Granulocyte-macrophage colony-stimulating factor promotes human blastocyst development in vitro

The cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) is synthesized in the female reproductive tract and has been implicated in the growth and development of the preimplantation embryo in rodent and livestock species. To examine the effect of GM-CSF on human embryo development in vitro, surplus frozen 2-4-cell embryos were cultured in media supplemented with 2 ng/ml recombinant human GM-CSF. The addition of cytokine increased the proportion of embryos that developed to the blastocyst stage from 30 to 76%. The developmental competence of these blastocysts, as assessed by hatching and attachment to extracellular matrix-coated culture dishes, was also improved by GM-CSF. The period in culture required for 50% of the total number of blastocysts to form was reduced by 14 h, and blastocysts grown in GM-CSF were found to contain approximately 35% more cells, due primarily to an increase in the size of the inner cell mass. The beneficial effect of GM-CSF was exerted in each of two sequential media systems (IVF-50/S2 and G1. 2/G2.2) and was independent of the formulation of recombinant cytokine that was used. These data indicate that GM-CSF may have a physiological role in promoting the development of the human embryo as it traverses the reproductive tract in vivo, and suggest that addition of this cytokine to embryo culture media may improve the yield of implantation-competent blastocysts in human in-vitro fertilization programmes.     

Placenta growth factor (PlGF) induces vascular endothelial growth factor (VEGF) secretion from mononuclear cells and is co-expressed with VEGF in synovial fluid

The aims of this study were (i) to determine whether PlGF, VEGF and PlGF/VEGF heterodimers are detected in synovial fluid (SF) and plasma samples from patients with a range of arthropathies; (ii) to describe whether any correlation exists between SF PlGF, VEGF and PlGF/VEGF heterodimer levels and the total and differential SF leucocyte counts; and (iii) to investigate the regulation of peripheral blood mononuclear cell (PBMC) VEGF secretion by stimuli relevant to inflammatory joints. PlGF, VEGF and PlGF/VEGF heterodimer levels were measured in the SF and plasma of patients with a range of arthropathies and normal controls by ELISA. Western blotting for PlGF was performed on SF from three patients with rheumatoid arthritis (RA) and primary inflammatory arthropathies. VEGF was quantified in cell culture supernatants after stimulation with lipopolysaccharide (LPS), PlGF or cobalt ions of PBMC isolated from RA patients and controls. PlGF and VEGF were detected in all SF samples. PlGF/VEGF heterodimers were detected in 10.2% of SF samples, most frequently in RA samples. Western blotting confirmed the presence of PlGF in RA SF. PlGF was detected in 52% of RA and 31% of control plasma samples, and VEGF was detected in 38% of RA and 38% of control plasma samples. PlGF/VEGF heterodimers were detected in 21% of RA samples and none of the control samples. In primary inflammatory arthropathy patients, SF PlGF and VEGF levels correlated significantly with the SF total leucocyte count and the neutrophil count. PlGF was the most potent inducer of PBMC VEGF production in both RA and control subjects. This is the first report of the detection of PlGF and PlGF/VEGF heterodimers in the SF of patients with inflammatory arthropathies, and we have shown for the first time that PlGF up-regulates PBMC VEGF production. PlGF may therefore play a key role in the production of VEGF in the inflammatory joint.     

Localisation of Hepatocyte Growth Factor and its Receptor (c-met) Protein and mRNA in Human Term Placenta

Abstract

Successful pregnancy depends upon placental growth and development, which follows a specific spatial and temporal sequence. Hepatocyte Growth Factor (HGF) is a potent mitogen, morphogen and motogen to both endothelial and epithelial cell types and is linked to a tyrosine kinase, proto-oncogene, c-met receptor. In ‘normal’ third trimester placentae (n=5) full thickness biopsies (obtained at Caesarean section), immunolocalisation and in situ hybridisation studies were performed for HGF and c-met, respectively. HGF immunoreactive protein was present in mesenchymal core, the vaculosyncytial membrane (syncytotrophoblast) and the vascular endothelial cells of villous trophoblast. The HGF mRNA was present particularly strongly in the perivascular stromal cells surrounding the villous vasculature and the amnion/chorionic membranes. Immunoreactive c-met protein was strongly localised to the endothelial cells lining the villous vasculature and the vasculosyncytial membrane. A relatively weak and diffuse hybridisation signal for c-met mRNA was present throughout the villous trophoblast, most pronounced in the vasculosyncytial membrane. These results indicate that HGF may serve as a paracrine mediator to control placental development and growth.     

The effects of group size on development and interferon-tau secretion by in-vitro fertilized and cultured bovine blastocysts.

The effects of culturing bovine embryos in groups were investigated. In the first experiment, 1000 oocytes were matured, fertilized and then cultured in groups of 40 in 25 microl of medium. From half of these groups, blastocysts were removed and cultured separately, while in the other half blastocysts were allowed to remain in the group culture microdrop. Blastocysts developed equally well in both groups, although hatching was reduced in those blastocysts removed from the culture droplet. In the second experiment, 1000 zygotes were cultured from the 8-cell stage to the blastocyst stage either individually or in groups of 40. Culture in groups increased the formation of blastocysts, the percentage of hatching blastocysts, the number of cells within blastocysts and the production of interferon-tau. In the final experiment, 1000 zygotes were cultured in groups up to the blastocyst stage. Two-thirds of these blastocysts were then cultured in groups of three, while the remaining blastocysts were cultured individually. Co-culture did not affect hatching or cell number but significantly elevated interferon-tau secretion. These results demonstrate that group culture either before or after blastocyst formation can alter the expression of a specific gene important for the establishment of pregnancy.     

Relationships between maternal plasma leptin, placental leptin mRNA and protein in normal pregnancy, pre-eclampsia and intrauterine growth restriction without pre-eclampsia

Leptin, an adipocyte hormone involved in energy homeostasis, is important in reproduction and pregnancy. Questions yet to be addressed include the source of higher leptin during pregnancy and its relationship to pregnancy outcome and fetal growth. The objective of this study was to investigate the relationship between placental leptin gene expression, placental leptin protein concentration and maternal plasma leptin concentration among control pregnant women, women with pre-eclampsia and women with growth-restricted infants. We also investigated the relationship between placental leptin expression and the placental expression of enzymes involved in cellular lipid balance: fatty acid translocase (CD36), carnitine palmitoyltransferase I (CPT-1B) and lipoprotein lipase (LPL). Placental leptin expression, placental protein and maternal plasma concentration were higher in pre-eclampsia than in controls but not in women with growth-restricted infants. Placental leptin expression and placental protein were higher in the preterm pre-eclamptic subjects, whereas maternal leptin was higher in the term pre-eclamptic subjects. The placental gene expression of CD36, CPT-1B and LPL were not different among the groups. This study suggests that despite similar failed placental bed vascular remodelling in pre-eclampsia and intrauterine growth restriction (IUGR), leptin gene expression is higher only in preterm pre-eclampsia.     

Expression of vascular endothelial growth factor (VEGF) under hypoxia in placenta with intrahepatic cholestasis of pregnancy and its clinically pathological significance

The expression of vascular endothelial growth factor (VEGF) under hypoxia in the placenta with intrahepatic cholestasis of pregnancy (ICP) was observed, and mechanisms of ICP fetal distress were discussed. Methods: Different culturing times were established in hypoxia incubator, and protein expressions of VEGF in placental tissue were observed using immunohistochemical S-P method. Results: After 4 h hypoxic culture, VEGF protein expression in ICP group was higher than the normal group with significant difference (P < 0.05). With the extension of hypoxic exposure, VEGF protein expression in both groups was suppressed, but no distinction in-between. Regression analyses indicated a noticeable effect of CG on VEGF expression, the higher the CG was, the lower the VEGF protein expression was (P < 0.05). Conclusions: Short term hypoxia induces up-regulation of VEGF expression in ICP placenta, and this adaptive change is probably a protective mechanism of fetus in ICP.     

Long-term culture of bovine trophoblastic cells

Summary Procedures for the isolation and cultivation of bovine cotyledonary trophoblastic cells from early second trimester placentas are described. Collagenase digestion yielded a single cell suspension of both uninucleate and binucleate cells with minimal contamination with other cell types. Optimal growth conditions were obtained through supplementation of medium with epidermal growth factor, insulin, transferrin, and selenium. Trophoblastic cells survived multiple passages, cryopreservation, and serum deprivation and have been maintained in culture for more than 14 mo. Two cell types were identified by phase microscopy: uninucleated trophoblastic cells and trophoblastic giant cells, which were predominantly binucleate cells. Binucleate cells were present in small numbers through all passages. This procedure provides a reliable method to obtain trophoblastic cell lines for studies of trophoblastic cell physiology and susceptibility to infectious and toxic agents.     

Synergistic protein secretion by mesenchymal stromal cells seeded in 3D scaffolds and circulating leukocytes in physiological flow

Mesenchymal stromal cells (MSC) play an important role in natural wound healing via paracrine and juxtacrine signaling to immune cells. The aim of this study was to identify the signaling factors secreted by preseeded cells in a biomaterial and their interaction with circulating leukocytes, in the presence of physiological biomechanical stimuli exerted by the hemodynamic environment (i.e. strain and shear flow). Electrospun poly(ε-caprolactone)-based scaffolds were seeded with human peripheral blood mononuclear cells (PBMC) or MSC. Protein secretion was analyzed under static conditions and cyclic strain. Subsequently, the cross-talk between preseeded cells and circulating leukocytes was addressed by exposing the scaffolds to a suspension of PBMC in static transwells and in pulsatile flow. Our results revealed that PBMC exposed to the scaffold consistently secreted a cocktail of immunomodulatory proteins under all conditions tested. Preseeded MSC, on the other hand, secreted the trophic factors MCP-1, VEGF and bFGF. Furthermore, we observed a synergistic upregulation of CXCL12 gene expression and a synergistic increase in bFGF protein production by preseeded MSC exposed to PBMC in pulsatile flow. These findings identify CXCL12 and bFGF as valuable targets for the development of safe and effective acellular instructive grafts for application in in situ cardiovascular regenerative therapies.     

The effect of epidermal growth factor on vascular endothelial growth factor secretion by endometrial stromal cells

Endometrial stromal cells undergo morphological and functional changes to facilitate oocyte implantation under regulation of various hormones and growth factors. We studied physiological induction by epidermal growth factor (EGF) of vascular endothelial growth factor (VEGF) in these cells. In human endometrial stromal cells, the effect of EGF, genistein, tryphostin AG1478 (a tyrosine kinase inhibitor), and wortmannin (a phosphatidylinositol 3-kinase inhibitor) on production of VEGF was examined: Total RNA was extracted and VEGF mRNA expression was quantified by Northern analysis. EGF induced production of VEGF by stromal cells in a time-dependent manner; the effect became significant after 12 h and increased further between 24 and 48 h (P<0.05). Dose dependency was also significant (P<0.01). Genistein, tryphostin AG1478, and wortmannin partially suppressed the increase in production induced by EGF (P<0.01, P< 0.01, P<0.01), respectively. Production of EGF by fertilized oocytes and trophoblasts has been reported in early pregnancy. VEGF is believed to be induced by EGF through mechanisms involving tyrosine kinase and phosphatidylinositol 3-kinase. The increase in VEGF may contribute to neovascularization that promotes proliferation of endometrium and placentation. Received: 6 September 2001 / Accepted: 21 May 2002     

A placenta-derived suppressor factor with a T-cell bias

PROBLEM: Functional and mechanistic aspects of immunosuppression by murine placental supernatants (MPS) were investigated. METHOD OF STUDY: MPS and a low molecular weight fraction of the supernatant (MPSf) were tested for suppressive action on T-cell reactivity in vitro and in vivo, on B-cell responses and on T-cell activation events. RESULTS: MPS and MPSf suppress mitogen-induced proliferation and mixed lymphocyte reactions of human and murine lymphocytes, antigen-induced proliferation of T cells in vitro and in vivo, proliferation of CD8+ lymphocytes, proliferation induced by cross-linking of surface CD3 and the in vivo response of mice to allogeneic stimuli. MPSf affects cell cycling of activated T cells and blocks interleukin (IL)-2 production. MPSf does not affect antibody production or the induction of MHC class II expression on B cells. CONCLUSIONS: MPSf is a potent inhibitor of T-cell responses in vitro and in vivo, with no demonstrable effect on B-cell function.     

Olanzapine plus fluoxetine treatment increases Nt-3 protein levels in the rat prefrontal cortex

Evidence is emerging for a role for neurotrophins in the treatment of mood disorders. In this study, we evaluated the effects of chronic administration of fluoxetine, olanzapine and the combination of fluoxetine/olanzapine on the brain-derived-neurotrophic factor (BDNF), nerve growth factor (NGF), and neurotrophin-3 (NT-3) in the rat brain. Wistar rats received daily injections of olanzapine (3 or 6 mg/kg) and/or fluoxetine (12.5 or 25 mg/kg) for 28 days, and we evaluated for BDNF, NGF and NT-3 protein levels in the prefrontal cortex, hippocampus and amygdala. Our results showed that treatment with fluoxetine and olanzapine alone or in combination did not alter BDNF in the prefrontal cortex (p = 0.37), hippocampus (p = 0.98) and amygdala (p = 0.57) or NGF protein levels in the prefrontal cortex (p = 0.72), hippocampus (p = 0.23) and amygdala (p = 0.64), but NT-3 protein levels were increased by olanzapine 6 mg/kg/fluoxetine 25 mg/kg combination in the prefrontal cortex (p = 0.03), in the hippocampus (p = 0.83) and amygdala (p = 0.88) NT-3 protein levels did not alter. Finally, these findings further support the hypothesis that NT-3 could be involved in the effect of treatment with antipsychotic and antidepressant combination in mood disorders.     

Expression of insulin-like growth factor in the placenta of intrauterine growth-retarded human fetuses

Many cases of intrauterine growth retardation (IUGR) are the result of placental and fetal tissue insufficiency. Insulin-like growth factor-I (IGF-I) is known to play a role in placental and fetal growth. An immunocytochemical study was performed to localize IGF-I peptides in human placenta and umbilical cords of normal (n = 3) and IUGR (n = 3) fetuses. The peripartum fetal conditions were evaluated as well. Immunoreactive IGF-I was detected in the cytotrophoblast, syncytiotrophoblast, amnion, endothelial cells of fetal capillaries and in the decidua in both normal and IUGR placental tissue. A more robust immunostaining and increased numbers of positively stained cells were found in the decidua of IUGR placenta (p < 0.001). Intense immunostaining was also found in endothelial cells, smooth muscle cells and fibroblasts of the umbilical vein. IGF-I immunoreactivity was also present in stroma (Hofbauer cells and/or fibroblasts) of IUGR villi. Our results indicate that expression of IGF-I is high in specific sites in placenta and umbilical cords, which indicates a paracrine and/or endocrine function. The increased expression of IGF-I in placenta of IUGR fetuses indicates its involvement in restoring normal growth by means of a positive feed-back mechanism.     

Influence of growth factors and cytokines on angiogenic function of endothelial progenitor cells: a review of in vitro human studies

Abstract

Growth factors and cytokines released at sites of injury and inflammation play an important role in stimulating endothelial progenitor cell (EPC) migration to these sites. A comparative analysis of the literature shows under neutral in vitro conditions (pH 7.4), several growth factors and cytokines influenced favorably indices of EPC angiogenic function. They included SDF-1, VEGF, PlGF, FGF-2, NGF and IL-1β. Others, e.g. TNF-α, have an unfavorable influence. SDF-1 and VEGF in combination increased chemotactic cell migration and reduced apoptosis caused by serum starvation. Under acidic conditions (pH 6.5), the biological activity of certain growth factors may be impaired, although TPO, SCF and IL-3 were each able to rescue EPCs from acidic exposure apoptosis, a combination of these three factors stimulated cell proliferation and prevented apoptosis. Possible combinations of growth factors and cytokines together with EPC transplantation may provide for a greater extent of vessel repair and new vessel formation.     

子痫前期患者白细胞介素-8和胎盘生长因子的表达及其意义

目的通过检测子痫前期患者羊水中白细胞介素-8（IL-8）和胎盘组织中胎盘生长因子（PLGF）的水平．以探讨IL-8和PLGF在子痫前期发病中的作用。方法采用酶联免疫吸附法和免疫组化法分别测定20例轻度子痫前期患者,22例重度子痫前期患者和30例正常妊娠妇女（对照组）的羊水中IL-8和胎盘组织中的PLGF的表达。结果子痫前期轻度组及重度组羊水IL-8均高于对照组,重度组羊水IL-8显著高于轻度组,差异均有统计学意义（P〈0．01）。子痫前期轻度组及重度组胎盘组织中PLGF表达量均低于对照组（P〈0．01）,而重度组低于轻度组（P〈0．01）。子痫前期轻、重度组及对照组胎盘组织中PLGF表达量与羊水IL-8水平均呈高度负相关（P〈0．01）。结论子痫前期患者羊水中IL-8增高,而胎盘组织中PLGF表达下降,可能与子痫前期的发生、发展有关。     

妊高征中胎盘血管内皮生长因子的表达及其与胎盘释放血管活性物质的关系

目的 探讨胎盘血管内皮生长因子（ＶＥＧＦ）在妊高征中的表达及其与胎盘释放前列环素（ＰＧＩ２）、血栓素Ａ２（ＴＸＡ２）的关系。方法 分别采用蛋白免疫印渍（Ｗｅｓｔｅｒｎ ｂｌｏｔ）和放免技术测定２５例孕晚期正常妊娠（ＮＬＰ）及２５例妊高征（ＰＩＨ）患者的胎盘ＶＥＧＦ表达及胎盘和血浆中ＰＧＩ２、ＴＸＡ２水平。结果 与ＮＬＰ比较,ＰＩＨ组胎盘ＶＥＧＦ的表达显著降低（Ｐ〈０．０１）;胎盘组织及血浆中ＰＧＩ２水平及ＰＧＩ２／ＴＸＡ２显著下降（Ｐ〈０．０１）,ＴＸＡ２水平显著升高（Ｐ〈０．０１）;ＰＩＨ中胎盘ＶＥＧＦ表达与其产生的ＰＧＩ２、ＰＧＩ２／ＴＸＡ２呈显著正相关（Ｐ〈０．０１）,与ＴＸＡ２呈显著负相关（Ｐ〈０．０１）,并与临床收缩期血压（Ｐ〈０．０１）和舒张期血压（Ｐ〈０．０５）呈显著负相关。结论 胎盘ＶＥＧＦ下降