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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

Addition of TNF-α and IL-6 triggered release of VEGF-A from endothelial tube cells.(A) The tube cells were treated with 47.5 and 42.4 pg/mL TNF-α and VEGF-A, respectively, and incubated for a 24 h. The medium was collected and subjected to SDS-PAGE. Half the gel was used for western blot analysis, probing with anti-VEGF-A antibodies, and the other half was identically loaded as a loading control, stained with Coomassie blue. (B) Semiquantitative analysis of the intensity of VEGF-A bands from the blot. Furthermore, TNF-α plus IL-6 independently stimulated HO-1 expression. (C) Analysis of HO-1 expression in HUVEC tube lysates was performed using immunoblotting. Equal protein loading was confirmed with GAPDH immunoreactivity. (D) Semiquantitative analysis of the intensities of HO-1 bands from the blot. § indicates p < 0.05 significance decrease from DEP (100 g/mL) to NAC+DEP (100 g/mL). (E) HO-1 mRNA expression response to TNF-α and IL-6 was quantified using qPCR, with normalization to 18S. HO-1 expressed in the negative control was defined as 1. Values are expressed as the fold-change compared to the negative control (n = 3). Significance was reached at *p < 0.05 as analyzed using Student’s t-test.
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pone.0131911.g004: Addition of TNF-α and IL-6 triggered release of VEGF-A from endothelial tube cells.(A) The tube cells were treated with 47.5 and 42.4 pg/mL TNF-α and VEGF-A, respectively, and incubated for a 24 h. The medium was collected and subjected to SDS-PAGE. Half the gel was used for western blot analysis, probing with anti-VEGF-A antibodies, and the other half was identically loaded as a loading control, stained with Coomassie blue. (B) Semiquantitative analysis of the intensity of VEGF-A bands from the blot. Furthermore, TNF-α plus IL-6 independently stimulated HO-1 expression. (C) Analysis of HO-1 expression in HUVEC tube lysates was performed using immunoblotting. Equal protein loading was confirmed with GAPDH immunoreactivity. (D) Semiquantitative analysis of the intensities of HO-1 bands from the blot. § indicates p < 0.05 significance decrease from DEP (100 g/mL) to NAC+DEP (100 g/mL). (E) HO-1 mRNA expression response to TNF-α and IL-6 was quantified using qPCR, with normalization to 18S. HO-1 expressed in the negative control was defined as 1. Values are expressed as the fold-change compared to the negative control (n = 3). Significance was reached at *p < 0.05 as analyzed using Student’s t-test.

Mentions: HO-1 has previously been shown to induce the secretion of VPF/VEGF-A [18, 40, 41]. Chao et al (2012) demonstrated that DEPs upregulate HO-1 and stimulate VEGFA secretion into the culture media [12]. Because our previous experiment only investigated whether TNF-α and IL-6 were upregulated, we wondered whether these cytokines affected permeability by directly influencing VEGFA levels. Therefore, we treated tube cells with exogenous TNF-α (47.5 pg/mL) and IL-6 (42.4 pg/mL) for 24 h to mimic the effects of 24-h exposure to 100 μg/mL DEPs. As seen in Fig 4A, the combination treatment with both cytokines for 24 h further increased the level of VEGF-A. Addition of Tin protoporphyrin IX (SnPP) (HO-1 inhibitor, 25 μM) might not affect the VEGF-A secretion. Quantification of the results is shown in Fig 4B, indicating that VEGF-A secretion was associated with a DEP-induced pro-inflammatory response but not directly correlated to HO-1.


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)

Addition of TNF-α and IL-6 triggered release of VEGF-A from endothelial tube cells.(A) The tube cells were treated with 47.5 and 42.4 pg/mL TNF-α and VEGF-A, respectively, and incubated for a 24 h. The medium was collected and subjected to SDS-PAGE. Half the gel was used for western blot analysis, probing with anti-VEGF-A antibodies, and the other half was identically loaded as a loading control, stained with Coomassie blue. (B) Semiquantitative analysis of the intensity of VEGF-A bands from the blot. Furthermore, TNF-α plus IL-6 independently stimulated HO-1 expression. (C) Analysis of HO-1 expression in HUVEC tube lysates was performed using immunoblotting. Equal protein loading was confirmed with GAPDH immunoreactivity. (D) Semiquantitative analysis of the intensities of HO-1 bands from the blot. § indicates p < 0.05 significance decrease from DEP (100 g/mL) to NAC+DEP (100 g/mL). (E) HO-1 mRNA expression response to TNF-α and IL-6 was quantified using qPCR, with normalization to 18S. HO-1 expressed in the negative control was defined as 1. Values are expressed as the fold-change compared to the negative control (n = 3). Significance was reached at *p < 0.05 as analyzed using Student’s t-test.
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Related In: Results  -  Collection

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pone.0131911.g004: Addition of TNF-α and IL-6 triggered release of VEGF-A from endothelial tube cells.(A) The tube cells were treated with 47.5 and 42.4 pg/mL TNF-α and VEGF-A, respectively, and incubated for a 24 h. The medium was collected and subjected to SDS-PAGE. Half the gel was used for western blot analysis, probing with anti-VEGF-A antibodies, and the other half was identically loaded as a loading control, stained with Coomassie blue. (B) Semiquantitative analysis of the intensity of VEGF-A bands from the blot. Furthermore, TNF-α plus IL-6 independently stimulated HO-1 expression. (C) Analysis of HO-1 expression in HUVEC tube lysates was performed using immunoblotting. Equal protein loading was confirmed with GAPDH immunoreactivity. (D) Semiquantitative analysis of the intensities of HO-1 bands from the blot. § indicates p < 0.05 significance decrease from DEP (100 g/mL) to NAC+DEP (100 g/mL). (E) HO-1 mRNA expression response to TNF-α and IL-6 was quantified using qPCR, with normalization to 18S. HO-1 expressed in the negative control was defined as 1. Values are expressed as the fold-change compared to the negative control (n = 3). Significance was reached at *p < 0.05 as analyzed using Student’s t-test.
Mentions: HO-1 has previously been shown to induce the secretion of VPF/VEGF-A [18, 40, 41]. Chao et al (2012) demonstrated that DEPs upregulate HO-1 and stimulate VEGFA secretion into the culture media [12]. Because our previous experiment only investigated whether TNF-α and IL-6 were upregulated, we wondered whether these cytokines affected permeability by directly influencing VEGFA levels. Therefore, we treated tube cells with exogenous TNF-α (47.5 pg/mL) and IL-6 (42.4 pg/mL) for 24 h to mimic the effects of 24-h exposure to 100 μg/mL DEPs. As seen in Fig 4A, the combination treatment with both cytokines for 24 h further increased the level of VEGF-A. Addition of Tin protoporphyrin IX (SnPP) (HO-1 inhibitor, 25 μM) might not affect the VEGF-A secretion. Quantification of the results is shown in Fig 4B, indicating that VEGF-A secretion was associated with a DEP-induced pro-inflammatory response but not directly correlated to HO-1.

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