Ozlem Ozbey

The immunohistochemical localization of notch receptors and ligands in human articular cartilage, chondroprogenitor culture and ultrastructural characteristics of these progenitor cells

The presence of progenitor/stem cells in human articular cartilage remains controversial. Therefore, we attempted to isolate and culture progenitor/stem cells and to investigate their phenotypic characteristics. Biopsies were obtained (with consent) from patients undergoing arthroscopic surgery. Full depth explants were fixed and cryosectioned or enzymatically digested and the resulting cells cultured and plated on fibronectin-coated dishes. Chondrocytes were cultured until colonies of >32 cells were present. Colonies were trypsinized and expanded in monolayer for pellet culture. Immunolocalization of Notch and its ligands were detected in vivo and in vitro using immunocytochemistry. In vitro studies investigated differences in immunolocalization of Notch and its associated ligands in colony-forming cells and small clusters of non-colony-forming cells. The ultrastructure of the chondroprogenitors was examined by scanning and transmission electron microscopy. Results revealed that the immunolocalization of Notch-1 and its ligand Delta were concentrated in regions closest to the articular surface. Notch-1 was also densely localized in the deeper zone of articular cartilage. Notch-2 immunolabeling was densely localized in all zones of articular cartilage. Jagged-1 was concentrated in the deeper regions of articular cartilage. Notch-1, Delta and Jagged-1 were more abundant in colony-forming cells than non-colony-forming chondrocytes in vitro. Notch-3, Notch-4 and Jagged-2 were absent from all regions of the articular cartilage tissues and cultured cartilage cells in vitro. Ultrastructurally, chondrocytes cultured in monolayer dedifferentiated to fibroblast-like cells with cell surface processes of varying lengths, pellet cultured cells varied in morphology, as flattened and rounded. In conclusion, we propose that adult human articular cartilage may contain cells having progenitor cell features.

Distribution of Notch family proteins in intrauterine growth restriction and hypertension complicated human term placentas.

Members of the Notch family have been detected in many developmental and cell specification processes during placental development. However, Notch protein expression in Intrauterine Growth Restriction (IUGR) and Pregnancy Induced Hypertension (PIH) is not clear. In this study we aimed to clarify the immunolocalization of Notch proteins in full-term placentas after IUGR and PIH in comparison with normal placentas. Formalin-fixed, paraffin-embedded term placentas obtained by caesarean operations were processed for immunohistochemical localization of Notch 1, 2, 4 and Jagged 2. Transmission electron microscopy was also performed. In normal term placentas, all Notch proteins were intensely immunostained in the brush border of cells of the syncytiotrophoblast layer of the basal (maternal) side and the chorionic plate (fetal) side. The endothelial cells were also intensely immunostained in both sides for Notch 1. However, in IUGR and PIH placentas, the immunoreactivities of all Notch proteins were decreased significantly in the brush border of cells of the syncytiotrophoblast layer and the reaction was generally observed in the cytoplasm of syncytiotrophoblast cells in the basal and chorionic plate sides. The reactivity in endothelial cells was also significantly decreased. Our results have shown that the immunoreactivity and localization of Notch proteins is altered in pathologic placentas. Therefore, we propose that deregulated expression of Notch proteins may contribute to the disruption of trophoblast differentiation, endothelial cell function and/or feto-maternal traffic down-regulation during pregnancy or vice versa in such pathologic conditions.

Expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and peroxiredoxin 6 (Prdx6) proteins in healthy and pathologic placentas of human and rat

A relationship has been shown between preeclampsia (PE) and intrauterine growth restriction (IUGR) and oxidative stress (OS). Since such pregnancies experience OS, we aimed to detect the distribution pattern and expression levels of a transcription factor, Nuclear factor erythroid 2-related factor-2 (Nrf2) that has a role in the regulation of antioxidant enzymes, and peroxiredoxin 6 (Prdx6) an antioxidant enzyme, in human healthy, IUGR, PE and in groups of rat healthy and IUGR placentas using immunohistochemistry and Western blotting. Both Nrf2 and Prdx6 immunoreactivities were weaker in human and rat IUGR group placentas compared to human and rat control group placentas, respectively. Nrf2 and Prdx6 were immunostained in labyrinth trophoblasts, decidua, giant, glycogen and fetal vessel endothelial cells in rat control and IUGR group placentas. Nrf2 and Prdx6 immunoreactivities were seen in the decidua, syncytiotrophoblasts, villous stromal cells, and vascular endothelium in human control, IUGR and PE group placentas. Results of Nrf2 and Prdx6 Western blotting applied for rat and human placentas were compatible with the results of Nrf2 and Prdx6 immunohistochemical observations with regard to rat and human placentas. Down-regulation of Nrf2 and Prdx6 proteins in human and rat IUGR group placentas may have led to the formation of OS which may have impaired proliferation and invasion of cytotrophoblasts.

Characterization of colony-forming cells in adult human articular cartilage.

Recent studies have shown that adult human articular cartilage contains stem-like cells within the native structure. In this study, we aimed to determine the localization of putative stem cell markers such as CD90, STRO-1, OCT-3/4, CD105 and CD166 in adult human articular cartilage tissue sections and demonstrate the expression of these markers within the expanded surface zone colony-forming (CF) cells and evaluate their differentiation potential. Biopsy samples were either fixed immediately for immunohistochemical analyses or processed for in vitro cell culture. Immunohistochemical and flow cytometry analyses were performed by using CD90, STRO-1, OCT-3/4, CD105 and CD166 antibodies. Isolated colony-forming (CF) cells were further stimulated, by using the appropriate growth factors in their pellet culture, to obtain cartilage, bone and adipose lineages. We observed that the expression of the stem cell markers were in various zones of the human adult cartilage. Flow cytometry results showed that in CF cells the expression of CD90 and CD166 was high, while OCT-3/4 was low. We also determined that CF cells could be stimulated towards cartilage, bone and adipose lineages. The results of this research support the idea that the resident stem-like cells in adult human articular cartilage express these putative stem cell markers, but further experimental investigations are needed to determine the precise localization of these cells.

The effects of water immersion and restraint stress on the expressions of apelin, apelin receptor (APJR) and apoptosis rate in the rat heart

Apelin has been identified as an endogenous ligand of the orphan G-protein-coupled apelin receptor (APJR). These receptors are widely expressed in the central nervous system and periphery and play a role in the regulation of fluid and glucose homeostasis, feeding behavior, vessel formation, cell proliferation and immunity. We aimed to investigate whether water immersion and restraint stress have effects on apelin and APJR expression and apoptosis in heart tissue of male Wistar rats. The cardiac tissues were obtained from control, water immersion and restraint stress (WIRS) and apelin antagonist (F13A)+WIRS groups of rats and embedded in paraffin wax. Immunohistochemical staining methods were used to localize apelin, APJR and TUNEL immunopositive cells. H-SCORE was used for semi-quantitative determinations. Apelin protein levels were determined by Western blot in the cardiac tissues and plasma corticosteroid levels were measured by enzyme immunoassay (EIA). Apelin immunolocalization was found especially in endothelial cells and mast cells and faintly in cardiomyocytes, APJR immunostaining was shown in endothelial cells and cardiomyocytes, and TUNEL reaction was observed in endothelial cells and in some fibroblasts. Apelin expression was significantly increased in the WIRS and F13A+WIRS groups compared to the control group. The APJR reaction was similar in all groups. The number of TUNEL-positive cells was significantly higher in the F13A+WIRS group than that of the control group. Our study showed that WIRS for 6h increased plasma corticosterone levels and cardiac apelin expression in rats. The increased levels of apelin inhibited stress-induced apoptosis in heart. These results may be important for the therapeutic approach to a variety of stress-related heart disease.

The effect of systemic corticosteroid treatment on the immunolocalisation of Notch-1, Delta, CD105 and CD166 in rat articular cartilage

We studied the immunolocalisation of the stem cell-specific markers Notch-1, Delta, CD105 and CD166 in rat articular cartilage and analysed the effect of systemic corticosteroid treatment on the patterns of distribution of cells labelling for these markers. Female Wistar rats were separated randomly into two groups: the control group (n=8) was injected with isotonic salt solution and the corticosteroid group (n=8) was injected with 10 mg/kg intramuscular corticosteroid (methylprednisolone) once a week for a period of 8 weeks. Femoral head specimens from each group were obtained at the end of the treatment and processed for routine histological and immunohistochemical examinations. Quantitative data were obtained by H-SCORE and statistical evaluations were performed. The immunolocalisation of all markers was more apparent in the superficial zone and decreased through the deeper zones in all groups. However, the intensity of labelling was much less obvious in the group treated with corticosteroid compared to control. H-SCORE analysis confirmed that in the group treated with corticosteroid, the intensity of Notch-1, Delta, CD105 and CD166 labelling had decreased significantly compared to control (p<0.05). In conclusion, based on the immunolocalisation of stem cell-specific markers Notch-1, Delta, CD105 and CD166, the data suggest that the stem cells may continue to exist in adult rat articular cartilage. It was also observed that systemic corticosteroid treatment may effect the immunolabelling intensity of these markers, suggesting that corticosteroid treatment may reduce the function and the regenerative capacity of these cells in articular cartilage.

Apelin-APJ system is responsible for stress-induced increase in atrial natriuretic peptide expression in rat heart

BACKGROUND The cardiovascular system is a primary target of stress and stress is the most important etiologic factor in cardiovascular diseases. Stressors increase expressions of atrial natriuretic peptide (ANP) and apelin in cardiac tissue. AIM The aim of the present study was to investigate whether stress-induced apelin has an effect on the expression of ANP in the right atrium of rat heart. METHODS The rats were divided into the control, stress and F13A+stress groups. In the stress and F13A+stress groups, the rats were subjected to water immersion and restraint stress (WIRS) for 6h. In the F13A+stress group, apelin receptor antagonist F13A, was injected intravenously immediately before application of WIRS. The plasma samples were obtained for the measurement of corticosterone and atrial natriuretic peptide. The atrial samples were used for immunohistochemistry and western blot analysis. RESULTS F13A administration prevented the rise of plasma corticosterone and ANP levels induced by WIRS. While WIRS application increased the expressions of apelin, HIF-1α and ANP in atrial tissue, while F13A prevented the stress-induced increase in the expression of HIF-1α and ANP. CONCLUSION Stress-induced apelin induces ANP expression in atrial tissue and may play a role in cardiovascular homeostasis by increasing ANP expression under WIRS conditions.

Characterization of Notch Signalling Pathway Members in Normal Prostate, Prostatic Intraepithelial Neoplasia (PIN) and Prostatic Adenocarcinoma

Prostate Cancer (PCa) holds the second place in terms of cancer-related mortality rate among men. The Notch signalling pathway regulates the proliferation and differentiation in embryonic and adult tissues and determines the cell fate. The body of knowledge in the present literature is currently controversial about the effect of the Notch pathway on prostatic cancer. Therefore, the present study aimed to examine the immunolocalization and expression levels of Notch1-4, Jagged1-2, Delta, HES1 and HES5 from among the members of the Notch signalling pathway in tissues of normal, prostatic intraepithelial neoplasia (PIN) and malignant prostate. The current study included a sample of 20 patients with localised prostatic adenocarcinoma, 18 patients with high grade PIN (H-PIN) and 18 normal prostatic tissue. Immunolocalisations of Notch1, 2, 3, 4, Jagged1, 2, Delta, HES1 and HES5 were identified through the immunohistochemical method. The findings of the present study showed that all in-scope members of the Notch signalling pathway were localised in PIN structures to a greater extent than in other tissues and from amongst these members, specifically Notch1, Notch4, Jagged1 and HES1 were at more significant levels. Consequently, the findings of the present study may indicate that the Notch signalling pathway can play a role especially in the formation of PIN structures.

Protective effect of zoledronic acid on corticosteroid-induced chondrocyte apoptosis in rat articular cartilage

OBJECTIVE The aim of this study was to assess the apoptotic effects of systemic corticosteroid application on the articular cartilage chondrocytes in vivo and to investigate the potential effects of zoledronic acid on corticosteroid-induced apoptosis. METHODS Twenty-four Wistar rats were randomly divided into 3 groups. In the control group, intramuscular isotonic salt solution was injected weekly. In the second group, a dose of 10 mg/kg intramuscular corticosteroid (methylprednisolone) injection was applied weekly for 8 weeks. In the third group, a dose of 10 mg/kg intramuscular corticosteroid (methylprednisolone) injection was applied weekly for 8 weeks and 0.1 mg/kg zoledronic acid was injected subcutaneously on days 0, 21 and 42. Femoral head specimens from each group were obtained at the end of the treatment and the TUNEL method was applied to detect apoptotic chondrocytes. Comparison analyses were performed using the ANOVA method and Tukeys test. RESULTS There was a significant difference between the corticosteroid group and two other groups (control group: p=0.005; corticosteroid + zoledronic acid group: p=0.047). Zoledronic acid treatment significantly decreased the number of corticosteroid-induced apoptotic chondrocytes in the joint cartilage (p<0.05). CONCLUSION Zoledronic acid may have the potential to prevent joint cartilage deterioration due to the corticosteroid-induced apoptosis of the chondrocytes.