Adhesion of endometrial cells labeled with 111 Indium-tropolonate to peritoneum: a novel in vitro model to study endometriosis

Objective: To evaluate, in a new original in vitro assay, putative factors that could modulate the adhesion of endometrial cells to peritoneum. Design: Prospective, controlled in vitro study. Setting: Academic research laboratory. Patient(s): Fourteen nonmenopausal women undergoing hysterectomy or laparoscopy for benign gynecologic indication. Intervention(s): Endometrial cells obtained from women with regular cycles without endometriosis were labeled with (111)Indium and confronted in vitro with mouse peritoneum in the presence of various cytokines and/or antiadhesive compounds. Main Outcome Measure(s): Radioactivity in (111)Indium-labeled endometrial cells. Result(s): The adhesion of human endometrial cells to mouse peritoneum was increased by treatment with pro-inflammatory cytokines (interleukin IL-1 beta, IL-6, TNF alpha, TGF-beta1). Whereas heparan sulfate had no effect on cell adhesion, a gel of ferric hyaluronate (Intergel) was able to counteract the pro-adhesive effect of cytokines. Interestingly, the pretreatment of peritoneum with cytokines, 24 hours before cell seeding in the presence of the ferric hyaluronate gel, restored the cytokine-promoting effect on cell adhesion. Conclusion(s): Proinflammatory cytokines promote the in vitro peritoneal adhesion of endometrial cells. An antiadhesive hyaluronate gel used in clinics decreases the adhesion in a dose-dependent manner and reduces cytokine bioavailability. (Fertil Steril((R)) 2003;79(Suppl 1):724-9. (C) 2003 by American Society for Reproductive Medicine.).Peer reviewe

In vitro and in vivo evaluation of [I-123]-VEGF(165) as a potential tumor marker

peer reviewedOne of the research challenges in oncology is to develop new biochemical methods for noninvasive tumor therapy evaluation to determine,whether the chemotherapeutics is effective. Vascular endothelial growth factor (VEGF) was labeled with radioiodine and evaluated in vitro as well as in vivo, using A2058, a melanoma cell line overexpressing VEGFR-1 and -2. Saturation binding analysis with [I-125]-VEGF resulted in a K-d of 0.1 nM. Internalization assays indicate the preserved ligand induced internalization and metabolization of the tracer. Biodistribution studies with [I-123]-VEGF in wild type and A2058 tumor-bearing athymic mice showed low background activity and a tumor to reference tissue ratio of maximum 6.12. These results suggest that [I-123]-VEGF is a potentially suitable tracer for tumor therapy evaluation. (c) 2005 Elsevier Inc. All rights reserved

Hypoxia is responsible for soluble vascular endothelial growth factor receptor-1 (VEGFR-1) but not for soluble endoglin induction in villous trophoblast

peer reviewedBACKGROUND: Pre-eclampsia is a pregnancy disorder characterized by a maternal endothelial cell dysfunction associated with low levels of circulating placental growth factor (PlGF) and increased levels of total vascular endothelial growth factor (VEGF), soluble VEGF receptor-1 (sVEGFR-1), and soluble endoglin, a transforming growth factor b1 and 3 coreceptor. Here, we tested the hypothesis that these altered levels of angiogenic cytokines and of the anti-angiogenic soluble forms of cytokine receptors could be the consequence of hypoxia. METHODS: Normal human umbilical vein endothelial cells, immortalized first trimester extravillous trophoblast cells (HTR8/SVneo) and first trimester placental villi explants (8–14 weeks) were used for culture under normoxia (20% O2) or hypoxia (1% O2). Culture media were collected for the measurement of cytokines by enzyme-linked immunosorbent assay. Total RNA was extracted for RT-PCR analysis. RESULTS: Under hypoxia, villous trophoblast expressed higher levels of VEGF, VEGFR-1, sVEGFR-1 and VEGFR-2 mRNAs (P < 0.001), and secreted more VEGF and sVEGFR-1 proteins (P < 0.05). In contrast, PlGF mRNA and protein were decreased in 1% O2 (P < 0.001), whereas endoglin (Eng) was not modulated. Additionally, sVEGFR-1 directly abolished VEGF/PlGF-induced angiogenesis in the rat aortic ring assay. CONCLUSIONS: Our results support the hypotheses that, in pre-eclampsia, (i) overproduction of VEGF family factors by pre-eclamptic placenta is a consequence of induced hypoxia; (ii) overproduction of sVEGFR-1 by hypoxic villous trophoblast accounts for maternal free VEGF depletion; (iii) low circulating level of free PlGF is not only related to sVEGFR-1 overproduction, but also to hypoxia induced mRNA down-regulation; (iv) Eng is not modulated by hypoxia.

Trophoblast Invasion and Placentation: Molecular Mechanisms and Regulation

Trophoblast invasion is a key process during human placentation. This event constitutes the basis of the conversion of the uterine spiral arteries, a process which allows an adequate vascular connection between the intervillous space and the maternal blood flow. Trophoblast invasion is transient, with stringent spatial and temporal control. Preeclampsia, a leading cause of maternal and fetal mortality and morbidity, is associated with decreased, shallow trophoblastic invasion. In this article, we review the molecular mechanisms of trophoblast invasion, and its mechanisms of regulation. Insights into the etiopathogenesis of preeclampsia will also be detailed.Peer reviewe

Automated method for the determination of a new matrix metalloproteinase inhibitor in ovine plasma and serum by coupling of restricted access material for on-line sample clean-up to liquid chromatography

A fully automated liquid chromatographic method was developed for the determination of Ro 28-2653, a new synthetic inhibitor of matrix metalloproteinases (MMPs), in ovine serum and plasma. The method was based on the coupling of a pre-column packed with restricted access material, namely LiChrospher RP-8 ADS (alkyl diol silica), for sample clean-up to an analytical column containing octyl silica stationary phase. One hundred mul of biological sample, to which 2-propanol was automatically added, were injected onto the ADS pre-column, which was then washed with a washing liquid consisting of a mixture of 25 mM phosphate buffer (pH 7.0) and acetonitrile (90: 10; v/v) for 10 min. By rotation of the switching valve, the analyte was then eluted in the back-flush mode with the LC mobile phase composed of a mixture of acetonitrile and 25 mM phosphate buffer (pH 7.0) (57:43; v/v). The UV detection was performed at 395 nm. The main parameters likely to influence the sample preparation technique were investigated. The method was then validated over a concentration range from 17.5 to 1950 ng/mI, the first concentration level corresponding to the lower limit of quantitation. At this concentration level, the mean bias and the R.S.D. value for intermediate precision were -2.4% and 4.2%, respectively. (C) 2004 Elsevier B.V. All rights reserved

Human endometrial epithelial cells modulate the activation of gelatinase a by stromal cells.

peer reviewedMetalloproteinases (MMPs) are central effectors in endometrial physiology. Their production is tightly regulated by ovarian steroids and cytokines. Using zymography, we investigated MMP-2 production by human endometrial cells treated with estradiol-17beta + progesterone (E(2)+P) and by various key cytokines in endometrial physiology (IL-1beta, LIF, TGF-beta, and TNF-alpha). No gelatinase activity was detected in the culture media of epithelial cells. In basal conditions, stromal cells produced the pro form of MMP-2. MMP-2 production/activation was not directly affected by cytokine treatment. Interestingly, activated MMP-2 was only detected after treatment of stromal cells with culture medium from epithelial cells. Cytokine treatment of epithelial cells increased the capacity of conditioned medium to stimulate stromal cells to activate MMP-2. As the tissue inhibitor of MMP-2 (TIMP-2) is a regulator of gelatinase A activity, its concentration was measured by ELISA. TIMP-2 production by stromal cells was not affected by cytokines or by epithelial cell-conditioned medium. These results strongly suggest that regulation of stromal MMP-2 activation involves soluble factor(s) derived from the epithelial compartment

Stimulation of matrix metalloproteinase-9 expression in human fibrosarcoma cells by synthetic matrix metalloproteinase inhibitors.

Enhanced expression and activation of matrix metalloproteinase-2 (MMP-2) and MMP-9 have been associated with tumor progression, invasion, and metastasis. The use of synthetic MMP inhibitors to block the proteolytic activity of these enzymes recently emerged as a potential therapeutic tool to treat cancer. In this study, we report that GI129471, a synthetic broad-spectrum MMP inhibitor, efficiently reduced the in vitro invasiveness of HT1080 cells through type IV collagen, a major component of basement membranes. This reduced invasion was paralleled by a complete inhibition of pro-MMP-2 activation; however, GI129471 strongly increased the amount of secreted pro-MMP-9, which could be subsequently activated through a plasminogen-dependent mechanism. Quantitative RT-PCR and northern blot analysis revealed that GI129471 specifically increased the MMP-9 mRNA steady-state level. Moreover, transient transfection of HT1080 cells with beta-galactosidase reporter vectors containing different lengths of the 5'-flanking region of the MMP-9 gene revealed an upregulation of the transcriptional activity of the corresponding promoter. Well-known modulators of MMP-9 expression such as Il-1beta and TNF-alpha were not involved in this upregulation. These findings emphasize the complexity of the regulation of MMP expression and the requirement for a detailed characterization of the potential adverse side effects associated with the use of broad-spectrum MMPIs

Role of endocrine status and cell type in adhesion of human endometrial cells to the peritoneum in nude mice

Objective: To investigate the role of different cellular types (epithelial and stromal endometrial cells and peritoneal cells) in the ectopic implantation of endometrium and to evaluate the importance of endocrine environment on the adhesion of endometrial cells to the peritoneum. Design: Experimental prospective study. Setting: University hospital, department of cell biology. Animal(s): One hundred one nude mice. Intervention(s): Monolayer culture of human epithelial and stromal endometrial cells obtained from patients undergoing hysterectomy or laparoscopy for benign disease. Intraperitoneal injection of cells into nude mice. Main Outcome Measure(s): Two weeks after cell injection, adhesion of endometrial cells was evaluated by histological and immunohistochemical examination. Result(s): Mixed cultures of stromal and epithelial cells, but not purified epithelial or stromal cells alone, adhered to the mouse peritoneum and led to endometriotic-like nodules. Pretreatment of cells with estrogen alone or with estrogen and progestin resulted in a higher percentage of animals developing endometriotic-like nodules, whereas treatment with progestin alone did not affect endometriotic implantation. Conclusion(s): Our data indicate that the success of endometrial cell implantation is dependent on the cooperativeness between stromal and epithelial endometrial cells, as well as on the endocrine environment of endometrial cells, but not that of recipient animals. The results emphasize the role of both endometrial cell types for ectopic implantation. (C) 2002 by American Society for Reproductive Medicine.Peer reviewe

Down-Regulation of Vascular Endothelial Growth Factor by Tissue Inhibitor of Metalloproteinase-2: Effect on in Vivo Mammary Tumor Growth and Angiogenesis

The tissue inhibitor of metalloproteinases-2 (TIMP-2) has at least two independent functions, i.e., regulation of matrix metalloproteinases and growth promoting activity. We investigated the effects of TIMP-2 overexpression, induced by retroviral mediated gene transfer, on the in vivo development of mammary tumors in syngeneic mice inoculated with EF43.fgf-4 cells. The EF43.fgf-4 cells established by stably infecting the normal mouse mammary EF43 cells with a retroviral expression vector for the fgf-4 oncogene, are highly tumorigenic and overproduce vascular endothelial growth factor (VEGF). Despite a promotion of the in vitro growth rate of EF43.fgf-4 cells overexpressing timp-2, the in vivo tumor growth was delayed. At day 17 post-cell injection, the volume of tumor derived from TIMP-2-overexpressing cells was reduced by 80% as compared with that obtained with control cells. Overexpression of TIMP-2 was associated with a down-regulation of VEGF expression in vitro and in vivo, a reduction of vessel size, density, and blood supply in the induced tumors. In addition, TIMP-2 completely inhibited the angiogenic activity of EF43.fgf-4 cell-conditioned medium in vitro using a rat aortic ring model. Our findings suggest that overexpression of TIMP-2 delays growth and angiogenesis of mammary carcinoma in vivo and that down-regulation of VEGF expression may play an important role in this TIMP-2-mediated antitumoral and antiangiogenic effects. Finally the in vivo delivery of TIMP-2, as assessed by i.v. injection of recombinant adenoviruses vectors, significantly reduced the growth of the EF43.fgf-4-induced tumors. This effect of TIMP-2 was shown to be equally comparable with that of angiostatin, a known potent inhibitor of angiogenesis

New radioiodinated carboxylic and hydroxamic matrix metalloproteinase inhibitor tracers as potential tumor imaging agents

Several studies have demonstrated a positive correlation between tumor progression and expression of extracellular proteinases such as matrix metalloproteinases (MMPs). MMP-2 and MMP-9 have become attractive targets for cancer research because of their increased expression in human malignant tumor tissues of various organs, providing a target for medical imaging techniques. Radioiodinated carboxylic and hydroxamic MMP inhibitors 2-(4'-[123]iodo-biphenyl-4-sulfonylainino)-3-(1H-indol-3-yl)-propionic acid (9) and 2-(4'-[I-123] iodo-biphenyl-4-sulfonylamino)-3-(1H-indol-3-yl)-propionamide (11) were synthesized by electrophilic aromatic substitution of the tributylstannyl derivatives and resulted in radiochemical yields of 60% +/- 5% (n - 3) and 70% +/- 5% (n = 6), respectively. In vitro zymography and enzyme assays showed high inhibition capacities of the inhibitors on gelatinases. In vivo biodistribution showed no long-terin accumulation in organs and the possibility to accumulate in the tumor. These results warrant further studies of radioiodinated carboxylic and hydroxamic MNIP inhibitor tracers as potential SPECT tumor imaging agents. (C) 2004 Elsevier Inc. All rights reserved