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P2X7 purinoceptors contribute to the death of Schwann cells transplanted into the spinal cord.

Luo J, Lee S, Wu D, Yeh J, Ellamushi H, Wheeler AP, Warnes G, Zhang Y, Bo X - Cell Death Dis (2013)

Bottom Line: Furthermore, ATP did not cause death of SCs isolated from P2X7R-knockout mice.All these results suggest that P2X7R is responsible for ATP-induced SC death in vitro.These results indicate that targeting P2X7R on SCs could be a potential strategy to improve the survival of transplanted cells.

View Article: PubMed Central - PubMed

Affiliation: 1] Centre for Neuroscience and Trauma, Blizard Institute, Queen Mary University of London, London E1 2AT, UK [2] Department of Physiology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.

ABSTRACT
The potential to use Schwann cells (SCs) in neural repair for patients suffering from neurotrauma and neurodegenerative diseases is well recognized. However, significant cell death after transplantation hinders the clinical translation of SC-based therapies. Various factors may contribute to the death of transplanted cells. It is known that prolonged activation of P2X7 purinoceptors (P2X7R) can lead to death of certain types of cells. In this study, we show that rat SCs express P2X7R and exposure of cultured SCs to high concentrations of ATP (3-5 mM) or a P2X7R agonist, 2'(3')-O-(4-benzoylbenzoyl)ATP (BzATP) induced significant cell death rapidly. High concentrations of ATP and BzATP increased ethidium uptake by SCs, indicating increased membrane permeability to large molecules, a typical feature of prolonged P2X7R activation. SC death, as well as ethidium uptake, induced by ATP was blocked by an irreversible P2X7R antagonist oxidized ATP (oxATP) or a reversible P2X7R antagonist A438079. oxATP also significantly inhibits the increase of intracellular free calcium induced by minimolar ATP concentrations. Furthermore, ATP did not cause death of SCs isolated from P2X7R-knockout mice. All these results suggest that P2X7R is responsible for ATP-induced SC death in vitro. When rat SCs were treated with oxATP before transplantation into uninjured rat spinal cord, 35% more SCs survived than untreated SCs 1 week after transplantation. Moreover, 58% more SCs isolated from P2X7R-knockout mice survived after being transplanted into rat spinal cord than SCs from wild-type mice. This further confirms that P2X7R is involved in the death of transplanted SCs. These results indicate that targeting P2X7R on SCs could be a potential strategy to improve the survival of transplanted cells. As many other types of cells, including neural stem cells, also express P2X7R, deactivating P2X7R may improve the survival of other types of transplanted cells.

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ATP induces SC death dose-dependently in vitro. (a) Phase contrast images showing SCs in culture with or without exposure to ATP for 30 min. (b) Flow cytometry cell viability assay showing the proportions of live cells after exposure to 3, 4, 5 mM ATP for 1 h. (c) The percentage of live SCs after being exposed to increasing concentrations of ATP or BzATP (200 μM) with or without oxATP (350 μM) or A438079 (100 μM).+P<0.05, ++P<0.01, +++P<0.001 (compared with the group without ATP); *P<0.05, **P<0.01, ***P<0.001 (compared between the corresponding groups with or without one of the antagonists), single factor AVNOA, n=3–7
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fig2: ATP induces SC death dose-dependently in vitro. (a) Phase contrast images showing SCs in culture with or without exposure to ATP for 30 min. (b) Flow cytometry cell viability assay showing the proportions of live cells after exposure to 3, 4, 5 mM ATP for 1 h. (c) The percentage of live SCs after being exposed to increasing concentrations of ATP or BzATP (200 μM) with or without oxATP (350 μM) or A438079 (100 μM).+P<0.05, ++P<0.01, +++P<0.001 (compared with the group without ATP); *P<0.05, **P<0.01, ***P<0.001 (compared between the corresponding groups with or without one of the antagonists), single factor AVNOA, n=3–7

Mentions: During an experiment looking for potential factors that might induce SC death, we exposed SCs to various concentrations of ATP. No obvious morphological change occurred to SCs exposed to ATP concentrations up to 1 mM (Figure 2a); however, SCs exposed to ATP concentrations higher than 2 mM underwent significant morphological changes within 10–15 min; the higher the concentration, the quicker the changes occurred. Cell processes started to withdraw and cells gradually rounded up (Figure 2a). Most of the SCs detached from the culture dishes after exposure to 5 mM ATP for 1 h. Cells were then dissociated, labeled with Annexin V Apoptosis Assay kit and subjected to flow cytometry to measure cell viability. No significant SC death occurred after exposure to 1 or 2 mM ATP (Figure 2c). However, at 3 mM cell death became significant and 4 and 5 mM ATP induced even more profound cell death (Figures 2b and c).


P2X7 purinoceptors contribute to the death of Schwann cells transplanted into the spinal cord.

Luo J, Lee S, Wu D, Yeh J, Ellamushi H, Wheeler AP, Warnes G, Zhang Y, Bo X - Cell Death Dis (2013)

ATP induces SC death dose-dependently in vitro. (a) Phase contrast images showing SCs in culture with or without exposure to ATP for 30 min. (b) Flow cytometry cell viability assay showing the proportions of live cells after exposure to 3, 4, 5 mM ATP for 1 h. (c) The percentage of live SCs after being exposed to increasing concentrations of ATP or BzATP (200 μM) with or without oxATP (350 μM) or A438079 (100 μM).+P<0.05, ++P<0.01, +++P<0.001 (compared with the group without ATP); *P<0.05, **P<0.01, ***P<0.001 (compared between the corresponding groups with or without one of the antagonists), single factor AVNOA, n=3–7
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3824653&req=5

fig2: ATP induces SC death dose-dependently in vitro. (a) Phase contrast images showing SCs in culture with or without exposure to ATP for 30 min. (b) Flow cytometry cell viability assay showing the proportions of live cells after exposure to 3, 4, 5 mM ATP for 1 h. (c) The percentage of live SCs after being exposed to increasing concentrations of ATP or BzATP (200 μM) with or without oxATP (350 μM) or A438079 (100 μM).+P<0.05, ++P<0.01, +++P<0.001 (compared with the group without ATP); *P<0.05, **P<0.01, ***P<0.001 (compared between the corresponding groups with or without one of the antagonists), single factor AVNOA, n=3–7
Mentions: During an experiment looking for potential factors that might induce SC death, we exposed SCs to various concentrations of ATP. No obvious morphological change occurred to SCs exposed to ATP concentrations up to 1 mM (Figure 2a); however, SCs exposed to ATP concentrations higher than 2 mM underwent significant morphological changes within 10–15 min; the higher the concentration, the quicker the changes occurred. Cell processes started to withdraw and cells gradually rounded up (Figure 2a). Most of the SCs detached from the culture dishes after exposure to 5 mM ATP for 1 h. Cells were then dissociated, labeled with Annexin V Apoptosis Assay kit and subjected to flow cytometry to measure cell viability. No significant SC death occurred after exposure to 1 or 2 mM ATP (Figure 2c). However, at 3 mM cell death became significant and 4 and 5 mM ATP induced even more profound cell death (Figures 2b and c).

Bottom Line: Furthermore, ATP did not cause death of SCs isolated from P2X7R-knockout mice.All these results suggest that P2X7R is responsible for ATP-induced SC death in vitro.These results indicate that targeting P2X7R on SCs could be a potential strategy to improve the survival of transplanted cells.

View Article: PubMed Central - PubMed

Affiliation: 1] Centre for Neuroscience and Trauma, Blizard Institute, Queen Mary University of London, London E1 2AT, UK [2] Department of Physiology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.

ABSTRACT
The potential to use Schwann cells (SCs) in neural repair for patients suffering from neurotrauma and neurodegenerative diseases is well recognized. However, significant cell death after transplantation hinders the clinical translation of SC-based therapies. Various factors may contribute to the death of transplanted cells. It is known that prolonged activation of P2X7 purinoceptors (P2X7R) can lead to death of certain types of cells. In this study, we show that rat SCs express P2X7R and exposure of cultured SCs to high concentrations of ATP (3-5 mM) or a P2X7R agonist, 2'(3')-O-(4-benzoylbenzoyl)ATP (BzATP) induced significant cell death rapidly. High concentrations of ATP and BzATP increased ethidium uptake by SCs, indicating increased membrane permeability to large molecules, a typical feature of prolonged P2X7R activation. SC death, as well as ethidium uptake, induced by ATP was blocked by an irreversible P2X7R antagonist oxidized ATP (oxATP) or a reversible P2X7R antagonist A438079. oxATP also significantly inhibits the increase of intracellular free calcium induced by minimolar ATP concentrations. Furthermore, ATP did not cause death of SCs isolated from P2X7R-knockout mice. All these results suggest that P2X7R is responsible for ATP-induced SC death in vitro. When rat SCs were treated with oxATP before transplantation into uninjured rat spinal cord, 35% more SCs survived than untreated SCs 1 week after transplantation. Moreover, 58% more SCs isolated from P2X7R-knockout mice survived after being transplanted into rat spinal cord than SCs from wild-type mice. This further confirms that P2X7R is involved in the death of transplanted SCs. These results indicate that targeting P2X7R on SCs could be a potential strategy to improve the survival of transplanted cells. As many other types of cells, including neural stem cells, also express P2X7R, deactivating P2X7R may improve the survival of other types of transplanted cells.

Show MeSH
Related in: MedlinePlus