Proteases Inhibition Assessment on PC12 and NGF Treated Cells after Oxygen and Glucose Deprivation Reveals a Distinct Role for Aspartyl Proteases

Hypoxia is a severe stressful condition and induces cell death leading to neuronal loss both to the developing and adult nervous system. Central theme to cellular death is the activation of different classes of proteases such as caspases calpains and cathepsins. In the present study we investigated the involvement of these proteases, in the hypoxia-induced PC12 cell death. Rat PC12 is a model cell line for experimentation relevant to the nervous system and several protocols have been developed for either lethal hypoxia (oxygen and glucose deprivation OGD) or ischemic preconditioning (IPS). Nerve Growth Factor (NGF) treated PC12 differentiate to a sympathetic phenotype, expressing neurites and excitability. Lethal hypoxia was established by exposing undifferentiated and NGF-treated PC12 cells to a mixture of N2/CO2 (93:5%) in DMEM depleted of glucose and sodium pyruvate for 16 h. The involvement of caspases, calpains and lysosomal cathepsins D and E to the cell death induced by lethal OGD was investigated employing protease specific inhibitors such as z-VAD-fmk for the caspases, MDL28170 for the calpains and pepstatin A for the cathepsins D and E. Our findings show that pepstatin A provides statistically significant protection from cell death of both naive and NGF treated PC12 cells exposed to lethal OGD. We propose that apart from the established processes of apoptosis and necrosis that are integral components of lethal OGD, the activation of cathepsins D and E launches additional cell death pathways in which these proteases are key partners

Self-care behaviors of adults with type 2 diabetes mellitus in Greece.

The purpose of this study was to examine self-care behaviors of adults with type 2 diabetes mellitus living in the Metropolitan Area of Thessaloniki in Northern Greece. The Summary of Diabetes Self-Care behaviors measurement was administered to 215 patients, out of which 177 were eligible to participate (87 males). Patients, aged 30 years or more, were recruited through a university hospital day-clinic. Older patients (>65 years), as well as those with "higher educational level" did not distribute their daily carbohydrate intake equally. Nevertheless, they were more likely to adapt to their physician's recommendations regarding medication and to regularly perform suggested blood glucose checking. Exercise patterns were more often found for higher educated, earlier diagnosed males. Younger patients were less likely to follow their healthcare professional's recommendations, regarding diet, medication intake, blood glucose checking, foot care and exercise compared to older patients. These results pose a higher risk for complications and morbidity in younger patients with type 2 diabetes mellitus, who most possibly will require intensive treatment in the future

Effect of 16 hours Oxygen-Glucose Deprivation (OGD) on % survival of PC12 cells in the absence or presence of the permeable calpain inhibitor MDL28170 (10 µM, 50 µM and 100 µM).

<p>The values are presented as mean ± SEM, *p<0.05, **p<0.01, ***p<0,001. <b>Panel a</b>, naive PC12 cells, <b>panel b</b> PC12 cells NGF treated.</p

Effect of 16 hours Oxygen-Glucose Deprivation (OGD) on % survival of PC12 cells in the absence or presence of different combinations of caspase, calpain and aspartyl protease inhibition (10 µM/10 µM/5 µM).

<p>The values are presented as mean ± SEM, *p<0.05, **p<0.01. <b>Panel a</b>, naive PC12 cells, <b>panel b</b> PC12 cells NGF treated.</p

Effect of 16 hours Oxygen-Glucose Deprivation (OGD) on % survival of PC12 cells in the absence or presence of 10 µM and 50 µM of the specific caspase inhibitor z-VAD-fmk.

<p>The values are presented as mean ± SEM, *p<0.05, **p<0.01. <b>Panel a</b>, naive PC12 cells, <b>panel b</b> PC12 cells NGF treated.</p

Effect of 16 hours Oxygen-Glucose Deprivation (OGD) on % survival of PC12 cells in the absence or presence of different concentrations of the aspartyl protease inhibitor pepstatin A (1 µM and 5 µM).

<p>The values are presented as mean ± SEM, *p<0.05, **p<0.01. <b>Panel a</b>, naive PC12 cells, <b>panel b</b> PC12 cells NGF treated.</p