Gozde Unek

Immunolocalization of PCNA, Ki67, p27 and p57 in normal and dexamethasone-induced intrauterine growth restriction placental development in rat.

Intrauterine growth restriction (IUGR) is a major clinical problem which causes perinatal morbidity and mortality. Although fetuses with IUGR form a heterogeneous group, a major etiological factor is abnormal placentation. Despite the fact that placental development requires the coordinated action of trophoblast proliferation and differentiation, there are few studies on cell cycle regulators, which play the main roles in the coordination of these events. Moreover it is still not determined how mechanisms of coordination of proliferation and differentiation are influenced by dexamethasone-induced IUGR in the placenta. The aim of the study was to investigate the spatial and temporal immunolocalization of proliferating cell nuclear antigen (PCNA), Ki67, p27 and p57 in normal and IUGR placental development in pregnant Wistar rats. The study demonstrated altered expressions of distinct cell cycle proteins and cyclin dependent kinase inhibitors (CKIs) in IUGR placental development compared to control placental development. We found reduced immunostaining of PCNA and Ki67 and increased immunostaining of p27 and p57 in the dexamethasone-induced IUGR placental development compared to control placental development. In conclusion, our data show that the cell populations in the placenta stain for a number of cell cycle related proteins and that these staining patterns change as a function of both gestational age and abnormal placentation.

Immunohistochemical distribution of cell cycle proteins p27, p57, cyclin D3, PCNA and Ki67 in normal and diabetic human placentas

The placenta is a regulator organ for many metabolic activities between mother and fetus. Therefore, fetal growth is directly related to the placental development. Placental development is a series of events that depend on the coordinated action of trophoblasts’ proliferation, differentiation and invasion. Studies on cell cycle related proteins which control these events are fairly limited. How placental tissue proliferation is affected by diabetes is not exactly known yet. Therefore in this study, the immunohistochemical localizations of cell cycle related proteins like PCNA, Ki67, cyclin D3, p27 and p57 in the differentiation, proliferation and apoptosis mechanisms of normal and diabetic placentas were investigated. Information on cell cycle related proteins that control these events is limited and how they are affected in diabetes mellitus is not fully understood yet. Therefore, in this study, to understand the role of cell cycle regulators in diabetic placentas we aimed to determine the spatio-temporal immunolocalizations of cell cycle regulators in diabetic and normal human term placentas. Term placentas were obtained from diabetic women and from normal pregnancies with informed consent following caesarean deliveries. Placental samples were stained via immunohistochemistry with PCNA, Ki67, cyclin D3, p27 and p57 antibodies and were examined by light microscopy. When compared to control placentas, PCNA, Ki67 and cyclin D3 staining intensities significantly increased in villous parts of diabetes group. Moreover, Ki67 and cyclin D3 stainings also significantly increased in basal plates and chorionic plate respectively. In chorionic plates, p27 and p57 staining intensities significantly decreased in diabetic group. p57 staining also significantly decreased in villous parts of diabetic placentas. Placental abnormalities seen in diabetic placentas could be associated with proliferation and cell cycle arrest mechanisms’ alterations occurred in diabetes mellitus.

Immunolocalization of cell cycle proteins (p57, p27, cyclin D3, PCNA and Ki67) in intrauterine growth retardation (IUGR) and normal human term placentas

Placental development involves a series of events that depend on the coordinated action of proliferation, differentiation and invasion of trophoblasts. Studies on cell cycle related proteins controlling these events are fairly limited. It is still not fully determined how placental tissue proliferation is affected by intrauterine growth retardation (IUGR). Information on cell cycle related proteins that control these events is limited and how they are affected in IUGR is not fully understood. The aim of this study was to understand the role of cell cycle regulators in IUGR placentas and to determine the spatio-temporal immunolocalization of these cell cycle regulators in human IUGR and normal term placentas. Placental samples were stained immunohistochemically with PCNA, Ki67, cyclin D3, p27 and p57 antibodies and were examined by light microscopy. In all regions of IUGR placentas, PCNA, Ki67 and cyclin D3 staining intensities were statistically significantly decreased compared to normal controls. p27 staining intensity of the IUGR group was statistically significantly increased in villous parts and chorionic plates in comparison with the normal term placentas. Moreover, p57 staining intensity was statistically significantly increased in all parts of the IUGR group compared to controls. The observed placental abnormalities in IUGR placentas may be associated with arrest mechanisms affecting cell proliferation and cell cycle alterations in IUGR.

The Presence of Kinesin Superfamily Motor Proteins KIFC1 and KIF17 in Normal and Pathological Human Placenta

Kinesin superfamily proteins (KIFs) are motor proteins that participate in chromosomal and spindle movements during mitosis and meiosis, and transport membranous organelles and macromolecules fundamental for cellular functions. Although the roles of KIFs in axonal and dendritic transports have been studied extensively, their role in intracellular transport in general is less well known. The diversity of kinesins suggests that each kinesin may have a specific function. Therefore, in this study we aimed to investigate the presence and cellular localization of KIFC1 and KIF17 in normal and pathological human placentas. First-trimester (22-56 days) and normal, preeclamptic (PE), and diabetic-term placental tissues were obtained and further studied by immunohistochemistry (IHC) and Western blot methods. KIFC1 was mainly localized to the syncytiotrophoblast both in early and term placental samples. However, a stronger immunoreactivity was observed both in PE and diabetic placentas compared to normal-term placentas. KIF17 was most intensively localized in developing vascular endothelium in early pregnancy. Even though KIF17 was moderately stained in the endothelium of villi from normal human-term placentas, stronger immunoreactivity was observed in all types of villi of both PE and diabetic placentas. Western blotting of tissue extracts confirmed the IHC results. Here, we demonstrate the presence of KIFC1 and KIF17 in human placenta for the first time. The intense expression of KIFC1 in syncytiotrophoblast and KIF17 in vascular endothelium suggests that both the proteins might be important in a cargo-transport system. An increased expression pattern of both KIFC1 and KIF17 in PE and diabetes might suggest that these proteins may be involved in complex trophoblast functions and placental pathologies. Further studies will clarify the physiological role of KIFs in human placental transport and development.

The PI3K/Akt and MAPK-ERK1/2 pathways are altered in STZ induced diabetic rat placentas.

Diabetic pregnancy is associated with complications such as early and late embryonic death, fetal growth disorders, placental abnormalities, and embryonal-placental metabolic disorders. Excessive apoptosis and/or changes of proliferation mechanisms are seen as a major event in the pathogenesis of diabetes-induced embryonic death, placental weight and structural anomalies. Akt and ERK1/2 proteins are important for placental and fetal development associated with cellular proliferation and differentiation mechanisms. The mechanism underlying the placental growth regulatory effects of hyperglycemia have not been elucidated. Moreover, it is still not determined how Akt and ERK1/2 proteins related proliferation and apoptosis mechanisms are influenced by Streptozotocin (STZ) induced diabetic rat placental development. The aim of this study was to investigate the expression levels and spatio-temporal immunolocalizations of Akt, p-Akt, ERK1/2 and p-ERK1/2 proteins in normal and STZ-treated diabetic rat placental development. In order to compose the diabetic group, pregnant females were injected with a single dose of 40 mg/kg STZ intraperitonally seven days before their sacrifice at 12th, 14th, 16th, 18th and 20th day of their gestation. We found that maternal diabetic environment led to a decrease in ERK1/2 and Akt phosphorylation during rat placental development. It could be said that MAPK-ERK1/2 and PI3K/Akt cell signaling pathways are affected from hyperglycemic conditions in rat placentas. In conclusion, hyperglycemia-induced placental and embryonal developmental abnormalities could be associated with reduction of Akt and ERK1/2 phosphorylation.

Glucocorticoid exposure altered angiogenic factor expression via Akt/mTOR pathway in rat placenta.

During pregnancy, glucocorticoids (GCs) are used for fetal lung maturation in women at risk of preterm labor. Exogenous GCs do not have exclusively beneficial effects and repeated use of GCs remains controversial. It has been observed that GC exposed rats have smaller placentas and intrauterine growth retarded fetuses. In this study, we questioned whether or not glucocorticoids effect placental angiogenesis mechanisms. One of the most important signaling pathways among several downstream of VEGFR-2 is PI3K/Akt which subsequently activates the mammalian target of rapamycin. Therefore, we hypothesized that overexposure to GCs may adversely affect placental angiogenesis mechanisms by regulating pro-angiogenic factors and their receptors via Akt/mTOR pathway. According to our results Dexamethasone, a synthetic glucocorticoid, administration led to a decrease in VEGF, PIGF expression during pregnancy. VEGFR2 expression was first decreased at gestational day 14 and afterwards increased at gestational days 16, 18 and 20 in rat placentas. These results are in accordance with the reduced phosphorylation of Akt, 4EBP1 and p70S6K. Dexamethasone injection also resulted in a reduction of VEGF, VEGFR1, and VEGFR2 mRNA expression at gestational days 14 and 20, but PIGF mRNA expression was not altered. Growth retarded fetuses seen in Dexamethasone treated pregnancies, may be a result of altered angiogenic factor expression of the placenta mediated via altered mTOR pathway signaling.

Triamcinolone up‐regulates GLUT 1 and GLUT 3 expression in cultured human placental endothelial cells

The placenta is a glucocorticoid target organ, and glucocorticoids (GCs) are essential for the development and maturation of fetal organs. They are widely used for treatment of a variety of diseases during pregnancy. In various tissues, GCs have regulated by glucose transport systems; however, their effects on glucose transporters in the human placental endothelial cells (HPECs) are unknown.

Mapping of CIP/KIP inhibitors, G1 cyclins D1, D3, E and p53 proteins in the rat term placenta

As cell cycle regulation is fundamental to the normal growth and development of the placenta, the aim of the present study was to determine the immunolocalizations of cell cycle related proteins, which have key roles in proliferation, differentiation and apoptosis during the development of the rat placenta. Here immunohistochemistry has been used to localize G1 cyclins (D1, D3, E), which are major determinants of proliferation, CIP/KIP inhibitors (p21, p27, p57), p53 as a master regulator and proliferating cell nuclear antigen in all cell types of the rat term placenta. The proportion of each cell type immunolabeled was counted. Cyclin D1 and cyclin D3 were present mostly in cells of the fetal aspect of the placenta, whereas the G1/S cyclin E was present only in the spongio- and labyrinthine trophoblast populations. Among the CIP/KIP inhibitors, p21 was present only in cells of the fetal aspect whereas p27 and p57 were found in all cell types studied. p53 was only found in a small proportion of cells with no co-localization of p53 and p21. The data suggest that the cells of the fetal side of the rat placenta still have some proliferation potential which is kept in check by expression of the CIP/KIP cell cycle inhibitors, whereas cells of the maternal aspect have lost this potential. Apoptosis is only marginal in the term rat placenta. In conclusion, proliferation and apoptosis in rat placental cells appears controlled mostly by the CIP/KIP inhibitors in late pregnancy.

The Effects of Glucocorticoids on Fetal and Placental Development

Glucocorticoids (GCs), steroid hormones produced predominantly by the adrenal gland, are key mediators of stress responses. Whilst the acute and chronic effects of pharmacological glucocorticoid excess are well-recognized (including induction of hyperglycemia, insulin resistance, hyperlipidemia, hypertension and dysphoria, with suppression of immune, inflammatory and cognitive processes), their role in the biology of the response to stress is more nuanced, with balanced homeostatic effects to facilitate short-term survival and recovery from challenge [1, 2]. In addition, glucocorticoids play an essential role in normal fetal development and are important for the development and maturation of various fetal tissues including the liver, lungs, gut, skeletal muscle and adipose tissue in preparation for extrauterine life. Glucocorticoids most notably act during late gestation to stimulate surfactant production by the lung. This action is critical to prepare the fetus for extrauterine life, and it is for this reason that synthetic glucocorticoid treatment is so widely used in preterm pregnancies where lung immaturity threatens neonatal viability. Although these treatments greatly improve survival [3], they are not without adverse effects.

Expression of glucocorticoid receptor and glucose transporter-1 during placental development in the diabetic rat.

In various tissues, glucocorticoids (GCs) are known to downregulate glucose transport systems; however, their effects on glucose transporters (GLUTs) in the placenta of a diabetic rat are unknown. Glucocorticoid hormone action within the cell is regulated by the glucocorticoid receptor (GR). Thus, this study was designed to investigate the relationship between GR and glucose transporter expression in the placenta of the diabetic rat. Our immunohistochemical results indicated that GR and glucose transporter protein 1 (GLUT 1) are expressed ubiquitously in the trophoblast and endothelial cells of the labyrinthine zone, where maternal fetal transport takes place in the rat placenta. Expression of GR in the junctional zone of the rat placenta was detected in giant cells, and in some spongiotrophoblast cells, but not in the glycogen cells. GLUT 1 was present, especially in glycogen cells during early pregnancy, and in the spongiotrophoblast cells of the junctional zone during late pregnancy. Amounts of GR and GLUT 1 protein were increased towards the end of gestation both in the control and the diabetic placenta. However, at days 17 and 19 of gestation, only the placental GR protein was significantly increased in the streptozotocin-induced diabetic rats compared to control rats. Diabetes led to a significant decrease in placental weight at gestation day 15. In contrast, at gestational days 17 and 21, the weights of the diabetic placenta were significantly increased as compared with the controls. Moreover, diabetes induced fetus intrauterine growth retardation at gestational days 13, 17 and 21. In conclusion, the localization pattern of GR and GLUT 1 proteins in the same cell types led us to believe that there might be a relationship between GR and GLUT 1 expressions at the cellular level. GLUT 1 does not play a pivotal role in diabetic pregnancies. However, placental growth abnormalities during diabetic pregnancy may be related to the amount of GR.