H2O2 INDUCES NECROPTOSIS IN MESOANGIOBLAST STEM CELLS

Research output: Contribution to journalBook/Film/Article review

Abstract

Stem cells are used in regenerative medicine, but their therapeutic efficacy is compromised by their huge death during the first days post-transplantation. Indeed, the microenvironment within damaged tissues is hostile for stem cell survival mainly due to oxidative stress. H2O2 may play a relevant role in inducing death of the injected cells. The aim of our study was to determine the mechanism of mesoangioblast (A6) cell death after an H2O2 treatment.FACS analysis with annV/PI showed that H2O2 induced a dose and time-dependent decrement in A6 viability. We have also found an increase in caspases 8, 9 and 3 activity after the treatment. To assess their involvement in cell death, the pan caspase inhibitor Z-VAD was used. Neither early apoptosis, nor late apoptosis/necrosis, nor necrosis were reduced, suggesting that the cell death induced by H2O2 was caspase-independent. Then, we tested whether H2O2 is responsible for the autophagy activation.To study autophagy we evaluated the expression of specific markers. H2O2 decreased beclin1, Atg5, Atg7 and the ratio LC3II/I, in a dose dependent way. At the same time it increased p62 protein expression indicating an impaired autophagic flux, also confirmed by the increase of pAKT, responsible for the activation of mTOR, a negative regulator of autophagy. According to these data A6 treatment with H2O2 seems to not induce nor apoptosis or autophagy. For this reason we hypothesized the activation of necroptosis, a specific form of caspase-independent, non-apoptotic or necrotic cell death. To confirm whether the observed cell death was due to enhanced necroptosis, the proportion of necrotic cells was determined by annV/PI staining. FACS analysis showed an increase in percentage of both late apoptotic/necrotic and necrotic cells, which were further increased by pretreatment with Z-VAD. To investigate the relationship between physiological autophagy and necroptosis, cells were treated with H2O2 in the presence of the autophagic inhibitor 3MA. AnnV/PI staining showed that the inhibition of autophagy by 3MA significantly enhanced necroptosis in A6 treated cells. Conversely, 3MA had no effect on apoptosis. In conclusion, our data indicate that the cytotoxicity of H2O2 in A6 mainly occurred via the induction of necroptosis, enhanced by both apoptosis and autophagy inhibition.
Original languageEnglish
Pages (from-to)3-3
Number of pages1
JournalEUROPEAN JOURNAL OF HISTOCHEMISTRY
Volumevolume 62/ supplement 1
Publication statusPublished - 2018

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Autophagy
Stem Cells
Cell Death
Apoptosis
Caspases
Necrosis
Staining and Labeling
Regenerative Medicine
Caspase Inhibitors
Caspase 9
Caspase 8
Caspase 3
Cell Survival
Oxidative Stress
Transplantation
Proteins

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H2O2 INDUCES NECROPTOSIS IN MESOANGIOBLAST STEM CELLS. / Sconzo, Gabriella; Geraci, Fabiana; Barreca, Maria Magdalena.

In: EUROPEAN JOURNAL OF HISTOCHEMISTRY, Vol. volume 62/ supplement 1, 2018, p. 3-3.

Research output: Contribution to journalBook/Film/Article review

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abstract = "Stem cells are used in regenerative medicine, but their therapeutic efficacy is compromised by their huge death during the first days post-transplantation. Indeed, the microenvironment within damaged tissues is hostile for stem cell survival mainly due to oxidative stress. H2O2 may play a relevant role in inducing death of the injected cells. The aim of our study was to determine the mechanism of mesoangioblast (A6) cell death after an H2O2 treatment.FACS analysis with annV/PI showed that H2O2 induced a dose and time-dependent decrement in A6 viability. We have also found an increase in caspases 8, 9 and 3 activity after the treatment. To assess their involvement in cell death, the pan caspase inhibitor Z-VAD was used. Neither early apoptosis, nor late apoptosis/necrosis, nor necrosis were reduced, suggesting that the cell death induced by H2O2 was caspase-independent. Then, we tested whether H2O2 is responsible for the autophagy activation.To study autophagy we evaluated the expression of specific markers. H2O2 decreased beclin1, Atg5, Atg7 and the ratio LC3II/I, in a dose dependent way. At the same time it increased p62 protein expression indicating an impaired autophagic flux, also confirmed by the increase of pAKT, responsible for the activation of mTOR, a negative regulator of autophagy. According to these data A6 treatment with H2O2 seems to not induce nor apoptosis or autophagy. For this reason we hypothesized the activation of necroptosis, a specific form of caspase-independent, non-apoptotic or necrotic cell death. To confirm whether the observed cell death was due to enhanced necroptosis, the proportion of necrotic cells was determined by annV/PI staining. FACS analysis showed an increase in percentage of both late apoptotic/necrotic and necrotic cells, which were further increased by pretreatment with Z-VAD. To investigate the relationship between physiological autophagy and necroptosis, cells were treated with H2O2 in the presence of the autophagic inhibitor 3MA. AnnV/PI staining showed that the inhibition of autophagy by 3MA significantly enhanced necroptosis in A6 treated cells. Conversely, 3MA had no effect on apoptosis. In conclusion, our data indicate that the cytotoxicity of H2O2 in A6 mainly occurred via the induction of necroptosis, enhanced by both apoptosis and autophagy inhibition.",
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T1 - H2O2 INDUCES NECROPTOSIS IN MESOANGIOBLAST STEM CELLS

AU - Sconzo, Gabriella

AU - Geraci, Fabiana

AU - Barreca, Maria Magdalena

PY - 2018

Y1 - 2018

N2 - Stem cells are used in regenerative medicine, but their therapeutic efficacy is compromised by their huge death during the first days post-transplantation. Indeed, the microenvironment within damaged tissues is hostile for stem cell survival mainly due to oxidative stress. H2O2 may play a relevant role in inducing death of the injected cells. The aim of our study was to determine the mechanism of mesoangioblast (A6) cell death after an H2O2 treatment.FACS analysis with annV/PI showed that H2O2 induced a dose and time-dependent decrement in A6 viability. We have also found an increase in caspases 8, 9 and 3 activity after the treatment. To assess their involvement in cell death, the pan caspase inhibitor Z-VAD was used. Neither early apoptosis, nor late apoptosis/necrosis, nor necrosis were reduced, suggesting that the cell death induced by H2O2 was caspase-independent. Then, we tested whether H2O2 is responsible for the autophagy activation.To study autophagy we evaluated the expression of specific markers. H2O2 decreased beclin1, Atg5, Atg7 and the ratio LC3II/I, in a dose dependent way. At the same time it increased p62 protein expression indicating an impaired autophagic flux, also confirmed by the increase of pAKT, responsible for the activation of mTOR, a negative regulator of autophagy. According to these data A6 treatment with H2O2 seems to not induce nor apoptosis or autophagy. For this reason we hypothesized the activation of necroptosis, a specific form of caspase-independent, non-apoptotic or necrotic cell death. To confirm whether the observed cell death was due to enhanced necroptosis, the proportion of necrotic cells was determined by annV/PI staining. FACS analysis showed an increase in percentage of both late apoptotic/necrotic and necrotic cells, which were further increased by pretreatment with Z-VAD. To investigate the relationship between physiological autophagy and necroptosis, cells were treated with H2O2 in the presence of the autophagic inhibitor 3MA. AnnV/PI staining showed that the inhibition of autophagy by 3MA significantly enhanced necroptosis in A6 treated cells. Conversely, 3MA had no effect on apoptosis. In conclusion, our data indicate that the cytotoxicity of H2O2 in A6 mainly occurred via the induction of necroptosis, enhanced by both apoptosis and autophagy inhibition.

AB - Stem cells are used in regenerative medicine, but their therapeutic efficacy is compromised by their huge death during the first days post-transplantation. Indeed, the microenvironment within damaged tissues is hostile for stem cell survival mainly due to oxidative stress. H2O2 may play a relevant role in inducing death of the injected cells. The aim of our study was to determine the mechanism of mesoangioblast (A6) cell death after an H2O2 treatment.FACS analysis with annV/PI showed that H2O2 induced a dose and time-dependent decrement in A6 viability. We have also found an increase in caspases 8, 9 and 3 activity after the treatment. To assess their involvement in cell death, the pan caspase inhibitor Z-VAD was used. Neither early apoptosis, nor late apoptosis/necrosis, nor necrosis were reduced, suggesting that the cell death induced by H2O2 was caspase-independent. Then, we tested whether H2O2 is responsible for the autophagy activation.To study autophagy we evaluated the expression of specific markers. H2O2 decreased beclin1, Atg5, Atg7 and the ratio LC3II/I, in a dose dependent way. At the same time it increased p62 protein expression indicating an impaired autophagic flux, also confirmed by the increase of pAKT, responsible for the activation of mTOR, a negative regulator of autophagy. According to these data A6 treatment with H2O2 seems to not induce nor apoptosis or autophagy. For this reason we hypothesized the activation of necroptosis, a specific form of caspase-independent, non-apoptotic or necrotic cell death. To confirm whether the observed cell death was due to enhanced necroptosis, the proportion of necrotic cells was determined by annV/PI staining. FACS analysis showed an increase in percentage of both late apoptotic/necrotic and necrotic cells, which were further increased by pretreatment with Z-VAD. To investigate the relationship between physiological autophagy and necroptosis, cells were treated with H2O2 in the presence of the autophagic inhibitor 3MA. AnnV/PI staining showed that the inhibition of autophagy by 3MA significantly enhanced necroptosis in A6 treated cells. Conversely, 3MA had no effect on apoptosis. In conclusion, our data indicate that the cytotoxicity of H2O2 in A6 mainly occurred via the induction of necroptosis, enhanced by both apoptosis and autophagy inhibition.

UR - http://hdl.handle.net/10447/358591

M3 - Book/Film/Article review

VL - volume 62/ supplement 1

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JO - EUROPEAN JOURNAL OF HISTOCHEMISTRY

JF - EUROPEAN JOURNAL OF HISTOCHEMISTRY

SN - 1121-760X

ER -