Oxygen-Glucose Deprivation (OGD) Modulates the Unfolded Protein Response (UPR) and Inflicts Autophagy in a PC12 Hypoxia Cell Line Model.

Hypoxia is the lack of sufficient oxygenation of tissue, imposing severe stress upon cells. It is a major feature of many pathological conditions such as stroke, traumatic brain injury, cerebral hemorrhage, perinatal asphyxia and can lead to cell death due to energy depletion and increased free radical generation. The present study investigates the effect of hypoxia on the unfolded protein response of the cell (UPR), utilizing a 16-h oxygen-glucose deprivation protocol (OGD) in a PC12 cell line model. Expression of glucose-regulated protein 78 (GRP78) and glucose-regulated protein 94 (GRP94), key players of the UPR, was studied along with the expression of glucose-regulated protein 75 (GRP75), heat shock cognate 70 (HSC70), and glyceraldehyde 3-phosphate dehydrogenase, all with respect to the cell death mechanism(s). Cells subjected to OGD displayed upregulation of GRP78 and GRP94 and concurrent downregulation of GRP75. These findings were accompanied with minimal apoptotic cell death and induction of autophagy. The above observation warrants further investigation to elucidate whether autophagy acts as a pro-survival mechanism that upon severe and prolonged hypoxia acts as a concerted cell response leading to cell death. In our OGD model, hypoxia modulates UPR and induces autophagy.

Low Dose Administration of Glutamate Triggers a Non-Apoptotic, Autophagic Response in PC12 Cells.

BACKGROUND/AIMS: Increasing amounts of the neurotransmitter glutamate are associated with excitotoxicity, a phenomenon related both to homeostatic processes and neurodegenerative diseases such as multiple sclerosis. METHODS: PC12 cells (rat pheochromocytoma) were treated with various concentrations of the non-essential amino acid glutamate for 0.5-24 hours. The effect of glutamate on cell morphology was monitored with electron microscopy and haematoxylin-eosin staining. Cell survival was calculated with the MTT assay. Expression analysis of chaperones associated with the observed phenotype was performed using either Western Blotting at the protein level or qRT-PCR at the mRNA level. RESULTS: Administration of glutamate in PC12 cells in doses as low as 10 µM causes an up-regulation of GRP78, GRP94 and HSC70 protein levels, while their mRNA levels show the opposite kinetics. At the same time, GAPDH and GRP75 show reduced protein levels, irrespective of their transcriptional rate. On a cellular level, low concentrations of glutamate induce an autophagy-mediated pro-survival phenotype, which is further supported by induction of the autophagic marker LC3. CONCLUSION: The findings in the present study underline a discrete effect of glutamate on neuronal cell fate depending on its concentration. It was also shown that a low dose of glutamate orchestrates a unique expression signature of various chaperones and induces cell autophagy, which acts in a neuroprotective fashion.

The immunohistochemical expression of CD24 and CD171 adhesion molecules in borderline ovarian tumors.

CD24 and CD171 are cell adhesion proteins, which have been shown to be overexpressed in several carcinomas and to be associated with a poor clinical outcome. Our aim was to determine the expression of these two adhesion molecules in ovarian borderline neoplasms. We investigated 50 ovarian borderline tumors (serous, mucinous and endometrioid) as well as 29 benign cystadenomas and 25 carcinomas, which were used as controls. Paraffin sections were stained immunohistochemically for CD24 and CD171, and their expression was recorded in a semi-quantitative manner. In normal epithelium and benign ovarian cystadenomas both the CD24 and CD171 expression was negative to low, while their expression was significantly increased in borderline and malignant ovarian tumors. High-grade carcinomas, and carcinomas with metastases to the omentum presented considerably higher CD24 expression than low-grade carcinomas, and carcinomas without metastases. In addition, a few borderline and many malignant tumors presented cytoplasmic CD24 immunoreactivity, whereas all benign and most borderline tumors showed apical localization of this molecule. In conclusion, borderline tumors and carcinomas of the ovary present increased expression of CD24 and CD171 in relation to their benign counterparts, as is the case in malignant tumors of other organs. Change of staining pattern of CD24 (apical to cytoplasmic) apparently relates to a more aggressive phenotype.

Comparison of ultrasound turbinate reduction, radiofrequency tissue ablation and submucosal cauterization in inferior turbinate hypertrophy.

Chronic nasal obstruction owed to chronic hypertrophic rhinitis is a common cause of nasal airway obstruction. In cases unresponsive to conservative treatment, various surgical techniques are commonly performed, but the issue of the optimal surgical procedure is still controversial. The objective of the present study was to evaluate the effectiveness and safety of ultrasound treatment of the hypertrophied inferior turbinates, which is a technique recently applied in rhinologic surgery. We aimed, also, to compare this method with the radiofrequency cold coblation turbinate reduction and the traditional submucosal monopolar inferior turbinate cauterization. We studied prospectively 60 patients with chronic hypertrophic rhinitis of nonallergic etiology, who underwent different surgical methods of turbinate reduction, divided into two groups: (1) in 30 patients, inferior turbinate volume reduction using ultrasound procedure on the left side and monopolar diathermy on the right was performed; (2) in 30 patients, radiofrequency coblation technique on the left side and ultrasound turbinate reduction on the right side was undertaken. Subjective evaluation of nasal obstruction and pain was performed using visual analog scales and objective evaluation of the surgical outcome was obtained using active anterior rhinomanometry and acoustic rhinometry. Examinations were performed preoperatively, and 1, 3 and 6 months after surgery. Both subjective and objective evaluation showed significant postoperative improvement in all cases. The best results were obtained with the ultrasound procedure, and second with the radiofrequency technique. The least improvement was observed in the electrocautery group, although its results did not differ significantly from the radiofrequency group. It may be, thus, concluded that ultrasound turbinate reduction is an effective and safe procedure for the management of chronic hypertrophic rhinitis, in patients failing to respond to medical treatment. Using this method, better results were obtained in decreasing subjective symptoms and nasal obstruction, in comparison with radiofrequency and electrocautery.

Mucosal changes in chronic hypertrophic rhinitis after surgical turbinate reduction.

Chronic nasal obstruction owed to chronic hypertrophic rhinitis is one of the most common problems encountered in rhinology. Various forms of conservative therapy have been used, but these are often ineffective and surgical reduction techniques have been successfully applied. However, the issue of postoperative histological changes in the nasal mucosa has not been adequately addressed. The aim of the present study was to comparatively evaluate the effect of the methods of submucosal monopolar diathermy, radiofrequency coblation and ultrasounds on the nasal mucosa. Sixty patients with chronic hypertrophic rhinitis of nonallergic etiology underwent different surgical methods of turbinate reduction, divided into two groups: (1) 30 patients underwent tissue volume reduction of the inferior nasal turbinates using ultrasound procedure on the left side and monopolar diathermy on the right; (2) 30 patients underwent radiofrequency coblation technique on the left side and ultrasound reduction on the right. We studied 20 preoperative specimens of the inferior turbinate mucosa taken randomly from both groups of patients, 5 from each side of each group. Normal nasal mucosal specimens taken from ten healthy persons were used as controls. Specimens of the inferior turbinate were taken after 1, 3, and 6 months after surgery, from the same patients. All specimens were examined by electron microscopy. Preoperative observation revealed degeneration of epithelial cells, loss of cilia, disruption of intercellular connections, edema, nasal mucus overproduction and inflammatory infiltration in chorium. Postoperative observations revealed decrease of intercellular edema, reduction of mucus, overproduction of collagen and degeneration of the epithelium to flattened stratified. Only specimens after use of ultrasounds showed islands with normally organized epithelium of columnar ciliated cells. It may be concluded that epithelial changes owed to chronic hypertrophic rhinitis do not significantly improve postoperatively after turbinate tissue volume reduction. Only in several cases operated with ultrasounds, regeneration of epithelium occurs, resulting to anatomical and functional restoration of the nasal physiology.

In vitro cardiomyogenic differentiation of adult human bone marrow mesenchymal stem cells. The role of 5-azacytidine.

The aim of this study is to investigate the ability of adult human bone marrow mesenchymal stem cells to differentiate towards a cardiomyogenic phenotype in vitro. Bone marrow samples have been aspirated from 30 patients undergoing open heart surgery. Mesenchymal stem cells were isolated and cultured in enriched medium. Second passaged cells were treated with 10 microM 5-azacytidine for 24 h. Selected surface antigens were analyzed by flow cytometry. Morphologic characteristics were analyzed by confocal and electron microscopy. Expression of cytoskeletal protein vimentin and muscle specific myosin heavy chain were analyzed by immunohistochemistry. Expression of alpha-cardiac actin, beta-myosin heavy chain and cardiac troponin-T was detected by reverse transcriptase polymerase chain reaction. Mesenchymal stem cells were spindle-shaped with irregular processes. Cells treated with 5-azacytidine have assumed a stick-like morphology. They were connecting with adjoining cells forming myotube-like structures. Numerous myofilaments were detected in induced cells running in a parallel fashion without forming sarcomeres that were immunohistochemically positive for myosin heavy chain and vimentin. The mRNAs of alpha-cardiac actin, beta-myosin heavy chain and troponin-T were expressed in both induced and uninduced cells. These results indicate that adult human bone marrow mesenchymal stem cells can differentiate towards a cardiomyogenic lineage in vitro.