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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 ethidium uptake by SCs. (a) Photomicrographs showing the morphological changes of SCs (phase contrast images) and ethidium fluorescence in SCs 20 min after exposure to various concentrations of ATP. Green arrows in the two photomicrographs for 3 mM ATP point to two fibroblasts. (b) Quantification of ethidium fluorescence intensities in SCs 20 min after exposure to various concentrations of ATP with or without oxATP (350 μM) or A438079 (100 μM) treatment. +++P<0.001 (compared with the group without ATP); ***P<0.001 (compared between the corresponding groups with and without one of the antagonists), single factor AVNOA, n=3. (c) Representative time course of ethidium uptake by SCs after exposure to different concentrations of ATP over 20 min
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fig3: ATP induces ethidium uptake by SCs. (a) Photomicrographs showing the morphological changes of SCs (phase contrast images) and ethidium fluorescence in SCs 20 min after exposure to various concentrations of ATP. Green arrows in the two photomicrographs for 3 mM ATP point to two fibroblasts. (b) Quantification of ethidium fluorescence intensities in SCs 20 min after exposure to various concentrations of ATP with or without oxATP (350 μM) or A438079 (100 μM) treatment. +++P<0.001 (compared with the group without ATP); ***P<0.001 (compared between the corresponding groups with and without one of the antagonists), single factor AVNOA, n=3. (c) Representative time course of ethidium uptake by SCs after exposure to different concentrations of ATP over 20 min

Mentions: Cell death induced by high concentrations of ATP is attributed to the prolonged activation of P2X7R, which leads to pore formation on cell membranes.12, 13 To test whether ATP can also induce pore formation in SCs, SCs were exposed to various concentrations of ATP in the presence of 10 μM ethidium bromide. Using time-lapse confocal microscopy, it was shown that a gradual increase in ethidium uptake into SCs occurred at ATP concentrations above 1 mM (Figure 3c). Under an epifluorescence microscope, we also observed that ethidium uptake occurred at ATP concentrations above 1 mM (Figures 3a and b). By comparing the corresponding bright-field and fluorescence images of the same microscopic field taken at 20 min after exposure to ATP, it is evident that the extent of ethidium uptake is correlated with the morphological changes of SCs (Figure 3a). Quantification of ethidium fluorescence intensities in SCs 20 min after the exposure to ATP shows that ethidium uptake is concentration-dependent (Figure 3b). After pretreatment of SCs with 350 μM oxATP for 2 h or 100 μM A438079 for 20 min, ATP at all tested concentrations did not induce ethidium uptake (Figure 3b), indicating the blockade of P2X7R prevents the pore formation on SCs.


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 ethidium uptake by SCs. (a) Photomicrographs showing the morphological changes of SCs (phase contrast images) and ethidium fluorescence in SCs 20 min after exposure to various concentrations of ATP. Green arrows in the two photomicrographs for 3 mM ATP point to two fibroblasts. (b) Quantification of ethidium fluorescence intensities in SCs 20 min after exposure to various concentrations of ATP with or without oxATP (350 μM) or A438079 (100 μM) treatment. +++P<0.001 (compared with the group without ATP); ***P<0.001 (compared between the corresponding groups with and without one of the antagonists), single factor AVNOA, n=3. (c) Representative time course of ethidium uptake by SCs after exposure to different concentrations of ATP over 20 min
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: ATP induces ethidium uptake by SCs. (a) Photomicrographs showing the morphological changes of SCs (phase contrast images) and ethidium fluorescence in SCs 20 min after exposure to various concentrations of ATP. Green arrows in the two photomicrographs for 3 mM ATP point to two fibroblasts. (b) Quantification of ethidium fluorescence intensities in SCs 20 min after exposure to various concentrations of ATP with or without oxATP (350 μM) or A438079 (100 μM) treatment. +++P<0.001 (compared with the group without ATP); ***P<0.001 (compared between the corresponding groups with and without one of the antagonists), single factor AVNOA, n=3. (c) Representative time course of ethidium uptake by SCs after exposure to different concentrations of ATP over 20 min
Mentions: Cell death induced by high concentrations of ATP is attributed to the prolonged activation of P2X7R, which leads to pore formation on cell membranes.12, 13 To test whether ATP can also induce pore formation in SCs, SCs were exposed to various concentrations of ATP in the presence of 10 μM ethidium bromide. Using time-lapse confocal microscopy, it was shown that a gradual increase in ethidium uptake into SCs occurred at ATP concentrations above 1 mM (Figure 3c). Under an epifluorescence microscope, we also observed that ethidium uptake occurred at ATP concentrations above 1 mM (Figures 3a and b). By comparing the corresponding bright-field and fluorescence images of the same microscopic field taken at 20 min after exposure to ATP, it is evident that the extent of ethidium uptake is correlated with the morphological changes of SCs (Figure 3a). Quantification of ethidium fluorescence intensities in SCs 20 min after the exposure to ATP shows that ethidium uptake is concentration-dependent (Figure 3b). After pretreatment of SCs with 350 μM oxATP for 2 h or 100 μM A438079 for 20 min, ATP at all tested concentrations did not induce ethidium uptake (Figure 3b), indicating the blockade of P2X7R prevents the pore formation on SCs.

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