Nuray Acar

Location of cell cycle regulators cyclin B1, cyclin A, PCNA, Ki67 and cell cycle inhibitors p21, p27 and p57 in human first trimester placenta and deciduas

Although placental development and implantation depend on the coordination of trophoblast proliferation, differentiation and invasion, little is known about the cell cycle regulators that govern the control of these events. The hypothesis that the coordinated expression of cell cycle progression and inhibition factors will determine whether cytotrophoblasts proliferate or undergo cell cycle arrest or cell cycle exit allowing subsequent differentiation was tested. The cell cycle promotors cyclin A, cyclin B1, PCNA, Ki67 and the cell cycle inhibitors p21, p27 and p57 were immunolocalized in tissue sections of first trimester pregnancies (weeks 6 and 9–12). Double staining with cytokeratin 7 allowed unambiguous identification of extravillous cytotrophoblast (EVT) in the decidua. Villous cytotrophoblasts were immunolabelled for Ki67 and cyclin A but only few were stained with anti-cyclin B1. The syncytiotrophoblast was devoid of immunoreactivity for any of the cell cycle progression factors. It expressed especially p21, whereas p27 and p57 were predominantly found in villous cytotrophoblasts. PCNA, Ki67, cyclin A and cyclin B1 were immunolocalized in proximal and distal EVTs of anchoring villi and in EVT which had invaded the upper decidual segments. All EVTs strongly expressed p27 and p57, but not p21. These data clearly suggest different functions for p21, p27 and p57 in placental development with distinct roles for p21 and p57 in syncytiotrophoblast and EVT differentiation, respectively. p27 appears to be involved in both the processes. The results may also challenge the concept of differential mitotic activity in the proximal and distal parts of the first trimester cytotrophoblast cell column, but more functional studies are clearly needed. The presence of p27 and p57 in EVT cells, which invade the deciduas deeply, may account for the loss of mitogenic potential of these cells.

Uterine FK506-binding protein 52 (FKBP52)–peroxiredoxin-6 (PRDX6) signaling protects pregnancy from overt oxidative stress

Immunophilin FK506-binding protein 52 (FKBP52) is a cochaperone that binds to the progesterone receptor (PR) to optimize progesterone (P4)-PR signaling. We recently showed that Fkbp52-deficient (Fkbp52−/−) mice have reduced uterine PR responsiveness and implantation failure which is rescued by excess P4 supplementation in a genetic background-dependent manner. This finding led us to hypothesize that FKBP52 has functions in addition to optimizing PR activity. Using proteomics analysis, we found that uterine levels of peroxiredoxin-6 (PRDX6), a unique antioxidant, are significantly lower in Fkbp52−/− mice than in WT and PR-null (Pgr−/−) mice. We also found that Fkbp52−/− mice with reduced uterine PRDX6 levels are susceptible to paraquat-induced oxidative stress (OS), leading to implantation failure even with P4 supplementation. The same dose of paraquat did not interfere with implantation in WT mice. Moreover, treatment with antioxidants α-tocopherol and N-acetylcysteine (NAC) attenuated paraquat-induced implantation failure in P4-treated Fkbp52−/− mice. Functional analyses using mouse embryonic fibroblasts show that Fkbp52 deficiency associated with reduced PRDX6 levels promotes H2O2-induced cell death, which is reversed by the addition of NAC or by forced expression of PRDX6, suggesting that Fkbp52 deficiency diminishes the threshold against OS by reducing PRDX6 levels. These findings provide evidence that heightened uterine OS in Fkbp52−/− females with reduced PRDX6 levels induces implantation failure even in the presence of excess P4. This study shows that FKBP52–PRDX6 signaling protects pregnancy from overt OS.

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.

Endogenous orexin-A modulates gastric motility by peripheral mechanisms in rats

Orexin-A (OXA) and orexin receptor type 1 (OX1R) are found in enteric nervous system and smooth muscle cells in the digestive tract. Fasting is a stimulant for OXA synthesis. The aim of the present study was to investigate central and peripheral effects of endogenous OXA on gastric motility. Endogenous OXA synthesis was induced by 36h fasting. Vagotomy was used to evaluate N.vagus-mediated effects of OXA. Gastric emptying and interdigestive gastric motility were measured by spectrophotometric and manometric methods, respectively. Rats were pretreated with OX1R antagonist SB-334867 prior to measurements. Plasma OXA concentration was assayed with radioimmunoassay while preproorexin (PPO) expression was determined with Western blotting in gastric and hypothalamic tissues. OXA immunoreactivity in antrum was determined with immunohistochemistry. Plasma OXA level, PPO protein expression and OXA immunoreactivity were significantly increased in response to 36h fasting. Endogenous OXA facilitated gastric emptying and inhibited gastric interdigestive motility. As these effects were abolished with SB-334867, it is likely that gastrokinetic effects of OXA are mediated via OX1R. Vagotomy did not alter OXA-mediated effects. According to current data, OXA is up-regulated both centrally and peripherally upon fasting. Endogenous OXA accelerates gastric emptying while it inhibits interdigestive motility.

Is there a relationship between PCNA expression and diabetic placental development during pregnancy

We aimed to investigate the distribution pattern of proliferating cell nuclear antigen (PCNA) by immunohistochemistry and Western blot in placentas of control and diabetic rats at different stages of pregnancy. It is still not clear how proliferation is coordinated and how this coordination is affected by diabetes in the placenta. Diabetes was induced by streptozocin on the first day of pregnancy. Animals were sacrificed on days 11, 13, 17 and 21 of pregnancy. In control placentas immunolabeling intensity of PCNA was the highest on days 11 and 13 of pregnancy and decreased with progression of pregnancy. In the diabetic groups immunolabeling was less intense on days 11 and 13 of pregnancy compared to controls. However, in parallel with placental weights, PCNA immunopositivity was more intense in diabetic groups than control groups on days 17 and 21 of pregnancy, and the difference was statistically significant on day 17. According to Western blot data, on days 11 and 13 of pregnancy the amount of PCNA was greater in control groups than in the diabetics, whereas it was greater in diabetic groups than the controls on days 17 and 21 of pregnancy. We conclude that PCNA may play a role in abnormal placenta formation resulting from diabetes.

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.