Oxidative stress induces necropolis in mouse mesoangioblast stem cells

Nowadays stem cells are extensively used in regenerative medicine, as they show a great potential in restoration of several pathologies such as ischaemic heart disease, stroke, degenerative disorders and so on. However, it is well known that their therapeutic efficacy is compromised not only due to a reduced homing towards the target site, but also for their massive death during the first several days post-transplantation caused by the cytotoxic environment. In fact, the microenvironment within damaged tissues is unfavourable for stem cell survival due to hypoxia, inflammatory mediators and above all oxidative stress, which is particularly detrimental. H2O2 that diffuses freely into and out of cells may play a significant role in inducing death of injected stem cells. The most common types of cell death are represented by apoptosis, autophagy and necrosis. Apoptosis is a process of programmed cell death that occurs in multicellular organisms generally by activating caspases, or enzymes that degrade proteins. Autophagy is an important mechanism of cell self-protection, which helps cells maintain the synthesis and degradation cycles and promotes cell survival through the lysosomal degradation mechanism. Finally, necrosis is known as a fortuitous and regulated means of cell death that is induced by non specific and non physiological stress. The aim of our study was to determine the mechanism of mesoangioblast cell death after an in vitro H2O2 treatment, focusing whether apoptosis, autophagy or necrosis were implied. Mouse mesoangioblasts are vessel associated progenitor stem cells, which are able to differentiate in almost all mesodermal tissues. FACS analysis with annexinV/sytox green demonstrated that H2O2 induced a dose- and time-dependent decrement in mesoangioblast viability. We have also found an increase in caspases 8, 9 and 3 activity after hydrogen peroxide treatment. To assess whether they were involved in causing cell death, the pan caspase inhibitor Z-VAD-fmk was used to inhibit caspase activity. Neither early apoptosis, nor late apoptosis/necrosis, nor necrosis were reduced, suggesting that the cell death induced by hydrogen peroxide was caspase-independent. For this reason we tested whether H2O2 is responsible for the autophagic pathway activation. To study autophagy we evaluated the expression of specific markers (e.g., LC3II, beclin1, p62, Atg7, Atg5). H2O2 decreased beclin1, Atg5, Atg7 levels and the ratio LC3II/I, in a dose dependent way. At the same time H2O2 increased p62 protein expression indicating an impaired autophagic flux, also confirmed by the increase of phospho AKT, responsible for the activation of mTOR, a negative regulator of autophagy. According to these data mesoangioblast treatment with hydrogen peroxide seems to not induce nor apoptosis or autophagy. For this reason we hypothesized the activation of necroptosis, which is a specific form of caspase-independent, non-apoptotic or necrotic cell death that is triggered by cell death ligands via a unique downstream signaling pathway. To confirm whether the observed cell death was due to enhanced necroptosis, the proportion of necrotic cells was determined by annexin V/sytox green 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-fmk. To investigate the relationship between physiological autophagy and necroptosis cells were treated with H2O2 in the presence of the autophagic inhibitor 3-MA. Annexin V/sytox green staining showed that the inhibition of autophagy by 3-MA significantly enhanced necroptosis in mesoangioblast treated cells. On the contrary, 3-MA had no effect on apoptosis. In conclusion, our in vitro data indicate that the cytotoxicity of H2O2 in mesoangioblast mainly occurred via the induction of necroptosis, enhanced by both apoptosis and autophagy inhibition.