Investigating the role of tumour cell derived iNOS on tumour growth and vasculature in vivo using a tetracycline regulated expression system.

Nitric oxide (NO) is a free radical signalling molecule involved in various physiological and pathological processes, including cancer. Both tumouricidal and tumour promoting effects have been attributed to NO, making its role in cancer biology controversial and unclear. To investigate the specific role of tumour-derived NO in vascular development, C6 glioma cells were genetically modified to include a doxycycline regulated gene expression system that controls the expression of an antisense RNA to inducible nitric oxide synthase (iNOS) to manipulate endogenous iNOS expression. Xenografts of these cells were propagated in the presence or absence of doxycycline. Susceptibility magnetic resonance imaging (MRI), initially with a carbogen (95% O2/5% CO2) breathing challenge and subsequently an intravascular blood pool contrast agent, was used to assess haemodynamic vasculature (ΔR2*) and fractional blood volume (fBV), and correlated with histopathological assessment of tumour vascular density, maturation and function. Inhibition of NO production in C6 gliomas led to significant growth delay and inhibition of vessel maturation. Parametric fBV maps were used to identify vascularised regions from which the carbogen-induced ΔR2* was measured and found to be positively correlated with vessel maturation, quantified ex vivo using fluorescence microscopy for endothelial and perivascular cell staining. These data suggest that tumour-derived iNOS is an important mediator of tumour growth and vessel maturation, hence a promising target for anti-vascular cancer therapies. The combination of ΔR2* response to carbogen and fBV MRI can provide a marker of tumour vessel maturation that could be applied to non-invasively monitor treatment response to iNOS inhibitors.

Impaired decidual natural killer cell regulation of vascular remodelling in early human pregnancies with high uterine artery resistance.

During human pregnancy, natural killer (NK) cells accumulate in the maternal decidua, but their specific roles remain to be determined. Decidual NK (dNK) cells are present during trophoblast invasion and uterine spiral artery remodelling. These events are crucial for successful placentation and the provision of an adequate blood supply to the developing fetus. Remodelling of spiral arteries is impaired in the dangerous pregnancy complication pre-eclampsia. We studied dNK cells isolated from pregnancies at 9-14 weeks' gestation, screened by uterine artery Doppler ultrasound to determine resistance indices which relate to the extent of spiral artery remodelling. dNK cells were able to promote the invasive behaviour of fetal trophoblast cells, partly through HGF. Cells isolated from pregnancies with higher resistance indices were less able to do this and secreted fewer pro-invasive factors. dNK cells from pregnancies with normal resistance indices could induce apoptotic changes in vascular smooth muscle and endothelial cells in vitro, events of importance in vessel remodelling, partly through Fas signalling. dNK cells isolated from high resistance index pregnancies failed to induce vascular apoptosis and secreted fewer pro-apoptotic factors. We have modelled the cellular interactions at the maternal-fetal interface and provide the first demonstration of a functional role for dNK cells in influencing vascular cells. A potential mechanism contributing to impaired vessel remodelling in pregnancies with a higher uterine artery resistance is presented. These findings may be informative in determining the cellular interactions contributing to the pathology of pregnancy disorders where remodelling is impaired, such as pre-eclampsia.

Fas ligand-induced apoptosis is regulated by nitric oxide through the inhibition of fas receptor clustering and the nitrosylation of protein kinase Cepsilon.

Apoptosis induced by the death-inducing ligand FasL (CD95L) is a major mechanism of cell death. Trophoblast cells express the Fas receptor yet survive in an environment that is rich in the ligand. We report that basal nitric oxide (NO) production is responsible for the resistance of trophoblasts to FasL-induced apoptosis. In this study we demonstrate that basal NO production resulted in the inhibition of receptor clustering following ligand binding. In addition NO also protected cells through the selective nitrosylation, and inhibition, of protein kinase Cepsilon (PKCepsilon) but not PKCalpha. In the absence of NO production PKCepsilon interacted with, and phosphorylated, the anti-apoptotic protein cFLIP. The interaction is predominantly with the short form of cFLIP and its phosphorylation reduces its recruitment to the death-inducing signaling complex (DISC) that is formed following binding of a death-inducing ligand to its receptor. Inhibition of cFLIP recruitment to the DISC leads to increased activation of caspase 8 and subsequently to apoptosis. Inhibition of PKCepsilon using siRNA significantly reversed the sensitivity to apoptosis induced by inhibition of NO synthesis suggesting that NO-mediated inhibition of PKCepsilon plays an important role in the regulation of Fas-induced apoptosis.

Trophoblast apoptosis is inhibited by hepatocyte growth factor through the Akt and beta-catenin mediated up-regulation of inducible nitric oxide synthase.

Excessive apoptosis of trophoblast cells is thought to be a contributing factor in complications of pregnancy such as pre-eclampsia. Hepatocyte growth factor (HGF) inhibits apoptosis in trophoblasts and we have investigated the signalling pathways through which this anti-apoptotic effect is mediated. Treatment of cells with HGF led to rapid phosphorylation of Akt while an Akt inhibitor blocked the protective effect of HGF. Glycogen synthase kinase-3beta (GSK-3beta) was found to be one of the downstream targets of Akt. HGF treatment inactivated GSK-3beta which in turn led to the activation of the transcription factor beta-catenin. Pharmacological inhibition of GSK-3beta, independently of HGF treatment, strongly increased both beta-catenin activity and cell survival, suggesting that beta-catenin alone has a pronounced anti-apoptotic effect. We also found that both HGF treatment and pharmacological activation of beta-catenin leads to increased expression of inducible nitric oxide synthase (iNOS). We suggest that the Akt mediated activation of beta-catenin leads to inhibition of trophoblast apoptosis following increased expression of iNOS.

Modulation of Graves' disease autoantibody stimulation of recombinant human thyrotrophin receptor.

BACKGROUND: The cAMP response of porcine thyrocytes to the immunoglobulin (Ig) fraction from Graves' sera is increased if these fractions are prepared with higher than usual concentrations of polyethylene glycol (PEG; 22.5% rather than 13.5%), leading to the suggestion that additional factors might exist in serum which influence the ability of autoantibodies to stimulate thyroid cells. METHODS: We characterised the stimulatory activity of fractions prepared by differential PEG precipitation of Graves' sera, using heterologous eukaryotic cells expressing recombinant human thyrotrophin (TSH) receptor. RESULTS: We found no evidence that material soluble in 13.5% PEG but precipitated by 22.5% PEG was stimulatory either on its own or in combination with the immunoglobulin-containing material precipitated by 13.5% PEG. Indeed, the stimulatory effect was reproduced by simply including low concentrations of PEG (1-4%) in the diluted sera from the majority of Graves' patients (12/18 tested) added to the target cells. Intriguingly, PEG had no effect on basal levels, with normal sera or stimulation by thyrotrophin. CONCLUSIONS: The increase in stimulation reported when using immunoglobulins prepared with higher concentrations of PEG is attributable to a direct effect of the extra PEG (which had not been removed from the preparations) during the stimulation of the cells. Because the effect is observed with recombinant heterologous cells, it must be caused by interaction between PEG and the autoantibodies and/or the receptor-it cannot involve any other thyroid-specific molecule, although an involvement of other molecules widely distributed on many cell types is possible.

A functional site on the human TSH receptor: a potential therapeutic target in Graves' disease.

OBJECTIVE: Identifying sites on the TSH-receptor that are involved in the pathological stimulation of the thyroid by autoantibodies in Graves' disease would aid the development of new therapies. We tested a series of monoclonal antibodies that recognize the native receptor for their ability to inhibit stimulation of the receptor in vitro. PATIENTS AND METHODS: Heterologous cells expressing the recombinant human TSH-receptor were stimulated with TSH or serum samples from 13 Graves' disease patients or the MRC Long-Acting Thyroid Stimulator standard B (LATS-B) and their cAMP responses measured. The effect on this stimulation of various doses of purified monoclonal antibodies with defined epitopes was determined. RESULTS: Antibodies against one epitope (residues 381-384) inhibited TSH-stimulated cyclic adenosine monophosphate (cAMP) production (1 microg/ml causing 50% inhibition of the response to 100 microU/ml TSH) and also inhibited cAMP production induced by sera from approximately 40% (6/14) of Graves' disease patients, including the MRC LATS-B standard. CONCLUSIONS: Residues 381-384 of the human TSH-receptor are important in the physiological and pathological stimulation of the thyroid. This opens the possibility of more specific therapy of some Graves' disease patients by agents directed against this epitope.

Nitric oxide protects human extravillous trophoblast cells from apoptosis by a cyclic GMP-dependent mechanism and independently of caspase 3 nitrosylation.

Apoptosis is thought to play an important regulatory role in placental development and inappropriate trophoblast apoptosis has been implicated in complications of pregnancy such as pre-eclampsia. Here we show that apoptosis of a human extravillous trophoblast-derived cell line (SGHPL-4) can be regulated by nitric oxide (NO). Nitric oxide produced exogenously by the addition of NO donors was able to delay or inhibit apoptosis induced by a combination of tumour necrosis factor alpha and actinomycin D and to suppress the activity of caspase 3. Treatment with hepatocyte growth factor (HGF) stimulated expression of the inducible isoform of NO synthase and was also able to protect SGHPL-4 cells from caspase 3 activation and apoptosis. The inhibition of basal NO production with NO synthase inhibitors was shown to sensitise cells to apoptotic stimuli and to reduce the level of endogenous caspase 3 nitrosylation. The anti-apoptotic effects of NO in these extravillous trophoblast cells appear to be mediated through the production of cyclic GMP as inhibitors of soluble guanylate cyclase inhibited the protective effect of both HGF and NO donors.

Thyroid-stimulating autoantibodies in Graves disease preferentially recognize the free A subunit, not the thyrotropin holoreceptor.

Graves disease is directly caused by thyroid-stimulating autoantibodies (TSAb's) that activate the thyrotropin receptor (TSHR). We observed upon flow cytometry using intact cells that a mouse mAb (3BD10) recognized the TSHR ectodomain with a glycosidylphosphatidylinositol (ECD-GPI) anchor approximately tenfold better than the same ectodomain on the wild-type TSHR, despite the far higher level of expression of the latter. The 3BD10 epitope contains the N-terminal cysteine cluster critical for TSAb action. Consequently, we hypothesized and confirmed that TSAb (but not thyrotropin-blocking autoantibodies [TBAb's]) also poorly recognize the wild-type TSHR relative to the ECD-GPI. Despite poor recognition by TSAb of the holoreceptor, soluble TSHR A subunits (known to be shed from surface TSHR) fully neutralized autoantibody-binding activity. These data indicate that the epitope(s) for TSAb's, but not for TBAb's, are partially sterically hindered on the holoreceptor by the plasma membrane, the serpentine region of the TSHR, or by TSHR dimerization. However, the TSAb epitope on the soluble A subunit is freely accessible. This observation, as well as other evidence, supports the concept that A subunit shedding either initiates or amplifies the autoimmune response to the TSHR, thereby causing Graves disease in genetically susceptible individuals.