Effects of transcranial direct current stimulation on the glutamatergic pathway in the male rat hippocampus after experimental focal cerebral ischemia.

This study aimed to assess the focal cerebral ischemia-induced changes in learning and memory together with glutamatergic pathway in rats and the effects of treatment of the animals with transcranial Direct Current Stimulation (tDCS). One hundred male rats were divided into five groups as sham, tDCS, Ischemia/Reperfusion (IR), IR + tDCS, and IR + E-tDCS groups. Learning, memory, and locomotor activity functions were evaluated by behavioral experiments in rats. Glutamate and glutamine levels, alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate receptor (AMPAR1), N-Methyl-D-Aspartate receptors (NMDAR1 and NMDAR2A), vesicular glutamate transporter-1 (VGLUT-1), and excitatory amino acid transporters (EAAT1-3) mRNA expressions in hippocampus tissues were measured. Ischemic areas were analyzed by TTC staining. The increase was observed in IR + tDCS, and IR + E-tDCS groups compared to the IR group while a significant decrease was observed in IR group compared to the sham in the locomotor activity, learning, and memory tests. While glutamate and glutamine levels, AMPAR1, NMDAR1, NMDAR2A, VGLUT1, and EAAT1 mRNA expressions were significantly higher in IR group compared to the sham group, it was found to be significantly lower in IR + tDCS and IR + E-tDCS groups compared to the IR group. EAAT2 and EAAT3 mRNA expressions were significantly higher in IR + tDCS and IR + E-tDCS groups compared to the IR group. Ischemic areas were significantly decreased in IR + tDCS and IR + E-tDCS groups compared to the IR group. Current results suggest that tDCS application after ischemia improves learning and memory disorders and these effects of tDCS may be provided through transporters that regulate glutamate levels.

Effects of Hyperglycemia on Angiogenesis in Human Placental Endothelial Cells.

The placenta is a temporary organ that provides communication between the mother and fetus. Maternal diabetes and abnormal placental angiogenesis may be linked. We investigated the angiogenesis mechanism resulting from VEGF and glucose stimulation in PECs obtained from human term placenta. Immunohistochemistry was performed to characterize PECs obtained from human term placenta. D-glucose was added to the medium containing PECs to establish normoglycemic and hyperglycemic conditions. The expression levels of VEGF, VEGFR-1 and VEGFR-2 genes and proteins in PECs from the control and experimental groups were analyzed by RT-PCR and Western blotting, respectively. With 48-hours incubation, gene expressions increased due to hyperglycemia, while protein levels increased due to the combined effect of VEGF and hyperglycemia. While VEGFR-2 gene expression and protein amounts increased in 24-hours due to the combined effect of VEGF and hyperglycemia, the effect of VEGF stimulation and glucose level on VEGFR-2 decreased in 48-hour incubation with time. VEGF, VEGFR-1 and VEGFR-2 genes and proteins were affected by hyperglycemic conditions in PECs. Hyperglycemia occurring in various conditions such as gestational diabetes mellitus and diabetes mellitus may affect VEGF, VEGFR-1 and VEGFR-2 genes and proteins of PECs derived from human term placenta.

Human placental trophoblast progenitor cells (hTPCs) promote angiogenesis and neurogenesis after focal cerebral ischemia in rats.

INTRODUCTION: Reduction of blood flow below a threshold value in brain regions locally or globally is called cerebral ischemia and proper treatment requires either the restoration of normal blood flow and/or the administration of neuroprotective therapies. Human trophoblast progenitor cells (hTPCs) give rise to the placenta and are responsible for the invasion and vascular remodeling of the maternal vessels within the uterus. Here, we tested whether hTPCs promoted to differentiate along neural lineages may exhibit therapeutic properties in the setting of cerebral ischemia in vivo. MATERIALS AND METHODS: Cerebral ischemia was generated in rats via middle cerebral artery occlusion and, after 24 h, hTPCs were injected systemically via tail vein. Animals were sacrified at Day 3 or 11. RESULTS: TTC staining indicated that infarct volumes were smaller in hTPC treated animals. Visible myelin recovery was observed in the hTPC injected group with Luxol Fast Blue staining. On Day 11 after hTPC transplantation, DLX5 and VEGF expression, as well as 2 and 10 d after hTPC transplantation, NKX2.2 were significantly increased; while LHX6, Olig1, PDGFRα, VEGFR1 and VEGFR2 showed trends toward improved expression in brain tissue via immunoblot analysis. Neuron-like differentiated cells were positive for both NeuN and Cresyl Violet staining. CONCLUSION: Here, we demonstrate for the first time that hTPCs enhance the expression of angiogenic and neurogenic factors in rat brain after stroke. Transplantation of hTPCs could form the basis of novel therapeutic approaches for the treatment of stroke in the clinical setting.

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.

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.

Effects of amnion derived mesenchymal stem cells on fibrosis in a 5/6 nephrectomy model in rats.

Chronic kidney disease (CKD) is characterized by disruption of the glomerulus, tubule and vascular structures by renal fibrosis. Mesenchymal stem cells (MSC) ameliorate CKD. We investigated the effects of human amnion derived MSC (hAMSC) on fibrosis using expression of transforming growth factor beta (TGF-ß), collagen type I (COL-1) and bone morphogenetic protein (BMP-7). We also investigated levels of urinary creatinine and nitrogen in CKD. We used a 5/6 nephrectomy (5/6 Nx) induced CKD model. We used 36 rats in six groups of six animals: sham group, 5/6 Nx group, 15 days after 5/6 Nx (5/6 Nx + 15) group, 30 days after 5/6 Nx (5/6 Nx + 30) group, transfer of hAMSC 15 days after 5/6 Nx (5/6 Nx + hAMSC + 15) group and transfer of hAMSC 30 days after 5/6 Nx (5/6 Nx + hAMSC + 30) group. We isolated 106 hAMSC from the amnion and transplanted them via the rat tail vein into the 5/6 Nx + hAMSC + 15 and 5/6 Nx + hAMSC + 30 groups. We measured the expression of BMP-7, COL-1 and TGF-ß using western blot and immunohistochemistry, and their gene expressions were analyzed by quantitative real time PCR. TGF-ß and COL-1 protein, and gene expressions were increased in the 5/6 Nx +30 group compared to the 5/6 Nx + hAMSC + 30 group. Conversely, both protein and gene expression of BMP-7 was increased in 5/6 Nx + hAMSC + 30 group compared to the 5/6 Nx groups. Increased TGF-ß together with decreased BMP-7 expression may cause fibrosis by epithelial-mesenchymal transition due to chronic renal injury. Increased COL-1 levels cause accumulation of extracellular matrix in CKD. Levels of urea, creatinine and nitrogen were increased significantly in 5/6 Nx + 15 and 5/6 Nx + 30 groups compared to the hAMSC groups. We found that hAMSC ameliorate CKD.

Does fresh or frozen embryo transfer affect imprinted gene expressions in human term placenta?

Our research aimed to compare the epigenetic alterations between placentae of in vitro fertilization (IVF) patients and spontaneous pregnancies. Additionally, the expression levels of proliferation markers (PCNA, Ki67) and glucose transporter proteins (GLUT1, GLUT3) were assessed in control and IVF placentae to examine the possible consequences of epigenetic alterations on placental development. Control group placentae were obtained from spontaneous pregnancies of healthy women (n = 16). IVF placentae were obtained from fresh (n = 16) and frozen (n = 16) embryo transfer pregnancies. A group of maternal and paternal imprint genes H19, IGF2, IGF2, IGF2R, PHLDA2, PLAGL1, MASH2, GRB10, PEG1, PEG3, and PEG10 were detected by Real-Time PCR. Additionally, PCNA, Ki67, GLUT1, and GLUT3 protein levels were assessed by immunohistochemistry and western blot. In the fresh embryo transfer placenta group (fETP), gene expression of paternal PEG1 and PEG10 was upregulated compared with the control group. Increased gene expression in paternal PEG1 and maternal IGFR2 genes was detected in the frozen embryo transfer placenta group (FET) compared with the control group. Conversely, expression levels of H19 and IGF2 genes were downregulated in the FET group. On the other hand, GLUT3 and PCNA expression was increased in FET group placentae. IVF techniques affect placental imprinted gene expressions which are important for proper placental development. Imprinted genes are differently expressed in fresh ET placentae and frozen ET placentae. In conclusion, these data indicate that altered imprinted gene expression may affect glucose transport and cell proliferation, therefore play an important role in placental development.

Rapamycin administration during normal and diabetic pregnancy effects the mTOR and angiogenesis signaling in the rat placenta.

OBJECTIVE: The mammalian target of rapamycin (mTOR) signaling pathway has newly been recommended to be a nutrient sensor in the placenta. It is speculated that mTORC1 may be activated in diabetes, associated with increased placental nutrient availability. Thus, we aimed to investigate the mTOR signaling pathway both in diabetic and non-diabetic placenta and searched for the alterations of angiogenic factors VEGF, VEGFR1 and VEGFR2. METHODS: Streptozotocin (STZ) was administered by intravenous injection in doses of 60 mg/kg body weight and STZ injected rats were exposed to Everolimus (Rapamycin analog) and sacrificed at gestational days 14 and 20. mTORC1 and mTORC2 target proteins and angiogenic factors were analyzed at protein and mRNA levels in the placenta. Soluble VEGF A and Insulin protein levels were determined in blood serum. RESULTS: Placenta and embryo weights were altered after STZ and/or Rapamycin administration. mTOR pathway inhibition was confirmed by decreased p70S6K (Thr389) phosphorylation levels. We found that maternal diabetic environment led to an increase in Akt phosphorylation at 14th and decrease at 20th gestational days. Serum levels of Insulin in 14 th and 20 th days of gestation were decreased in Rapamycin and diabetic groups. On the other side serum levels of Soluble VEGF were increased in 14 th and decreased in 20 th days of pregnancy. CONCLUSION: According to our results, it might be suggested that angiogenesis related proteins will be related with placental growth regulation and mTOR may be a candidate pathway mediating the process in normal and diabetic pregnancy.

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.

Human Trophoblast Progenitor Cells Express and Release Angiogenic Factors.

Trophoblast stem cells develop from polar trophectoderm and give rise to the cells that generate the placenta. Trophoblast cells are responsible for the uterine invasion and vascular remodeling during the implantation of the embryo. However this knowledge is not yet to be confirmed for trophoblast progenitor cells (TPCs). In this study, we aimed to demonstrate that human TPCs (hTPCs) express and release angiogenic factors for the first time. TPCs were isolated from the term placenta. Then immunophenotyping was performed by FACS method by analyzing caudal type homeobox 2 (CDX2) and eomesodermin (EOMES). Immunofluorescence staining of CDX2 and EOMES was performed on these cells. Lastly, angiogenesis-related proteins were detected by western blot and ELISA assays. The isolated cells were positive for trophoblast stem cell markers CDX2 and EOMES in 92.5% and 92.7% of cells, respectively showing the characteristics of TPCs. The investigation of vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor 1 (VEGFR1), and vascular endothelial growth factor receptor 2 (VEGFR2) at protein and mRNA level in comparison with human umbilical vein endothelial cells (HUVECs), revealed that human TPCs (hTPCs) have higher levels of VEGF and VEGFR1 transcripts. Additionally soluble forms of VEGF and VEGFR1 were detected in supernatants of hTPCs. With this information, TPCs seem to be promising for regenerative cell therapies by increasing angiogenesis.