G.St.J. Whitley

Cellular and Molecular Regulation of Spiral Artery Remodelling: Lessons from the Cardiovascular Field

A number of important changes take place in the maternal uterine vasculature during the first few weeks of pregnancy resulting in increased blood flow to the intervillous space. Vascular endothelial and smooth muscle cells are lost from the spiral arteries and are replaced by fetal trophoblast cells. Failure of the vessels to remodel sufficiently is a common feature of pregnancy pathologies such as early pregnancy loss, intrauterine growth restriction and pre-eclampsia. There is evidence to suggest that some vascular changes occur prior to trophoblast invasion, however, in the absence of trophoblasts remodelling of the spiral arteries is reduced. Until recently our knowledge of these events has been obtained from immunohistochemical studies which, although extremely useful, can give little insight into the mechanisms involved. With the development of more complex in vitro models a picture of events at a cellular and molecular level is beginning to emerge, although some caution is required in extrapolating to the in vivo situation. Trophoblasts synthesise and release a plethora of cytokines and growth factors including members of the tumour necrosis factor family. Studies suggest that these factors may be important in regulating the remodelling process by inducing both endothelial and vascular smooth muscle cell apoptosis. In addition, it is evident from studies in other vascular beds that the structure of the vessel is influenced by factors such as flow, changes in the composition of the extracellular matrix, the phenotype of the vascular cells and the local immune cell environment. It is the aim of this review to present our current knowledge of the mechanisms involved in spiral artery remodelling and explore other possible pathways and cellular interactions that may be involved, informed by studies in the cardiovascular field.

Synthesis of NG, NG Dimethylarginine by Human Endothelial Cells

Endothelial cells synthesise nitric oxide from L-arginine and this process can be inhibited by NG, NG dimethylarginine (asymmetric dimethylarginine; ADMA), a constituent of plasma and urine. In the present study cultures of human umbilical vein endothelial cells have been shown to release ADMA. Over 7 days 5.1 ± 2.8; μg ADMA was produced by 107 cells (n = 8). These results suggest the presence of a metabolic pathway that may regulate NO synthesis within the endothelium.

Inhibition of nitric oxide synthase by antisense techniques: investigations of the roles of NO produced by murine macrophages.

An antisense approach to block nitric oxide (NO) synthesis was developed, complementing the widely used chemical inhibitors and overcoming problems associated with their use in studying the roles of NO. Murine macrophage cell lines (J774.2) were generated expressing a 500 bp sequence from inducible NO synthase (iNOS) in either the antisense or sense orientation, driven by the SV40 promoter/enhancer region. Messenger RNA derived from the transfected sequences was detected by a specific cDNA probe. Cells expressing sense and antisense iNOS RNA were characterized further. The antisense lines produced 22–97% less NO than the sense lines on stimulation with lipopolysaccharide (LPS) in the range 1 ng ml−1–10 μg ml−1, as determined by nitrite production. One antisense line in particular, A10, expressed substantially less iNOS protein on LPS stimulation as determined by western blot analysis. Adhesion of the antisense line, A10, to cytokine‐stimulated murine endothelial cells (sEnd.1 line) was significantly higher than adhesion of the sense lines. There was a negative correlation between the amount of NO produced, as determined by nitrite accumulation, and the level of adhesion of the transfected lines. This indicates an anti‐adhesive role of NO, produced by macrophages during the 15 min of the assay, in adhesion to endothelial cells. This novel approach allowed the roles of NO in adhesion to be investigated with the substantial advantage that the contribution of NO produced rapidly by activated macrophages could be studied separately from that produced in a continuous manner by endothelial cells. These lines, and the extension of this approach, will be of great use in dissecting the contributions of NO produced by different cell types to its many potential functions.

SGHTL-34, a thyrotrophin-responsive immortalised human thyroid cell line generated by transfection

Normal human thyrocytes were immortalised by electroporation-mediated transfection with the plasmid pSV3neo. One resultant cell line (SGHTL-34) contains approximately 10 copies of simian virus 40 (SV40) early region incorporated into the genome and has been in continuous monolayer culture for 9 months. SGHTL-34 cells grow rapidly (doubling time 30 h) and contain cytokeratin filaments. They demonstrate a morphological change in response to thyrotrophin (TSH) and possess a TSH- and forskolin-sensitive adenylate cyclase, the thresholds for stimulation being 10 microU/ml bovine TSH- and forskolin-sensitive development of this novel human thyrocyte cell line may allow further study of the regulation of human thyroid growth and differentiation.

Expression of intercellular adhesion molecule-1 (ICAM-1) on human thyroid cell lines correlated with their binding of lymphoblasts

We have investigated the expression of intercellular adhesion molecule-1 (ICAM-1) by novel functional human thyroid cell lines (designated SGHTL). ICAM-1 is constitutively expressed and it is rapidly upregulated in response to each of the recombinant cytokines: gamma-interferon, interleukin-1 and tumour necrosis factor. This contrasts with the more slowly increased expression of major histocompatibility complex (MHC) class II antigens in response to gamma-interferon alone. We have also demonstrated binding of activated lymphocytes to SGHTL cells: this interaction is increased following treatment with these cytokines and is inhibited by monoclonal antibodies directed against ICAM-1 or lymphocyte function-associated antigen-1 (LFA-1) but not by antibodies against CD2 or MHC class II antigens. Hence, we conclude that the binding of lymphoblasts to human thyroid cells involves an LFA-1- and ICAM-1-dependent pathway as well as other basal and cytokine-inducible pathway(s). These do not appear to involve MHC class II antigens, CD2 or an LFA-1 ligand other than ICAM-1.

Live cell image analysis of cell-cell interactions reveals the specific targeting of vascular smooth muscle cells by fetal trophoblasts

In early pregnancy, fetal trophoblasts selectively invade and remodel maternal spiral arteries. A healthy pregnancy is dependent on this adaptation to allow sufficient maternal blood to reach the placenta and the developing fetus. However, little is known of the role played by trophoblasts in this adaptation process. In this study, the interactions between trophoblast cells (TC) and vascular smooth muscle cells (VSMC) were examined using novel live cell image analysis methods which allow quantitative assessment of the behaviour of these two cell types in co-culture. TC and VSMC were simultaneously tracked in co-culture and, for each cell type, directionality, speed and the cell-cell interaction were assessed. The overall migratory behaviour of TC was markedly different in the presence of VSMC with co-cultured TC migrating further with directional movement while mono-cultured TC moved more randomly. Furthermore, TC were shown to specifically target VSMC, suggesting that invading TC may initiate targeted vascular remodelling. Analysis of movement behaviour and cell-cell attraction will be useful in other co-culture systems in addition to answering important questions in the reproductive field.

Elevated glucocorticoid metabolism in placental tissue from first trimester pregnancies at increased risk of pre-eclampsia

BACKGROUND The local actions of glucocorticoids in the placenta can be modulated by 11β-hydroxysteroid dehydrogenase (11βHSD) enzymes, which catalyse inter-conversion of cortisol with its inert metabolite, cortisone, and are known to be expressed in the term placenta and decidua. However, the expression and activity of these enzymes have not been well characterised in the first trimester placenta. The aim of this study was to compare 11βHSD2 expression and activity in first trimester placental tissue from pregnancies at either relatively low or high risk of developing pre-eclampsia as determined by Doppler ultrasound. METHODS Enzyme expression was assessed by western blot analysis and immunohistochemistry while 11βHSD enzyme activities were quantified using radiometric conversion of [3H]-cortisol in the presence of NADP(+) or NAD(+). RESULTS 11βHSD2 was expressed in syncytiotrophoblast of first trimester placenta, and there was no difference in the level of expression of placental 11βHSD2 protein between 9 high pre-eclampsia risk and 14 low pre-eclampsia risk pregnancies. NAD(+)-dependent cortisol oxidation was elevated 3-fold in placental tissue from pregnancies at higher risk of pre-eclampsia than in normal pregnancies (50.9 ± 15.9 versus 18.3 ± 1.9 pmol cortisone/mg protein.10 min, n = 11 & 12, respectively; P < 0.05). CONCLUSIONS Expression of 11βHSD2 is thought to protect the fetus from exposure to maternal cortisol. While other studies have suggested that 11βHSD2 is down regulated in term pre-eclamptic placentae, our study suggests that there is increased cortisol inactivation in first trimester placenta prior to week 10 of gestation, from pregnancies at higher risk of developing pre-eclampsia.

An analogue of creatine increases TRH-stimulated prolactin secretion and phosphoinositide hydrolysis in rat pituitary tumour cells

Prolactin (PRL)-secreting GH3 cells were grown, in vitro, with the creatine analogue beta-guanidinopropionic acid (GPA) added to the culture medium. After 5 days there was a small increase in basal and greatly increased thyrotropin-releasing hormone (TRH)-stimulated PRL secretion. The site of action of GPA is at the TRH-induced hydrolysis of phosphoinositides, since increased amounts of mono, bis and tris/tetrakis inositol phosphates were found in treated cells, while the PRL secretion induced by a phorbol ester or a calcium ionophore, treatments which mimic the second messages generated by inositol phospholipid hydrolysis, were not enhanced by GPA. The mechanism by which GPA increases phospholipase C activity has not been fully elucidated but may involve the activity of a controlling G protein.

Permeability Changes in the Human Endothelial Cell Line, SGHEC-7, Caused by IL-1β, TNFα and Substance P

Vascular microvessels normally have low permeability to macromolecules. During an inflammatory reaction, however, this barrier function is lost and fluid leaks from the vessel into the tissues. Soluble mediators, such as cytokines an vasoactive hormones are thought to contribute to this process. Recent technical advances in endothelial cell (EC) culture and the production of porous membranes have allowed the study of EC permeability in vitro (Albelda et al., 1988; Burke-Gaffney and Keenan, 1993). Permeability of EC monolayer grown on porous membranes has been shown to increase following stimulation with recombinant human interleukin-1α(rHuIL-1α) and -β and recombinant human tumor necrosis factor-α(rHuTNFα) (Burke-Gaffney and Keenan, 1993). In vivo substance P dilates human arterial and venous beds in skeletal muscle (McEwan et al., 1988) and skin (Foreman et al., 1983). Substance P has also been shown to dilate isolated human large vessels where the responses seem to be endothelium dependent in arteries (Luscher and Vanhoutte, 1988) but not in all veins (Luu et al., 1992) and may contribute to oedema formation by increasing vascular permeability.

Characterization of Novel Human Endothelial Cell Lines

Leukocyte-endothelial cell interactions are centra to the normal re-circulation of lymphocytes and to pathological situations such as inflammation. In vitro studies aimed at dissecting molecular processes underlying these interactions would benefit greatly from a stable, reproducible, homogeneous source of human endothelial cells. Here we report the characterization of a novel series of human endothelial cell lines (designated “SGHEC”) regarding the expression and release of adhesion molecules and their binding of lymphocytes. SGHEC expressed significant levels of intercellular adhesion molecule-1 (ICAM-1) which increased after cytokine stimulation. Vascular cell adhesion molecule-1 (VCAM-1) and E-selectin were not detectable on unstimulated SGHEC but substantial levels were expressed after stimulation with either TNFα or IL-1β but not with IFN-γ. The increased expression of ICAM-1 and VCAM-1 was evident after 4h stimulation and was even higher after 24h; E-selectin was maximal after 4h and returned almost to basal levels by 24h. Substantial quantities of immunoreactive ICAM-1 and VCAM-1 also accumulated as soluble material in the supernatants of TNFα-stimulated SGHEC (VCAM-1) was substantially higher than ICAM-1), but E-selectin remained below the limits of detection. The quantities suggested that this release is stimulated by the cytokine and is not merely a loss of a fixed proportion of the cell-surface content, thus providing support for the idea of a physiological function for these soluble molecules.