Contribution of Human Trophoblast Progenitor Cells to Neurogenesis in Rat Focal Cerebral Ischemia Model.

OBJECTIVE: : A decrease in the blood flow below a current level in the brain results in ischemia. Studies demonstrated that human trophoblast progenitor cells (hTPCs) contribute to the treatment of many diseases. Therefore, hTPCs might be a promising source to repair ischemia in cerebral ischemia models. For this purpose, we evaluated the expression of many neurogenesis markers by performing hTPC transplantation after focal cerebral ischemia in rats. METHODS: : hTPCs, isolated from the term placentae, were characterized by immunofluorescent staining and differentiated into neuron-like cells. Differentiation was confirmed with immunostaining of GFAP and NeuN proteins. Cerebral ischemia models were generated in rats via middle cerebral artery occlusion and, after 24 hours, hTPCs were injected via the tail vein. Animals were sacrificed on day 3 or day 11. Immunohistochemical analysis was performed with proteins associated with neurogenesis and neuronal development, such as DLX2, DLX5, LHX6, NGN1, and NGN2, Olig1, Olig2, and PDGFRα. RESULTS: : According to our results, hTPCs may alleviate ischemic damage in the brain and contribute to the neurogenesis after ischemia. CONCLUSIONS: : Based on our findings, this topic should be further investigated as the hTPC-based therapies may be a reliable source that can be used in the treatment of stroke and ischemia.

Effects of Human Placental Amnion Derived Mesenchymal Stem Cells on Proliferation and Apoptosis Mechanisms in Chronic Kidney Disease in the Rat.

BACKGROUND AND OBJECTIVES: The feature of chronic kidney failure (CKF) is loss of kidney functions due to erosion of healthy tissue and fibrosis. Recent studies showed that Mesenchymal stem cells (MSCs) differentiated into tubular epithelial cells thus renal function and structures renewed.Furthermore, MSCs protect renal function in CKF. Therefore, we aimed to investigate whether human amnion-derived mesenchymal stem cells (hAMSCs) can repair fibrosis and determine the effects on proliferation and apoptosis mechanisms in chronic kidney failure. METHODS AND RESULTS: In this study, rat model of CKF was constituted by applying Aristolochic acid (AA). hAMSCs were isolated from term placenta amnion membrane and transplanted into tail vein of rats. At the end of 30 days and 60 days of recovery period, we examined expressions of PCNA, p57 and Parp-1 by western blotting. Immunoreactivity of PCNA, Ki67, IL-6 and Collagen type I were detected by immunohistochemistry. Besides, apoptosis was detected by TUNEL. Serum creatinine and urea were measured. Expressions of PCNA and Ki67 increased in hAMSC groups compared with AA group. Furthermore, expressions of PARP-1 apoptosis marker and p57 cell cycle inhibitory protein increased in AA group significantly according to control, hAMSC groups and sham groups. IL-6 proinflammatory cytokine increased in AA group significantly according to control, hAMSCs groups and sham groups. Expressions of Collagen type I protein reduced in hAMSCs groups compared to AA group. After hAMSC treatment, serum creatinine and urea levels significantly decreased compared to AA group. After injection of hAMSC to rats, Masson’s Trichrome and Sirius Red staining showed fibrosis reduction in kidney. CONCLUSIONS: According to our results hAMSCs can be ameliorate renal failure.

Effect of glucocorticoids on mechanisms of placental angiogenesis.

The benefits of antenatal glucocorticoid (GC) treatment to promote human fetal lung maturation are well established. However, reports have emerged indicating that maternal exposure to high concentrations of circulating GCs alters placental and fetal development. Because many adult-onset metabolic and cardiovascular disorders have their origins in utero, the importance of prenatal conditions should be considered in detail. Therefore, this review aims to present an overview of the GC effect on placental and fetal development, specifically with regard to mechanisms of placental angiogenesis. We assumed that GC overexposure affects fetal development by altering placental angiogenesis. Disturbances in the development of the villous tree and pathological changes in the villous vascular system with insufficient uteroplacental blood flow have been linked to the pathogenesis of intrauterine growth retardation. Moreover, low birth weight is a serious risk factor known to correlate with an increased risk of adult-onset diseases. Although there have been many circumstances in which maternal GCs are elevated, we focused on exogenous synthetic GCs that are applied for therapeutic reasons. However, some questions about the use of steroids remain unanswered, which will require further studies that lead us to review alterations in placental angiogenesis under the perspective of GC overexposure.

The proliferation mechanism of normal and pathological human placentas.

The placenta, which is a regulator organ for many metabolic activities between mother and fetus, is critical in influencing the outcome of pregnancy. Therefore, fetal growth is directly related to the placental development. Placental development depends on the coordinated action of trophoblast proliferation, differentiation and invasion. Studies on cell cycle related proteins that control these events are limited. Abnormal placental development is linked to various pregnancy pathologies such as preeclampsia, intrauterine growth restriction, diabetes mellitus and gestational trophoblastic diseases. The cell cycle mechanism of human placenta should be well understood for a healthy pregnancy outcome. Moreover, how cell cycle related proteins that control placental development are affected in pregnancy pathologies is not fully understood yet. Therefore, the aim of this review is to address the currently available knowledge on cell cycle regulatory proteins involved in human placental development and on the expression differences of these proteins in pathological placentas.

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.

LIMK1 regulates human trophoblast invasion/differentiation and is down-regulated in preeclampsia.

Successful human pregnancy requires extensive invasion of maternal uterine tissues by the placenta. Invasive extravillous trophoblasts derived from cytotrophoblast progenitors remodel maternal arterioles to promote blood flow to the placenta. In the pregnancy complication preeclampsia, extravillous trophoblasts invasion and vessel remodeling are frequently impaired, likely contributing to fetal underperfusion and maternal hypertension. We recently demonstrated in mouse trophoblast stem cells that hypoxia-inducible factor-2 (HIF-2)-dependent Lim domain kinase 1 (LIMK1) expression regulates invasive trophoblast differentiation by modulating the trophoblast cytoskeleton. Interestingly, in humans, LIMK1 activity promotes tumor cell invasion by modulating actin and microtubule integrity, as well as by modulating matrix metalloprotease processing. Here, we tested whether HIF-2α and LIMK1 expression patterns suggested similar roles in the human placenta. We found that LIMK1 immunoreactivity mirrored HIF-2α in the human placenta in utero and that LIMK1 activity regulated human cytotrophoblast cytoskeletal integrity, matrix metallopeptidase-9 secretion, invasion, and differentiation in vitro. Importantly, we also found that LIMK1 levels are frequently diminished in the preeclampsia setting in vivo. Our results therefore validate the use of mouse trophoblast stem cells as a discovery platform for human placentation disorders and suggest that LIMK1 activity helps promote human placental development in utero.

The expression of cell cycle related proteins PCNA, Ki67, p27 and p57 in normal and preeclamptic human placentas.

Placenta is a transitional area making many physiological activities between mother and fetus and therefore, it is a critical organ influencing the outcome of pregnancy. Fetal growth is directly related to placental development. Accurate placental development depends on coordinated action of trophoblasts' proliferation, differentiation and invasion. Information on cell cycle related proteins that control these events is limited and how they are affected in preeclampsia is not fully understood yet. Therefore, in this study, in order to understand the role of cell cycle regulators in preeclamptic placentas we aimed to determine the spatio-temporal immunolocalizations of cell cycle regulators in preeclamptic and normal human term placentas. Term placentas were obtained from women diagnosed with preeclampsia and from normal pregnancies with informed consent following cesarean deliveries. Placental samples were stained via immunohistochemistry with PCNA, Ki67, p27, p57, vimentin and cytokeratin 7 antibodies and were examined by light microscopy. PCNA and Ki67 staining intensities significantly increased in villous parts, significantly decreased in basal plates of PE group and did not change in chorionic plates. Staining intensities of cell cycle inhibitors p27 and p57 significantly increased in all parts of preeclamptic placentas compared to control. Placental abnormalities of preeclamptic placentas might be associated with proliferation and cell cycle arrest mechanisms' alterations occurred in preeclampsia.

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.

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.

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.

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. In the present study, HPECs were cultured 24 h in the presence or absence of 0.5, 5 and 50 µmol · l(-1) of synthetic GC triamcinolone (TA). The glucose carrier proteins GLUT 1, GLUT 3 and GC receptor (GR) were detected in the HPECs. We showed increased expression of GLUT 1 and GLUT 3 proteins and messenger RNA (mRNA) levels (p < 0.05) after 24-h cell culture in the presence of 0.5, 5 and 50 µmol · l(-1) of TA. In contrast, GR protein and mRNA expressions were down-regulated (p < 0.05) with 0.5, 5 and 50 µmol · l(-1) of TA 24-h cell culture. The results demonstrate that GCs are potent regulators of placental GLUT 1 and GLUT 3 expression through GR. Excessive exposure to GCs causes maternal and fetal hypoglycemia and diminished fetal growth. We speculate that to compensate for fetal hypoglycemia and diminished fetal growth, the expression of placental endothelial glucose transporters might be increased.

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.

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.

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 expression of Akt and ERK1/2 proteins decreased in dexamethasone-induced intrauterine growth restricted rat placental development.

The placenta is a complicated tissue that lies between maternal and fetal compartments. Although the architecture of the human and rodent placentas differ a little in their details, their overall structures and the molecular mechanisms of placental developments are thought to be very similar. In rats, fetal-placental exposure to maternally administered glucocorticoids decreases birth weight and placental weight. The mechanism underlying the placental growth inhibitory effects of glucocorticoids have not been elucidated. Moreover it is still not determined that how Akt and ERK1/2 proteins related proliferation and apoptosis mechanisms are influenced by dexamethasone-induced IUGR (Intrauterine Growth Restriction) placentas. 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 dexamethasone treated placental development in pregnant Wistar rats. Pregnant rats were subcutaneously injected with 100 µg/kg dexamethasone 21-acetate in 0.1 ml 10% ethanol on day 10 and 12 of gestation. Afterwards injection was continued as 200 µg/kg until they were killed on day 12 (injection started on day 10), 14, 16, 18 and 20 (injections started on day 12) of pregnancy. Placental and embryonal tissues were collected for immunohistochemistry and Western blot analysis. We found that maternal dexamethasone treatment led to a decrease in ERK1/2 and Akt activation during rat placental development. The decrease in Akt and ERK1/2 activations may result with cell survival inhibition or apoptosis stimulation. Hence, dexamethasone induced placental and embryonal developmental abnormalities could be associated with reduction of Akt and ERK1/2 activation.