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

Effect of long-acting β₂-agonists and corticosteroids on glycosaminoglycans, proteoglycans and matrix metalloproteinases of connective tissue levels, in primary cell cultures of human lungs

Primary cell cultures of ASMC were established from control objects, asthma and COPD patients. The cell cultures were developed with 5% FCS in the absence and presence of 10⁻⁸ M formoterol and 10⁻⁸ Μ of their combination for 0, 12 and 24 hours cell culture media were collected separately. From the cell layers GAG’s were isolated and purified by lipid extraction, digestion with pronase enzyme, DNA digestion and precipitation by the addition of ethanol quantitation. Fractionation was achieved by electrophoresis on cellulose acetate membranes. Enzymatic treatment with specific GAG degrading enzymes confirmed each GAG quantitation of HA was performed by ELISA, determination of HA molecular mass, RNA isolation, RT-PCR and western blot analysis. Asthma and COPD patients secreted lower amounts of HA, due to decreased gene and protein expression of HAS-1 and HAS-2, increased expression of HYAL1, decreased expression of CD44 and lack of the HA receptor RHAMM. The effect of corticosteroids and long-acting β₂-agonists reverse all the above results.Αναπτύχθηκαν πρωτογενείς καλλιέργειες λείων μυϊκών κυττάρων από αεραγωγούς φυσιολογικών πνευμόνων, ασθενών με άσθμα και ΧΑΠ. Οι καλλιέργειες αναπτύχθηκαν σε 5% FCS απουσία και παρουσία 10⁻⁸ Μ του συνδυασμού τους για 0, 12 και 24 ώρες. Ακολούθησε διαχωρισμός του υπερκείμενου θρεπτικού υλικού από το κυτταρικό στρώμα. Σε κάθε δείγμα πραγματοποιήθηκε απομόνωση των GAG’s με απολιπίδωση, εξαντλητική πέψη με προνάση, πέψη με DNose, και εν τέλει οι ολικές GAG’s συλλέχθηκαν με καταβύθιση με αιθανόλη. Πραγματοποιήθηκε ποσοτικός προσδιορισμός των GAG’s, ανάλυση με ηλεκτροφορική σε οξικές κυτταρίνες, ταυτοποίηση με ειδικά ένζυμα, μέτρηση HA με ELISA, προσδιορισμός μοριακής μάζας ΗΑ, απομόνωση RNA και RT-PCR καθώς και ανοσοαποτύπωση κατά western. Διαπιστώθηκε μείωση του ΗΑ στους ασθενείς με άσθμα και ΧΑΠ. Η μείωση οφείλεται σε μειωμένη έκφραση της HAS-1 και HAS-2 και σε αυξημένη έκφραση της HYAL-1 η έκφραση του υποδοχέα CD44 μειώνεται και ο υποδοχέας RHAMM εκφράζεται μόνο στου υγιείς. Η επίδραση των κορτικοστεροειδών και των β₂-αδρενεργικών αγωνιστών αναστρέφει όλο τα παραπάνω

COPD Exacerbations Are Associated With Proinflammatory Degradation of Hyaluronic Acid

BACKGROUND: COPD is characterized by chronic airway inflammation and remodeling, with serious modifications of the extracellular matrix (ECM). Hyaluronic acid (HA) is an abundant ECM molecule in the lung with various biologic functions that depend on its molecular weight (MW). High-MW HA exhibits antiinflammatory and immunosuppressive effects, whereas low-MW HA is proinflammatory. In this study, we investigated whether acute exacerbations of COPD (AECOPDs), which affect patient quality of life and survival, are associated with altered HA turnover in BAL. METHODS: We used BAL from patients with stable COPD (n = 53) or during AECOPD (n = 44) matched for demographics and clinical characteristics and BAL from control subjects (n = 15). HA, HA synthase-1 (HAS-1), and hyaluronidase (HYAL) values were determined by enzyme-linked immunosorbent assay, and HYAL activity was determined by HA zymography. The MW of HA was analyzed by agarose electrophoresis. RESULTS: Levels of HA, HAS-1, and HYAL were significantly increased in BAL of patients with stable COPD and during exacerbations compared with control subjects. HYAL activity was significantly increased in BAL of patients with AECOPD, resulting in an increase of low-MW HA during exacerbations. In patients with AECOPD, we also observed a significant negative correlation of HA and HYAL levels with FEV1 % predicted but not with diffusing capacity of lung for carbon monoxide % predicted, indicating that increased HA degradation may be more associated with airway obstruction than with emphysema. CONCLUSIONS: AECOPDs are associated with increased HYAL activity in BAL and subsequent degradation of HA, which may contribute to airway inflammation and subsequent lung function decline during exacerbations

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 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 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 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

Steroids and beta2-agonists regulate hyaluronan metabolism in asthmatic airway smooth muscle cells

Glycosaminoglycans (GAGs), especially hyaluronic acid (HA), regulate tissue flexibility, cell motility, and inflammation. Airway smooth muscle cells (ASMCs) of patients with asthma exhibit abnormal HA metabolism, which contributes to inflammation and remodeling. Here, we investigated the effects of glucocorticoids and long-acting beta(2)-agonists (LABAs) on GAG synthesis and HA metabolism by human primary ASMCs. ASMCs were isolated from airway specimens of 10 patients without asthma and 11 patients with asthma. ASMCs were incubated with glucocorticoids, LABAs, or their combination, as well as with their specific receptor antagonists. Secreted and deposited total GAGs were measured by [(3)H]-glucosamine incorporation. The expression of specific GAGs was determined by ELISA and electrophoresis. The expression of HA synthases (HAS), of hyaluronidases (HYALs), and of the HA receptor CD44 was determined by RT-PCR, immunoblotting in cell cultures, and immunohistochemistry in tissue sections of asthmatic lungs. In serum-activated asthmatic ASMCs, glucocorticoids and LABAs significantly inhibited the increased secretion and deposition of total GAGs, but they stimulated secreted and deposited HA of high molecular mass. This effect was attributed to increased mRNA and protein expression of HAS-1 and to the reduced expression of HYAL-1. Furthermore, drug treatment stimulated the expression of CD44 receptors in asthmatic ASMCs. These effects of the drugs were eliminated by their respective receptor inhibitors. Our findings indicate that the combination of glucocorticoids with LABAs counteracts the pathologic degradation of HA, and thereby may reduce the proinflammatory potential of asthmatic ASMCs