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Biomedical subjects

S K Smith

Publications and source records attributed to S K Smith.

At least 19 recordsLinked to original sources
PubMed2003

Expression of the VEGF and angiopoietin genes in endometrial atypical hyperplasia and endometrial cancer.

Angiogenesis is critical for the growth and metastasis of endometrial cancer and is therefore an important therapeutic target. Vascular endothelial growth factor-A (VEGF-A) is a key molecule in angiogenesis, but the identification of related molecules and the angiopoietins suggests a more complex picture. We investigated the presence of transcripts for VEGF-A, VEGF-B, VEGF-C, VEGF-D, Angiopoietin-1 and Angiopoietin-2 in benign endometrium, atypical complex hyperplasia (ACH) and endometrioid endometrial carcinoma using in situ hybridisation. We confirmed the presence of VEGF-A mRNA in the epithelial cells of cancers examined (13 out of 13), but not in benign endometrium or ACH. We also demonstrate, using quantitative polymerase chain reaction, that levels of VEGF-B mRNA are significantly lower in endometrial cancer than benign endometrium. We conclude that loss of VEGF-B may contribute to the development of endometrial carcinoma by modulating availability of receptors for VEGF-A.

Adult↗
PubMed2003

Vascular endothelial growth factor-D is an independent prognostic factor in epithelial ovarian carcinoma.

We assessed the presence of vascular endothelial growth factor (VEGF)-C, VEGF-D and their receptor VEGFR-3 by immunohistochemistry in 59 epithelial ovarian carcinomas, 11 borderline tumours and 20 benign cystadenomas. VEGF-C and VEGF-D were generally expressed in tumour cells and also in endothelia adjacent to tumour nests which showed a strong staining for them. VEGFR-3 was expressed in lymphatic and vascular endothelial cells adjacent to tumour nests. Immunoreactivity was significantly more frequent as lesions progressed from a benign tumour to advanced carcinoma. A strong correlation was found between VEGF-C and VEGF-D detected in carcinoma and VEGFR-3 detected in neighbouring endothelial cells. Increased expression of VEGF-C, VEGF-D and VEGFR-3 was significantly associated with lymph node metastasis and peritoneal metastasis outside the pelvis. There was a significant correlation between the high levels of VEGF-C and VEGF-D proteins, and poor survival. The presence of VEGF-D was an independent prognostic indicator by multivariate analysis. We conclude that VEGF-C, VEGF-D and VEGFR-3 play an important role in lymphatic spread and intraperitoneal tumour development in ovarian carcinoma. Since VEGF-D was found to be an independent predictor of poor outcome, its measurement, together with other prognostic markers may improve prospective identification of patients with a poor prognosis.

Adenocarcinoma, Clear Cell↗
PubMed2003

The effect of RU486 on the gene expression profile in an endometrial explant model.

Administration of RU486 in vivo during the receptive phase rapidly renders the endometrium non-receptive to the implanting embryo. In order to identify key pathways responsible for endometrial receptivity we have used cDNA arrays to monitor gene expression changes in short-term endometrial explants in response to RU486. Endometrial biopsies from five normal fertile women at mid-secretory phase were cultured in the presence of estradiol and progesterone with or without RU486 for 12 h. cDNA arrays were produced containing approximately 1000 sequence-verified clones which included genes known to be important in angiogenesis, apoptosis, cell signalling, extracellular matrix remodelling and cell cycle regulation. cDNA probes from the paired endometrial samples were hybridized to the arrays and hybridization signals were quantified. A total of 12 genes displayed significant changes in expression; six were up-regulated and six down-regulated following RU486 treatment. For five of these genes this is the first report suggesting that they are regulated by steroids in the endometrium. JAK1 and JNK1 were two of the genes shown by the arrays to be down-regulated in RU486-treated endometrial explants. This was confirmed by real time RT-PCR. JAK1 immunoreactivity was localized to both glandular epithelium and the stroma of normal endometrium and staining was much stronger in the luteal phase of the cycle. These results show that components of two important signalling pathways in endometrium-the JAK/STAT pathway, and the JNK pathway-are altered by RU486. Genes whose expression is controlled by these pathways are likely to be involved in the mechanism by which steroids render the endometrium receptive to the implanting embryo.

Adult↗
PubMed2002

Placental vessel adaptation during gestation and to high altitude: changes in diameter and perivascular cell coverage.

The objective of this study was to determine how human placental vascular structures change during gestation and whether this would be altered by external factors such as reduced ambient oxygen. To achieve this, several experiments were carried out: Vessel profile diameter was measured and the presence of perivascular cells (pericytes or smooth muscle cells) noted. This was carried out in normal human first trimester and term placentae, and in term placentae obtained from high altitude and an ethnically matched lowland population. In addition, to characterize endothelial cells in human placenta a panel of endothelial markers anti-CD 105, CD31, CD34, Von Willebrand factor (vWF), Ulex europaeus agglutinin 1 (UEA I), Peanut agglutinin (PNA), Dolichos biflorus agglutinin (DBA) and Bandeieraea simplicifolia agglutinin 1 (BS 1) was used. The proportion of vessels associated with perivascular cells rises during gestation from 37 per cent in the first trimester to 63 per cent at term (P<0.0001) and vessels with perivascular cells have a larger median diameter at term. In placentae obtained at high altitude, the vessels are dilated and are less frequently associated with perivascular cells. The absence of perivascular cells may allow remodelling of capillaries and this is likely to be physiological important in the first trimester but also under physiological or pathological stress.

Actins↗
PubMed2002

Soluble gp130 is up-regulated in the implantation window and shows altered secretion in patients with primary unexplained infertility.

Members of the IL-6 family of cytokines, which includes leukemia inhibitory factor (LIF) and IL-11, play important roles in implantation. The activity of these cytokines is modified by soluble receptors such as the IL-6 receptor (sIL-6R). gp130 is a signal transduction molecule common to the receptor complexes of this family, and its soluble form (sgp130) antagonizes their actions. The purpose of this study was to determine whether secretion of IL-6, LIF, sIL-6R, and sgp130 was different in the endometrium of women with primary unexplained infertility compared with normal fertile women. Endometrial biopsies were taken between d LH+6 and +13 and cultured in serum-free medium for 4 h. Secretion of IL-6, LIF, sIL-6R, and sgp130 was measured in the supernatant by ELISA. We also measured the secretion of IL-6, sIL-6R, and sgp130 by endometrial biopsies taken throughout the menstrual cycle in normal fertile women. Secretion of sgp130 increased 20-fold between d 20 and 26 of the cycle, coinciding with the implantation window (proliferative phase, median, 27.0 pg/ml.mg; range, 23-36; d 20-26, median, 501.5 pg/ml x mg; range, 26.1-1344; P = 0.03). RT-PCR showed that none of the known splice variants of gp130 were present in endometrium, indicating that sgp130 is produced by proteolytic cleavage of the membrane-bound form. IL-6 secretion varied considerably between patients and was greatest during the secretory phase and at menstruation. No significant change was seen in sIL-6R during the cycle. Between LH+6 and +13, secretion of sgp130 was significantly reduced in the infertile group (median, 93.1 pg/ml.mg; range, 28.5-256; compared with the fertile group, median, 223 pg/ml x mg; range, 63-534; U-statistic = 37; P = 0.017). Secretion of IL-6, LIF, and sIL-6R did not differ between the two groups. Immunolocalization of gp130, IL-6R, and the LIF receptor showed that the glandular epithelium and also endothelial cells are targets for IL-6 and LIF. These findings show that during a normal menstrual cycle, sgp130 secretion is greatly increased between d LH+6 and +13, due to proteolytic cleavage of membrane-bound gp130. Infertile patients show reduced secretion of sgp130 compared with fertile controls during this period, which coincides with the implantation window.

Adult↗
PubMed2002

Expression of CD105 (endoglin) in arteriolar endothelial cells of human endometrium throughout the menstrual cycle.

The cellular mechanisms underlying normal and pathological endometrial bleeding are not well understood, although abnormalities in the structure of endometrial blood vessels may lead to menstrual disorders. Endothelial cells in different organs are heterogeneous and differ in structure, function, antigen composition, metabolic properties and responses to growth factors. Immunostaining was performed with anti-CD105, CD31, CD34 and von Willebrand factor (vWF), and lectin binding with Ulex europaeus agglutinin 1 (UEA 1), Bandeieraea simplicifolia agglutinin 1 (BS 1), Dolichos biflorus agglutinin (DBA) and Peanut agglutinin (PNA) to characterize endothelial cells in human endometrium throughout the menstrual cycle. Serial sections fixed with formalin were stained with primary antibodies and lectins after antigen retrieval. Positive staining for CD31, CD105 and vWF was confined to the vascular endothelium. Endothelial expression of CD31 was observed in all types of vessel, including single cells, and strong staining was found during the early proliferative and mid-secretory phases. Anti-vWF stained arterioles and veins, but there was little positive staining of capillaries. In contrast, staining for CD105 was confined to the arterioles. Although anti-CD34 strongly stained endothelial cells of small vessels and capillaries, staining was also observed on some non-endothelial stromal cells. Strong positive staining for UEA 1 was observed in endothelial cells of all types of vessel throughout the menstrual cycle. Binding of PNA, DBA and BS 1 was confined to the apical region of glandular epithelial cells. This study demonstrates that the differential binding of anti-CD31, CD34, CD105, vWF and UEA 1 distinguishes between endometrial populations of endothelial cells.

Adult↗
PubMed2002

Streamlined approach to functional analysis of promoter-region polymorphisms.

We have developed a rapid method for identifying functional promoter-region polymorphisms. Using a modified pGL3 luciferase expression T-vector, we can amplify by PCR, clone, identify allelic pairs of a polymorphic gene promoter region, and prepare plasmids for cell culture 10 promoters (20 allele pairs) per week per researcher. By utilizing 96-well plate technology and an internal control plasmid expressing secreted alkaline phosphatase, each of these allele pairs can be tested for relative promoter activity in each of three cell lines (HEK293t, TE671, and JEG3) with similar resources.

Alleles↗
PubMed2001

Physical properties of erythrocyte ghosts that determine susceptibility to secretory phospholipase A2.

Artificial membranes may be resistant or susceptible to catalytic attack by secretory phospholipase A(2) (sPLA(2)) depending on the physical properties of the membrane. Living cells are normally resistant but become susceptible during trauma, apoptosis, and/or a significant elevation of intracellular calcium. Intact erythrocytes and ghosts were studied to determine whether the principles learned from artificial systems apply to biological membranes. Membrane properties such as phospholipid and/or protein composition, morphology, and microscopic characteristics (e.g. fluidity) were manipulated by preparing ghosts under different experimental conditions such as in the presence or absence of divalent cations with or without ATP. The properties of each membrane preparation were assessed by biochemical and physical means (fluorescence spectroscopy and electron and two-photon microscopy using the membrane probes bis-pyrene and laurdan) and compared with sPLA(2) activity. The properties that appeared most relevant were the degree of phosphatidylserine exposure on the outer face of the membrane and changes to the membrane physical state detected by bis-pyrene and laurdan. Specifically, vulnerability to hydrolysis by sPLA(2) was associated with an increase in bilayer order apparently reflective of expansion of membrane regions of diminished fluidity. These results argue that the general principles identified from studies with artificial membranes apply to biological systems.

Erythrocyte Membrane↗
PubMed2001

Mechanisms by which intracellular calcium induces susceptibility to secretory phospholipase A2 in human erythrocytes.

Exposure of human erythrocytes to the calcium ionophore ionomycin rendered them susceptible to the action of secretory phospholipase A(2) (sPLA(2)). Analysis of erythrocyte phospholipid metabolism by thin-layer chromatography revealed significant hydrolysis of both phosphatidylcholine and phosphatidylethanolamine during incubation with ionomycin and sPLA(2). Several possible mechanisms for the effect of ionomycin were considered. Involvement of intracellular phospholipases A(2) was excluded since inhibitors of these enzymes had no effect. Assessment of membrane oxidation by cis-parinaric acid fluorescence and comparison to the oxidants diamide and phenylhydrazine revealed that oxidation does not participate in the effect of ionomycin. Incubation with ionomycin caused classical physical changes to the erythrocyte membrane such as morphological alterations (spherocytosis), translocation of aminophospholipids to the outer leaflet of the membrane, and release of microvesicles. Experiments with phenylhydrazine, KCl, quinine, merocyanine 540, the calpain inhibitor E-64d, and the scramblase inhibitor R5421 revealed that neither phospholipid translocation nor vesicle release was required to induce susceptibility. Results from fluorescence spectroscopy and two-photon excitation scanning microscopy using the membrane probe laurdan argued that susceptibility to sPLA(2) is a consequence of increased order of membrane lipids.

Biological Transport↗
PubMed2001

Cross-protection studies with gut membrane glycoprotein antigens from Haemonchus contortus and Teladorsagia circumcincta.

Gel profiles of the peanut and ConA lectin binding integral membrane glycoproteins of Teladorsagia circumcincta and Haemonchus contortus were compared and found to be considerably different. However, some of the Teladorsagia polypeptides were recognized by antisera specific for Haemonchus amino-, metallo- or aspartyl peptidases, enzymes which are known to be protective antigens for that parasite. As expected, an experimental vaccine containing these Haemonchus proteases was extremely effective against homologous challenge, reducing egg and worm counts by more than 99% and 92%, respectively, but it did not provide any useful cross-protection against either T. circumcincta, Trichostrongylus axei or Cooperiaoncophora. A reciprocal experiment, where sheep were immunized with the equivalent glycoproteins from T. circumcincta, showed that, while they were not protected against homologous challenge, there was some cross-protection against Haemonchus as measured by a significant reduction in worm egg output.

Animals↗
PubMed2001

Fetal endocrine signals and preterm labor.

Increased uterine contractility at term and preterm results from activation and then stimulation of the myometrium. Activation can be provoked by mechanical stretch of the uterus and by an endocrine pathway resulting from increased activity of the fetal hypothalamic-pituitary-adrenal axis. Cortisol, derived from the fetal adrenal in cases of intrauterine compromise or from the maternal adrenal in response to stress, or generated locally from cortisone in choriodecidual trophoblasts, provides a crucial link to uterine stimulation. Cortisol contributes to the increased production of prostaglandins (PGs) by fetal membranes and the decidua through the upregulation of PG synthase and the downregulation of PG dehydrogenase enzymes. Cortisol also stimulates placental corticotropin-releasing hormone (CRH) output, although CRH may both relax and stimulate uterine activity depending on the distribution and affinity of its receptor subtypes. Other agents such as cytokines may intercede in this sequence to stimulate PGs and/or CRH, giving rise to a cascade phenomenon that results in preterm birth.

Corticotropin-Releasing Hormone↗
PubMed2001

Angiogenic growth factor messenger ribonucleic acids in uterine natural killer cells.

Angiogenesis is essential for endometrial growth and repair, and disruption of this process may lead to common disorders of women, including menorrhagia and endometriosis. In pregnancy, failure of the endometrial spiral arterioles to undergo remodeling leads to preeclampsia. Here we report that in addition to vascular endothelial growth factor A (VEGF-A), human endometrium expresses messenger ribonucleic acids (mRNAs) encoding VEGF-C, placenta growth factor (PlGF), the angiopoietins, angiopoietin 1 (Ang1) and Ang2, and the receptors VEGFR-3 (Flt-4), Tie 1, and Tie 2. Levels of VEGF-C, PlGF, and Tie 2 changed during the menstrual cycle. Intense hybridization for VEGF-C and PlGF mRNAs was found in uterine nature killer cells in secretory phase endometrium and for Ang2 mRNA in the same cells in the late secretory phase. Interleukin-2 (IL-2) and IL-15 up-regulated VEGF-C, but not PlGF or Ang2, mRNA levels in isolated NK cells. Conditioned medium from decidual NK cells did not induce human umbilical vein endothelial cell apoptosis. These results indicate that human endometrium expresses a wide range of angiogenic growth factors and that uterine nature killer cells may play an important role in the abnormal endometrial angiogenesis that underlies a range of disorders affecting women.

Angiogenesis Inducing Agents↗
PubMed2001

Distribution of vascular endothelial growth factor (VEGF) and its binding sites at the maternal-fetal interface during gestation in pigs.

Pigs show epitheliochorial placentation, in which the maternal uterine epithelium and the fetal trophectoderm become closely apposed. There is no invasion of trophoblast into the maternal tissue, and nutrient and waste exchange take place across two epithelial layers beneath which a complex network of capillaries forms. Later in gestation, the epithelial cells become indented by blood vessels, which greatly reduces the distance for diffusion between the two circulatory systems. Vascular endothelial growth factor is a secreted homodimeric angiogenic growth factor that is involved in physiological and pathological angiogenesis. Its receptors are generally restricted to endothelial cells. Ligand binding, in situ hybridization and immunohistochemistry were carried out in pig placenta throughout gestation to investigate the possible role of vascular endothelial growth factor and its receptors in non-invasive placentation. In situ hybridization and immunohistochemistry revealed that mRNA and immunoreactivity for vascular endothelial growth factor were localized in both maternal and fetal epithelial cells at the maternal-fetal interface and over the maternal glands, although the signal was generally weaker in the maternal glands. Ligand binding was used to localize for vascular endothelial growth factor receptors; no binding was observed over the maternal glands, but very strong binding was localized to the endometrial blood vessels. At the interface between maternal and fetal tissue, a similar pattern was observed whereby the numerous small capillaries at the bases of the two apposed epithelia bound vascular endothelial growth factor specifically. It is concluded that vascular endothelial growth factor produced by the maternal and fetal epithelial layers promotes the growth of capillaries locally, which would facilitate the development of two vascular networks for the efficient transfer of nutrients and waste products.

Animals↗
PubMed2001

The regulation and localization of angiopoietin-1, -2, and their receptor Tie2 in normal and pathologic human placentae.

BACKGROUND: Angiopoietin-1 (Ang-1) and its antagonist angiopoietin-2 (Ang-2) act on the endothelial cell Tie-2 receptor to regulate vascular integrity and remodeling. The local balance of these factors and the level of other angiogenic factors determine whether blood vessels grow, are maintained or regress. Profound angiogenesis and vascular remodeling occur in the placenta and this is altered in preeclampsia, a major cause of maternal and fetal morbidity and mortality. MATERIALS AND METHODS: The mRNAs encoding Ang-1, Ang-2, and Tie-2 were detected and localized in human placentae throughout gestation. The mechanism of regulation angiopoietin mRNAs level was determined by explant culture in ambient and reduced oxygen, and in the presence of actinomycin D. RESULTS: In situ hybridization showed that Ang-2 mRNA was abundant in the syncytiotrophoblast in the first trimester of human pregnancy. Ang-1 mRNA could not be detected by in situ hybridization, but was by reverse transcriptase-polymerase chain reaction (RT-PCR) and Northern blotting. Placental vascular structure is altered in preeclampsia and intrauterine growth restriction, conditions where feto-placental oxygenation is perturbed. In villous explant cultures, a reduction in oxygen tension significantly raised the levels of Ang-2 mRNA, and this was dependent on transcription. However, similar experiments showed that the stability of the Ang-1 message was greatly reduced under these conditions. Thus, hypoxia has opposite effects on Ang-1 and Ang-2 mRNA levels. Placentae obtained from women with preeclampsia had reduced levels of Ang-2 mRNA compared to gestationally matched controls. There was no difference in the levels of Ang-1 mRNAs. CONCLUSIONS: These data show that the relative levels of Ang-1 and Ang-2 mRNA are regulated by local oxygen tension by different mechanisms and that this may be important during normal human placentation.

Actins↗