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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

DEP dissociate VEGF-R2 from adherens junction connection.(A) After 24 h DEP (1, 10 and 100 g/mL) exposure, the tube cells were lysed and harvested. The untreated cells (0 g/mL) were defined as negative control. The level of adheren junctional proteins VEGF-R2, VE-cadherin (VE-Cad), -catenin and cytoskeleton actin were determined by Western blot. Equal amount of protein loading was confirmed based on total GAPDH expression. (B) Quantification showed VEGF-R2 decrease in a dose dependent manner as the relative fold change to the control. Again, the level of VE-cadherin, -catenin and cytoskeleton actin activities are unaffected by DEP. (C) Images show the distribution of VEGF-R2 in response to DEP (10 and 100 g/mL) and DEP (100 g/mL) plus NAC exposure for 24 h. Nuclei were stained with 10 mM DAPI. Shown is a representative image from three independent experiments. Scale bar = 10 m. Magnification is 400X. (D) To confirm whether DEP change VEGF-R2 expression, the cDNA were probed with VEGFR2 primers and the activity were analyzed using QPCR after exposure with DEP for 24 h. The level of mRNA normalized to 18s gene expression was presented as the fold change relative to the untreated control (0 μg/mL). Means ± SD, n = 3. (E) Effects of DEP on the secretion of VEGF-R2. Secretion of VEGF-R2 of capillary tube cells related to the DEP exposure. After 24 h DEP (1, 10 and 100 g/mL) exposure, the supernatant/medium were collected and loaded (40 L/well) onto the SDS-PAGE. The expression of secreted VEGF-R2 was tested and measured by using western blot. Coomassie blue stain was defined as the loading control. The supernatant collected from the cells treated without DEP were defined as negative control.
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pone.0131911.g007: DEP dissociate VEGF-R2 from adherens junction connection.(A) After 24 h DEP (1, 10 and 100 g/mL) exposure, the tube cells were lysed and harvested. The untreated cells (0 g/mL) were defined as negative control. The level of adheren junctional proteins VEGF-R2, VE-cadherin (VE-Cad), -catenin and cytoskeleton actin were determined by Western blot. Equal amount of protein loading was confirmed based on total GAPDH expression. (B) Quantification showed VEGF-R2 decrease in a dose dependent manner as the relative fold change to the control. Again, the level of VE-cadherin, -catenin and cytoskeleton actin activities are unaffected by DEP. (C) Images show the distribution of VEGF-R2 in response to DEP (10 and 100 g/mL) and DEP (100 g/mL) plus NAC exposure for 24 h. Nuclei were stained with 10 mM DAPI. Shown is a representative image from three independent experiments. Scale bar = 10 m. Magnification is 400X. (D) To confirm whether DEP change VEGF-R2 expression, the cDNA were probed with VEGFR2 primers and the activity were analyzed using QPCR after exposure with DEP for 24 h. The level of mRNA normalized to 18s gene expression was presented as the fold change relative to the untreated control (0 μg/mL). Means ± SD, n = 3. (E) Effects of DEP on the secretion of VEGF-R2. Secretion of VEGF-R2 of capillary tube cells related to the DEP exposure. After 24 h DEP (1, 10 and 100 g/mL) exposure, the supernatant/medium were collected and loaded (40 L/well) onto the SDS-PAGE. The expression of secreted VEGF-R2 was tested and measured by using western blot. Coomassie blue stain was defined as the loading control. The supernatant collected from the cells treated without DEP were defined as negative control.

Mentions: The cytoplasmic tail of VE-cadherin is known to interact with VEGF-R2 and β-catenin and form a complex linking the actin cytoskeleton to the cell membrane to stabilize cell shape and adherens junctions [47]. Depolarization of actin cytoskeleton network and disruption of VEGF-R2-VE-cadherin formed complex might not only affect endothelial cell-cell contact, but also regulate cell survivability [12, 47–49]. Hence, here we assess whether VEGF-R2 and other junctional proteins change in response to DEP. As shown in Fig 7A, VE-cadherin and actin are unaffected by DEP exposure, but only VEGF-R2 is decreased slightly. According to a quantitation analysis (Fig 7B), VEGF-R2 expresses at 87.5% and 75% intensity of control at 10 and 100 μg/mL respectively, but remains unchanged at 1 μg/mL. Furthermore, fluorescence images show the morphology of VEGF-R2 that is outlining the cell junctions fairly regularly in the negative control. Image of DEP (10 μg/mL) treated tube cells indicate a loss in sharpness of border (arrows) to the VEGF-R2 staining. At 100 μg/mL DEP treated tube cells, which have a similar VEGF-R2 performance to 10 μg/mL, but has more discontinuity patterns on the cell-cell boarder. Addition of NAC mitigates this VEGF-R2 alternation. On the other hand, our qPCR result reveals that VEGF-R2 mRNA level is unaffected (Fig 7D). This interesting result implies that VEGF-R2 may be secreted to the supernatant/medium. As shown in Fig 7E, western blot indicated that sVEGF-R2 (soluble VEGF-R2) and VEGF-A in the medium are dramatically increased in a DEP dose dependent manner.


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)

DEP dissociate VEGF-R2 from adherens junction connection.(A) After 24 h DEP (1, 10 and 100 g/mL) exposure, the tube cells were lysed and harvested. The untreated cells (0 g/mL) were defined as negative control. The level of adheren junctional proteins VEGF-R2, VE-cadherin (VE-Cad), -catenin and cytoskeleton actin were determined by Western blot. Equal amount of protein loading was confirmed based on total GAPDH expression. (B) Quantification showed VEGF-R2 decrease in a dose dependent manner as the relative fold change to the control. Again, the level of VE-cadherin, -catenin and cytoskeleton actin activities are unaffected by DEP. (C) Images show the distribution of VEGF-R2 in response to DEP (10 and 100 g/mL) and DEP (100 g/mL) plus NAC exposure for 24 h. Nuclei were stained with 10 mM DAPI. Shown is a representative image from three independent experiments. Scale bar = 10 m. Magnification is 400X. (D) To confirm whether DEP change VEGF-R2 expression, the cDNA were probed with VEGFR2 primers and the activity were analyzed using QPCR after exposure with DEP for 24 h. The level of mRNA normalized to 18s gene expression was presented as the fold change relative to the untreated control (0 μg/mL). Means ± SD, n = 3. (E) Effects of DEP on the secretion of VEGF-R2. Secretion of VEGF-R2 of capillary tube cells related to the DEP exposure. After 24 h DEP (1, 10 and 100 g/mL) exposure, the supernatant/medium were collected and loaded (40 L/well) onto the SDS-PAGE. The expression of secreted VEGF-R2 was tested and measured by using western blot. Coomassie blue stain was defined as the loading control. The supernatant collected from the cells treated without DEP were defined as negative control.
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Related In: Results  -  Collection

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pone.0131911.g007: DEP dissociate VEGF-R2 from adherens junction connection.(A) After 24 h DEP (1, 10 and 100 g/mL) exposure, the tube cells were lysed and harvested. The untreated cells (0 g/mL) were defined as negative control. The level of adheren junctional proteins VEGF-R2, VE-cadherin (VE-Cad), -catenin and cytoskeleton actin were determined by Western blot. Equal amount of protein loading was confirmed based on total GAPDH expression. (B) Quantification showed VEGF-R2 decrease in a dose dependent manner as the relative fold change to the control. Again, the level of VE-cadherin, -catenin and cytoskeleton actin activities are unaffected by DEP. (C) Images show the distribution of VEGF-R2 in response to DEP (10 and 100 g/mL) and DEP (100 g/mL) plus NAC exposure for 24 h. Nuclei were stained with 10 mM DAPI. Shown is a representative image from three independent experiments. Scale bar = 10 m. Magnification is 400X. (D) To confirm whether DEP change VEGF-R2 expression, the cDNA were probed with VEGFR2 primers and the activity were analyzed using QPCR after exposure with DEP for 24 h. The level of mRNA normalized to 18s gene expression was presented as the fold change relative to the untreated control (0 μg/mL). Means ± SD, n = 3. (E) Effects of DEP on the secretion of VEGF-R2. Secretion of VEGF-R2 of capillary tube cells related to the DEP exposure. After 24 h DEP (1, 10 and 100 g/mL) exposure, the supernatant/medium were collected and loaded (40 L/well) onto the SDS-PAGE. The expression of secreted VEGF-R2 was tested and measured by using western blot. Coomassie blue stain was defined as the loading control. The supernatant collected from the cells treated without DEP were defined as negative control.
Mentions: The cytoplasmic tail of VE-cadherin is known to interact with VEGF-R2 and β-catenin and form a complex linking the actin cytoskeleton to the cell membrane to stabilize cell shape and adherens junctions [47]. Depolarization of actin cytoskeleton network and disruption of VEGF-R2-VE-cadherin formed complex might not only affect endothelial cell-cell contact, but also regulate cell survivability [12, 47–49]. Hence, here we assess whether VEGF-R2 and other junctional proteins change in response to DEP. As shown in Fig 7A, VE-cadherin and actin are unaffected by DEP exposure, but only VEGF-R2 is decreased slightly. According to a quantitation analysis (Fig 7B), VEGF-R2 expresses at 87.5% and 75% intensity of control at 10 and 100 μg/mL respectively, but remains unchanged at 1 μg/mL. Furthermore, fluorescence images show the morphology of VEGF-R2 that is outlining the cell junctions fairly regularly in the negative control. Image of DEP (10 μg/mL) treated tube cells indicate a loss in sharpness of border (arrows) to the VEGF-R2 staining. At 100 μg/mL DEP treated tube cells, which have a similar VEGF-R2 performance to 10 μg/mL, but has more discontinuity patterns on the cell-cell boarder. Addition of NAC mitigates this VEGF-R2 alternation. On the other hand, our qPCR result reveals that VEGF-R2 mRNA level is unaffected (Fig 7D). This interesting result implies that VEGF-R2 may be secreted to the supernatant/medium. As shown in Fig 7E, western blot indicated that sVEGF-R2 (soluble VEGF-R2) and VEGF-A in the medium are dramatically increased in a DEP dose dependent manner.

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