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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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Cell mediators from SAEC exposure can pass through the Transwell membrane. SAEC were grown in the apical Transwell chamber without the presence of HMVEC in the basolateral well. HMVEC were grown in the basolaeral well without the presence of SAEC in the apical chamber. The apical chamber was exposed to DM or 1.2 μg/ml MWCNT for 24 h. Apical and basolateral media were collected for analysis by VEGFA ELISA. All values given are the mean ± standard error. SAEC only VEGFA levels increased from 89.04 ± 2.27 pg/ml to 194.04 ± 23.85 pg/ml in the apical well and 20.00 ± 2.06 pg/ml to 123.33 ± 8.13 pg/ml in the basolateral chamber. HMVEC only VEGFA levels were not above the level of detection of the ELISA assay.
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Figure 3: Cell mediators from SAEC exposure can pass through the Transwell membrane. SAEC were grown in the apical Transwell chamber without the presence of HMVEC in the basolateral well. HMVEC were grown in the basolaeral well without the presence of SAEC in the apical chamber. The apical chamber was exposed to DM or 1.2 μg/ml MWCNT for 24 h. Apical and basolateral media were collected for analysis by VEGFA ELISA. All values given are the mean ± standard error. SAEC only VEGFA levels increased from 89.04 ± 2.27 pg/ml to 194.04 ± 23.85 pg/ml in the apical well and 20.00 ± 2.06 pg/ml to 123.33 ± 8.13 pg/ml in the basolateral chamber. HMVEC only VEGFA levels were not above the level of detection of the ELISA assay.

Mentions: MWCNT were noted in vivo to invoke an acute inflammatory response in the lung after aspiration exposure followed by a persistent fibrotic response [12]. To determine whether cellular mediators released following SAEC exposure could pass through the Transwell membrane to the underlying HMVEC, SAEC were cultured in the apical Transwell chamber without HMVEC in the basolateral chamber (SAEC alone). Conversely, HMVEC were cultured in the basolateral chamber without SAEC in the apical chamber (HMVEC alone). Each culture system was exposed to either DM or 1.2 μg/ml MWCNT in the apical well for 24 h. Following exposure, media was removed from both the apical and basolateral chambers. Expression levels of vascular endothelial growth factor A (VEGFA) in the apical and basolateral chambers following MWCNT exposure were analyzed by an enzyme-linked immunosorbent assay (ELISA) (Figure 3). In SAEC alone cultures, VEGFA levels increased from 89.04 ± 2.27 pg/ml to 194.04 ± 23.85 pg/ml in the apical chamber after MWCNT exposure, while protein levels increased from 20.00 ± 2.06 pg/ml to 123.33 ± 8.13 pg/ml in the basolateral chamber. Neither DM nor MWCNT exposure to the apical chamber of the culture consisting of HMVEC alone in the basolateral chamber gave appreciable VEGFA levels above the threshold of detection of the ELISA assay. As VEGFA protein levels increased in the basolateral chamber after SAEC exposure, it was concluded that cellular mediators secreted by SAEC were capable of passing through the Transwell membrane to the basolateral chamber, thus potentially affecting the underlying endothelial layer.


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)

Cell mediators from SAEC exposure can pass through the Transwell membrane. SAEC were grown in the apical Transwell chamber without the presence of HMVEC in the basolateral well. HMVEC were grown in the basolaeral well without the presence of SAEC in the apical chamber. The apical chamber was exposed to DM or 1.2 μg/ml MWCNT for 24 h. Apical and basolateral media were collected for analysis by VEGFA ELISA. All values given are the mean ± standard error. SAEC only VEGFA levels increased from 89.04 ± 2.27 pg/ml to 194.04 ± 23.85 pg/ml in the apical well and 20.00 ± 2.06 pg/ml to 123.33 ± 8.13 pg/ml in the basolateral chamber. HMVEC only VEGFA levels were not above the level of detection of the ELISA assay.
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Figure 3: Cell mediators from SAEC exposure can pass through the Transwell membrane. SAEC were grown in the apical Transwell chamber without the presence of HMVEC in the basolateral well. HMVEC were grown in the basolaeral well without the presence of SAEC in the apical chamber. The apical chamber was exposed to DM or 1.2 μg/ml MWCNT for 24 h. Apical and basolateral media were collected for analysis by VEGFA ELISA. All values given are the mean ± standard error. SAEC only VEGFA levels increased from 89.04 ± 2.27 pg/ml to 194.04 ± 23.85 pg/ml in the apical well and 20.00 ± 2.06 pg/ml to 123.33 ± 8.13 pg/ml in the basolateral chamber. HMVEC only VEGFA levels were not above the level of detection of the ELISA assay.
Mentions: MWCNT were noted in vivo to invoke an acute inflammatory response in the lung after aspiration exposure followed by a persistent fibrotic response [12]. To determine whether cellular mediators released following SAEC exposure could pass through the Transwell membrane to the underlying HMVEC, SAEC were cultured in the apical Transwell chamber without HMVEC in the basolateral chamber (SAEC alone). Conversely, HMVEC were cultured in the basolateral chamber without SAEC in the apical chamber (HMVEC alone). Each culture system was exposed to either DM or 1.2 μg/ml MWCNT in the apical well for 24 h. Following exposure, media was removed from both the apical and basolateral chambers. Expression levels of vascular endothelial growth factor A (VEGFA) in the apical and basolateral chambers following MWCNT exposure were analyzed by an enzyme-linked immunosorbent assay (ELISA) (Figure 3). In SAEC alone cultures, VEGFA levels increased from 89.04 ± 2.27 pg/ml to 194.04 ± 23.85 pg/ml in the apical chamber after MWCNT exposure, while protein levels increased from 20.00 ± 2.06 pg/ml to 123.33 ± 8.13 pg/ml in the basolateral chamber. Neither DM nor MWCNT exposure to the apical chamber of the culture consisting of HMVEC alone in the basolateral chamber gave appreciable VEGFA levels above the threshold of detection of the ELISA assay. As VEGFA protein levels increased in the basolateral chamber after SAEC exposure, it was concluded that cellular mediators secreted by SAEC were capable of passing through the Transwell membrane to the basolateral chamber, thus potentially affecting the underlying endothelial layer.

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