tube cells contributed to a pro-inflammatory response to DEP-induced intracellular ROS generation. Endothelial oxidative stress induced the release of TNF-α and IL-6 from tube cells, subsequently stimulating the secretion of VEGF-A independent of HO-1. Our data suggests that DEP-induced intracellular ROS and release of the pro-inflammatory cytokines TNF- α and IL-6, which would contribute to VEGF-A secretion and disrupt cell-cell borders and increase vasculature permeability. Addition of NAC suppresses DEP-induced ROS efficiently and reduces subsequent damages by increasing endogenous glutathione.

No MeSH data available.


Related in: MedlinePlus

DEPs affected the expression of intracellular proteins.(A) After a 24-h exposure to DEPs ± NAC, endothelial tube cells were lysed, and cytoplasmic and nuclear proteins were extracted for western blot analysis. The 8 lanes of the immunoblot represent samples exposed to different concentrations of DEPs and DEPs+NAC. Equal loading of cytoplasmic and nuclear proteins was confirmed with GAPDH and Lamin B1 immunoreactivities, respectively. (B) Semiquantitative analysis of the band intensities. Significance was reached at *p < 0.05, **p < 0.01, and ***p < 0.001, as analyzed using Student’s t-test.
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pone.0131911.g003: DEPs affected the expression of intracellular proteins.(A) After a 24-h exposure to DEPs ± NAC, endothelial tube cells were lysed, and cytoplasmic and nuclear proteins were extracted for western blot analysis. The 8 lanes of the immunoblot represent samples exposed to different concentrations of DEPs and DEPs+NAC. Equal loading of cytoplasmic and nuclear proteins was confirmed with GAPDH and Lamin B1 immunoreactivities, respectively. (B) Semiquantitative analysis of the band intensities. Significance was reached at *p < 0.05, **p < 0.01, and ***p < 0.001, as analyzed using Student’s t-test.

Mentions: The respiratory system and capillaries have been shown to respond to DEP exposure by secreting the pro-inflammatory cytokines TNF-α and IL-6 [7, 38, 39]. To examine whether DEP-induced ROS triggered the release of these cytokines from in vitro endothelial tubes and NAC reduces this activity, cultures were treated for 24 h with 1, 10, or 100 μg/mL DEPs ± NAC. As determined by comparison with an enzyme-linked immunosorbent assay (ELISA)-generated standard curve, negative control HUVEC tubes released 16.6 pg/mL TNF-α (Fig 2A) and 11.8 pg/mL IL-6 (Fig 2B) into the medium. No significant difference was observed in cytokine levels when cells were treated with 1 μg/mL DEPs. However, at 10 μg/mL DEPs, 22.7 ± 0.6 pg/mL TNF-α and 21.8 ± 0.6 pg/mL IL-6 was found in the medium. Moreover, after a 24-h exposure to 100 μg/mL DEPs, tube cells released 47.5 ± 2.3 pg/mL TNF-α and 42.4 ± 3.6 pg/mL IL-6, about 4 times that of the untreated control. The presence of NAC significantly suppressed the release of both cytokines. With this ROS scavenger, secretion was reduced to 20.4 ± 5.2 pg/mL TNF-α and 20.2 ± 0.84 pg/mL IL-6 in samples treated with 100 μg/mL DEPs, comparable to levels of these cytokines in cells treated with 10 μg/mL DEPs without the addition of NAC, strongly indicating that DEP-induced ROS mediated the secretion of these cytokines from endothelial tube cells. However, as shown in Fig 2C, no significant upregulation of IL-1β was observed in response to DEPs. Furthermore, as shown in Fig 3, immunoblotting results supported that DEPs dramatically increased levels of cytoplasmic HO-1 and slightly increased levels of TNF-α and IL-6. IκB-α was reduced in a dose-dependent manner following treatment with DEPs, while IL-1β was not observed. In nuclear extracts, nuclear p65 was elevated in response to DEP exposure. Addition of NAC significantly reduces HO-1, TNF-α, IL-6, IκB-α, and p65 expression. Quantification of the blots of DEP treated samples is shown in Fig 3B.


Protective Effects of N-Acetyl Cysteine against Diesel Exhaust Particles-Induced Intracellular ROS Generates Pro-Inflammatory Cytokines to Mediate the Vascular Permeability of Capillary-Like Endothelial Tubes.

Tseng CY, Chang JF, Wang JS, Chang YJ, Gordon MK, Chao MW - PLoS ONE (2015)

DEPs affected the expression of intracellular proteins.(A) After a 24-h exposure to DEPs ± NAC, endothelial tube cells were lysed, and cytoplasmic and nuclear proteins were extracted for western blot analysis. The 8 lanes of the immunoblot represent samples exposed to different concentrations of DEPs and DEPs+NAC. Equal loading of cytoplasmic and nuclear proteins was confirmed with GAPDH and Lamin B1 immunoreactivities, respectively. (B) Semiquantitative analysis of the band intensities. Significance was reached at *p < 0.05, **p < 0.01, and ***p < 0.001, as analyzed using Student’s t-test.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4492618&req=5

pone.0131911.g003: DEPs affected the expression of intracellular proteins.(A) After a 24-h exposure to DEPs ± NAC, endothelial tube cells were lysed, and cytoplasmic and nuclear proteins were extracted for western blot analysis. The 8 lanes of the immunoblot represent samples exposed to different concentrations of DEPs and DEPs+NAC. Equal loading of cytoplasmic and nuclear proteins was confirmed with GAPDH and Lamin B1 immunoreactivities, respectively. (B) Semiquantitative analysis of the band intensities. Significance was reached at *p < 0.05, **p < 0.01, and ***p < 0.001, as analyzed using Student’s t-test.
Mentions: The respiratory system and capillaries have been shown to respond to DEP exposure by secreting the pro-inflammatory cytokines TNF-α and IL-6 [7, 38, 39]. To examine whether DEP-induced ROS triggered the release of these cytokines from in vitro endothelial tubes and NAC reduces this activity, cultures were treated for 24 h with 1, 10, or 100 μg/mL DEPs ± NAC. As determined by comparison with an enzyme-linked immunosorbent assay (ELISA)-generated standard curve, negative control HUVEC tubes released 16.6 pg/mL TNF-α (Fig 2A) and 11.8 pg/mL IL-6 (Fig 2B) into the medium. No significant difference was observed in cytokine levels when cells were treated with 1 μg/mL DEPs. However, at 10 μg/mL DEPs, 22.7 ± 0.6 pg/mL TNF-α and 21.8 ± 0.6 pg/mL IL-6 was found in the medium. Moreover, after a 24-h exposure to 100 μg/mL DEPs, tube cells released 47.5 ± 2.3 pg/mL TNF-α and 42.4 ± 3.6 pg/mL IL-6, about 4 times that of the untreated control. The presence of NAC significantly suppressed the release of both cytokines. With this ROS scavenger, secretion was reduced to 20.4 ± 5.2 pg/mL TNF-α and 20.2 ± 0.84 pg/mL IL-6 in samples treated with 100 μg/mL DEPs, comparable to levels of these cytokines in cells treated with 10 μg/mL DEPs without the addition of NAC, strongly indicating that DEP-induced ROS mediated the secretion of these cytokines from endothelial tube cells. However, as shown in Fig 2C, no significant upregulation of IL-1β was observed in response to DEPs. Furthermore, as shown in Fig 3, immunoblotting results supported that DEPs dramatically increased levels of cytoplasmic HO-1 and slightly increased levels of TNF-α and IL-6. IκB-α was reduced in a dose-dependent manner following treatment with DEPs, while IL-1β was not observed. In nuclear extracts, nuclear p65 was elevated in response to DEP exposure. Addition of NAC significantly reduces HO-1, TNF-α, IL-6, IκB-α, and p65 expression. Quantification of the blots of DEP treated samples is shown in Fig 3B.

Bottom Line: Previous studies using in vitro endothelial tubes as a simplified model of capillaries have found that DEP-induced ROS increase vascular permeability with rearrangement or internalization of adherens junctional VE-cadherin away from the plasma membrane.Our data suggests that DEP-induced intracellular ROS and release of the pro-inflammatory cytokines TNF- α and IL-6, which would contribute to VEGF-A secretion and disrupt cell-cell borders and increase vasculature permeability.Addition of NAC suppresses DEP-induced ROS efficiently and reduces subsequent damages by increasing endogenous glutathione.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, College of Engineering, Chung Yuan Christian University, Zhongli district, Taoyuan city, Taiwan; Center of Nanotechnology, Chung Yuan Christian University, Zhongli district, Taoyuan city, Taiwan.

ABSTRACT
Exposure to diesel exhaust particles (DEP) is associated with pulmonary and cardiovascular diseases. Previous studies using in vitro endothelial tubes as a simplified model of capillaries have found that DEP-induced ROS increase vascular permeability with rearrangement or internalization of adherens junctional VE-cadherin away from the plasma membrane. This allows DEPs to penetrate into the cell and capillary lumen. In addition, pro-inflammatory cytokines are up-regulated and mediate vascular permeability in response to DEP. However, the mechanisms through which these DEP-induced pro-inflammatory cytokines increase vascular permeability remain unknown. Hence, we examined the ability of DEP to induce permeability of human umbilical vein endothelial cell tube cells to investigate these mechanisms. Furthermore, supplementation with NAC reduces ROS production following exposure to DEP. HUVEC tube cells contributed to a pro-inflammatory response to DEP-induced intracellular ROS generation. Endothelial oxidative stress induced the release of TNF-α and IL-6 from tube cells, subsequently stimulating the secretion of VEGF-A independent of HO-1. Our data suggests that DEP-induced intracellular ROS and release of the pro-inflammatory cytokines TNF- α and IL-6, which would contribute to VEGF-A secretion and disrupt cell-cell borders and increase vasculature permeability. Addition of NAC suppresses DEP-induced ROS efficiently and reduces subsequent damages by increasing endogenous glutathione.

No MeSH data available.


Related in: MedlinePlus