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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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Related in: MedlinePlus

SAEC exposure to MWCNT increases the expression of secreted inflammatory mediators in coculture. SAEC and HMVEC were grown in the apical and basolateral chambers, respectively, of a co-culture system and SAEC exposed to 1.2 μg/ml MWCNT for 24 hours. Apical and basolateral media were collected and assayed by ELISA for VEGFA, sICAM1, and sVCAM1 protein expression. All values given are the mean ± standard error. VEGFA levels (A) increased from 87.15 ± 6.58 pg/ml to 114.96 ± 14.89 pg/ml in the basolateral chamber and 313.45 ± 27.85 pg/ml to 378.38 ± 21.89 pg/ml in the apical chamber. sICAM1 levels (B) increased from 107.99 ± 6.14 pg/ml to 123.76 ± 3.00 pg/ml in the basolateral chamber and 44.13 ± 3.04 pg/ml to 55.56 ± 2.63 pg/ml in the apical chamber. sVCAM1 levels (C) increased from 38.77 ± 7.52 pg/ml to 46.07 ± 11.07 pg/ml in the basolateral chamber and 43.69 ± 6.38 pg/ml to 85.29 ± 11.64 pg/ml in the apical chamber. * p < 0.05 above control.
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Figure 4: SAEC exposure to MWCNT increases the expression of secreted inflammatory mediators in coculture. SAEC and HMVEC were grown in the apical and basolateral chambers, respectively, of a co-culture system and SAEC exposed to 1.2 μg/ml MWCNT for 24 hours. Apical and basolateral media were collected and assayed by ELISA for VEGFA, sICAM1, and sVCAM1 protein expression. All values given are the mean ± standard error. VEGFA levels (A) increased from 87.15 ± 6.58 pg/ml to 114.96 ± 14.89 pg/ml in the basolateral chamber and 313.45 ± 27.85 pg/ml to 378.38 ± 21.89 pg/ml in the apical chamber. sICAM1 levels (B) increased from 107.99 ± 6.14 pg/ml to 123.76 ± 3.00 pg/ml in the basolateral chamber and 44.13 ± 3.04 pg/ml to 55.56 ± 2.63 pg/ml in the apical chamber. sVCAM1 levels (C) increased from 38.77 ± 7.52 pg/ml to 46.07 ± 11.07 pg/ml in the basolateral chamber and 43.69 ± 6.38 pg/ml to 85.29 ± 11.64 pg/ml in the apical chamber. * p < 0.05 above control.

Mentions: To determine the levels of inflammatory mediators in co-culture, VEGFA and two other secreted proteins known to have a role in the inflammatory process, soluble intracellular adhesion molecular 1 (sICAM-1) and soluble vascular cell adhesion molecule 1 (sVCAM-1), were assayed for their protein levels in co-culture media after epithelial exposure. SAEC and HMVEC were grown in co-culture for 72 h, serum-starved overnight, and SAEC exposed to DM or 1.2 μg/ml for 24 h. Following exposure, media were removed from both the apical and basolateral chambers of the co-culture and assayed for these inflammatory protein markers by ELISA. Protein levels of VEGFA increased significantly from 87.15 ± 6.58 pg/ml in the basolateral chamber to 114.96 ± 14.89 pg/ml after MWCNT exposure (Figure 4A). VEGFA protein levels also increased significantly from 313.45 ± 27.85 pg/ml in the apical chamber to 378.38 ± 21.89 pg/ml after MWCNT exposure (Figure 4A). Significant increases in sICAM-1 and sVCAM-1 were also noted. sICAM-1 protein levels increased significantly in the apical well from 44.13 ± 3.04 pg/ml to 55.56 ± 2.63 pg/ml, as well as in the basolateral chamber from 107.99 ± 6.14 pg/ml to 123.76 ± 3.00 pg/ml, following MWCNT exposure (Figure 4B). sVCAM-1 protein levels increased significantly in the apical well from 43.69 ± 6.38 pg/ml to 85.29 ± 11.64 pg/ml after epithelial exposure to MWCNT (Figure 4C). There was no significant increase in sVCAM-1 levels in the basolateral chamber after exposure with an increase of 38.77 ± 7.52 pg/ml to 46.07 ± 11.07 pg/ml.


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 secreted inflammatory mediators in coculture. SAEC and HMVEC were grown in the apical and basolateral chambers, respectively, of a co-culture system and SAEC exposed to 1.2 μg/ml MWCNT for 24 hours. Apical and basolateral media were collected and assayed by ELISA for VEGFA, sICAM1, and sVCAM1 protein expression. All values given are the mean ± standard error. VEGFA levels (A) increased from 87.15 ± 6.58 pg/ml to 114.96 ± 14.89 pg/ml in the basolateral chamber and 313.45 ± 27.85 pg/ml to 378.38 ± 21.89 pg/ml in the apical chamber. sICAM1 levels (B) increased from 107.99 ± 6.14 pg/ml to 123.76 ± 3.00 pg/ml in the basolateral chamber and 44.13 ± 3.04 pg/ml to 55.56 ± 2.63 pg/ml in the apical chamber. sVCAM1 levels (C) increased from 38.77 ± 7.52 pg/ml to 46.07 ± 11.07 pg/ml in the basolateral chamber and 43.69 ± 6.38 pg/ml to 85.29 ± 11.64 pg/ml in the apical chamber. * p < 0.05 above control.
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Figure 4: SAEC exposure to MWCNT increases the expression of secreted inflammatory mediators in coculture. SAEC and HMVEC were grown in the apical and basolateral chambers, respectively, of a co-culture system and SAEC exposed to 1.2 μg/ml MWCNT for 24 hours. Apical and basolateral media were collected and assayed by ELISA for VEGFA, sICAM1, and sVCAM1 protein expression. All values given are the mean ± standard error. VEGFA levels (A) increased from 87.15 ± 6.58 pg/ml to 114.96 ± 14.89 pg/ml in the basolateral chamber and 313.45 ± 27.85 pg/ml to 378.38 ± 21.89 pg/ml in the apical chamber. sICAM1 levels (B) increased from 107.99 ± 6.14 pg/ml to 123.76 ± 3.00 pg/ml in the basolateral chamber and 44.13 ± 3.04 pg/ml to 55.56 ± 2.63 pg/ml in the apical chamber. sVCAM1 levels (C) increased from 38.77 ± 7.52 pg/ml to 46.07 ± 11.07 pg/ml in the basolateral chamber and 43.69 ± 6.38 pg/ml to 85.29 ± 11.64 pg/ml in the apical chamber. * p < 0.05 above control.
Mentions: To determine the levels of inflammatory mediators in co-culture, VEGFA and two other secreted proteins known to have a role in the inflammatory process, soluble intracellular adhesion molecular 1 (sICAM-1) and soluble vascular cell adhesion molecule 1 (sVCAM-1), were assayed for their protein levels in co-culture media after epithelial exposure. SAEC and HMVEC were grown in co-culture for 72 h, serum-starved overnight, and SAEC exposed to DM or 1.2 μg/ml for 24 h. Following exposure, media were removed from both the apical and basolateral chambers of the co-culture and assayed for these inflammatory protein markers by ELISA. Protein levels of VEGFA increased significantly from 87.15 ± 6.58 pg/ml in the basolateral chamber to 114.96 ± 14.89 pg/ml after MWCNT exposure (Figure 4A). VEGFA protein levels also increased significantly from 313.45 ± 27.85 pg/ml in the apical chamber to 378.38 ± 21.89 pg/ml after MWCNT exposure (Figure 4A). Significant increases in sICAM-1 and sVCAM-1 were also noted. sICAM-1 protein levels increased significantly in the apical well from 44.13 ± 3.04 pg/ml to 55.56 ± 2.63 pg/ml, as well as in the basolateral chamber from 107.99 ± 6.14 pg/ml to 123.76 ± 3.00 pg/ml, following MWCNT exposure (Figure 4B). sVCAM-1 protein levels increased significantly in the apical well from 43.69 ± 6.38 pg/ml to 85.29 ± 11.64 pg/ml after epithelial exposure to MWCNT (Figure 4C). There was no significant increase in sVCAM-1 levels in the basolateral chamber after exposure with an increase of 38.77 ± 7.52 pg/ml to 46.07 ± 11.07 pg/ml.

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