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Multi-walled carbon nanotubes induce human microvascular endothelial cellular effects in an alveolar-capillary co-culture with small airway epithelial cells.

Snyder-Talkington BN, Schwegler-Berry D, Castranova V, Qian Y, Guo NL - Part Fibre Toxicol (2013)

Bottom Line: Nanotechnology, particularly the use of multi-walled carbon nanotubes (MWCNT), is a rapidly growing discipline with implications for advancement in a variety of fields.While many studies showed adverse effects to the vascular endothelium upon MWCNT exposure, in vitro results often do not correlate with in vivo effects.Following exposure of the epithelial layer to MWCNT, the effects to the endothelial barrier were determined.

View Article: PubMed Central - HTML - PubMed

Affiliation: Pathology and Physiology Research Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, 1095 Willowdale Road, Morgantown, WV 26505-2888, USA.

ABSTRACT

Background: Nanotechnology, particularly the use of multi-walled carbon nanotubes (MWCNT), is a rapidly growing discipline with implications for advancement in a variety of fields. A major route of exposure to MWCNT during both occupational and environmental contact is inhalation. While many studies showed adverse effects to the vascular endothelium upon MWCNT exposure, in vitro results often do not correlate with in vivo effects. This study aimed to determine if an alveolar-capillary co-culture model could determine changes in the vascular endothelium after epithelial exposure to MWCNT.

Methods: A co-culture system in which both human small airway epithelial cells and human microvascular endothelial cells were separated by a Transwell membrane so as to resemble an alveolar-capillary interaction was used. Following exposure of the epithelial layer to MWCNT, the effects to the endothelial barrier were determined.

Results: Exposure of the epithelial layer to MWCNT induced multiple changes in the endothelial cell barrier, including an increase in reactive oxygen species, actin rearrangement, loss of VE-cadherin at the cell surface, and an increase in endothelial angiogenic ability. Overall increases in secreted VEGFA, sICAM-1, and sVCAM-1 protein levels, as well as increases in intracellular phospho-NF-κB, phospho-Stat3, and phospho-p38 MAPK, were also noted in HMVEC after epithelial exposure.

Conclusion: The co-culture system identified that alveolar-capillary exposure to MWCNT induced multiple changes to the underlying endothelium, potentially through cell signaling mediators derived from MWCNT-exposed epithelial cells. Therefore, the co-culture system appears to be a relevant in vitro method to study the pulmonary toxicity of MWCNT.

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SAEC exposure to MWCNT increases the expression of cellular inflammatory signals in HMVEC. Two biological replicates of SAEC and HMVEC were grown in the apical and basolateral chambers, respectively, of a co-culture system, and SAEC was exposed to DM or 1.2 μg/ml MWCNT for 1, 6 or 24 h. HMVEC were lysed and protein levels of intracellular inflammatory signals read at an absorbance of 450 nm using a PathScan Inflammation Multi-Target Sandwich ELISA from Cell Signaling Technology. All values given are the mean ± standard error. NF-κB had a significant decrease in absorbance over time (DM, 1.152 ± 0.051; 1 h, 0.713 ± 0.048; 6 h, 0.6795 ± 0.0145; 24 h, 0.7445 ± 0.0055). Phospho-NF-κB (Ser536) (DM, 0.341 ± 0.013; 1 h, 1.0065 ± 0.0855; 6 h, 0.944 ± 0.187; 24 h, 1.068 ± 0.026) and phospho-Stat3 (Tyr705) (DM, 0.96 ± 0.107; 1 h, 1.483 ± 0.079; 6 h, 1.7265 ± 0.1895; 24 h, 1.446 ± 0.07) had significant increases in absorbance over time, while phospho-p38 (Thr180/Tyr182) (DM, 0.5245 ± 0.0185; 1 h, 0.673 ± 0.047; 6 h, 0.6645 ± 0.0255; 24 h, 0.6285 ± 0.0345) had a significant increase at 1 and 6 h.* p < 0.05. Phospho-SAPK/JNK (Thr183/Tyr185) (DM, 0.101 ± 0.011; 1 h, 0.112 ± 0.011; 6 h, 0.0905 ± 0.0025; 24 h, 0.08 ± 0.002) and phospho-IκB-α (Ser32) (DM, 0.0765 ± 0.0025; 1 h, 0.082 ± 0.013; 6 h, 0.0695 ± 0.0035; 24 h, 0.066 ± 0.002) did not have a significant increases in absorbance.
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Figure 7: SAEC exposure to MWCNT increases the expression of cellular inflammatory signals in HMVEC. Two biological replicates of SAEC and HMVEC were grown in the apical and basolateral chambers, respectively, of a co-culture system, and SAEC was exposed to DM or 1.2 μg/ml MWCNT for 1, 6 or 24 h. HMVEC were lysed and protein levels of intracellular inflammatory signals read at an absorbance of 450 nm using a PathScan Inflammation Multi-Target Sandwich ELISA from Cell Signaling Technology. All values given are the mean ± standard error. NF-κB had a significant decrease in absorbance over time (DM, 1.152 ± 0.051; 1 h, 0.713 ± 0.048; 6 h, 0.6795 ± 0.0145; 24 h, 0.7445 ± 0.0055). Phospho-NF-κB (Ser536) (DM, 0.341 ± 0.013; 1 h, 1.0065 ± 0.0855; 6 h, 0.944 ± 0.187; 24 h, 1.068 ± 0.026) and phospho-Stat3 (Tyr705) (DM, 0.96 ± 0.107; 1 h, 1.483 ± 0.079; 6 h, 1.7265 ± 0.1895; 24 h, 1.446 ± 0.07) had significant increases in absorbance over time, while phospho-p38 (Thr180/Tyr182) (DM, 0.5245 ± 0.0185; 1 h, 0.673 ± 0.047; 6 h, 0.6645 ± 0.0255; 24 h, 0.6285 ± 0.0345) had a significant increase at 1 and 6 h.* p < 0.05. Phospho-SAPK/JNK (Thr183/Tyr185) (DM, 0.101 ± 0.011; 1 h, 0.112 ± 0.011; 6 h, 0.0905 ± 0.0025; 24 h, 0.08 ± 0.002) and phospho-IκB-α (Ser32) (DM, 0.0765 ± 0.0025; 1 h, 0.082 ± 0.013; 6 h, 0.0695 ± 0.0035; 24 h, 0.066 ± 0.002) did not have a significant increases in absorbance.

Mentions: In addition to secreted inflammatory factors, a number of intracellular inflammatory proteins were altered in HMVEC after SAEC exposure to MWCNT. SAEC and HMVEC were grown in the apical and basolateral chambers, respectively, of a co-culture system, serum-starved overnight, and SAEC exposed to DM or 1.2 μg/ml for 24 h. Following exposure, HMVEC were lysed and cell lysates assayed at absorbance 450 nm for protein levels of NF-κB, phospho-SAPK/JNK, phospho-NF-κB p65, phospho-p38, phospho-IκB-α, and phospho-Stat3 using a Pathscan Inflammation Multi-Target Sandwich ELISA from Cell Signaling Technology. Although a significant decrease (p < 0.05) was seen in overall NF-κB levels in HMVEC after SAEC exposure to MWCNT (DM, 1.152 ± 0.051; 1 h, 0.713 ± 0.048; 6 h, 0.6795 ± 0.0145; 24 h, 0.7445 ± 0.0055), there was a significant increase (p < 0.05) in phospho-NF-κB p65 (Ser536) (DM, 0.341 ± 0.013; 1 h, 1.0065 ± 0.0855; 6 h, 0.944 ± 0.187; 24 h, 1.068 ± 0.026) and phospho-Stat3 (Tyr705) (DM, 0.96 ± 0.107; 1 h, 1.483 ± 0.079; 6 h, 1.7265 ± 0.1895; 24 h, 1.446 ± 0.07) at 1, 6 and 24 h post-exposure (Figure 7). A significant increase (p < 0.05) in phospho-p38 MAPK (Thr180/Tyr182) (DM, 0.5245 ± 0.0185; 1 h, 0.673 ± 0.047; 6 h, 0.6645 ± 0.0255; 24 h, 0.6285 ± 0.0345) was also noted at 1 and 6 hrs post-exposure (Figure 7a). No significant change (p > 0.05) was seen in the levels of phospho-SAPK/JNK (Thr183/Tyr185) (DM, 0.101 ± 0.011; 1 h, 0.112 ± 0.011; 6 h, 0.0905 ± 0.0025; 24 h, 0.08 ± 0.002) or phospho-IκB-α (Ser32) (DM, 0.0765 ± 0.0025; 1 h, 0.082 ± 0.013; 6 h, 0.0695 ± 0.0035; 24 h, 0.066 ± 0.002) in HMVEC after SAEC exposure to MWCNT (Figure 7).


Multi-walled carbon nanotubes induce human microvascular endothelial cellular effects in an alveolar-capillary co-culture with small airway epithelial cells.

Snyder-Talkington BN, Schwegler-Berry D, Castranova V, Qian Y, Guo NL - Part Fibre Toxicol (2013)

SAEC exposure to MWCNT increases the expression of cellular inflammatory signals in HMVEC. Two biological replicates of SAEC and HMVEC were grown in the apical and basolateral chambers, respectively, of a co-culture system, and SAEC was exposed to DM or 1.2 μg/ml MWCNT for 1, 6 or 24 h. HMVEC were lysed and protein levels of intracellular inflammatory signals read at an absorbance of 450 nm using a PathScan Inflammation Multi-Target Sandwich ELISA from Cell Signaling Technology. All values given are the mean ± standard error. NF-κB had a significant decrease in absorbance over time (DM, 1.152 ± 0.051; 1 h, 0.713 ± 0.048; 6 h, 0.6795 ± 0.0145; 24 h, 0.7445 ± 0.0055). Phospho-NF-κB (Ser536) (DM, 0.341 ± 0.013; 1 h, 1.0065 ± 0.0855; 6 h, 0.944 ± 0.187; 24 h, 1.068 ± 0.026) and phospho-Stat3 (Tyr705) (DM, 0.96 ± 0.107; 1 h, 1.483 ± 0.079; 6 h, 1.7265 ± 0.1895; 24 h, 1.446 ± 0.07) had significant increases in absorbance over time, while phospho-p38 (Thr180/Tyr182) (DM, 0.5245 ± 0.0185; 1 h, 0.673 ± 0.047; 6 h, 0.6645 ± 0.0255; 24 h, 0.6285 ± 0.0345) had a significant increase at 1 and 6 h.* p < 0.05. Phospho-SAPK/JNK (Thr183/Tyr185) (DM, 0.101 ± 0.011; 1 h, 0.112 ± 0.011; 6 h, 0.0905 ± 0.0025; 24 h, 0.08 ± 0.002) and phospho-IκB-α (Ser32) (DM, 0.0765 ± 0.0025; 1 h, 0.082 ± 0.013; 6 h, 0.0695 ± 0.0035; 24 h, 0.066 ± 0.002) did not have a significant increases in absorbance.
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Figure 7: SAEC exposure to MWCNT increases the expression of cellular inflammatory signals in HMVEC. Two biological replicates of SAEC and HMVEC were grown in the apical and basolateral chambers, respectively, of a co-culture system, and SAEC was exposed to DM or 1.2 μg/ml MWCNT for 1, 6 or 24 h. HMVEC were lysed and protein levels of intracellular inflammatory signals read at an absorbance of 450 nm using a PathScan Inflammation Multi-Target Sandwich ELISA from Cell Signaling Technology. All values given are the mean ± standard error. NF-κB had a significant decrease in absorbance over time (DM, 1.152 ± 0.051; 1 h, 0.713 ± 0.048; 6 h, 0.6795 ± 0.0145; 24 h, 0.7445 ± 0.0055). Phospho-NF-κB (Ser536) (DM, 0.341 ± 0.013; 1 h, 1.0065 ± 0.0855; 6 h, 0.944 ± 0.187; 24 h, 1.068 ± 0.026) and phospho-Stat3 (Tyr705) (DM, 0.96 ± 0.107; 1 h, 1.483 ± 0.079; 6 h, 1.7265 ± 0.1895; 24 h, 1.446 ± 0.07) had significant increases in absorbance over time, while phospho-p38 (Thr180/Tyr182) (DM, 0.5245 ± 0.0185; 1 h, 0.673 ± 0.047; 6 h, 0.6645 ± 0.0255; 24 h, 0.6285 ± 0.0345) had a significant increase at 1 and 6 h.* p < 0.05. Phospho-SAPK/JNK (Thr183/Tyr185) (DM, 0.101 ± 0.011; 1 h, 0.112 ± 0.011; 6 h, 0.0905 ± 0.0025; 24 h, 0.08 ± 0.002) and phospho-IκB-α (Ser32) (DM, 0.0765 ± 0.0025; 1 h, 0.082 ± 0.013; 6 h, 0.0695 ± 0.0035; 24 h, 0.066 ± 0.002) did not have a significant increases in absorbance.
Mentions: In addition to secreted inflammatory factors, a number of intracellular inflammatory proteins were altered in HMVEC after SAEC exposure to MWCNT. SAEC and HMVEC were grown in the apical and basolateral chambers, respectively, of a co-culture system, serum-starved overnight, and SAEC exposed to DM or 1.2 μg/ml for 24 h. Following exposure, HMVEC were lysed and cell lysates assayed at absorbance 450 nm for protein levels of NF-κB, phospho-SAPK/JNK, phospho-NF-κB p65, phospho-p38, phospho-IκB-α, and phospho-Stat3 using a Pathscan Inflammation Multi-Target Sandwich ELISA from Cell Signaling Technology. Although a significant decrease (p < 0.05) was seen in overall NF-κB levels in HMVEC after SAEC exposure to MWCNT (DM, 1.152 ± 0.051; 1 h, 0.713 ± 0.048; 6 h, 0.6795 ± 0.0145; 24 h, 0.7445 ± 0.0055), there was a significant increase (p < 0.05) in phospho-NF-κB p65 (Ser536) (DM, 0.341 ± 0.013; 1 h, 1.0065 ± 0.0855; 6 h, 0.944 ± 0.187; 24 h, 1.068 ± 0.026) and phospho-Stat3 (Tyr705) (DM, 0.96 ± 0.107; 1 h, 1.483 ± 0.079; 6 h, 1.7265 ± 0.1895; 24 h, 1.446 ± 0.07) at 1, 6 and 24 h post-exposure (Figure 7). A significant increase (p < 0.05) in phospho-p38 MAPK (Thr180/Tyr182) (DM, 0.5245 ± 0.0185; 1 h, 0.673 ± 0.047; 6 h, 0.6645 ± 0.0255; 24 h, 0.6285 ± 0.0345) was also noted at 1 and 6 hrs post-exposure (Figure 7a). No significant change (p > 0.05) was seen in the levels of phospho-SAPK/JNK (Thr183/Tyr185) (DM, 0.101 ± 0.011; 1 h, 0.112 ± 0.011; 6 h, 0.0905 ± 0.0025; 24 h, 0.08 ± 0.002) or phospho-IκB-α (Ser32) (DM, 0.0765 ± 0.0025; 1 h, 0.082 ± 0.013; 6 h, 0.0695 ± 0.0035; 24 h, 0.066 ± 0.002) in HMVEC after SAEC exposure to MWCNT (Figure 7).

Bottom Line: Nanotechnology, particularly the use of multi-walled carbon nanotubes (MWCNT), is a rapidly growing discipline with implications for advancement in a variety of fields.While many studies showed adverse effects to the vascular endothelium upon MWCNT exposure, in vitro results often do not correlate with in vivo effects.Following exposure of the epithelial layer to MWCNT, the effects to the endothelial barrier were determined.

View Article: PubMed Central - HTML - PubMed

Affiliation: Pathology and Physiology Research Branch, Health Effects Laboratory Division, National Institute for Occupational Safety and Health, 1095 Willowdale Road, Morgantown, WV 26505-2888, USA.

ABSTRACT

Background: Nanotechnology, particularly the use of multi-walled carbon nanotubes (MWCNT), is a rapidly growing discipline with implications for advancement in a variety of fields. A major route of exposure to MWCNT during both occupational and environmental contact is inhalation. While many studies showed adverse effects to the vascular endothelium upon MWCNT exposure, in vitro results often do not correlate with in vivo effects. This study aimed to determine if an alveolar-capillary co-culture model could determine changes in the vascular endothelium after epithelial exposure to MWCNT.

Methods: A co-culture system in which both human small airway epithelial cells and human microvascular endothelial cells were separated by a Transwell membrane so as to resemble an alveolar-capillary interaction was used. Following exposure of the epithelial layer to MWCNT, the effects to the endothelial barrier were determined.

Results: Exposure of the epithelial layer to MWCNT induced multiple changes in the endothelial cell barrier, including an increase in reactive oxygen species, actin rearrangement, loss of VE-cadherin at the cell surface, and an increase in endothelial angiogenic ability. Overall increases in secreted VEGFA, sICAM-1, and sVCAM-1 protein levels, as well as increases in intracellular phospho-NF-κB, phospho-Stat3, and phospho-p38 MAPK, were also noted in HMVEC after epithelial exposure.

Conclusion: The co-culture system identified that alveolar-capillary exposure to MWCNT induced multiple changes to the underlying endothelium, potentially through cell signaling mediators derived from MWCNT-exposed epithelial cells. Therefore, the co-culture system appears to be a relevant in vitro method to study the pulmonary toxicity of MWCNT.

Show MeSH
Related in: MedlinePlus