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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 induced intracellular ROS generation in capillary-like tube cells and caused cytotoxicity.(A) DEPs were incubated in the cell-free system for 2 h. Cm-H2DCFDA assays were then carried out. ROS production is shown for each sample. (B) HUVECs were exposed to DEPs ± NAC for 24 h. Intracellular ROS is shown. (C) Tube cells were exposed to DEPs with or without the addition of NAC. Cytotoxicity was measured as described in the Methods.
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pone.0131911.g001: DEPs induced intracellular ROS generation in capillary-like tube cells and caused cytotoxicity.(A) DEPs were incubated in the cell-free system for 2 h. Cm-H2DCFDA assays were then carried out. ROS production is shown for each sample. (B) HUVECs were exposed to DEPs ± NAC for 24 h. Intracellular ROS is shown. (C) Tube cells were exposed to DEPs with or without the addition of NAC. Cytotoxicity was measured as described in the Methods.

Mentions: Chao et al (2012) demonstrated that tube cells exposed to 1 μg/mL DEPs generates H2O2 (0.18 nmol). At 10 and 100 μg/mL concentrations of DEPs, production of H2O2 in tube cells was increased to 0.51 and 2.05 nmol, respectively [12]. Thus, cytotoxicity might be a consequence of exposure to DEP-induced oxidative stress. However, whether these free radicals are generated from DEPs before they contact the cells or after they are taken up by cells and induce sequential intracellular oxidative damage remains unclear. Therefore, we used Cm-H2DCFDA to evaluate this pathway. As shown in Fig 1A, changes in relatively fluorescence units (RFU) could be used to demonstrate the level of DEP-induced ROS in the cell-free model. From these data, the increased RFU was observed only in the H2O2-containing sample with a time-dependent manner. Other samples containing DEPs (10 or 100 μg/mL) ± NAC (10 mM) showed no detectable changes in RFU. In contrast, Cm-H2DCFDA analysis showed that DEP induced intracellular ROS generation in tube cells in a dose-dependent manner (Fig 1B). Addition of NAC, oxidative stress was completely blocked. Fig 1C shows cytotoxicity curves for HUVEC tube cells treated with DEPs (1, 5, 10, 50, or 100 μg/mL) ± NAC. Cytotoxicity in tube cells exposed to 1 μg/mL DEP reached 12%; additionally, 5 and 10 μg/mL DEPs induced cell death in 19% and 22% of cells, respectively. Higher doses of DEPs (50 and 100 μg/mL) also induced greater cell death (45% and 49%, respectively). Thus, cytotoxicity was increased in a dose-dependent manner, similar to the results shown in Fig 1B, suggesting that intracellular ROS might affect cell survival. These experiments not only confirm the results generated by Chao et al (2012), but also suggest that cytotoxicity is directly caused by the free radicals generated in tube cells [12].


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 induced intracellular ROS generation in capillary-like tube cells and caused cytotoxicity.(A) DEPs were incubated in the cell-free system for 2 h. Cm-H2DCFDA assays were then carried out. ROS production is shown for each sample. (B) HUVECs were exposed to DEPs ± NAC for 24 h. Intracellular ROS is shown. (C) Tube cells were exposed to DEPs with or without the addition of NAC. Cytotoxicity was measured as described in the Methods.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131911.g001: DEPs induced intracellular ROS generation in capillary-like tube cells and caused cytotoxicity.(A) DEPs were incubated in the cell-free system for 2 h. Cm-H2DCFDA assays were then carried out. ROS production is shown for each sample. (B) HUVECs were exposed to DEPs ± NAC for 24 h. Intracellular ROS is shown. (C) Tube cells were exposed to DEPs with or without the addition of NAC. Cytotoxicity was measured as described in the Methods.
Mentions: Chao et al (2012) demonstrated that tube cells exposed to 1 μg/mL DEPs generates H2O2 (0.18 nmol). At 10 and 100 μg/mL concentrations of DEPs, production of H2O2 in tube cells was increased to 0.51 and 2.05 nmol, respectively [12]. Thus, cytotoxicity might be a consequence of exposure to DEP-induced oxidative stress. However, whether these free radicals are generated from DEPs before they contact the cells or after they are taken up by cells and induce sequential intracellular oxidative damage remains unclear. Therefore, we used Cm-H2DCFDA to evaluate this pathway. As shown in Fig 1A, changes in relatively fluorescence units (RFU) could be used to demonstrate the level of DEP-induced ROS in the cell-free model. From these data, the increased RFU was observed only in the H2O2-containing sample with a time-dependent manner. Other samples containing DEPs (10 or 100 μg/mL) ± NAC (10 mM) showed no detectable changes in RFU. In contrast, Cm-H2DCFDA analysis showed that DEP induced intracellular ROS generation in tube cells in a dose-dependent manner (Fig 1B). Addition of NAC, oxidative stress was completely blocked. Fig 1C shows cytotoxicity curves for HUVEC tube cells treated with DEPs (1, 5, 10, 50, or 100 μg/mL) ± NAC. Cytotoxicity in tube cells exposed to 1 μg/mL DEP reached 12%; additionally, 5 and 10 μg/mL DEPs induced cell death in 19% and 22% of cells, respectively. Higher doses of DEPs (50 and 100 μg/mL) also induced greater cell death (45% and 49%, respectively). Thus, cytotoxicity was increased in a dose-dependent manner, similar to the results shown in Fig 1B, suggesting that intracellular ROS might affect cell survival. These experiments not only confirm the results generated by Chao et al (2012), but also suggest that cytotoxicity is directly caused by the free radicals generated in tube cells [12].

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