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G E Lash

Publications and source records attributed to G E Lash.

At least 19 recordsLinked to original sources
PubMed2006

Effect of low oxygen concentrations on trophoblast-like cell line invasion.

The applicability of trophoblast-like cell lines to the study of trophoblast function has been widely debated. The present study investigated the effect of oxygen on the invasiveness, apoptosis, proliferation and secreted proteases of four different trophoblast cell lines; HTR-8/SVneo, SGHPL-4, JEG3 and JAR. All experiments were performed at 20% and 3% oxygen for 24, 48 and 72h. Immunostaining for integrins alpha1, alpha6 and beta3, cytokeratin 7 and HLA-G was used to determine the phenotype of the different cell lines. Invasion was assessed using the Matrigel invasion assay. Immunostaining for M30 and Ki67 determined levels of apoptosis and proliferation, respectively. Gelatin and casein/plasminogen zymography were performed on conditioned media to determine levels of secreted matrix metalloproteinase (MMP) 2 and MMP9 and urokinase plasminogen activator (uPA), respectively. None of the cell lines immunostained for all markers normally expressed by extravillous trophoblast cells. Invasiveness of HTR-8/SVneo and JEG3 cells cultured in 3% oxygen was increased after 24h but was inhibited by 72h in culture. Invasion of SGHPL-4 cells was inhibited after culture in 3% oxygen for 24h. Invasion by JAR cells was not affected by changes in oxygen concentration. The different cell lines also displayed different responses to culture period in 3% oxygen with respect to apoptosis, proliferation and secreted proteases. Care should be taken before results obtained using cell lines as a model for EVT are extrapolated to extravillous trophoblast cell behaviour in vivo.

Animals↗
PubMed2003

Vascular endothelial growth factor is a chemoattractant for trophoblast cells.

A role for angiogenic growth factors in trophoblast invasion has been postulated. Directional motility (chemotaxis) is an important function of trophoblast cells. We have previously shown that vascular endothelial growth factor (VEGF) increases the random movement of trophoblast cells although placental growth factor (PlGF) has no effect. Heparin inhibited this effect of VEGF. Motility of trophoblast cells has been proposed to be mediated by a nitric oxide (NO) pathway. We hypothesized that VEGF but not PlGF would be chemotactic for trophoblast cells. Chemotaxis of a first trimester extravillous trophoblast cell line, SGHPL-4, and primary isolates of first trimester and term trophoblast cells was measured using a Boyden chamber. Initial experiments to optimize the time of the experiment and identify a positive control were performed. Subsequent experiments ran for 20 h, used 0.5 per cent FBS or 10 ng/ml PDGF as negative and positive controls and were performed in triplicate. VEGF (1, 10 and 100 ng/ml+/-1 microg/ml heparin or +/-100 microM L-NAME) and PlGF (1, 10, 100 ng/ml) were tested. The chamber was placed in a 5 per cent CO(2) in air, 37 degrees C incubator. The number of cells in the lower chamber were counted. There was a dose dependent increase in chemotactic motility of the SGHPL-4 cell line and term trophoblast cells in response to VEGF. PlGF had no effect on the movement of the first trimester trophoblast cell line but did increase the motility of the term trophoblast cells in a dose dependent manner. Heparin increased the cellular motility of both cell types alone. It also further enhanced the chemoactivity of VEGF on the term trophoblast cells but not the cell line. L-NAME did not affect the VEGF-stimulated motility of the first trimester cell line. However, in the term trophoblast cells L-NAME increased the directional cellular motility in the absence of, or in the presence of low concentrations of VEGF. In conclusion, the first trimester and term trophoblast cells appeared to respond differently to the various factors tested in the present study that may reflect differential cellular function as gestation progresses.

Adult↗
PubMed2003

Effects of hypoxia on heme oxygenase expression in human chorionic villi explants and immortalized trophoblast cells.

Although hypoxia induces heme oxygenase (HO)-1 protein and mRNA expression in many cell types, hypoxia has also been shown to decrease HO-1 mRNA and protein expression. We tested the hypothesis that 24-h preexposure to hypoxia in human placental preparations suppresses HO protein expression and enzymatic function. Immortalized HTR-8/SVneo first-trimester trophoblast cells and explants of normal human chorionic villi (CV) from term placentas were cultured for 24 h in 1%, 5%, or 20% O(2). HO protein levels were determined by Western blot analysis, and microsomal HO activity was measured. HO-2 protein content was decreased by 17% and 5% in human trophoblast cells after 24-h exposure to 1% and 5% O(2), respectively, versus 20% O(2). In contrast, HO-2 protein content in CV explants was unaffected by changes in oxygenation. HO-1 protein content, which was barely detectable in both biological systems, was not affected by changes in oxygenation. Similarly, HO enzymatic activity was unchanged in both preparations after 24-h exposure to 1%, 5%, or 20% O(2). The above data do not support the hypothesis that hypoxia in the human placenta suppresses both HO protein content and HO protein function. The present observations reinforce the necessity to determine both HO protein expression and function.

Blotting, Western↗
PubMed2003

Effect of glucose and oxygen deprivation on heme oxygenase expression in human chorionic villi explants and immortalized trophoblast cells.

Although hypoxia induces heme oxygenase (HO)-1 mRNA and protein expression in many cell types, recent studies in our laboratory using human placental tissue have shown that a preexposure to hypoxia does not affect subsequent HO enzymatic activity for optimized assay conditions (20% O2; 0.5 mM NADPH; 25 microM methemalbumin) or HO-1 protein content. One of the consequences of impaired blood flow is glucose deprivation, which has been shown to be an inducer of HO-1 expression in HepG2 hepatoma cells. The objective of the present study was to test the effects of a 24-h preexposure to glucose-deprived medium, in 0.5 or 20% O2, on HO protein content and enzymatic activity in isolated chorionic villi and immortalized HTR-8/SVneo first-trimester trophoblast cells. HO protein content was determined by Western blot analysis, and microsomal HO enzymatic activity was measured by assessment of the rate of CO formation. HO enzymatic activity was increased (P < 0.05) in both placental models after 24-h preexposure to glucose-deficient medium in 0.5 or 20% O2. Preexposure (24 h) in a combination of low O2 and low glucose concentrations decreased the protein content of the HO-1 isoform by 59.6% (P < 0.05), whereas preexposure (24 h) to low glucose concentration alone increased HO-2 content by 28.2% in chorionic villi explants (P < 0.05). In this preparation, HO enzymatic activity correlated with HO-2 protein content (r = 0.825). However, there was no correlation between HO-2 protein content and HO enzymatic activity in HTR-8/SVneo trophoblast cells preexposed to 0.5% O2 and low glucose concentration for 24 h. These findings indicate that the regulation of HO expression in the human placenta is a complex process that depends, at least in part, on local glucose and oxygen concentrations.

Cell Hypoxia↗
PubMed2002

Relationship between tissue damage and heme oxygenase expression in chorionic villi of term human placenta.

Heme oxygenase (HO) catalyzes the oxidation of heme to carbon monoxide (CO), biliverdin, and iron and is thought to play a role in protecting tissues from oxidative damage. There are three isoforms of HO: HO-1 (inducible), HO-2 (constitutive), and HO-3 (unknown function). Preeclampsia is characterized by an inadequately perfused placenta and areas of tissue damage. We hypothesized that damaged areas of placentas from women with PE and uncomplicated pregnancies are associated with an alteration in HO expression. Compared with microsomes isolated from morphologically normal and peri-infarct chorionic villi of pathological placentas, microsomes from infarcted chorionic villi from the same placentas had decreased HO activity measured under optimized assay conditions. There was no correlation between microsomal HO levels and activity and tissue damage in uncomplicated pregnancies. Whereas there was no significant difference in HO-1 protein levels across all regions of uncomplicated and mildly preeclamptic pregnancies, HO-2 protein levels were decreased (P < 0.05) in peri-infarct regions and infarcted chorionic villi of mildly preeclamptic pregnancies. Immunohistochemical analysis revealed an apparent decrease in both HO-1 and HO-2 protein expression in damaged tissues. HO-1 and HO-2 were immunolocalized in the syncytiotrophoblast layer of the chorionic villi, the underlying cytotrophoblast, and in the vascular endothelium. This study suggests that the ability of the chorionic villi to oxidize heme to CO, biliverdin, and iron may be compromised in areas of tissue damage in the placenta of women with preeclampsia.

Blotting, Western↗
PubMed2002

Vascular endothelial growth factor and placental growth factor release in cultured trophoblast cells under different oxygen tensions.

The oxygen status of the placenta during pregnancy is unclear although it has been hypothesised that in pre-eclampsia large regions of the placenta are hypoxic. Circulating levels of vascular endothelial growth factor (VEGF) are increased in women with pre-eclampsia, while circulating placental growth factor (PlGF) levels are decreased. We hypothesise that secreted levels of VEGF are increased in cultures of trophoblast cells under lowered oxygen conditions while secreted levels of PlGFare alternatively regulated. Primary isolates of first trimester and term cytotrophoblasts cells were cultured in 20 and 5% oxygen for 24h. There was a significant increase in the levels of VEGF secreted fromfirst trimester and term cytotrophoblast cells cultured under lowered oxygen conditions compared to the controls while there was a significant decrease in the secreted levels of PIGF in the same cell populations (as measured by ELISA). In first trimester and term trophoblast cells the presence of VEGF (121, 165 and 189) and PlGF (132 and 152) mRNA were demonstrated in both groups by reverse transcription-polymerase chain reaction (RT-PCR). These altered levels of secreted VEGF andPIGF may be released as compensatory molecules in the pathogenesis of diseases such as pre-eclampsia and intrauterine growth restriction.

Base Sequence↗
PubMed2001

Effect of hypoxia on cellular adhesion to vitronectin and fibronectin.

Cellular invasion of extracellular matrix (ECM) occurs during normal and pathological settings. For cells to invade, they must adhere to the underlying substratum, break down barrier molecules, and detach from the substratum prior to migrating through the ECM. We previously demonstrated that incubation under reduced oxygen levels increases the in vitro invasiveness of trophoblast and breast carcinoma cells, an effect linked to elevated expression of the cell surface receptor for urokinase-type plasminogen activator (uPAR). This study examined the role of oxygen, integrins and the urokinase-type plasminogen activator (uPA) system on the adhesion of trophoblast and breast carcinoma cells to the ECM molecules vitronectin and fibronectin. Compared to exposure to 20 and 5% oxygen, exposure to 1% oxygen decreased adhesion of these cells to vitronectin and fibronectin, an effect that was reversible by re-exposure to 20% oxygen. Incubation in 1% oxygen also resulted in reduced expression of surface alpha(5) integrin. Furthermore, adhesion to vitronectin and fibronectin was reduced by compounds that interfere with integrin function, such as EDTA, anti-integrin antibodies, or by antibodies that interfere with the binding of pro-uPA to uPAR, soluble uPAR, soluble vitronectin, phosphatidylinositol-specific phospholipase C, as well as plasminogen activator inhibitor-1. These findings suggest an important role for oxygen in the regulation of cellular invasion, possibly in part through its effects on integrin and uPAR-mediated mechanisms of adhesion.

Cell Adhesion↗
PubMed2001

Endogenous carbon monoxide formation by chorionic villi of term human placenta.

Carbon monoxide (CO) is a novel messenger that is proposed to play a complementary role with nitric oxide in the regulation of placental haemodynamics. In a previous study, CO formation from exogenous haem has been measured in the microsomal fraction of chorionic villi as an index of haem oxygenase activity. The objective of the present study was to determine whether endogenous CO is formed by dissected chorionic villi of term human placenta, to which no exogenous substrate or co-factor had been added. Each sample of freshly isolated chorionic villi (approximately 0.4 g) of term human placenta from caesarean delivery was incubated in a sealed vial containing 1 ml of Krebs' solution (pH 7.4) at 37 degrees C. CO formation was determined by quantitating, using a gas-chromatographic method, the amount of CO released into the headspace gas of the incubation vial. There was time-dependent formation of endogenous CO in chorionic villi incubated at 37 degrees C during a 60-min time course. CO formation was found to be minimal in chorionic villi samples incubated at 4 degrees C and was increased relative to tissue weight. The data demonstrate that there is endogenous CO formation by chorionic villi of term human placenta.

Carbon Monoxide↗
PubMed2001

Localization and distribution of platelet activating factor receptors in the mouse ovary and oviduct during the estrous cycle and early pregnancy.

PROBLEM: This study determined platelet activating factor (PAF) binding to ovaries and oviduct tissues to identify the location of PAF tissue interaction. METHOD: Mouse ovaries and oviducts taken during the estrous cycle and days 1, 4 and 7 post-conception (pc) were analyzed by [3H]PAF binding using frozen sections and autoradiography. RESULTS: For the outer epithelium of estrous and metestrous ovaries there was no significant difference in PAF binding (P>0.05); however, both stages were significantly different from proestrous (P<0.001). Ovarian stroma PAF binding was significantly higher (P<0.001) at estrous than metestrous, with no PAF binding at proestrous. Binding of PAF to estrous ovarian follicles was significantly greater (P<0.001) than at proestrous and metestrous. Estrous oviduct binding of PAF was significantly increased for stroma, inner epithelium and lumen (P<0.001 in all cases). At days 1 and 7 pc, all ovarian tissues had the greatest PAF binding with day 4 pc failing to bind PAF except for a significant decrease in corpora luteal binding (P<0.001). Oviduct binding of PAF was greatest at day 1 pc. CONCLUSION: Ovarian and oviduct PAF receptor expression corresponds to peak embryo PAF synthesis and establishes the basis for PAF PAF receptor mediated early pregnancy signaling system.

Animals↗
PubMed2000

Investigation of an in vitro model of trophoblast invasion.

OBJECTIVES: To re-investigate methods for visualising cytotrophoblast invasion using the Matrigel invasion model. STUDY DESIGN: Cytotrophoblast cells were isolated from pooled first trimester placentae and cultured on Matrigel-coated transwells in media supplemented with either 5 ng/ml vascular endothelial growth factor (VEGF) or 10 ng/ml epidermal growth factor (EGF). Invasion was visualised using standard and confocal fluorescent microscopy. RESULTS: The purity of the enriched cytotrophoblast cells was 84-100%. Immunofluorescent cytokeratin staining examined with an inverted fluorescent microscope suggested that cytokeratin-positive cells were present on the underside of the membrane, having invaded through the Matrigel on the upper surface. However, confocal microscopy indicated that these cells did not invade through the Matrigel, and no viable cells were identified in the culture media below the membrane. CONCLUSION: These results suggest that cytokeratin-positive staining on Matrigel-coated transwells is not necessarily indicative of cell invasion, and that similar studies should be interpreted with caution depending on the method of quantification.

Biocompatible Materials↗
PubMed1999

The effects of angiogenic growth factors on extravillous trophoblast invasion and motility.

There is accumulating evidence that deficient trophoblast invasion of the placental bed spiral arteries is crucial to the pathogenesis of pre-eclampsia and intrauterine growth restriction. However, the factors which regulate the process of trophoblast invasion remain unclear. We have investigated whether extravillous trophoblast invasion and motility are mediated by the angiogenic growth factors, vascular endothelial growth factor (VEGF) and placental growth factor (PlGF). The SGHPL-4 extravillous trophoblast cell line was utilized. Expression of mRNA for the receptors of VEGF and PlGF (KDR and flt-1) was determined using the reverse transcriptase polymerase chain reaction. An in vitro model of invasion assessed the number and length of trophoblast processes invading into an extracellular matrix. The motility of cells under standard culture conditions was also quantified. The effect of the addition of VEGF and PlGF (+/-heparin) on trophoblast invasion and motility was determined. The effect of VEGF and PlGF (+/-heparin) on SGHPL-4 cell proliferation was assessed by cell counts at 24, 48 and 72 h post-addition of growth factor. The SGHPL-4 cells expressed mRNA for the flt-1 but not the KDR receptor. The addition of VEGF resulted in a significant decrease in the number of trophoblast processes formed (P< 0.02); this effect was not influenced by the addition of heparin. However, there was no effect on the length of processes formed in response to VEGF (+/-heparin). The addition of PlGF had no effect on either the number or the length of processes formed. The addition of VEGF increased the motility of the SGHPL-4 cells (P< 0.002); the addition of heparin prevented this VEGF-induced increase in motility. The addition of PlGF had no effect on SGHPL-4 motility (+/-heparin). Neither growth factor had any effect on the proliferative ability of SGHPL-4 cells. Contrary to our hypothesis, we did not find that the angiogenic growth factors, VEGF and PlGF, mediated the in vitro invasion of trophoblast cells into an extracellular matrix. However, VEGF did increase trophoblast motility. Our findings of an effect of VEGF on trophoblast motility (and possibly invasion) suggests the presence of functional receptors, which can mediate the actions of VEGF. Caution must be exercised before any extrapolation to the in vivo situation, however, it could be speculated that the increased motility in response to VEGF may be an initial response to attract trophoblast cells to the decidua, and that VEGF might then limit the degree to which trophoblast cells invade.

Cell Movement↗
PubMed1997

Synthesis of early pregnancy factor using red deer (Cervus elaphus) as a delayed implantation model.

PURPOSE: This study measured serum early pregnancy factor (EPF) in pregnant red deer (Cervus elaphus) and ascertained whether EPF synthesis is associated with implantation. METHODS: Serial serum samples were taken from mated hinds up to 42 days postconception and analyzed for EPF activity using the rosette inhibition test. EPF activity was then correlated with calving records and stages of preimplantation development. RESULTS: EPF was detected in all pregnant animals, with a twin pregnancy giving increased EPF activity. Three animals gave an EPF response following fertilization but failed to continue beyond the preimplantation embryo stage. The increase in EPF synthesis previously associated with implantation in other mammals occurred at the blastocyst stage in red deer. CONCLUSIONS: EPF synthesis in red deer (Cervus elaphus) is consistent with the preimplantation period, as occurs in other mammals. However, the second phase of the biphasic increase in early pregnancy factor production is associated with blastocyst formation, not implantation.

Animals↗
PubMed1997

Induction of early pregnancy factor activity in vitro by platelet-activating factor in mice.

The rosette inhibition test was used to determine early pregnancy factor activity in culture media from oestrous mouse ovaries and oviducts stimulated in vitro for early pregnancy factor production under different experimental conditions. Embryo conditioned media, platelet-activating factor and cortical granule release media could all stimulate the production of early pregnancy factor by oestrous mouse ovaries and oviducts. This stimulation was completely blocked by the presence of BN 52021, a platelet-activating factor receptor antagonist. This study indicates that platelet-activating factor is the 'ovum factor' released by the zygote on fertilization to initiate the synthesis of early pregnancy factor.

Animals↗
PubMedDate not supplied

Endothelial monocyte-activating polypeptide-2 is increased in pregnancy but is not further increased in preeclampsia.

OBJECTIVES: Endothelial monocyte-activating polypeptide-2 (EMAP-2) is a novel protein that demonstrates potent proinflammatory activity in vivo and in vitro. The purpose of this study was to investigate the expression of EMAP-2 in normal pregnancy and in pregnancies complicated with preeclampsia and to test the hypothesis that EMAP-2 is a causative agent of the endothelial activation of preeclampsia. METHODS: Expression of EMAP-2 in the placenta was investigated using reverse transcriptase-polymerase chain reaction to detect mRNA, and immunohistochemistry with an EMAP-2-specific polyclonal antiserum was carried out to detect protein. Levels of circulating EMAP-2 protein were measured in the blood of nonpregnant, normal pregnant, and preeclamptic individuals using a specific enzyme-linked immunoabsorbent assay and the molecular forms present assessed by Western blotting. RESULTS: EMAP-2 is transcribed and translated by the placenta troughout pregnancy and in preeclampsia and can be detected in the plasma of nonpregnant and pregnant individuals. Levels of circulating EMAP-2 antigen are raised in pregnancy compared with nonpregnant controls; however, levels in patients with preeclampsia are identical to those in normal pregnant individuals. CONCLUSION: While circulating levels of EMAP-2 are increased in pregnancy, there is no evidence that EMAP-2 is involved directly in the pathogenesis of preeclampsia.

Adult↗
PubMedDate not supplied

Vascular endothelial growth factor but not placental growth factor promotes trophoblast syncytialization in vitro.

OBJECTIVE: The object of this study was to determine the effect of epithelial growth factor (EGF), vascular endothelial growth factor (VEGF), and placental growth factor (PlGF) on the differentiation of first-trimester and term cytotrophoblasts. METHODS: The first-trimester trophoblasts were isolated from villous tissue obtained at suction termination (n = 5), and the term trophoblasts were isolated from placentas (n = 6) at elective cesarean. Cultured cells were stimulated with EGF, VEGF, or PlGF at 0.5, 5, and 50 ng/mL, in the presence or absence of N(G)-nitro-L-arginine methyl ester hydrochloride (10(-4) M). Syncytialized trophoblasts were identified by immunostaining with antidesmosomal protein and anti-cytokeratin-7, whereas nuclei were counted in each syncytia using hematoxylin. RESULTS: Without treatment, background levels of syncytialization were significantly higher in term preparations than first-trimester cells. With VEGF and EGF, the number and size of syncytia increased significantly for the first-trimester cytotrophoblasts (P <.05). Neither VEGF nor EGF had any effect on the syncytialization of cultured cells at term. Nitric oxide showed no involvement in syncytial induction, and PlGF had no effect on syncytialization of cytotrophoblasts, from either the first or third trimester. CONCLUSION: Both EGF and VEGF appeared to enhance the in vitro syncytialization of first trimester cytotrophoblasts.

Cell Differentiation↗