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J E Cartwright

Publications and source records attributed to J E Cartwright.

15 recordsLinked to original sources
PubMed2002

Dimethylarginine dimethylaminohydrolase I enhances tumour growth and angiogenesis.

Angiogenesis is a prerequisite for tumour progression and is highly regulated by growth factors and cytokines a number of which also stimulate the production of nitric oxide. Asymmetric dimethylarginine is an endogenous inhibitor of nitric oxide synthesis. Asymmetric dimethylarginine is metabolised by dimethylarginine dimethylaminohydrolase. To study the effect of dimethylarginine dimethylaminohydrolase on tumour growth and vascular development, the rat C6 glioma cell line was manipulated to overexpress the rat gene for dimethylarginine dimethylaminohydrolase I. Enhanced expression of dimethylarginine dimethylaminohydrolase I increased nitric oxide synthesis (as indicated by a two-fold increase in the production of cGMP), expression and secretion of vascular endothelial cell growth factor, and induced angiogenesis in vitro. Tumours derived from these cells grew more rapidly in vivo than cells with normal dimethylarginine dimethylaminohydrolase I expression. Immunohistochemical and magnetic resonance imaging measurements were consistent with increased tumour vascular development. Furthermore, dimethylarginine dimethylaminohydrolase activity was detected in a series of human tumours. This data demonstrates that dimethylarginine dimethylaminohydrolase plays a pivotal role in tumour growth and the development of the tumour vasculature by regulating the concentration of nitric oxide and altering vascular endothelial cell growth factor production.

Amidohydrolases↗
PubMed2002

Transforming growth factor-beta1 regulates hepatocyte growth factor-induced trophoblast motility and invasion.

During placental development extravillous trophoblasts invade the uterine wall in a tightly regulated manner dependent on both pro- and anti-invasive molecules. We have shown using the extravillous trophoblast cell line, SGHPL-4, that both cellular invasion and motility are stimulated by hepatocyte growth factor (HGF). It has previously been demonstrated that transforming growth factor=beta1 (TGF-beta1), produced by the decidua, inhibits extravillous trophoblast proliferation and invasion. It was the aim of this study to determine whether TGF-beta1 could modulate HGF-induced motility and invasion and, if so, examine the mechanism involved. TGF-beta1 significantly inhibited the growth of SGHPL-4 cells stimulated with 10 per cent serum. HGF-stimulated trophoblast cell invasion and motility were significantly inhibited by TGF-beta1. Neither HGF nor TGF-beta1 had an effect on SGHPL-4 cell growth under the conditions used for the invasion and motility experiments (0.5 per cent serum). Previous studies suggest that both HGF-stimulated trophoblast invasion and motility may be regulated by the production of nitric oxide. TGF-beta1 was found to significantly decrease HGF-induced iNOS expression therefore suggesting a novel mechanism by which TGF-beta1 could regulate motility and invasion.

Blotting, Western↗
PubMed2000

Endogenously produced nitric oxide inhibits endothelial cell growth as demonstrated using novel antisense cell lines.

Proliferation of endothelial cells is a vital component of vascular repair and angiogenesis. The endothelial cell mediator, nitric oxide (NO) has been reported both to inhibit and to promote endothelial cell proliferation. In this study we have generated cell lines which constitutively express antisense RNA to a region of inducible nitric oxide synthase (iNOS) from a murine endothelial cell line, sEnd-1. In response to stimulation with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) these antisense cells had no detectable RNA for endogenous iNOS, barely detectable iNOS protein and produced 82% less NO than did the control transfected line. Stimulation of the control transfected line caused significant NO production and inhibition of cell growth whereas for the antisense line, producing little NO in response to stimulation, proliferation remained the same as for unstimulated cells. No differences in cell death were observed between unstimulated and LPS/IFN-gamma stimulated cells. The data presented in this study directly demonstrate that NO derived endogenously from iNOS inhibits proliferation of endothelial cells. This approach overcomes problems in other studies where NO donors or non-isoform specific inhibitors of NO synthase have been used.

Animals↗
PubMed1999

Hepatocyte growth factor regulates human trophoblast motility and invasion: a role for nitric oxide.

1. The expression of hepatocyte growth factor (HGF) is essential for normal placental development although its function is unknown. In this study we examined the effect of HGF on trophoblast cell motility and invasion of fibrin gels and investigated the possible role of nitric oxide (NO) in this process. 2. The human extravillous trophoblast cell line SGHPL-4 express both the constitutive and inducible isoforms of nitric oxide synthase (NOS). 3. HGF significantly stimulates cell motility in monolayer culture, the invasion of fibrin gels and the production of guanosine 3':5'-cyclic monophosphate (cyclic GMP). 4. Invasion, motility and cyclic GMP production were inhibited by Ng-monomethyl-L-arginine (L-NMMA). 5. Cell motility was also significantly inhibited by the inducible NOS specific inhibitor 1400 W. 6. Neither 8 Br-cyclic GMP nor the NO donor spermine-NO had any significant effect on basal trophoblast cell motility. 7. The data presented in this study demonstrate a direct effect of trophoblast-derived NO synthesis on trophoblast cell function and support the idea that HGF is involved in the regulation of trophoblast invasion through mechanisms that involve the production of NO. However neither exogenous NO nor activation of cyclic GMP-dependent pathways alone are sufficient to stimulate trophoblast cell motility.

Arginine↗
PubMed1999

Regulation of macrophage nitric oxide synthesis by endothelial cells: a role for NG,NG-dimethylarginine.

NG,NG-dimethylarginine is an endogenous inhibitor of nitric oxide synthesis produced by endothelial cells and found in the plasma and urine of normal adults. We have examined the ability of NG, NG-dimethylarginine, produced by endothelial cells (SGHEC-7), to regulate the production of nitric oxide by lipopolysaccharide-stimulated mouse macrophage cells (J774.2). Stimulation of SGHEC-7 or J774.2 cells with lipopolysaccharide had no effect on their release of NG,NG-dimethylarginine into the culture supernatant. Stimulation of J774.2 cells with lipopolysaccharide for 24 h significantly stimulated nitric oxide production by J774.2 but not SGHEC-7 cells. When lipopolysaccharide-stimulated J774.2 cells were co-cultured with endothelial cells for 24 h, there was a significant inhibition of nitrite accumulation. The inhibition observed was dependent on the endothelial cell number (12 +/- 5% [mean +/- SEM] following incubation with 0.6 x 105 cells, up to 47 +/- 8% with 4.8 x 105 cells). The inhibitory effect of endothelial cells was prevented by incubation with increasing concentrations of L-arginine; the IC50 was 2.9 +/- 0.6 mM arginine. Western blot analysis indicated that the expression of inducible nitric oxide synthase was not inhibited by co-culture with SGHEC-7 cells. The results presented here demonstrate that NG,NG-dimethylarginine synthesized by endothelial cells may inhibit nitric oxide synthase in adjacent cells and play a role in the regulation of nitric oxide synthesis by macrophages.

Animals↗
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

Endothelial cell adhesion molecule expression and lymphocyte adhesion to endothelial cells: effect of nitric oxide.

Interactions between circulating leukocytes and vascular endothelial cells are of fundamental importance in controlling normal recirculation and migration of cells into sites of inflammation. Nitric oxide (NO), which is synthesized by vascular endothelial cells, has been reported to decrease the binding of platelets, monocytes, macrophages, and neutrophils to endothelial cells. Using NO donors and inhibitors of the enzyme NO synthase, we found no evidence that physiologically relevant levels of NO alter adhesion of purified lymphocytes to an endothelial cell line derived from human umbilical vein endothelial cells (SGHEC-7). In addition, NO donors did not alter the cell surface expression of VCAM-1, ICAM-1, or E-selectin on SGHEC-7 cells.

Cell Adhesion↗
PubMed1997

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

1. 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. 2. 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. 3. Messenger RNA derived from the transfected sequences was detected by a specific cDNA probe. Cells expressing sense and antisense iNOS RNA were characterized further. 4. 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 micrograms 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. 5. 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 and anti-adhesive role of NO, produced by macrophages during the 15 min of the assay, in adhesion to endothelial cells. 6. 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. 7. 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.

Animals↗
PubMed1995

The expression and release of adhesion molecules by human endothelial cell lines and their consequent binding of lymphocytes.

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; CD54) which increased after stimulation with tumor necrosis factor-alpha (TNF alpha), interleukin-1 beta (IL-1 beta), or interferon-gamma (IFN-gamma). Vascular cell adhesion molecule-1 (VCAM-1; CD106) and E-selectin (CD62E) were not detectable on unstimulated SGHEC but substantial levels were expressed after stimulation with either TNF alpha or IL-1 beta but not with IFN-gamma. The increased expression of ICAM-1 and VCAM-1 was evident after 4 h stimulation and was even higher after 24 h; E-selectin was maximal after 4 h and returned almost to basal levels by 24 h. Substantial quantities of immunoreactive ICAM-1 and VCAM-1 also accumulated as soluble material in the supernatants of TNF alpha-stimulated SGHEC (VCAM-1 was substantially higher than ICAM-1), but E-selectin remained below the limits of detection. Various quantitative data suggest that this is a controlled release regulated by cytokine and provide support for a physiological function for these soluble molecules. Primary human lymphocytes and lymphoblastoid cell lines expressing lymphocyte function-associated antigen-1 (LFA-1) bound to SGHEC; this binding increased substantially after activation of either cell type. The binding was inhibited by monoclonal antibodies against LFA-1 and, to a lesser extent, ICAM-1, thus demonstrating the importance of these molecules in the observed binding; neither anti-VCAM-1 nor anti-E-selectin antibodies affected the binding. From these various data, we conclude that LFA-1/ICAM-1 interactions are partially responsible for the binding of lymphocytes to endothelial cells. The SGHEC lines should prove useful in investigating leukocyte-endothelial interactions and the mechanism of release of soluble adhesion molecules.

Cell Adhesion↗
PubMed1990

Cytokines in skin lesions of psoriasis.

Cytokine levels were compared in aqueous extracts of stratum corneum from psoriatic lesions and normal heel. Samples from heel contained high levels of interleukin-1 alpha (IL-1 alpha) and beta measured in immunoassays, although only the IL-1 alpha was biologically active. No other cytokines could be detected in heel samples. Interleukin-1 (IL-1) levels were dramatically reduced in lesional samples. A neutrophil chemoattractant was found in all lesional extracts, and was demonstrated to be mainly interleukin-8 (IL-8) using a specific neutralizing antiserum. Tumor necrosis factor alpha (TNF-alpha) and beta (TNF-beta), and interferon alpha (IFN-alpha) and gamma (IFN-gamma) were detected in lesional extracts using immunoassays, however, no equivalent biological activities could be detected. Interleukins 2 (IL-2), 4 (IL-4), and 6 (IL-6), granulocyte and granulocyte/macrophage colony stimulating factor (GM-CSF), could not be detected in any samples. IL-8 is therefore the only biologically active cytokine shown in this study to be elevated in psoriatic lesional extracts, and may therefore play a role in the pathogenesis of the disease.

Chemotaxis, Leukocyte↗
PubMed1989

Demonstration of cytokines in biological medicines produced in mammalian cell lines.

Commercially produced biological medicines may contain cytokines secreted by mammalian cell lines. Several such cell lines were found to produce interleukin 6 and, after stimulation, to secrete interleukin 1, tumour necrosis factor, granulocyte colony-stimulating factor, and granulocyte-macrophage colony-stimulating factor. High levels of interleukin 6 were detected in several vaccines and rDNA-derived proteins, and certain vaccines contained interleukin 1, granulocyte colony-stimulating factor, and granulocyte-macrophage colony-stimulating factor. Some preparations of human monoclonal antibodies were also found to contain interleukin 1 and tumour necrosis factor. Cytokines may contribute to certain types of adverse reactions to these products.

Animals↗
PubMedDate not supplied

Trophoblast invasion of spiral arteries: a novel in vitro model.

Extravillous trophoblasts invade the uterine wall (interstitial invasion) and the spiral arteries (endovascular invasion), replacing the cells of the vessel wall and creating a high-flow low-resistance vessel. We have developed a novel model to allow the interactions between the invading trophoblast cells and the cells of the spiral artery to be directly examined. Unmodified (non-placental bed) spiral arteries were obtained from uterine biopsies at caesarean section. Fluorescently labelled trophoblasts were seeded on top of artery segments embedded in fibrin gels (to study interstitial invasion) or perfused into the lumen of arteries mounted on a pressure myograph (to study endovascular invasion). Trophoblasts were incubated with the vessels for 3-5 days prior to cryo-sectioning. Both interstitial and endovascular interactions/invasion could clearly be detected and a comparison of the extravillous trophoblast cell line, SGHPL-4 and primary first trimester cytotrophoblasts showed both to be invasive in this model. This novel method will prove useful in an area where in vitro studies have been hampered by the lack of suitable models directly examining cellular interactions during invasion.

Adult↗