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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 angiogenic potential of HMVEC. A. SAEC and HMVEC were grown in the apical and basolateral chambers, respectively, of a co-culture system, and SAEC were exposed to either DM or 1.2 μg/ml MWCNT for 24 h. HMVEC were removed from the co-culture, rinsed thoroughly in serum free media, and plated in serum free media on Matrigel plugs. Images of capillary-like formation were captured 4 h after plating at both 4X and 10X magnifications in DM (a,b) and MWCNT (c,d) exposures. Three separate angiogenesis assays were performed, and a representative image is shown. B. The number of tube-forming cells in DM or MWCNT-exposed co-cultures was determined by counting the number of tube-forming cells in 6 randomly chosen 20 × 20 mm squares in the 4X images of three separate angiogenesis assays. The average number ± standard error of tube-forming cells in the DM control was 5.91 ± 0.81, and the mean ± standard error of tube-forming cells in the MWCNT-exposed co-culture was 22.50 ± 1.73. * p < 0.05.
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Figure 6: SAEC exposure to MWCNT increases the angiogenic potential of HMVEC. A. SAEC and HMVEC were grown in the apical and basolateral chambers, respectively, of a co-culture system, and SAEC were exposed to either DM or 1.2 μg/ml MWCNT for 24 h. HMVEC were removed from the co-culture, rinsed thoroughly in serum free media, and plated in serum free media on Matrigel plugs. Images of capillary-like formation were captured 4 h after plating at both 4X and 10X magnifications in DM (a,b) and MWCNT (c,d) exposures. Three separate angiogenesis assays were performed, and a representative image is shown. B. The number of tube-forming cells in DM or MWCNT-exposed co-cultures was determined by counting the number of tube-forming cells in 6 randomly chosen 20 × 20 mm squares in the 4X images of three separate angiogenesis assays. The average number ± standard error of tube-forming cells in the DM control was 5.91 ± 0.81, and the mean ± standard error of tube-forming cells in the MWCNT-exposed co-culture was 22.50 ± 1.73. * p < 0.05.

Mentions: A role of endothelial cells is to reform the vasculature and, upon injury, respond through physiological angiogenesis to form new blood vessels in a restricted manner [42]. Pathological angiogenesis is a known hallmark of a variety of diseases, such as rheumatoid arthritis and cancer, and is necessary for the progression of pulmonary diseases, such as fibrosis [39,42]. To determine if epithelial exposure to MWCNT increased the angiogenic potential of the endothelium, SAEC and HMVEC were grown in the apical and basolateral chambers, respectively, of a co-culture system, and SAEC were exposed to either DM or 1.2 μg/ml MWCNT for 24 h. Following exposure, HMVEC were removed from the co-culture system, rinsed with serum free media, and placed into serum free media on a Matrigel plug where they were allowed to form capillary-like structures for 4 h. HMVEC cells from DM exposed co-cultures had minimal tube formation and the majority remained as single cells 4 hours after plating (Figure 6A, a-b). Conversely, HMVEC from MWCNT exposed co-cultures had extensive capillary-like structure formation (Figure 6A, c-d). To determine the average number of tubes formed in both DM and MWCNT exposed co-cultures, 6 20 × 20 mm squares were randomly chosen in the 4X images of 3 independent angiogenesis assays and the number of tubes counted. There was a significant increase (p < 0.05) in the average number of tubes in the MWCNT-exposed co-culture (22.50 ± 1.73) than in the DM exposed control (5.91 ± 0.81) (Figure 6B). As HMVEC had been removed from the co-culture system and rinsed free of co-culture cellular mediators, this enhanced angiogenic ability was possibly due to cellular signaling from SAEC after MWCNT exposure affecting innate changes in the endothelial barrier.


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 angiogenic potential of HMVEC. A. SAEC and HMVEC were grown in the apical and basolateral chambers, respectively, of a co-culture system, and SAEC were exposed to either DM or 1.2 μg/ml MWCNT for 24 h. HMVEC were removed from the co-culture, rinsed thoroughly in serum free media, and plated in serum free media on Matrigel plugs. Images of capillary-like formation were captured 4 h after plating at both 4X and 10X magnifications in DM (a,b) and MWCNT (c,d) exposures. Three separate angiogenesis assays were performed, and a representative image is shown. B. The number of tube-forming cells in DM or MWCNT-exposed co-cultures was determined by counting the number of tube-forming cells in 6 randomly chosen 20 × 20 mm squares in the 4X images of three separate angiogenesis assays. The average number ± standard error of tube-forming cells in the DM control was 5.91 ± 0.81, and the mean ± standard error of tube-forming cells in the MWCNT-exposed co-culture was 22.50 ± 1.73. * p < 0.05.
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Figure 6: SAEC exposure to MWCNT increases the angiogenic potential of HMVEC. A. SAEC and HMVEC were grown in the apical and basolateral chambers, respectively, of a co-culture system, and SAEC were exposed to either DM or 1.2 μg/ml MWCNT for 24 h. HMVEC were removed from the co-culture, rinsed thoroughly in serum free media, and plated in serum free media on Matrigel plugs. Images of capillary-like formation were captured 4 h after plating at both 4X and 10X magnifications in DM (a,b) and MWCNT (c,d) exposures. Three separate angiogenesis assays were performed, and a representative image is shown. B. The number of tube-forming cells in DM or MWCNT-exposed co-cultures was determined by counting the number of tube-forming cells in 6 randomly chosen 20 × 20 mm squares in the 4X images of three separate angiogenesis assays. The average number ± standard error of tube-forming cells in the DM control was 5.91 ± 0.81, and the mean ± standard error of tube-forming cells in the MWCNT-exposed co-culture was 22.50 ± 1.73. * p < 0.05.
Mentions: A role of endothelial cells is to reform the vasculature and, upon injury, respond through physiological angiogenesis to form new blood vessels in a restricted manner [42]. Pathological angiogenesis is a known hallmark of a variety of diseases, such as rheumatoid arthritis and cancer, and is necessary for the progression of pulmonary diseases, such as fibrosis [39,42]. To determine if epithelial exposure to MWCNT increased the angiogenic potential of the endothelium, SAEC and HMVEC were grown in the apical and basolateral chambers, respectively, of a co-culture system, and SAEC were exposed to either DM or 1.2 μg/ml MWCNT for 24 h. Following exposure, HMVEC were removed from the co-culture system, rinsed with serum free media, and placed into serum free media on a Matrigel plug where they were allowed to form capillary-like structures for 4 h. HMVEC cells from DM exposed co-cultures had minimal tube formation and the majority remained as single cells 4 hours after plating (Figure 6A, a-b). Conversely, HMVEC from MWCNT exposed co-cultures had extensive capillary-like structure formation (Figure 6A, c-d). To determine the average number of tubes formed in both DM and MWCNT exposed co-cultures, 6 20 × 20 mm squares were randomly chosen in the 4X images of 3 independent angiogenesis assays and the number of tubes counted. There was a significant increase (p < 0.05) in the average number of tubes in the MWCNT-exposed co-culture (22.50 ± 1.73) than in the DM exposed control (5.91 ± 0.81) (Figure 6B). As HMVEC had been removed from the co-culture system and rinsed free of co-culture cellular mediators, this enhanced angiogenic ability was possibly due to cellular signaling from SAEC after MWCNT exposure affecting innate changes in the endothelial barrier.

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