H2O2 INDUCES NECROPTOSIS IN MESOANGIOBLAST STEM CELLS

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.